Profiles of Drug Substances Vol 39
Profiles of Drug Substances Vol 39
Profiles of Drug Substances Vol 39
ix
CHAPTER ONE
Azithromycin
Ahmed H.H. Bakheit*, Badraddin M.H. Al-Hadiya,
Ahmed A. Abd-Elgalil
*Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Kingdom of
Saudi Arabia
Research Center, College of Pharmacy, King Saud University, Riyadh, Kingdom of Saudi Arabia
Contents
Background 2
1. Description 2
1.1 Nomenclature 2
1.2 Formulae 3
1.3 Elemental analysis 4
1.4 Appearance 4
2. Methods of Preparation of Azithromycin 4
3. Physical Characteristics 5
3.1 Specific optical rotation 5
3.2 Ionization constant 6
3.3 Solubility characteristics 6
3.4 Partition coefficient 6
3.5 Particle morphology 6
3.6 Crystallographic properties 7
3.7 Hygroscopicity 7
3.8 Thermal methods of analysis 9
3.9 Spectroscopy 12
3.10 Mass spectrometry 17
4. Methods of Analysis 17
4.1 Compendial methods of analysis 17
4.2 Electrochemical methods of analysis 24
4.3 Spectroscopic methods of analysis 25
4.4 Chromatographic methods of analysis 28
4.5 Determination in body fluids and tissues 30
5. Stability 30
6. Clinical Applications 33
6.1 An overview 33
6.2 Antimicrobial spectrum susceptibility 33
6.3 Mechanism of action 33
6.4 Resistance to macrolides 34
6.5 Actions other than antimicrobial effects 34
Profiles of Drug Substances, Excipients, and Related Methodology, Volume 39 # 2014 Elsevier Inc. 1
ISSN 1871-5125 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800173-8.00001-5
2 Ahmed H.H. Bakheit et al.
BACKGROUND
A team of researchers at the Croatian pharmaceutical company Pliva,
led by Dr. Slobodan okic, discovered azithromycin in 1980. It was pat-
ented in 1981. Pfizer launched azithromycin under Plivas license in other
markets under the brand name Zithromax in 1991. After several years, the
U.S. Food and Drug Administration approved AzaSite, an ophthalmic for-
mulation of azithromycin, for the treatment of eye infections [1].
1. DESCRIPTION
1.1. Nomenclature
1.1.1 Systematic chemical names [25]
(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-(2,6-dideoxy-3-C-3-O-
dimethyl-a-L-ribo-hexopyranosyloxy)-2-ethyl-3,4,10-trihydroxy-3,5,6,
8,10,12,14-heptamethyl-11-(3,4,6-tride-oxy-3-dimethylamino-b-D-xylo-
hexopyranosyloxy)-1-oxa-6-aza-cyclopentadecan-15-one dehydrate.
(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-
3,5,6,8,10,12,14-heptamethyl-15-oxo-11-{[3,4,6-trideoxy-3-
(dimethylamino)-b-D-xylo-]oxy}-1-oxa-6-azacyclopentadec-13-yl
2,6-dideoxy-3-C-methyl-3-O-methyl-a-L-ribo-hexopyranoside.
(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-dideoxy-3-C-
methyl-3-O-methyl-a-lribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-tri-
hydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-
(dimethylamino)-b-D-xylo-hexopyranosyl]oxy]-1-oxa-6-
azacyclopentadecan-15-one.
(2R,3S,4R,5R,8R,11R,13S,14R)-11-(((2S,3S,4S,6R)-4-(dimethylamino)-
3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-ethyl-3,4,10-
trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimeth-
yltetrahydro-2H-pran-2-yl)oxy)-3,5,6,8,10,14-hexamethyl-1-oxa-6-
azacyclopentadecan-15-one.
N-methyl-11-aza-10-deoxo-10-dihydroerythromycin A; 9-deoxo-9a-
methyl-9a-aza-homoerythromycin A; CP-62993; XZ-450; Azitrocin;
Sumamed; Trozocina; Zithromax; Zitromax.
Azithromycin 3
HO BASE HO
O OCH3 O OCH3
O O
O OH O OH
O O
(1)
pTsCl, BASE
NMe2 NMe2
H
N OH N
HO O O
HO O HO O
HO O
Reductor
HO
Agent HO
O OCH3 O OCH3
O O
O OH O OH
O O
(4) (3)
Formaldehyde
Formic acid Rh/C, H2,
Formaldehyde, acetic acid
NMe2
H3C
N OH
HO O
HO O
HO
O OCH3
O
O OH
O
(5)
Scheme 1.1 Beckmanns rearrangement to obtain the intermediary (6,9-iminoether) for
preparation of Azithromycin.
3. PHYSICAL CHARACTERISTICS
3.1. Specific optical rotation [5,21]
[a] 20
45 to 49 (anhydrous substance) (C 1 in anhydrous ethanol R)
[a]20 37 (C 1 in CHCl3)
6 Ahmed H.H. Bakheit et al.
NMe2 NMe2
O
N
OH O
HO HO O
HO O O O HO O
HO S ONH2 HO
O
O OCH3 O OCH3
O O
NaHCO3
O OH O OH
O O
(3)
(1)
NMe2
H3C
N OH
HO O
HO O
HO
O OCH3
O
O OH
O
(5)
Scheme 1.2 An alternate synthetic method for Azithromycin.
microscope, D-6000, Japan). The samples were sputter coated with gold
before examination, and they found that the internal crystal structure appears
to be the same, as is evident from the similar enthalpy of fusion for all three
samples [26].
3.7. Hygroscopicity
Azithromycin was found to exhibit pseudopolymorphism and can exist as
monohydrate and dihydrate. The anhydrous form of AZI seemed to be
unstable since it converted to dihydrate on storage at room temperature.
On the other hand, monohydrate in the presence of moisture can convert
8 Ahmed H.H. Bakheit et al.
to the more stable dihydrate form. Therefore, the most stable form of AZI is
dehydrate [25].
Allen et al. [29] found that azithromycin was obtained as hygroscopic
monohydrate when it was prepared by crystallization from ethanol and
water, and nonhygroscopic dehydrate azithromycin was prepared by crys-
tallization from tetrahydrofuran and aliphatic (C5C7) hydrocarbon in the
presence of at least two molar equivalents of water.
Azithromycin 9
100,000
80,000
60,000
40,000
20,000
0
3 10 20 30 40
2-Theta-scale
Figure 1.3 An experimental powder X-ray diffraction pattern of azithromycin
dehydrate [8].
exo
10
mW
0 2 4 6 8 10 12 14 16 18 20 min
Temperature (C)
Figure 1.4 DSC thermograms of different forms of AZI. Upper curve depicts the com-
mercial sample of AZI; middle curve shows the endotherm of dihydrate (DH) and lower
curve shows the endotherm of monohydrate (MH). The thermograms were generated
using a sealed pan.
101
100
99
98 4.3816%
Mass %
97
AZC
96
95
94
93
92
25 45 65 85 105 125 145 165 185
Temperature (C)
Figure 1.5 TGA thermogram of AZC.
2
mg
Figure 1.6 TGA thermograms of different forms of AZI. Upper curve indicates stoichio-
metric weight loss of two water molecules in CS, middle curve indicates weight loss
of two water molecules in DH, and lower curve indicates weight loss of one water
molecule in MH.
3.8.5 Boiling point, enthalpy of vapor, flash point, and vapor pressure
The calculated value of the boiling point of azithromycin under a pressure of
760 mmHg was 822.1 C. The enthalpy of vapor calculated value was
135.99 KJ/mol. The value of flash point was found to be 451 C, and the
vapor pressure was calculated to be 251 1031 mmHg at 25 C [31].
3.9. Spectroscopy
3.9.1 UV/Vis spectroscopy
The ultraviolet spectrum of azithromycin dihydrate in methanol and mobile
phase (methanol: acetonitrile: phosphate buffer pH 6.7: tetrahydrofuran,
15:25:60:2.5, v/v) shown in Figure 1.7. The figures were recorded using
a double beam Model GBC 916UV VIS spectrophotometer (GBC Scien-
tific Equipment Pty Ltd., Melbourne, Victoria, Australia). The values of
wavelength maximum in nanometer (lmax) are 201.6 nm on methanol
and 199.2 nm on mobile phase. The spectra of azithromycin in methanol
(Figure 1.7A) and mobile phase (Figure 1.7B) were shown below.
A
0.8
201.6
0.6
Absorbance
0.4
0.2
0
192 202 212 222 232 242
Wavelength (nm)
0.2 199.2
0.16
Absorbance
0.12
0.08
0.04
0
195 205 215 225 235 245
Wavelength (nm)
Figure 1.7 (A) The ultraviolet spectrum of AZI dihydrate in methanol. (B) The ultraviolet
spectrum of AZI dihydrate in mobile phase.
14 Ahmed H.H. Bakheit et al.
75
70
65
% Transmittance
1251.57
1282.43
60
1268.93
55
50 3496.31
3561.88
45 1344.14
1083.80
3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800
Wavenumber (cm-1)
Figure 1.8 Infrared absorption spectrum of azithromycin.
10A 9 8 7 6 5 4 3 2 1
1
Figure 1.9 Full H NMR spectrum of azithromycin in CDCl3.
13
3.9.3.2 C NMR spectrum [33]
13
The C NMR spectrum of azithromycin was recorded by broadband pro-
ton spin decoupling at ambient probe temperature using an internal
Azithromycin 15
Table 1.2 Assignments for the resonance bands observed in the 1H NMR spectrum of
azithromycin in CDCl3
See Figure 1.10 reference 33
Chemical shift Number of Multiplicity and coupling
(ppm) protons constant (J) Assignment
9.44 1 (1H, C2dOH)
5.13 1 Doublet (5 Hz) C100 dH
4.74 1 Doublet (6 Hz) C11dOH
4.68 1 Doublet of doublets (10 Hz) C13dH
4.43 1 Doublet (7 Hz) C10 dH
4.29 1 Doublet of doublets C3dH
(5, 2 Hz)
4.08 1 Doublet of quartet C50 dH
(10, 6 Hz)
3.68 1 Doublet (5 Hz) C11dH
3.65 1 Doublet (7 Hz) C5dH
3.55-3.47 1 Multiple C50 dH
3.35 1 Singlet C12dOH
3.34 3 Singlet C30 dOCH3
3.23 1 Doublet of doublets C20 dH
(10, 7 Hz)
3.03 1 app. triplet (10 Hz) C40 dH
2.89 1 Singlet C6dOH
2.782.72 1 Multiple C2dH
2.69 1 Quartet (7 Hz) C10dH
2.52 1 Doublet (10 Hz) C9dH
2.462.41 Multiple C30 dH
2.35 1 Doublet (15 Hz) C20 dH
2.31 3 Singlet C1dH3
2.28 6 Singlet C30 dN(CH3)2
2.15 1 Doublet (11 Hz) C40 dOH
2.071.95 2 Multiple C4dH and
C8dH
Continued
16 Ahmed H.H. Bakheit et al.
Table 1.2 Assignments for the resonance bands observed in the 1H NMR spectrum of
azithromycin in CDCl3cont'd
Chemical shift Number of Multiplicity and coupling
(ppm) protons constant (J) Assignment
2.02 1 Doublet (10 Hz) C9dH
1.931.85 1 Multiple C13dCH2dCH3
1.79 1 Doublet (15 Hz) C7dH
1.671.63 1 Multiple C40 dH
1.58 1 Doublet of doublets C200 dH
(15, 5 Hz)
1.511.43 1 Multiple C13dCH2dCH3
1.32 3 Doublet (5 Hz) C500 dCH3
1.31 3 Singlet C6dCH3
1.24 3 Singlet C300 dCH3
1.261.21 2 Multiple C7dH and
C40 dH
1.22 3 Doublet (6 Hz) C50 dCH3
1.19 3 Doublet (7 Hz) C2dCH3
1.08 1 Doublet (7 Hz) C10dCH3
1.08 3 Singlet C12dCH3
1.04 3 Doublet (7 Hz) C4dCH3
0.90 3 Doublet (7 Hz) C8dCH3
0.89 3 Triplet (7 Hz) C13dCH2dCH3
deuterium lock. All chemical shift values are reported in ppm to the
nearest 0.01 ppm. An internal reference of dC 77.0 was used for CDCl3.
Assignments were supported by DEPT editing and determined either on
the basis of unambiguous chemical shift, by patterns observed in 2D exper-
iments (HMQC) or by analogy to fully interpreted spectra for related
compounds.
Azithromycin is shown in Figure 1.11 (full 13C NMR spectrum). The
assignments for the observed bands are provided in Table 1.3, which are
consistent with the 13 carbon contents of azithromycin.
Azithromycin 17
HO NMe2
N 9 7 3
O 1
HO 5
HO O
11 5
HO 13 3 OMe
O 1 3
O 1 OH
5
O O
4. METHODS OF ANALYSIS
4.1. Compendial methods of analysis
4.1.1 Identification
4.1.1.1 IR spectrum of Azithromycin
The IR spectra of the drug were obtained in the solid state using 90 g/l solu-
tions in methylene chloride [21,34]
200 175 150 125 100 75 50 25
13
Figure 1.11 Full C NMR spectrum of azithromycin in CDCl.
Azithromycin 19
Table 1.3 Assignments for the resonance bands observed in the 13C NMR spectrum of
azithromycin
Chemical shift (ppm) Assignments Chemical shift (ppm) Assignment
178.9 (C1) 42.3 (2C, C4 and C7)
0
102.9 (C1 ) 40.3 (C30 dN(CH3)2)
94.5 (C100 ) 36.2 (C1)
83.3 (C5) 34.7 (C200 )
78.1 (C400 ) 28.7 (C40 )
77.6 (C3) 27.6 (C6dCH3)
77.4 (C13) 26.7 (C8)
73.6 (C6) 22.0 (C8dCH3)
73.6 (C11) 21.6 (C300 dCH3)
72.9 (C300 ) 21.3 (C50 dCH3)
70.8 (C20 ) 21.3 (C13dCH2)
70.1 (C9) 18.2 (C500 dCH3)
68.7 (C50 ) 16.2 (C12dCH3)
65.9 (C30 ) 14.6 (C2dCH3)
65.5 (C500 ) 11.2 (C13dCH2dCH3)
62.4 (C10) 9.0 (C4dCH3)
49.4 (C300 -OCH3) 7.3 (C10dCH3)
45.3 (C2)
[M-H-desosamine]-
571.1700
100
[M-H]-
90 747.4300
80
Relative abundance
70
[M-H-desosamine-cladinose]-
60
50 396.1000 499.6300
40
668.6800
30
212.9100
20
10
0
100 150 200 250 300 350 400 450 500 550 600 650 700 750
m/z
[M(d5)-D]-
[M(d5)-D-DNSO2ND2]+ 751.4675
100
243.1004 [M(d5)-D-desosamine]-
90
80 573.2704
Relative abundance
70
60 [M(d5)-D-desosamine-cladinose]-
50
397.1729
40
30
20
10
0
100 150 200 250 300 350 400 450 500 550 600 650 700 750
m/z
Figure 1.12 Negative ion ESI mass spectra of azithromycin (MW 748): CID product ion
spectra (MS/MS) of [M H] at m/z 747 and the fully exchanged [M(d5)-D] at m/z 751.
Deuteration was achieved by liquid phase H/D exchange method. MS and MS/MS exper-
iments were performed on a TSQ Quantum Ultra AM mass spectrometer.
Azithromycin 21
OH(D)
O O
O
:O O O H HO
O O
:
H (D)HO O
O O O +
O N (D) HO
(D)HO
O HO HO
N (D) N
(D)
HO HO N
(D) (D) desosamine (neutral)
N m/z = 571 (574) 176 (177)
m/z = 747 (751)
O
OH (D) O
O
O
(D) HO
+
HO
O N
HO
cladinose (neutral) (D)
175 (176)
m/z = 396 (398)
Figure 1.13 Proposed CID fragmentation mechanisms for the major fragment ions from
deprotonated azithromycin at m/z 747 determined from H/D exchange patterns, high-
resolution mass measurements, and MS/MS experiments. Numbers in parentheses refer
to deuterated fragmentations. The proposed site of deprotonation is based on the most
acidic proton of the lactone ring.
(J) 13-O-decladinosylazithromycin,
Azithromycin 23
4.2.2 Coulometry
A variant of the Karl Fischer water determination was described [25]. By
heating the drug substance, the contained water was transferred into a titra-
tion cell by a carrier gas. The automated system consisted of an oven sample
processor and a coulometer.
0.16
0.08
i-(mAcm2)
0.08
0.16
acid were used as dilute solvents and 75 ! 100 sulfuric acid was used as col-
ored solvent. The samples were analyzed at the wavelength of 482 nm.
Huakan et al. [58] developed a spectrophotometric method for the deter-
mination of azithromycin based on the charge transfer reaction between
azithromycin as donor and alizarin as acceptor in ethanol solution. The com-
position ratio and stability constant of charge transfer complex were 11 and
4.8 103, respectively. The apparent molar absorptivity of complex at
546 nm is 5.79 103 L mol1 cm1.
Suhagia et al. [59] developed a simple and sensitive spectrophotometric
method for the determination of azithromycin in its pharmaceutical dosage
forms. In the method, azithromycin is oxidized with potassium permanga-
nate to liberate formaldehyde, which is determined in situ using acetyl ace-
tone, in the presence of ammonium acetate. An yellow colored chromogen
was obtained, having an absorption maxima at 412 nm. The method is
found to be linear in the concentration range of 1075 mg/ml, with regres-
sion coefficient of 0.9978.
Shu-xia et al. [60] established a method for the determination of
azithromycin tablets dissolution based on chargetransfer reaction with aliz-
arin red. Methods of the dissolution test were conducted, using phosphate
Azithromycin 27
buffer solution as medium, with a stirring speed of 100 rpm/min. The solu-
tion was withdrawn after 45 min. The absorbance of dissolution solutions
was measured at 538 nm. Dissolution limit is 75% of the labeled amount.
Result Good linear correlation was achieved at the range of 50250 mg/ml
azithromycin (r 0.9996).
Paula et al. [61] proposed a new method for simple and fast spectropho-
tometric determination of azithromycin in pharmaceutical formulations.
The method is based on the charge transfer reaction between the azithromycin
and quinalizarin in methanol medium. In order to achieve maximum sensi-
tivity, the effect of some chemical variables such as the type of solvent, reagent
concentration, and reaction time was evaluated. The reaction was character-
ized in terms of stability of the product formed and its stoichiometry, and the
apparent molar absorptivity and association constant were derived. Best con-
ditions for the analytical determination of azithromycin were observed in
methanol medium with a quinalizarin concentration of 50 mg L1. At these
conditions, the radical anion (absorbing species) was formed in the medium
immediately after mixing of the reagents and showed maximum absorption
at 564 nm. The method presented a limit of detection of 0.35 mg L1 and
a limit of quantification of 1.2 mg L1.
Sayed et al. [62] developed two simple, accurate, precise, and rapid spec-
trophotometric and conductometric methods for the estimation of erythro-
mycin thiocyanate(I), clarithromycin (II), and azithromycin dihydrate(III) in
both pure and pharmaceutical dosage forms. The spectrophotometric proce-
dure depends on the reaction of rose Bengal and copper with the cited drugs to
form stable ternary complexes which were extractable with methylene chlo-
ride, and the absorbances were measured at 558, 557, and 560 nm for (I), (II),
and (III), respectively. The conductometric method depends on the forma-
tion of an ion-pair complex between the studied drug and rose Bengal.
Ashour et al. [63] developed and validated new, simple, and rapid spec-
trophotometric for the assay of two macrolide drugs, azithromycin (AZT)
and erythromycin (ERY), in pure and pharmaceutical formulations. The
method was based on the reaction of AZT and ERY with sodium 1,2-
naphthoquinone-4-sulphonate in alkaline medium at 25 C to form an
orange-colored product of maximum absorption peak at 452 nm.
4.3.2 Spectrofluorimetry
El-Rabbat et al. [64] described a simple spectrofluorometric method for the
analysis of four macrolide antibiotics. The method is based on the conden-
sation of 10% (w/v) malonic acid and acetic acid anhydride under
28 Ahmed H.H. Bakheit et al.
the catalytic effect of tertiary amine groups of the studied macrolides. The
relative fluorescence intensity of the condensation product was measured at
397/452 nm (excitation/emission) for azithromycin dihydrate and at 392/
445 nm (for clarithromycin, erythromycin ethylsuccinate, and
roxithromycin.
Almeida et al. [65] proposed a fluorescence method for azithromycin
determination in pharmaceutical formulations. The method is based on
the synchronous fluorescence (Dl 30 nm, 482 nm) produced when
azithromycin is derivatized in strong acidic medium (9.0 mol L1 HCl).
Khashaba [66] analyzed the macrolides (erythromycin, erythromycin
esters, azithromycin dihydrate, clarithromycin, and roxithromycin) by a
simple spectrofluorimetric method based on the oxidation by cerium (VI)
in the presence of sulfuric acid and monitoring the fluorescence of cerium
(III) formed at lex 255 nm and lem 348 nm.
4.3.3 Colorimetry
Hunfeld et al. [67] used a newly developed colorimetric microdilution
method to analyze the activity of 12 antimicrobial agents against nine Borrelia
burgdorferi isolates, including all three genospecies pathogenic for humans. In
addition, in vitro antimicrobial resistance patterns of Borrelia valaisiana and
Borrelia bissettii tick isolates were investigated. The applied test system is
based upon color changes that occur in the presence of phenol red and result
from the accumulation of nonvolatile acid produced by actively metaboliz-
ing spirochetes. After 72 h of incubation, minimal inhibitory concentrations
(MICs) were determined from the decrease of absorbance by software-
assisted calculation of growth curves. MIC values were lowest for azlocillin
(MIC, 0.125 mg/ml), ceftriaxone (MIC range, 0.0150.06 mg/ml), and
azithromycin (MIC range, 0.0150.06 mg/ml). Whereas tobramycin
(MIC range, 864 mg/ml) exhibited little activity, spectinomycin (MIC
range, 0.252 mg/ml) showed in vitro antimicrobial activity against
B. burgdorferi.
Haleem et al. [68] developed a simple, accurate, and rapid spectropho-
tometric method for the estimation of azithromycin by the acidic hydrolysis
of the drug with sulfuric acid and monitoring the absorbance at 482 nm.
5. STABILITY
El-Gindy et al. [75] developed a validated stability-indicating HPLC
method for the analysis of azithromycin (AZ) and its related compounds
in raw materials and capsules. The stability of AZ was studied under accel-
erated acidic, alkaline, and oxidative conditions. The major peak detected
from the degradation of AZ in alkaline and acidic conditions was
decladinosylazithromycine, while azithromycin N-oxide was detected from
the oxidative degradation. Long-term stability studies for capsule and oral
suspension were also carried out.
Table 1.4 HPLC methods for the analysis of azithromycin
Column Sample matrix Mobile phase composition Detection References
Gamma-alumina Raw material Phosphate bufferacetonitrile, Amperometric guard: [35]
adjusted to pH 11.0 with potassium 0.70 V screen:
hydroxide 0.85 V
LiChroCART C18, 5 mm Raw material Phosphate bufferacetonitrile UV 215 nm [36]
methanol, adjusted to pH 8.0 with
phosphoric acid
Nova-Pack C18, 4 mm Raw material Ammonium acetateacetonitrile Amperometric guard: [37]
methanol tetrahydrofuran, mobile 0.7 V screen: 0.8 V
phase pH 7.27.4
XTerra RP C18, 5 mm Raw material Phosphate bufferwater UV 215 nm [38]
acetonitrile, adjusted to pH 6.5
with potassium hydroxide
Phenomenex Synergi C18, 4 mm Raw material, dosage Gradient elution, phosphate UV 210 nm [39]
forms bufferacetonitrilemethanol,
adjusted to pH 7.0 with potassium
hydroxide
YMC-Park ODS-AP C18, 5 mm Rats plasma Phosphate bufferacetonitrile, Amperometric detect: [40]
adjusted to pH 7.2 with potassium 0.95 V
hydroxide
Continued
Table 1.4 HPLC methods for the analysis of azithromycincont'd
Column Sample matrix Mobile phase composition Detection References
Nova-Pack C18, 4 mm Human tears and Phosphate buffersodium Amperometric guard: [41]
plasma perchlorateacetonitrilemethanol, 0.7 V screen:
adjusted to pH 7.0 with phosphoric 0.85 V
acid
Radial-Pak Resolve Silica Rats blood plasma, Ammonium acetateacetonitrile Coulometric guard: [42]
cartridge, 5 mm serum, and human methanol, adjusted to pH 7.0 with 0.90 V
urine acetic acid
C18 (250 mm 4.6 mm, 5 mm) Azithromycin syrup Acetonitrile 0.067 mol L1 UV 210 nm [43]
K2HPO3 (pH adjusted to 6.5)
(4060)
ODS-C18 column Eye drops Acetonitrile and 0.1 mol L1 UV 215 nm [44]
(150 mm 4.6 mm, 5 mm) KH2PO4 as the mobile phase
(3070)
CAPCELL PAK C(18) Azithromycin Acetonitrile phosphate buffer UV 210 nm [45]
MGIIcolumn (250 mm 4.6 mm, capsules (to dissolve dipotassium hydrogen
5 mm) phosphate 8.7 g diluting to 1000 ml
with water, and adjust pH to 8.2
with phosphoric acid) (60:40)
Dikma Technologies Diamonsil Ammonium dihydrogen phosphate UV 210 nm [46]
C18 column (150 mm 4.6 mm, (0.045 M, pH 3.0 adjusted by
5 mm) phosphoric acid):acetonitrile 47:15
(v/v)
Azithromycin 33
6. CLINICAL APPLICATIONS
6.1. An overview
Azithromycin is the member of macrolide antibiotics. It is semisynthetic
derivatives of erythromycin. Azithromycin differs from erythromycin by
the addition of a methyl-substituted nitrogen atom into the lactone ring.
This structural modification improved acid stability and tissue penetration
and broaden the spectrum of activity. Macrolides generally cover a wide
range of Gram-positive and Gram-negative bacterial species including intra-
cellular pathogens such as Chlamydia and Legionella. They express their
antibiotic activity by binding to the 50S ribosome subunit and inhibit pro-
tein synthesis [78,79].
Azithromycin pharmacology and therapeutics aspects were given below
in more details.
binding is 50% at very low plasma concentrations and less at higher concen-
trations. Azithromycin undergoes some hepatic metabolism (demethylation)
to inactive metabolites, but biliary excretion is the major route of elimina-
tion. Only 12% of drug is excreted unchanged in the urine. The elimination
half-life (t1/2), 4068 h, is prolonged because of extensive tissue sequestra-
tion and binding [79,85].
REFERENCES
[1] Z. Banic Tomisic, The story of azithromycin, Kemija u Industriji 60 (12) (2011)
603617.
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CHAPTER TWO
Cefdinir
Abdullah A. Al-Badr, Fahad A. Alasseiri
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457,
Riyadh, Saudi Arabia
Contents
1. Description 42
1.1 Nomenclature 42
1.2 Formulae 42
1.3 Elemental analysis 43
1.4 Appearance 43
2. Uses and Applications 43
3. Methods of Preparation 43
4. Physical Characteristics 54
4.1 Ionization constant 54
4.2 Solubility 54
4.3 X-ray powder diffraction 54
4.4 Thermal methods of analysis 54
5. Spectral Properties 55
5.1 Ultraviolet spectroscopy 55
5.2 Vibrational spectroscopy 55
5.3 Nuclear magnetic resonance spectrometry 59
5.4 Mass spectrometry 60
6. Methods of Analysis 60
6.1 Compendial methods 60
6.2 Spectrophotometric methods 92
6.3 Polarographic method 94
6.4 Voltammetric methods 95
6.5 Chromatographic methods 96
7. Pharmacokinetics 102
8. Stability 106
Acknowledgments 108
References 108
Profiles of Drug Substances, Excipients, and Related Methodology, Volume 39 # 2014 Elsevier Inc. 41
ISSN 1871-5125 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800173-8.00002-7
42 Abdullah A. Al-Badr and Fahad A. Alasseiri
1. DESCRIPTION
1.1. Nomenclature
1.1.1 Systematic chemical names
(6R,7R)-7-[[(2Z)-(2-Amino-4-thiazolyl)(hydroxyimino)acetyl]amino]-
3-ethenyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid.
Syn-7-[2-(2-amino-4-thiazolyl)-2-hydroxyiminoacetamido]-3-vinyl-3-
cephem-4-carboxylic acid.
()(6R,7R)-7-[2-(2-amino-4-thiazolyl)glyoxylamido]-8-oxo-3-vinyl-
5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 72-(Z)-oxime.
7-[(2-Amino-1,3-thiazol-4-yl)-2-[(Z)-hydroxyimino]acetamido]-3-vinyl-
3-cephem-4-carboxylic acid.
7b-[2-(2-aminothiazol-4-yl)-2-(Z)-hydroximinoacetamido]-3-vinyl-
3-cephem-4-carboxylic acid.
5-Thia-1-azabicyclo[4,2,0]oct-2-ene-carboxylic acid, 7-[[(2-amino-4-
thiazolyl)(hydroxyimino)acetyl]amino-]-3-ethenyl-8-oxo-,[6R-[6a,
7b(Z)]]-; [1,2].
1.2. Formulae
1.2.1 Empirical formula, molecular weight, and CAS number
C14H13N5O5S2 395.42 91832-40-5
S
O
H2N H H
N N S
H
N N
OH
O
O OH
Cefdinir 43
1.4. Appearance
White to slightly brownish-yellow solid [1].
3. METHODS OF PREPARATION
Gonzalez et al. [8] described the synthesis of cefdinir which is reported
in the literature [913] as follows.
7-Aminocepalosporanic acid 1 was treated with sodium hydroxide and
was then reacted with phenyl acetyl chloride in acetone, water, and
triethylamine and finally by diphenyldiazomethane to give the diprotected
compound 2. The alcohol 2 was treated with phosphorous tribromide in
tetrahydrofuran and then by triphenyl phosphine in ethyl acetate and finally
by formaldehyde in sodium carbonate and dichloromethane in water to
yield the 3-ethenyl compound 3. Compound 3 was reacted with phospho-
rous pentachloride in pyridine and dichloromethane. The product obtained
was treated with methanol and water to form 4. The amino group in 4 was
acylated with 4-bromoacetoacetyl bromide and methane sulfonic acid in
ethyl acetate, and compound 5 is produced. Compound 5 reacted with
sodium nitrite in acetaldehyde and water to yield 6. The latter compound
6 was cyclized by reaction with thiourea in N,N-dimethylacetamide to pro-
duce 7. Compound 7 reacted with trifluoroacetic acid in anisole to give the
free acid, cefdinir 8, Scheme 2.1.
44 Abdullah A. Al-Badr and Fahad A. Alasseiri
O
(1) NaOH (1) PBr 3 / THF
H2 N S (2) PhCH 2 COCl NH (2) Ph 3 P/ CH 3 COOC2 H 5
acetone-water/TEA S (3) H 2 CO/ Na 2 CO3
(3) Ph 2 C N 2 CH 2 Cl 2 - H 2 O
N O CH 3 N OH
O O
O
O OH O O
1 2
O
(1) PCl 5 / pyridine O O
NH S CH 2 Cl 2 HCl H 2 N S Br
(2) CH 3 OH Br
N CH 2 (3) H 2 O CH 3 SO3 H/ CH 3 COOC2 H 5
N CH 2
O O
O O O O
3
4
O O O O
Br Br S
NH S NH S H2 N C NH 2
NaNO2
CH 3 COH- H 2 O N CH 3 CON( CH 3 ) 2
N CH 2 OH N CH 2
O O
O O O O
5
6
S O S O
H2N H2N
N NH S N NH S
N CF3 COOH/ anisole N
OH N CH 2 OH N CH 2
O O
O O O OH
8
7 Cefdinir
O O O O O O
NaNO2 SO2 Cl 2 Cl
H3 C O CH 3 H3 C O CH 3 O CH 3
CH 3 COOH/ H 2 O CH 3 COOH
N N
OH OH
1 2 3
S S S
O Ph 3 CCl H O
H2N Ph 3 C N
( C2 H 5 )3 N, CHCl 3
H2 N NH 2 N N NaOH
O CH 3 O CH 3
N N dioxane/ H 2 O
OH O
4 5 CPh 3
HCl.H 2 N S
H S S
O N H
Ph 3 C N O Ph 3 C N
N Na N
O O O CH( Ph) 2 NH S
N 7 N (1) CF3 COOH/anisole
O CPh3 O N
6 POCl 3 / PhN(CH3)2 (2) HCOOH 90%
O
CH 2 Cl 2
CPh3
O O
CHPh 2
8
S O
H2 N
N NH S
N
OH N
O
9 O OH
Cefdinir
O O
NH NH S
S
(1) PBr 3 / THF
(2) Ph3 P/ CH 3 COOC2 H 5 N CH 2 (1) PCl5/pyridine//CH2Cl2
N OH
O (3) H 2 CO/ NaCO3 O (2) CH3OH (3) H2O
CH 2 Cl 2 - H 2 O
O O O O
3 4
HCl.H 2 N S S
S H O
H O (Ph)3C N
Ph 3 C N
N CH 2 N O Na N NH
O S
N O CPh3 N
6 (1) CF3COOH/anisole
O O N CH 2
POCl 3 / Ph- N( CH 3 ) 2 / CH 2 Cl 2 O (2) HCOOH 90%
O
CPh3
5 O O
S O
H2 N
N NH S
N
OH N CH 2
O
O OH
8
Cefdinir
O O O O
Br Br
H2 N O O NH
S S NH S
Br N
OH OH
N N N
O
2 NaNO2
O O
O OR O OR O OR
1 3 4
S O
H2 N S
C O
S N C H2 N
NH S C
C N N C
H2N NH 2 Deprotection NH S
OH
N N
O OH
N
O
O OR
O OH
5 6
Cefdinir
1 2 3
S O
S H
O Ph 3 C N
Ph 3 C NH C
C N
N OH NH
H2 N S N S
N O CPh O
Girard T 5 3
N CPh 3 N
O 1-Hydroxybenzotriazole O
O O CHPh 2 O O CHPh 2
4 6
S O
H2 N
N
NH S
N
HCOOH / CH 3 COOH OH
N
O
7 O OH
Cefdinir
S O
H2 N S H2 N
S N
O N
H2 N O NH
N N S
R O OH O
N 2 CPh 3 N
O O p-TsOHDMAc
Base / CH3CON(CH3)2
CPh3 p-TsOH
1 O O
3
S N
S R= S , N
O
H2N N N
N O
HN S O
N S
Acid OH O P [OR] 2 , O P [OR] 2
N
O R = C1C4 alkyl or phenyl with
3-cephem derivative
4 O OH
Cefdinir
S O
H2 N S H2N
S O N N
H2 N O NH
N O OH N S
R O
N 2 N
CPh 3
O (1) Base / CH3CON(CH3)2 O CH 3 SO3 HDMAc
CPh3 (2) CH3SO3H
1 O O
3
S N
S O R= S , N
H2 N N N
N O
HN S
N O S
Acid OH O P [OR]2 , O P [OR] 2
N
O R = C1C4 alkyl or phenyl with
4 3-cephem derivative
O OH
Cefdinir
H2 N
S H2N S
N
N N N
O O
S S S O
S S H2N N O OH
N OH
2 N S 4
O O
N S
Ph3P/CH2Cl2 O (1) THF/water
O
CH 3 ( C2 H 5 ) 3 N
(2) O N CH 3
CH 3
1 3 (3) NH 4 Cl/ K 2 CO 3
S O
H2 N
N
NH S
N
OH
N
O
5
O OH
Cefdinir
O
H2 N S O C CH
3
N
N N S S THF/ H 2 O/ TEA
+
O H2N NH 4 Cl/ K 2 CO3
S O N
O OH
1 2
H2 N O
S S
N O
S N N
O O
NH S O OH O OH
N I II
OH N S
O N
O CH 3
O OH O OH
3
III
CPh 3
O
N H2 N S
S S
( Bu) 3 N
S + N
N O N O CH 3 CON( CH 3 ) 2
H2 N O OH
1
2
S O S O
H2 N H2N
N N
NH NH
S S
N N
O HCl/HCOOH OH
N N
O O
CPh 3
O OH O OH
3 4
Cefdinir
O O
O O Br
H2N S NH
Br S
Br
NaNO2
N 2 N CH3COOH / CH2Cl2
O O O O
Si( CH 3 ) 3
H3 C N H2 N NH 2
O O CHPh 2 H O O CHPh 2
CH 3 COOC2 H 5
1 3
H2N
O O
S O
Br N
NH S
S NH S
N
OH H2 N NH 2 N
N OH CF3COOH/anisole
N
O CH 3 CON( CH 3 ) 2
O
O O CHPh 2
O O CHPh 2
4 5
H2 N
S O
N
NH S
N
OH N
O
O OH
6
Cefdinir
S S S
O O O
H2 N H2 N HCl H 2 N
N ONa N OH N Cl
PCl5
N N N
OH O CH 3 CH2Cl2 O CH 3
O O
1 2 3
S
O S
H2 N
H2N O
S H2 N
N
N NH N
O N S
N S
O OH N
O Hydrolysis
4 OH N
O N
N,O-bistrimethylsilyl O O
acetamide CH 3
NSi(CH3)3
O OH O OH
H3C OSi(CH3)3
5 6
Cefdinir
Method 1
H2N S S
O H2 N O N N
N
N N N CH 3
N N CH 3
1 OH + H3C S S S
N S S S N
O O
CPh 3 CPh 3
1 2 3
H2N S
O N N
O
N CH 3 / H 3 C C N( CH 3 ) 2
(1) S S
H2N S O ( CH 3)3 Si NH S N
(CH 3)3 Si HN C CH 3 O
2 N N 3
O CPh 3
O O
O
H 3 C C N(CH 3)2
O OH O OSi(CH3 ) 3 (2) p-TSAH2O / H 3 C C OC2 H 5
4 5
H2 N S S H2 N S
O H2 N O
O
N N N
NH NH NH
S NH 3 / CH 2 Cl 2 / H 2 O S CF3 COOH N S
N N
O O OH
N N N
CPh 3 CPh 3
O p-TSA 2DMAc O O
O OH O OH O OH
6 7 8
Cefdinir
Method 2
H2N S S
O H2 N
O
N N N N
N N N TPP / TEA N
OH CH 3 S
N
+ H3 C S CH 2 Cl 2
S S S N S CH 3
O O
O CH 3 H3 C O
9 2 10
H2 N S
S H2 N S
H2 N O O
N
O N N
NH NH
O OH S S
N N
4 O NH 4 Cl / K 2 CO3 OH
N N
THF H2O / TEA H 2 O, H 2 SO 4 O
O CH 3O
O OH HN(C2H5)3 O OH
11 8
Cefdinir
S S S O
O O
H2N H2 N H2N
N O C2 H 5 N N OH
ONa
NaOH Acetic anhydride
N N N
OH C2H5OH OH O CH 3
1 2 3 O
H2N S
S N S O
O O
S S H2 N S H2 N
S S
N S O OH N NH
N N S
4 N 6
N N
Ph3P/(C2H5)3N O CH 3 O N
THF
C O
O H3 C O
O OH
5
7
S
O
H2 N
N NH S
N
Hydrolysis OH N
O
O OH
8
Cefdinir
4. PHYSICAL CHARACTERISTICS
4.1. Ionization constant
pKa: 8.70
4.2. Solubility
Slightly soluble in dilute hydrochloric acid, sparingly soluble in 0.1 M pH 7
phosphate buffer [2].
5. SPECTRAL PROPERTIES
5.1. Ultraviolet spectroscopy
The ultraviolet absorption spectrum of cefdinir in ethanol was recorded
using a Shimadzu ultravioletvisible spectrophotometer 1601 PC. The
ultraviolet spectrum is shown in Figure 2.3 and cefdinir exhibited three
maxima at 200.5, 223.5, and 287 nm.
1668
Amide C O
1520 NH
11611, 1429
Acid C O
Table 2.3 The proton-nuclear magnetic resonance assignments for the spectrum of
cefdinir
S 13 O
14
H2N 11
N 12 10 NH 6 5 S
1
N 7
OH N 9
O 2
3 8
O 4 OH
6. METHODS OF ANALYSIS
6.1. Compendial methods
6.1.1 Japanese pharmacopeia methods [24]
6.1.1.1 Cefdinir
Cefdinir contains not less than 900 mg (potency) per mg. The potency of
cefdinir is expressed as mass (potency) of cefdinir (C14H13N5O5S2).
16
7.8
15
7.6
14
7.4
13
12
7.2
2.25
7.143 1.00 11.343
11
7.0
6.951
6.928
10
1.11 6.916 9.510
6.893 1.05 9.494
6.8
7.143
0.91 6.682 6.951
8
6.928
6.6
6.916
2.25 6.893
7
1.11 6.682
0.91 5.814
5.804
6.4
6
1.06 5.798
5.788
6.2
1.04
5.330
5.308
5.204
4
1.14 5.194
6.0
1.14 3.855
3.820
3 3.587
5.814
5.804 3.551
0.94 2.513
5.8
1.06 5.798
2
5.788
5.610
1
5.6
1.09 5.575
0
5.4
5.330
1.10
-1
5.308
5.204
5.2
1.04 5.194
-2
-3
Figure 2.6 The expanded 1H-NMR spectrum of cefdinir (5.07.4 ppm) in DMSO-d6.
5.0 ppm
ppm
62 Abdullah A. Al-Badr and Fahad A. Alasseiri
3.855
3.820
3.587
3.551
4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.513
2.5 2.4 2.3 2.2 2.1 ppm
1.14
1.14
0.94
Figure 2.7 The expanded 1H-NMR spectrum of cefdinir (2.44.0 ppm) in DMSO-d6.
Table 2.4 The 13C nuclear magnetic resonance assignments for the spectrum of cefdinir
S 13 O
14
H2N 11
N 12 10 NH 6 5 S
1
N 7
OH N 9
O 2
3 8
O 4 OH
170
168.500
168.206
165
163.914
163.775
13
160
168.206
163.914
163.775
163.232
155
148.487
147.380
13
150
131.980
148.487 125.531
147.380 125.305
124.763
145
124.227
143.575 117.205
142.844
106.822
140
210 200 190 180 170 160 150 140 130 120 110 100
90
135
80
131.980
130
70
62.595
58.761
60
125.531 57.863
125
124.763 39.941
50
124.227 39.861
39.773
39.694
40
39.607
120
39.440
117.205
30 39.273
39.106
115
20
23.223
10
110
0
106.822
ppm
ppm
Abdullah A. Al-Badr and Fahad A. Alasseiri
Cefdinir 65
Table 2.5 Summary of assignment for the fragmentation ions observed in the mass
spectrum of cefdinir
Fragment
Relative
m/z intensity (%) Formula Ions
395 8 C14H13N5O5S2 S O
H2N
N NH S
N
OH N
O
O OH
271 12 C8H7S2N4O3 S
O O
H2N S
N N
H
N
OH
243 30 C7H7S2N4O2 S O
H2N
N N
H S
N H
OH
Table 2.5 Summary of assignment for the fragmentation ions observed in the mass
spectrum of cefdinircont'd
Fragment
Relative
m/z intensity (%) Formula Ions
183 6 C5H3N4SO2 S O
H2N
N N
N O
170 8 C7H8NSO2 S
N CH2
O OH
152 18 C7H6SNO S
C O
137 13 C6H5SN2 S
H2N
N
126 68 C6H8NO2 O
HO
HN
and perform the test. Prepare the control solution with 2.0 mL of Standard
Lead Solution (not more than 10 ppm).
(2) Related substancesDissolve about 0.1 g of cefdinir in 10 mL of
0.1 mol/L phosphate buffer solution, pH 7.0. Pipet 3 mL of this solution,
add tetramethylammonium hydroxide TS, pH 5.5, to make exactly
20 mL, and use this solution as the sample solution. Perform the test with
10 mL of the sample solution as directed under the liquid chromatography,
in the general method <2.01> according to the following conditions:
determine the areas of each peak by the automatic integration method
and calculate the amounts of their peaks by the area percentage method;
the amount of E-isomer having the relative retention time 1.5 to cefdinir
is not more than 0.8%, and the amount of total peak areas other than cefdinir
is not more than 3.0%.
Operating conditions
Detector: An ultraviolet absorption photometer (wavelength:
254 nm).
Column: A stainless steel column 4.6 mm in inside diameter and
15 cm in length, packed with octadecyl silanized silica gel for liquid
chromatography (5 mm in particle diameter).
Column temperature: A constant temperature of about 40 C.
Mobile phase A: To 1000 mL of tetramethylammonium hydroxide
TS, pH 5.5, add 0.4 mL of 0.1 mol/L disodium dihydrogen eth-
ylenediamine tetraacetate TS.
Mobile phase B: To 500 mL of tetramethylammonium hydroxide
TS, pH 5.5 add 300 mL of acetonitrile for liquid chromatography
and 200 mL of methanol, and add 0.4 mL of 0.1 mol/L disodium
dihydrogen ethylenediamine tetraacetate TS.
Flowing of the mobile phase: Control the gradient by mixing the mobile
A and B as directed in the following table.
Time after injection of the sample Mobile phase A Mobile phase B
(min) (Vol. %) (Vol. %)
02 95 5
222 95 ! 75 5 ! 25
2232 75 ! 50 25 ! 50
3237 50 50
3738 50 ! 95 50 ! 5
3858 95 5
Cefdinir 69
Flow rate: 1.0 mL/min. The retention time of cefdinir is about 22 min
under this condition.
Time span of measurement: About 40 min after injection of the sample
solution.
System suitability
Test for required detection: Pipet 1 mL of the sample solution, add
tetramethylammonium hydroxide TS, pH 5.5, to make exactly 100 mL,
and use this solution as the test solution for system suitability. Pipet 1 mL
of the test solution for system suitability, add tetramethylammonium
hydroxide TS, pH 5.5, to make exactly 10 mL. Confirm that the peak area
of cefdinir obtained from 10 mL of this solution is equivalent to 713% of
that obtained from 10 mL of the test solution for system suitability.
System performance: Dissolve 0.03 g of Cefdinir Reference Standard
and 2 mg of cefdinir lactam ring-cleavage lactones in 3 mL of 0.1 mol/L
phosphate buffer solution, pH 7.0, add tetramethylammonium hydroxide
TS, pH 5.5, to make 20 mL. When the procedure is run with 10 mL of this
solution under the above operating conditions, peak 1 and peak 2 of cefdinir
lactam ring-cleavage lactones separated into four peaks, cefdinir, peak 3, and
peak 4 of remaining cefdinir lactam ring-cleavage lactones are eluted in this
order. Relative retention time of peak 3 of cefdinir lactam ring-cleavage lac-
tone to the retention time of cefdinir is not less than 1.09. The number of
theoretical plates and the symmetry factor of the peak of cefdinir are not less
than 7000 steps and not more than 3.0, respectively.
System repeatability: When the test is repeated 3 with 10 mL of the
test solution for system suitability under the above operating conditions,
the relative standard deviation of the peak areas of cefdinir is not more
than 2.0%.
Water Carry out this test according to the general method <2.48 > not
more than 2.0% (1 g, volumetric titration, direct titration). Use a mixture of
formamide and methanol for water determination (2:1) instead of using
methanol alone for water determination).
Assay Weigh accurately an amount of Cefdinir and Cefdinir
Reference Standard equivalent to about 20 mg (potency), dissolve each in
0.1 mol/L phosphate buffer solution, pH 7.0, to make exactly 100 mL,
and use these solutions as the sample solution and the standard solution.
Perform the test with 5 mL of the sample solution and the standard solution
as directed under Liquid Chromatography <2.01 > according to the follo-
wing conditions, and calculate the peak areas, AT and As, of cefdinir of the
solutions.
70 Abdullah A. Al-Badr and Fahad A. Alasseiri
AT
Amount mg potencyof cef dinir C14 H13 N5 O5 S2 W s 1000
AS
System suitability
System performance: Dissolve 2 mg of Cefdinir Reference Standard and
5 mg of cefdinir lactam ring-cleavage lactones in 10 mL of 0.1 mol/L phos-
phate buffer solution, pH 7.0. When the procedure is run with 5 mL of this
solution under the above operating conditions, peak 1 and peak 2 of cefdinir
lactam ring-cleavage lactones separated into four peaks, cefdinir, peak 3 and
peak 4 of remaining cefdinir lactam ring-cleavage lactones are eluted in this
order. The resolution between the peak 2 of cefdinir lactam ring-cleavage
lactone and that of cefdinir is not less than 1.2. The number of theoretical
plates and the symmetry factor of the peak of cefdinir are not less than 2000
steps and not more than 1.5, respectively.
System repeatability: When the test is repeated 6 with 5 mL of the stan-
dard solution under the above operating conditions, the relative standard
deviation of the peak areas of cefdinir is not more than 1.0%.
Containers and storage: ContainersTight containers. Storage
Light-resistant containers.
Operating conditions
Proceed as directed in the Assay under Cefdinir.
System suitability
System performance: When the procedure is run with 20 mL of the standard
solution under the above operating conditions, the number of theoretical
plates and the symmetry factor of the peak of cefdinir are not less than
2000 and not more than 2.0, respectively.
System repeatability: When the test is repeated 6 with 20 mL of the
standard solution under the above operating conditions, the relative standard
deviation of the peak area of cefdinir is not more than 1.0%.
Assay Weigh accurately not less than five Cefdinir capsules, take out the
contents, and powder. Wash the empty capsules with a little amount of
diethyl ether, if necessary, allow to stand at a room temperature to vaporize
the adhering diethyl ether, and weigh accurately the mass of the capsules to
calculate the mass of the contents. Weigh accurately an amount of the con-
tents, equivalent to about 0.1 g (potency) of cefdinir according to the labeled
amount, add 70 mL of 0.1 mol/L phosphate buffer solution, pH 7.0, shake
for 30 min, and add 0.1 mol/L phosphate buffer solution, pH 7.0, to make
exactly 100 mL. Centrifuge this solution at 3000 revolutions per minute for
10 min, pipet 4 mL of the supernatant liquid, add 0.1 mol/L phosphate
buffer solution, pH 7.0, to make exactly 20 mL, and use this solution as
the sample solution. Separately, weigh accurately an amount of Cefdinir
Reference Standard, equivalent to about 20 mg (potency), dissolved in
0.1 mol/L phosphate buffer solution, pH 7.0, to make exactly 100 mL,
and use this solution as the standard solution. Proceed as directed in the Assay
under Cefdinir.
System suitability
System performance: When the procedure is run with 20 mL of the standard
solution under the above operating conditions, the number of theoretical
plates and the symmetry factor of the peak of cefdinir are not less than
2000 and not more than 2.0, respectively.
System repeatability: When the test is repeated 6 with 20 mL of the
standard solution under the above operating conditions, the relative standard
deviation of the peak area of cefdinir is not more than 1.0%.
Particle size Carry out this test according to the general method
<6.03>. It meets the requirement of fine granules of the Powders.
Assay Powder, if necessary, and weigh accurately an amount of Cefdinir
Fine Granules, equivalent to about 0.1 g (potency) of cefdinir according to
the labeled amount, add 70 mL of 0.1 mol/L phosphate buffer solution,
pH 7.0, shake for 30 min, and add 0.1 mol/L phosphate buffer solution,
pH 7.0, to make exactly 100 mL. Centrifuge at 3000 revolutions per minute
for 10 min, pipet 4 mL of the supernatant liquid, add 0.1 mol/L phosphate
buffer solution, pH 7.0, to make 20 mL, and use this solution as the
sample solution. Separately, weigh accurately an amount of Cefdinir Refer-
ence Standard, equivalent to about 20 mg (potency), dissolve in 0.1 mol/L
phosphate buffer solution, pH 7.0, to make exactly 100 mL, and use this
solution as the standard solution. Proceed as directed in the Assay under
Cefdinir.
Amount mg potencyof cefdinirC14 H13 N5 O5 S2 W S AT =AS 5
100ri =rs
in which ri is the peak response for each impurity and rs is the sum of the
responses of all the peaks. The limits for the impurities are specified in
Table 2.6.
Assay
Buffer solutionDissolve about 7.1 g of anhydrous dibasic sodium phosphate
in 500 mL of water (Solution A). Dissolve about 6.8 g of monobasic
Cefdinir 77
AU C S D 900 100
AS L
in which AU and AS are the absorbances obtained from the Test solution and
the Standard solution, respectively; CS is the concentration, in mg/mL, of the
Standard solution; D is the dilution factor of the Test solution; 900 is the vol-
ume, in mL, of Medium; 100 is the conversion factor to percentage; and L is
the capsule label claim, in mg.
TolerancesNot less than 80% (Q) of the labeled amount of
C14H13N5O5S2 is dissolved in 30 min.
Uniformity of dosage units Carry out this test according to the general
method <905>: meet the requirements.
Related compounds
M Phosphate buffer solution
Solution ADissolve 14.2 g of sodium phosphate, dibasic, anhydrous, in
water, and dilute with water to 1000.0 mL.
Solution BDissolve 6.8 g of potassium phosphate, monobasic, in
water, and dilute with water to 500.0 mL.
Solution CCombine 1000 mL of Solution A with 500 mL of Solution
B. Verify the pH of 7.0 0.1, and adjust, if necessary using Solution A or
Solution B.
Dilute phosphoric acid solutionDilute phosphoric acid (1 in 1 0) with
water and mix.
0.1 M Disodium ethylenediamine tetraacetate (EDTA)Transfer about 3.
72 g of disodium ethylenediamine tetraacetate into a 100-mL volumetric
flask. Dissolve in and dilute with water to volume and mix.
0.1% Tetramethylammonium hydroxide solutionDilute 20 mL of
tetramethylammonium hydroxide (10% in water) with water to make
2000 mL and mix. Adjust with Dilute phosphoric acid solution to a pH of
5.5 0.1.
Mobile phase ATransfer 0.4 mL of 0.1 M EDTA to 1000 mL of 0.1%
Tetramethylammonium hydroxide solution and mix.
Mobile phase BMix 250 mL of 0.1% Tetramethylammonium hydroxide
solution, 150 mL of acetonitrile, and 100 mL of methanol. Add 0.2 mL of
0.1 M EDTA and mix.
Mobile phaseUse variable mixtures of Mobile phase A and Mobile phase B
as directed in the Chromatographic system.
Standard solution 1Dissolve an appropriate amount of USP Cefdinir RS
in 0.1 M Phosphate buffer solution (Solution C) to obtain a solution having a
Cefdinir 81
a suitable volumetric flask. Dissolve in and dilute with 0.1 M Phosphate buffer
solution to obtain a solution having a known concentration of about
0.05 mg/mL of cefdinir.
Resolution solutionAccurately weigh a known amount of USP Cefdinir
RS and m-hydroxybenzoic acid into a suitable volumetric flask. Dissolve in
0.1 M Phosphate buffer solution to obtain a solution having a known concen-
tration of about 0.05 mg/mL of cefdinir and about 0.175 mg/mL of
m-hydroxybenzoic acid.
Chromatographic system (see Chromatography, in the general method
<621 >)The liquid chromatography is equipped with a 254-nm detector
and a 3.9-mm 150-mm column that contains 4-mm packing L1. The flow
rate is maintained at about 1.4 mL/min. Chromatograph the Resolution
Cefdinir 85
solution and record the peak area response as directed for Procedure. The res-
olution between cefdinir and m-hydroxybenzoic acid is greater than 3.0; the
tailing factor of the cefdinir peak is not more than 2.0; and the relative stan-
dard deviation for replicate injections of the Standard preparation is not more
than 1.0%.
ProcedureSeparately inject equal volumes (about 15 mL) of the Standard
preparation and the Assay preparation into the chromatograph, record the
chromatograms, and measure the responses for the major peaks. Calculate
the percentage of cefdinir (C14H13N5O5S2) based on the label claim, in
the portion of Capsules taken by the formula:
CS rU
100
CU rS
AU C S d D 900 100
AS W U L
in which AU and AS are the absorbances obtained from the Test solution and
Standard solution, respectively; CS is the concentration, in mg/mL, of cefdinir
in the Standard solution; d is the density, in mg/mL, of the Oral Suspension
obtained by dividing the weight of Oral Suspension taken by 5 mL; D is the
dissolution factor used, if necessary, to prepare the Test solution; 900 is the
volume, in mL, of Medium; 100 is the conversion factor to percentage;
WU is the weight, in mg, of Oral Suspension taken; and L is the label
claim, in mg.
TolerancesNot less than 80% (Q) of the labeled amount of
C14H13N5O5S2 is dissolved in 30 min.
Uniformity of dosage units: Carry out this test as directed in the gen-
eral method <905>For Oral Suspension packaged in single-unit con-
tainers: meets the requirements.
Deliverable volume: Carry out this test as directed in the general
method <698>For Oral Suspension packaged in multiple-unit con-
tainers: meets the requirements.
pH: When this test is carried out as directed in the general method
<791>Between 3.5 and 4.5.
Loss on drying: This experiment should carried out as directed in the
general method <731>Dry about 1 g of powder over phosphorous pent-
oxide in a vacuum oven not exceeding 5 mm of mercury at 70 for 44.5 h:
it loses not more than 1.0%.
Related compounds
M Phosphate buffer solution
Solution ADissolve in and dilute with water 14.2 g of anhydrous dibasic
sodium phosphate to 1000.0 mL.
Solution BDissolve in and dilute with water 6.8 g of monobasic potas-
sium phosphate to 500.0 mL.
Solution CMix 1000 mL of Solution A with 500 mL of Solution B.
Verify a pH of 7.0 0.1.
Dilute phosphoric acid solutionDilute phosphoric acid with water (1 in
10) and mix.
0.1 M Disodium ethylenediaminetetraacetate (EDTA)Transfer about
3.72 g of disodium ethylenediaminetetraacetate into a 100-mL volumetric
flask. Dissolve in and dilute with water to volume and mix.
88 Abdullah A. Al-Badr and Fahad A. Alasseiri
100=F C S =C U r U =r S
than 3.0; the tailing factor of the cefdinir peak is not more than 2.0;
and the relative standard deviation for replicate injections of the Standard
preparation is not more than 1.0%.
ProcedureSeparately inject equal volumes (about 15 mL) of the Standard
preparation and the Assay preparation into the chromatograph, record the
chromatograms, and measure the responses for the major peaks. Calculate
the percentage of cefdinir (C14H13N5O5S2) in the portion of Oral Suspen-
sion taken by the formula:
100C S =C U r U =r S
in which CS is the concentration, in mg/mL, of cefdinir in the Standard solu-
tion; CU is the concentration of cefdinir in the Assay preparation; and rU and rS
are the peak responses of cefdinir in the Assay preparation and the Standard
preparation, respectively.
through the origin. The assay method was validated by low values of
percent relative standard deviation ranging from 0.57 to 0.91 and standard
error ranging from 0.33 to 0.52, indicating accuracy and precision of the
methods. The accuracy of the method was further proved by excellent
recovery data. The reproducibility of the results confirmed the ruggedness
of the method.
Sankar et al. [29] developed a simple spectrophotometric method for the
determination of cefdinir in pure form and in its pharmaceuticals. It is based
on formation of red complex with ferric chloride and 2,20 -bipyridyl having
absorption maxima at 530 nm. The chromogen obeys Beers law in the con-
centration range of 0.52.5 mg/mL.
Sankar et al. [30] developed two simple and sensitive visible spectropho-
tometric methods (A and B) for the estimation of cefdinir in pure and in
pharmaceutical dosage forms. Method A is based on the oxidation followed
by complexation between the cefdinir and 1,10-phenanthroline in presence
of ferric chloride to form a blood red colored chromogen with lmax at
520 nm. In method B, cefdinir reacts with FolinCiocalteu reagent an alka-
line media to form a blue colored chromogen at lmax at 710 nm. Beers law is
obeyed in the concentration range of 0.32.4 mg/mL and 1.57.5 mg/mL
for methods A and B, respectively. Results obtained are reproducible and
are statistically validated and are suitable for the analysis of cefdinir in bulk
and in pharmaceutical dosage forms.
Singh et al. [31] described a nonextractive spectrophotometric method
for the content assay of cefdinir in formulations. The method is based on
complexation of cefdinir and iron under reducing conditions in a buffered
medium (pH 11) to form a magenta colored donoracceptor complex
(lmax 550 nm; apparent molar absorptivity 3720 L/mol/cm). No other
cephalosporins, penicillins, and common excipients interfere under the test
conditions. The Beers law is followed in the concentrations range
8160 mg/mL.
Narala and Saraswathi [32] described three spectrophotometric methods
(A, B, and C) for the determination of cefdinir. The methods are based on
the oxidation of the drug with Fe(III) and the estimation of the Fe(II) pro-
duced after chelation with either 1,10-phenanthroline or 2,20 -Bipyrdyl or
potassium ferricyanide at 512, 510, and 700 nm. The Beers law was obeyed
in the concentration range of 28, 824, and 412 mg/mL for cefdinir for
the method A, B, and C, respectively. The results of the methods were val-
idated statistically and applied to the determination of cefdinir in bulk and in
pharmaceutical formulations without any interference from excipients.
94 Abdullah A. Al-Badr and Fahad A. Alasseiri
The optimal experimental parameters for the drug assay were accumulation
potential 0.3 V (vs. Ag/AgCl, 3 M KCl), accumulation time 15 s,
frequency 120 Hz, pulse amplitude 0.07 V, and scan increment 10 mV
in phosphate buffer pH 2. The first peak current showed a linear dependence
with the drug concentration over the range 1.88 108 to 12 108 M. The
achieved limit of detection and limit of quantification were 5 109 M
(0.2 ng/mL) and 1.7 109 M (0.67 ng/mL), respectively. The procedure
was applied to the assay of the drug in capsules form with mean percentage
recoveries of 99.7. Applicability to assay the drug in urine samples was illus-
trated. The peak current was linear, with the drug concentration in the range
0.080.7 ng/mL of the urine.
ranging from 0.05 to 5 mg/mL for serum samples. The method is accurate
and precise for pharmacokinetic and bioequivalence studies of cefdinir.
Snali et al. [45] determined the pKa value of cefdinir precisely in water
and methanolwater binary mixture (20% v/v) using spectrophotometric
titration and high-performance liquid chromatography, respectively. The
chromatographic procedure has been developed for the determination of
cefdinir in drug formulation. This method was validated for specificity, pre-
cision, linearity, range, accuracy, limit of detection, and limit of quantitation
as per the ICH guidelines. The method can be used for routine analysis of the
drug and as an alternative tool for drug quality control laboratories.
Li et al. [46] developed a high-performance liquid chromatography
coupled with online solid-phase extraction (SPE) and ultraviolet detection
for the determination of cefdinir in beagle dog plasma. After simple pre-
treatment for plasma with 6% perchloric acid, a volume of 100 mL upper
layer of the plasma sample was injected into the self-made online SPE extrac-
tion. The analytes were retained on the trap column (Lichrospher C18,
4.6 mm 3.7 cm, 25 mm) and the biological matrix was washed out with
the solvent (20 mM potassium dihydrogen phosphate adjusted to pH 3) at
a flow rate of 2 mL/min. By rotation of the switching valve, the target
analytes could be eluted from trap column to analytical column in the
pack-flush mode by the mobile phase (methanol:acetonitrile:20 mM potas-
sium dihydrogen phosphate adjusted pH 3 (11.25:6.75:82) at a flow rate of
1.5 mL/min), and then separated on the analytical column (Ultimate XB
C18, 5 cm 4.6 mm, 5 mm). The complete cycle of the online SPE
preconcentration, purification, and HPLC separation of the analytes was
4 min. The UV detection was performed at 286 nm. The method showed
good performance in terms of specificity, linearity, detection and quantifi-
cation limits, precision, and accuracy. The method was applied to the anal-
ysis of the drug in beagle dog plasma to support the preclinical
pharmacokinetic trials.
Sankar et al. [47] developed a simple, fast, and precise reversed-phase
high-performance liquid chromatographic method for estimation of the
cefdinir in bulk and in pharmaceutical dosage forms. The analysis was carried
out using a Partisil C18 octadecylsilane column (15 cm 4.6, 5 mm) in
isocratic mode, with mobile phase comprising acetonitrile and water in
the ratio 60:40 (v/v). The flow rate was 1 mL/min and effluents were mon-
itored at 240 nm. The retention time of cefdinir was 1.9 min. The method
produced linear response in the concentration range of 0.550 mg/mL and
the percentage recovery ranged from 98.6 to 100.15. The method was
Cefdinir 99
by paper disk, cup, and agar-well method using Providencia stuartii ATCC
43665 and Providencia stuartii ATCC 43664 (a sensitive mutant of P.
stuartii ATCC 43665) and antibiotic medium Number 1 (Difco). P. stuartii
ATCC 43665 was used for assaying high concentrations of cefdinir
(>0.13 mg/mL) and P. stuartii ATCC 43664 for lower concentrations.
The lowest detectable concentration of cefinir in human plasma was
0.016 mg/mL for the cup-plate and agar-well method and 0.03 mg/mL
for the disc-plate method using P. stuartii ATCC 43664. Plasma and urine
levels of cefdinir in human after oral administration were detected by HPLC.
The detection limits in plasma and urine were 0.4 and 0.5 mg/mL,
respectively.
7. PHARMACOKINETICS
Cefdinir exhibits broad range in vitro activity against Gram-positive
and Gram-negative aerobes. It is stable to hydrolysis by many of the com-
mon b-lactamases. The pharmacokinetic parameters of the drug in children
are similar to those obtained in adults using similar mg/m2 doses (300,
600 mg in adults 7.14 mg/kg in children, respectively) [57].
The terminal elimination half-life t of cefdinir increases in patients with
chronic renal failure to 11 that of healthy controls. Hemodialysis effec-
tively removes cefdinir and t during hemodialysis decreases to one sixth
of that in tests without dialysis but is still longer than in healthy subjects.
100 Mg of the drug once a day is sufficient dose for hemodialysis
patient [58].
A cefdinir dosage of 25 mg/kg once daily would be ineffective for treat-
ment of acute otitis media caused by penicillin-nonsusceptible Streptococcus
pneumonia strain [59].
Cefdinir is administered orally to patients with chronic renal failure
undergoing continuous ambulatory peritoneal dialysis to investigate changes
in serum concentration, excretion rate into the dialysate and serum protein
binding of cefdinir. The serum concentration level of cefdinir was found to
be dose dependent in patients given the drug for 414 day. Cefdinir
inhibited 90100% of the clinical isolates such as S. aureus, S. epidermidis,
E. coli, and its antibacterial activity was stronger than that of amoxicillin
and cofactor against clinical isolates [60].
The relative bioavailability and bioequivalence of cefdinir dispersible
tablets were studied in healthy volunteers. The pharmacokinetic parameter
of the test and reference dispersible tablets was bioequivalent [61].
The pharmacokinetic interaction between cefdinir and captopril or
quinapril was studied in rats. Captopril and quinapril and/or their metabo-
lites have a major impact on the disposition of cefdinir in rats, probably by
competition at the plasma protein binding level and at the tubular anionic
carrier level. This mechanism also is relevant in humans [62].
Cefdinir 103
of 3 mg/kg of the drug with Cmax ranging from 0.57 to 0.89 mg/mL except
1 case in which the absorption of the drug was poor. Recovery rates in urine
averaged at 13.4% with a large individual variation. Ten children with
12 bacterial infections were treated with 9 mg/kg/day of cefdinir fine gran-
ules. Clinical responses were good in 10 patients and fair in 1 patient, with an
efficacy rate of 90.9% [73].
Cefdinir is safe and effective for treating skin infections caused by
S. aureus and S. pyogenes and many Gram-negative pathogens. The pharma-
cokinetic, dosing schedule, adverse event profile, and efficacy data for
cefdinir in adults and pediatrics populations in the treatment of the uncom-
plicated skin structure infections [74].
The administration of cefdinir to healthy subjects after high protein diet
and 1-phenylalanine load was studied to explore the induction of intestinal
peptide transporter. Six healthy subjects had normal protein and high pro-
tein diets and L-phenylalanine for 12 days in a randomized three-way cross-
over study. A single dose of cefdinir (100 mg) was given on the 13th day.
Serial plasma samples were collected and measured by HPLC. Urinary urea
nitrogen levels were increased by high protein. Plasma trough Phenylalanine
levels were increased. However, Cmax, Tmax, AUClast in the high protein
and Phenylalanine groups were not different for normal protein group.
Intestinal peptide transporter did not seem to be affected by the high protein
diet and phenylalanine load with the subjects, duration and doses
examined [75].
8. STABILITY
Payne and Amyes [76] purified fourteen plasmid-encoded extended-
spectrum b-lactamases from Escherichia coli transconjugates of original clinical
isolates. The Vmax Km and Vmax/Km were each determined for cefdinir and
other related antibiotics as substrates with eight of these enzymes and with
the narrow-spectrum b-lactamase TEM-I. The relative rates of hydrolysis of
cefdinir was also determined for the remaining six enzymes. Cefdinir had
Vmax/Km of relative rate of hydrolysis values either equal to or lower than
other related antibiotics for the enzymes tested. Cefdinir was more stable to
the 15 b-lactamases tested than cefixime.
Mimura et al. [77] estimated the grinding effect on the solid-state stability
of cefdinir by use of microcalorimetry. Ground samples of cefdinir were
prepared and microcalorimetry was performed to estimate the effect of
grinding on the solid-state stability of cefdinir. High-performed liquid chro-
matographic analysis was also performed to interpret the microcalorimetric
Cefdinir 107
ACKNOWLEDGMENTS
The authors wish to thank Mr. Tanvir A. Butt, secretary of the Department of
Pharmaceutical Chemistry, College of Pharmacy, King Saud University, for his secretarial
assistance in typing of this profile.
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Cefdinir 111
Curcumin
Maria L.A.D. Lestari, Gunawan Indrayanto
Faculty of Pharmacy, Airlangga University, Dharmawangsa Dalam, Surabaya, Indonesia
Contents
1. General Information 113
1.1 Solubility 114
1.2 Chemical name 114
1.3 Synonym 114
1.4 CAS number 114
1.5 pKa and coefficient partition 114
1.6 Structural formula 114
1.7 Melting point 116
1.8 Molecular formula and molecular weight 116
2. Stability of Curcumin 116
2.1 Aqueous stability of curcumin 116
2.2 Thermal and photochemical stability of curcumin 117
3. Polymorphism 118
4. Spectroscopy 120
4.1 Ultraviolet/visible and fluorescence 120
4.2 Infrared and Raman Spectroscopy 122
4.3 Mass spectrometry 122
4.4 Nuclear magnetic resonance 131
5. Analysis of Nonbiological Sample 135
5.1 UV fluorometric analysis 135
5.2 Chromatography 138
6. Bioavailability, Metabolite Studies, and Bioanalysis 161
References 198
1. GENERAL INFORMATION
Curcumin is one of the main substances found in the rhizome of
Curcuma longa (L) and other Curcuma spp. Commercially curcumin contains
about 77% besides two other related compounds, that is, deme-
thoxycurcumin and bis-demethoxycurcumin. These compounds belong
to the group of diarylheptanoids. Together, these three compounds are
Profiles of Drug Substances, Excipients, and Related Methodology, Volume 39 # 2014 Elsevier Inc. 113
ISSN 1871-5125 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800173-8.00003-9
114 Maria L.A.D. Lestari and Gunawan Indrayanto
1.1. Solubility
Curcumin is practically insoluble in water at acidic and neutral pH but sol-
uble in polar and nonpolar organic solvents as well as in alkali or in extremely
acidic solvents such as glacial acetic acid [46].
1.3. Synonym
Diferuloylmethane [2]
A
O O
H3CO OCH3
HO OH
B O O
OCH3
HO OH
C O O
HO OH
Figure 3.1 Chemical structure of (A) curcumin, (B) demethoxycurcumin, (C) bis-
demethoxycurcumin.
116 Maria L.A.D. Lestari and Gunawan Indrayanto
2. STABILITY OF CURCUMIN
2.1. Aqueous stability of curcumin
Aqueous media here refers to the acidic or alkaline and biological media.
The stability of curcumin is pH dependent, which is proven by change of
the color of curcumin solution in various pH values. At pH < 1, curcumin
solutions are red in color due to the presence of the protonated form. At
pH 17, curcumin solutions are yellow with the majority of the molecules
being in the neutral form. At pH values higher than 7.5, curcumin solutions
exhibit a color change to orange red. Furthermore, for the buffer system
being used, curcumin forms complexes with borate, citrate, and phthalate,
while being inert toward KCl, KH2PO4, and NaHCO3 [19].
Kinetic degradation of curcumin was investigated in various buffer sys-
tems at pH 111 at 31.5 oC and in fixed ionic systems. Results showed that
the degradation of curcumin followed second-order kinetics. In addition, at
pH 7.07.8, different concentrations of phosphate buffer did not show sig-
nificant change in the rate of the degradation process. In another study [20],
degradation of curcumin in 0.1 M buffer solutions of pH 310 (using citrate,
phosphate, and carbonate buffers) at 37 oC followed apparent first-order
reaction kinetics at constant ion strength.
In these studies, three degradation products were found: vanillin, ferulic
acid (FA), and feruloyl methane. Vanillin was found to be the major degra-
dation product and the amount increased as the incubation time was
extended. It should also be noted that at pH 710 and a temperature of
31.5 oC, the primary degradation products of curcumin were FA and
feruloylmethane [19].
In biological media such as 0.1 M phosphate buffer or serum-free
medium pH 7.2 (both at 37 oC), 90% of curcumin was rapidly degraded
within 3 h of incubation. The degradation products were found to be van-
illin, FA, and feruloylmethane. This fast degradation is slowed down when
the biological media contains fetal calf serum. Similar results were also found
Curcumin 117
A CHO B CH=CHCOOH
C CH=CHCOCH3
D
O
H3CO
OH
OCH3 OCH3 OCH3
HO
OH OH OH
E F OH CH3 G H
O O O
H3CO H3CO
OH O CH3
H OH
HO HO
HO HO
Figure 3.2 Degradation products of curcumin: (A) vanillin, (B) ferulic acid, (C) feruloyl meth-
ane, (D) vanillic acid, (E) ferulic aldehyde, (F) 4-vinylguaiacol, (G) p-hydroxybenzaldehyde,
(H) p-hydroxybenzoic acid.
118 Maria L.A.D. Lestari and Gunawan Indrayanto
3. POLYMORPHISM
Tonnesen et al. [28] reported the crystal structure of curcumin which
was obtained via a crystallization process from ethanol at
70 oC. Commercially available curcumin was reported to be characterized
by the monoclinic space group P2/n (Form-1). Similar findings were
Curcumin 119
5000
4500
4000
3500
3000
Intensity
2500
2000
1500
1000
500
0
0 5 10 15 20 25 30 35 40
2q
Figure 3.3 X-ray powder diffraction pattern of commercially curcumin obtained from
Curcuma longa (turmeric) containing at least 70% of curcumin (Sigma-Aldrich Chemie
GmbH, Germany). Scanning was done from 0.06o to 40o, 2y at a step size of 0.04o and
step time of 0.5 s.
4. SPECTROSCOPY
4.1. Ultraviolet/visible and fluorescence
In acetone-bicarbonate buffer (pH 11), curcumin showed an absorption max-
imum at 520 nm [31]. The absorption band is found to exhibit an asymmetric
shape in these nonpolar solvents: chloroform, acetic acid, benzene, toluene,
and carbon tetrachloride. This asymmetric shape indicates a difference in
the solutesolvent interactions relative to those existing in polar solvents
(methanol, acetone, normal amyl methyl ketone, ethanol, tetrahydrofuran,
acetyl acetone, and acetic acid). Beers law was obeyed for curcumin over
the concentration range of 0.112 ppm or 0.115 ppm. Various maximum
wavelengths were observed in different solvent systems, ranging from
408 nm (in carbon tetrachloride) to 430 nm (DMSO). On the average, the
maximum wavelength was found at 418 nm. The absorption maximum of
curcumin in methanol and ethanol was around 420 nm, and the solution
exhibited a bright yellow color owing to its pp* excitation; the np*
transition was observed at 426 nm in methanol [32,33].
Curcumin 121
(m/z MH: 309.11) using direct analysis in real time MS. Hiserodt
et al. [75] optimized two different types of MS, the particle beam EI-mass
spectra and thermospray mass spectra. The thermospray LCMS provided
information for molecular weight but with limited fragmentation, while
particle beam interface was used to obtain EI-mass spectra of nonvolatile
components.
In another study, the fragmentation behavior of curcumin and two other
compounds was observed using ion trap LCMS/MS and confirmed with
sustained off-resonance irradiation fragmentation (Fourier Transform Ion
Cyclotron Resonance (FTICR) SORI-MS/MS) [43].
5.2. Chromatography
5.2.1 TLC/HPTLC
The use of TLC for qualitative analysis of curcumin is described in the Euro-
pean Pharmacopoeia [87]. In this method, fluorescein and thymol are dis-
solved in methanol and used as the reference solution. The test solution is
also dissolved in methanol. The mobile phase is a mixture of glacial acetic
acid and toluene (80:20 v/v). The dry TLC plate is sprayed with 0.4 g/L
dichloroquinonechlorimide in 2-propanol. The plate is then exposed to
ammonia vapor until the bluish-violet zone (for thymol) and yellow zone
in the lower part (for fluorescein) is observed. Two yellowish brown to
brown zones between the thymol and fluorescein zone indicate the presence
of curcumin and demethoxycurcumin.
For quantitative analysis, both TLC and HPTLC methods have been
reported. In the TLC method reported by Pothitirat et al. [82], the peaks
of curcumin, demethoxycurcumin, and demethoxycurcumin were not well
separated, although those three peaks can be distinguished from each other
based on their retardation factor (Rf). In the HPTLC method, it was found
that use of the LiChrosphere HPTLC plate successfully resolved the prob-
lem of broadness in the spot. The curcumin peak was also well separated, as
shown by its relatively high Rf apart from the peak of demethoxycurcumin
and bis-demethoxycurcumin. However, peaks of demethoxycurcumin
and bis-demethoxycurcumin were not completely separated [88]. Good
separation for the peaks of curcumin, demethoxycurcumin, and bis-
demethoxycurcumin was observed when the silica gel plate was used with
a mobile phase consisting of chloroform and methanol. In this method,
each peak was completely separated from each other [8991]. Similar results
were also obtained, where the combination of chloroform:MeOH 19:1
(v/v) gave the best result in the separation of curcuminoid compounds [92].
A summary of the TLC as well as HPTLC methods is provided in Table 3.9.
5.2.2 HPLC
An initial HPLC method for the determination of curcumin using HPLC
was reported by Asakawa et al. [93]. A Nucleosil C18 column was used with
a mobile phase consisting of acetonitrile, methanol, and water. However,
this method did not successfully separate curcumin from two other com-
pounds in curcuminoid, and a long running time of 40 min was needed.
Later on, Tonnesen et al. [7] reported a HPLC method which was able to
separate three different components of curcuminoid. Two different
Table 3.9 HPTLC analysis of curcumin
LOD, LOQ,
Compound Sample Solvent Chromatographic condition % recovery Reference
Curcumin, demethoxycurcumin, Rhizome of MeOH Stationary phase: Precoated silica gel LOD: n/a [82]
bis-demethoxycurcumin C. longa aluminum plate 60F254 LOQ: n/a
Mobile phase: CHCl3:benzene: Rec: n/a
MeOH 80:15:5
l 420 nm
Curcumin, demethoxycurcumin, Rhizome of MeOH Stationary phase: LOD: [88]
bis-demethoxycurcumin C. longa LiChrosphere 60F254 C: 40 ng
CHCl3:MeOH 98:2 DC: 40 ng
Temperature: 25 1 oC BDC: 20 ng
RH: 3540% LOQ:
l366 nm C: 100 ng
DC: 100 ng
BDC: 100 ng
Rec:
C: 99.79
DC: 96.97
BDC: 99.48
Curcumin, demethoxycurcumin, Rhizome of MeOH Stationary phase: LOD: n/a [90]
bis-demethoxycurcumin C. longa HPTLC silica gel 60F254 LOQ: n/a
CHCl3:MeOH 48:2 Rec:
Temperature: 25 5 oC C: 97.3
RH: 50% DC: 92.9
l 425 nm BDC: 95.4
Continued
Table 3.9 HPTLC analysis of curcumincont'd
LOD, LOQ,
Compound Sample Solvent Chromatographic condition % recovery Reference
Curcumin Rhizome of MeOH Stationary phase: LOD: [89]
C. longa Precoated silica gel aluminum 50 ng/spot
plate 60F254 LOQ:
Toluene:CHCl3:MeOH 5:4:1 200 ng/spot
Temperature: 25 2 oC Rec:
RH: 60 5% 98.55100.71
l 430 nm
Curcumin, demethoxycurcumin, Gel MeOH Stationary phase: LOD: [91]
bis-demethoxycurcumin Precoated silica gel 60F254 C:
CHCl3:MeOH:glacial acetic 100 ng/spot
acid 7.5:2.0:0.5 DC:
Temperature: n/a 45.0 ng/spot
RH: n/a BDC:
l 430 nm 52.0 ng/spot
LOQ:
C:
250 ng/spot
DC:
170 ng/spot
BDC:
80 ng/spot
Rec:
C: 100.8
DC: 99.23
BDC: 100.12
Curcumin 141
between the pH of the eluate and the percent recovery. The only consid-
eration is that higher pH is associated with concentrated NaOH, which led
to the large background noise during detection at 1.20 V.
In another publication, Lechtenberg et al. [108] reported the use of a
DAD for both qualitative and quantitative analysis of curcumin,
demethoxycurcumin, and bis-demethoxycurcumin in rhizome and curry
powder. In their method, a combination of phosphate, b-cyclodextrin
(b-CD) hydrate and sodium hydroxide was applied to obtain an optimal
analysis. In the optimization process, it was observed that increasing concen-
tration of phosphate improved the separation of curcuminoid compounds,
but prolonged the time of analysis. From the use of three different cyclodex-
trins, b-CD hydrate gave better separation with short migration time
(<6 min), and at a concentration of 14 mM, optimum separation was
achieved. Due to its similar chromophore with the curcuminoid com-
pounds, 3,4-dimethoxy-trans-cinnamic acid was chosen as the internal stan-
dard. Standard and sample solutions were found to be stable for at least 2 h.
Addition of methyl-b-CD (100 mg in water/MeOH, 1:1 v/v) to the stan-
dard/sample solution was needed to maintain stability in methanolic
solution.
Marakova et al. [109] optimized the combination of complexing buffer,
carrier anion, counter-ion, and pH of the buffer to determine curcuminoid
compounds. Compared to the previous CE-DAD method, the use of (3-
cyclohexylamino)-1-propanesulfonic acid (CAPS) gave better results com-
pared to the use of phosphate as the carrier electrolyte. In addition, more
effective resolution was achieved with the HP-b-CD compared to native
b-CD or ionizable TMA-b-CD. Furthermore, higher HP-b-CD levels
were able to reduce adsorption of curcuminoid onto the capillary wall
and also maintained stability of curcuminoid in the alkaline separation envi-
ronment. In term of pH, higher pH values were preferred due to their ability
to facilitate ionization of curcuminoid, thus increasing separation efficiency
and resolution, as well as shortening analysis time. In addition, coating the
fused-silica capillary tube with hydroxyethylcellulose (molecular weight
30,000) could replace the need to rinse the capillary in order to suppress
electro-osmotic flow. Connection with the DAD also enabled a spectral
purity check of the sample.
For the MEKC method, Watanabe et al. [110] reported the successful
separation of three components of curcuminoid using a butyl acrylatebutyl
methacrylatemethacrylic acid copolymer sodium salt solution containing
50% dimethyl sulfoxide as the running buffer. As comparison, a HPLC
Curcumin 161
method was also used and it was found that analysis with MEKC was faster
than when using HPLC. In the earlier development, 25 mM SDS solution
in 30 mM borate buffer at pH 8.0 was applied, but could not separate
curcuminoid component.
As an alternative to the CE method, Nhujak et al. [111] investigated the
application of MEEKC to analyze curcuminoid compounds. In MEEKC, a
microemulsion is used as the carrier electrolyte. This microemulsion consists
of oil droplets stabilized in aqueous solution by a surfactant to stabilize and to
give negative charge to the microemulsion. The principle of separation is
based on partitioning of the analyte between an aqueous phase and a
pseudo-stationary phase of oil droplets. Since curcumin is unstable in basic
media, an acidic buffer was preferred. The organic cosolvent in the separa-
tion process is needed to partition the analytes in the organic-aqueous phase.
In this research, three organic cosolvents were investigated, acetonitrile,
methanol, ethanol, and isopropanol at concentrations of 030%. Based on
the results obtained, the use of isopropanol at 25% yielded good separation
of the curcuminoid compounds. In the selection of appropriate alcohols, it
was observed that higher number of carbon atoms in the aliphatic chain
resulted in greater retention factor of analytes. The influence of temperature
was also studied, since increasing temperature will reduce the viscosity of the
micelle and enhance faster migration. However, above 25 oC, Joule heating
caused decreasing efficiency (N) or increasing in thermal dispersion (Ht) as
well as slight decrease in a. As a result, the resolution of the compounds
became worse. Therefore, the temperature used for the analysis process
was set at 25 oC.
A summary of the CE as well as MEKC and MEEKC methods is detailed
in Table 3.11.
A B
O OH O OH
H3CO OCH3 H3CO OCH3
HO OH HO OH
C D O O
O OH
H3CO OCH3
H3CO OCH3
- HO OH
O3SO OH
E
O OH
H3CO OCH3
OOC
O
HO
HO O OH
OH
200 mL
sample 80 mL
deionized water then
vortex-mixed for
20 s, then 40 mL IS
solution was added,
vortex-mixed again
for 20 s.
Add 500 mL
extracted solution
(EtOAc:
MeOH 95:5), then
vortex-mixed 30 s,
centrifuged 5 min
(13,500 rpm).
420 mL of organic
layer was removed,
evaporated, then
dissolved in 200 mL
mobile phase,
injected to HPLC
(20 mL).
Curcumin Column (HPLC-UV): Pig plasma b-Estradiol Extraction by EtOAc Rec: 6678% [50]
Symmetry shield C18, (25250 ng/mL in Intra- and interday
Waters, USA plasma): precision: <15%
(3.9 150 mm; 5 mm);
guard column: C18, 1 mL blank
Pnenomenex, USA plasma 20 mL
(3.0 4 mm) methanolic (range
Mobile phase: 0.1% concentrations:
citric acid pH 3.0 1.2512.5 mL/
(adjusted by 45% mL) 100 mL citrate
KOH): tetrahydrofuran buffer
(50/50, v/v) pH 3.0 2 mL
Detection: UV 280 nm EtOAc, then samples
were shaken,
Continued
Table 3.12 Summary of bioanalysis's method of curcumin, related compounds, and its metabolitescont'd
Validation data
Biological of recovery/accuracy/
Chromatography sample/ Internal standard Preparation of standard, bias, precision,
Analytes (samples) conditions matrices (IS) sample extraction others results Reference
centrifuged 1200 g
(10 min).
1.65 mL EtOAc was
removed,
evaporated, then
dissolved in 100 mL
MeOH, 50 mL was
injected.
1 mL plasma 20 mL
IS then vortex-
mixed for 10 s.
After vortex-mixing
100 mL. 1 M HCl
was added, mixed,
then 5 mL EtOAc,
vortex-mixed 5 min,
centrifuged 1500 g
for 10 min.
4 mL of organic layer
was taken,
evaporated, then
residue was dissolved
in mobile phase
(100 mL), vortex-
mixed, centrifuged
then 40 mL was
injected into HPLC.
Continued
Table 3.12 Summary of bioanalysis's method of curcumin, related compounds, and its metabolitescont'd
Validation data
Biological of recovery/accuracy/
Chromatography sample/ Internal standard Preparation of standard, bias, precision,
Analytes (samples) conditions matrices (IS) sample extraction others results Reference
Curcumin, tetra- Column (LCMS/ Rat plasma Salbutamol Standards: Curcumin, Bias: [53]
hydro-curcumin MS): C18 Phenomenex THC, and IS were Curcumin:
(THC); (curcumin Luna (250 4.6 mm) dissolved in MeOH Intraday: 2.01% to
administrated as Mobile phase: (50 mg/mL); calibration 6.00%
curcumin and ACN:0.05% acetic acid standards, QC samples Interday: 2.67% to
curcumin in water (70:30) were prepared by 9.52%
phospholipid complex) Detection: MRM spiking stock solutions THC:
(positive ion mode) into blank plasma Intraday: 1.30% to
Curcumin: m/z Samples: 4.32%
369.3285.1 Interday: 5.05% to
THC: m/z 0.1 mL aliquot 1.48%
373.2137.1 plasma 50 mL RSD precision:
IS: m/z 240.2148.1 phosphate buffer Curcumin:
(pH 6.86, 0.1 M) Intraday: 2.56% to
1000 U 13.52%
b-glucoronidase, Interday: 3.7712%
incubated
1 h at THC:
37 C. Intraday: 6.3613.75%
Then 50 mL mobile Interday: 3.9112.47%
phase containing
1 mg/mL IS, vortex-
mixed 30 s, then add
1 mL EtOAc,
vortex-mixed again
for 1 min, and
ultrasonificated for
15 min, centrifuged
6 min (15,000 g).
The organic layer
was taken,
evaporated, then
dissolved in 100 mL
mobile phase, 10 mL
was injected
into HPLC.
Curcumin Column (HPLC-UV/ Rat plasma n/r Standard: Curcumin Rec.: 82.7084.29% [54]
(administrated as Vis): Waters was dissolved in MeOH RSD precision:
phospholipid complex) m-Bondapack C18 (0.1 ppm) Intraday: 2.783.56%
(300 4.6 mm) Samples: Interday: 2.123.24%
Mobile phase:
MeOH:2% acetic acid: 1 mL serum 5 mL
ACN (5:30:65) MeOH, shaken
Detection: UV 425 nm vigorously
2530 min
(7580 C), then
diluted with MeOH
(10 mL).
Centrifugation
10 min (5000 rpm),
20 was injected
into HPLC.
Continued
Table 3.12 Summary of bioanalysis's method of curcumin, related compounds, and its metabolitescont'd
Validation data
Biological of recovery/accuracy/
Chromatography sample/ Internal standard Preparation of standard, bias, precision,
Analytes (samples) conditions matrices (IS) sample extraction others results Reference
Curcumin, dimethoxy- Column (LCMS/ Rat plasma Honokiol Standard: Bias: [55]
curcumin (DC) MS): Zorbax Extend- Curcuminoids and IS Curcumin:
C18 (150 4.6 mm; were dissolved in Intraday: 5% to 12%
5 mm). MeOH (1 mg/mL) Interday: 0.68%
Mobile phase: ACN: Samples: DC:
1 mM HCOOH Intraday: 0.4% to
(70:30, v/v) 150 mL blood 12%
Detection: MRM samples were Interday: 0.816%
(negative ion mode) centrifuged RSD precision:
Curcumin: m/z (3000 g, 5 min). Intraday: 1% to 18%
367217 50 mL plasma IS Interday: 0.519%
DC: m/z 337217 Solution (0.1 mg/ DC:
mL), vortex-mixed Intraday: 0.618%
and centrifuged. Interday: 0.317%
20 mL was injected.
Curcumin (curcumin Column (HPLC-UV/ Rat plasma 4-Hydroxy- Standard: Curcumin Rec: [56]
administrated as Vis): Waters benzophenone and IS were dissolved in 94.997.1%
solubilized curcumin m-Bondapack C18 MeOH (1 mg/mL); RSD precision:
and its micellar (300 3.9 mm) calibrations were Intraday: 2.032. 6%
formulations) Mobile phase: prepared by adding Interday: 4.4312.7%
ACN:1% citric acid stock solutions into
(w/v) pH 3 (adjusted by blank plasma
concentrated NaOH) Samples:
(55:45, v/v)
Detection: UV/Vis: 100 mL
428 and 300 nm plasma 50 mL IS,
mixed then 250 mL
ACN was added,
vortex-mixed,
centrifuged
14,000 g for 5 min,
100 mL supernatant
was injected.
Curcumin and its Column (HPLC-UV, Rat n/r Curcumin and its Quantitative validation [57]
metabolites in rats quantitative): Atlantis plasma, metabolites were data: n/r
dC18 (150 4.6 mm, intestinal extracted using SPE Curcumin,
3 mm), guard column mucosa, (1 mL Oasis HLB desmethoxy curcumin,
(20 3 mm, 3 mm) and liver cartridge, Waters). bis-desmethoxy
Mobile phase: Plasma: curcumin, tetra-
A: 10 mM ammonium hydrocurcumin, hexa
acetate pH 4.5 B: 1 mL plasma was hydrocurcumin,
ACN: Gradient: initial loaded curcumin glucoronide,
95% A, progressing to SPE was washed with and curcumin sulfate
55% A (20 min) and 5% MeOH:H2O:glacial were detected in
A (33 min) acetic acid (25:25:1) plasma, intestinal
Detection: UV 280 nm and eluted with 1 mL mucosa, and liver
Column (LCMS/MS, MeOH containing
identification 2% glacial acetic acid.
Continued
Table 3.12 Summary of bioanalysis's method of curcumin, related compounds, and its metabolitescont'd
Validation data
Biological of recovery/accuracy/
Chromatography sample/ Internal standard Preparation of standard, bias, precision,
Analytes (samples) conditions matrices (IS) sample extraction others results Reference
metabolite):
Conditions same as Mucosa and liver:
quantitative except
A consisted of 5 mM Suspended and
ammonium acetate homogenized using
pH 4.5 KCl then extracted
Metabolites were with acetone:formic
detected using MRM acid (9:1), then
centrifuged;
supernatant was
taken and evaporated
to dryness under
nitrogen.
Curcumin, COG, CS Column (HPLC-UV/ Human b-17-Estradiol Extraction curcumin: Rec: [58]
Vis): m-Bondapack C18 plasma acetate Curcumin at 1 mg/mL
(250 4.6 mm, 10 mm) 200 mL was 95.14%
Mobile phase: plasma 80 mL (RSD 2.75)
A: 0.1% acetic acid: water 20 mL IS
MeOH:H2O vortex-mixed.
(0.1:65:35) The solution was
B: MeOH, gradient extracted thrice using
elution 100% A to by vortexing using
100% B in 15 min 1 mL mixture of
Detection: Vis 420 nm EtOAc:MeOH
(95:5) for 3 min.
Sample was
centrifuged
3000 rpm (15 min,
4 C), organic layer
was collected,
evaporated to dryness
then dissolved in
100 mL MeOH.
Hydrolysis of curcumin
conjugates:
Same as above,
samples were then
mixed with
b-glucoronidase
(50 mL, 446 U, in
0.1 M phosphate
buffer pH 6.8) and
sulfatase (45 mL,
52 U, in Na acetate
buffer pH 5), then
incubated for 3.5 h.
Samples were
extracted as
described above.
Continued
Table 3.12 Summary of bioanalysis's method of curcumin, related compounds, and its metabolitescont'd
Validation data
Biological of recovery/accuracy/
Chromatography sample/ Internal standard Preparation of standard, bias, precision,
Analytes (samples) conditions matrices (IS) sample extraction others results Reference
Curcumin Column (HPLC-UV/ Human n/a Extraction: Validation data: n/r [59]
Vis): plasma
C18 ODS 5 mL blood was
Phenomenex centrifuged 10 min
(250 4.6 mm, 5 mm) (2000 g).
Mobile phase: MeOH 2.5 g plasma was
Detection: UV 420 nm extracted thrice using
EtOAc (3, 2, 2 mL).
Pooled extract was
evaporated to dryness
under N2, then
residue was dissolved
in 2 mL MeOH by
vortexing.
Curcumin, piperin Column (HPLC- Human b-17-Estradiol Standards: Stock Accuracy: [60]
UV/Vis): plasma acetate solutions were prepared Intraday:
Chromolith Speed by dissolving each of Curcumin:
ROD RP-18 (50 4/ the analytes in MeOH 94.5 2.0%
6 mm) (1 mg/mL); calibration Piperin: 98.0 4.8%
Mobile phase: ACN: standards, QC samples Interday:
MeOH: were prepared by Curcumin:
TFA:H2O spiking stock solutions 94.0 2.5%
(17.6:35.3:0.1:47, into blank plasma Piperin: 97.2 5 1%
v/v/v/v) Samples: RSD precision:
Detection: Intraday:
Curcumin: 415 nm 100 mL IS Curcumin: 5.7 2.1%
Piperin: 335 nm (1500 ng/mL) Piperin: 5.4 2.7%
IS: 280 nm 0.5 mL distilled Interday:
water 100 mL Curcumin: 5.9 2.7%
phosphate buffer Piperin: 6.1 3.0%
pH 3.4 6 mL
extraction solvent
(EtOAc:
propanol 9:1, v/v)
were added to
0.5 mL of samples,
then vortex-mixed,
then shaken well in a
rotating mixer for
15 min, centrifuged
15 min (100 g).
The upper organic
layer was taken,
evaporated then
dissolved in 100 mL
MeOH, and 40 mL
was injected.
Continued
Table 3.12 Summary of bioanalysis's method of curcumin, related compounds, and its metabolitescont'd
Validation data
Biological of recovery/accuracy/
Chromatography sample/ Internal standard Preparation of standard, bias, precision,
Analytes (samples) conditions matrices (IS) sample extraction others results Reference
Curcumin Column (HPLC- Rat plasma Emodin Standards: Curcumin Accuracy and (RSD [61]
(administrated as PDA): Diamonsil C18 and IS were dissolve in precision)
curcumin liposome) (100 4.6 mm, 5 mm) ACN, at concentrations Intraday:
Mobile phase: ACN: 0.5 and 1.5 mg/mL, 94.15106.23%
5% acetic acid (75:25, respectively. (1.747.51%)
v/v) Calibration standards Interday:
Detection: UV 420 nm and QC samples were 100.92107.05%
prepared by spiking (3.379.24%)
appropriate solutions
into blank plasma
Samples:
Samples were
prepared in 96-well
format plate (1 mL,
Variant, USA).
90 mL rat blank
plasma 10 mL
standard working
solutions, 100 mL
aliquot plasma
samples and QC
samples were
pipetted into 96-well
plates.
Then add 100 mL
ACN containing IS
(0.15 mg/mL),
capped, vortex-
mixed then
centrifuged 2500 g
15 min.
350 mL supernatant
was transferred to the
96-well plates, 50 mL
was injected
into HPLC.
Curcumin, tetra- Column (LCMS/ Mouse Hesperitin Standards: Standard Accuracy (RSD) [62]
hydrocurcumin MS): Beta basic C8 plasma, solutions of curcumin. Intraday:
(THC), 1,7-bis(3,4, (50 2.1 mm, 5 mm) cell THC, TMC, Curcumin:
dimethoxyphenyl)-4,4- with guard column medium DMCHC, and IS were 99.7 (6.22)103.1
dimethyl-1,6 Beta basic C8 javelin prepared in ACN; (14.01)%
heptadiene-3,5 dione (10 2.1 mm, 2 mm) standard solution in the THC: 90.3 (5.64)
(TMC), 1,7-bis(3,4, Mobile phase: 50% range of 109.9 (5.92)%
dimethoxyphenyl)-4- ACN containing 0.1% 1010,000 ng/mL TMC: 86.2 (9.23)
cyclohexyl-1,6 formic acid were prepared by 99.5 (7.87)%
heptadiene-3,5 dione Detection by SRM: dilutions. Calibration DMCHC: 97.9 (9.35)
(DMCHC) Curcumin: m/z standards and QC 101.2 (5.96)%.
Continued
Table 3.12 Summary of bioanalysis's method of curcumin, related compounds, and its metabolitescont'd
Validation data
Biological of recovery/accuracy/
Chromatography sample/ Internal standard Preparation of standard, bias, precision,
Analytes (samples) conditions matrices (IS) sample extraction others results Reference
369177 samples were prepared Interday:
THC: m/z 373137 by spiking the solutions Curcumin:
TMC: m/z 425191 into mouse plasma or 92.7 (7.61)98.5
DMCHC: m/z cell medium (5.02)%
465191 Samples: THC: 89.9 (5.36)
IS: m/z 303153 105.5 (7.51)%
200 mL RPMI cell TMC: 88.5 (15.10)
medium, or 100 mL 100.1 (3.99)%
plasma that DMCHC: 95.64
contained IS (9.25)100.5 (5.78)%
(1000 ng/mL) were
extracted with 1 mL
EtOAc for 60 min
mechanical shaking.
After extraction,
samples were
centrifuged 2 min
(11,000 g), and
place on dry ice for
1 min.
Supernatant of
EtOAc were
evaporated to dryness
under nitrogen,
reconstituted in
100 mL ACN 50%,
and centrifuged
2 min (11,000 g);
20 mL supernatant
was used for analysis.
Curcumin Column (LCMS/ Rat plasma Biochanin Standard: Accuracy (RSD [63]
MS): Supelco precision):
Discovery C18 Primary stock 96.33107.67%
(50 4.6 mm; 5 mm) solutions were (0.715.97%)
Mobile phase:0.01 M prepared by
ammonium acetate dissolving 5 mg in
pH 5.5:ACN (10:90) 0.2 mL DMSO and
Detection: MRM appropriate dilutions
(negative ion mode): were made with
Curcumin: m/z MeOH. Calibration
367217 standards and QC
IS: m/z 283268 samples were
prepared by spiking
working solutions
into control pooled
rat plasma.
Continued
Table 3.12 Summary of bioanalysis's method of curcumin, related compounds, and its metabolitescont'd
Validation data
Biological of recovery/accuracy/
Chromatography sample/ Internal standard Preparation of standard, bias, precision,
Analytes (samples) conditions matrices (IS) sample extraction others results Reference
Samples:
100 mL
plasma 10 mL IS
solution (MeOH,
1 mg/mL), mixed for
15 s.
Add 2 mL mixture of
CH2Cl2:EtOA (1:1,
v/v) then vortex-
mixed 3 min,
centrifuged 5 min
(2000 g).
1.6 mL organic layer
was removed,
evaporated, then
dissolved in 200 mL
mobile phase, 10 mL
was injected into LC.
Curcumin Column (LCMS/ Rat plasma Nimesulide Standard: Curcumin Accuracy (RSD [64]
(administrated as free MS): Chromolith and IS were dissolved in precision):
curcumin and as solid (50 4.6 mm, 5 mm) MeOH (100 mg/mL); Intraday: 102.43105.
lipid nanoparticle) Mobile phase: calibration standard and 67% (23.55%)
ACN:10 mM QC were prepared by Interday:
ammonium acetate spiking the appropriate 105.7108.5%
buffer (80:20, v/v) working solutions into (2.053.55%)
Detection: MRM plasma
(negative): Samples:
Curcumin: m/z
367217 100 mL rat
IS: m/z 307229 plasma 10 mL IS
solution 200 mL
b-glucoronidase,
incubate 37 C for
1 h.
Curcumin was
extracted by using
2 mL diethyl ether by
vortexing (1 min)
then cold centrifuged
(4 C, 5 min,
5000 rpm).
Supernatant was
evaporated to dryness
in nitrogen (50 C)
and residue was
dissolved in mobile
phase.
Continued
Table 3.12 Summary of bioanalysis's method of curcumin, related compounds, and its metabolitescont'd
Validation data
Biological of recovery/accuracy/
Chromatography sample/ Internal standard Preparation of standard, bias, precision,
Analytes (samples) conditions matrices (IS) sample extraction others results Reference
Curcumin, curcumin Column (HPLC-UV/ Rat plasma Emodin Standard: Stock Bias: [65]
di-decanoate (CurDD) Vis): C18 solutions of curcumin Curcumin: 2.77% to
Phenomenex CurDD and IS were 3.15%
(250 4.6 mm, 5 mm) prepared in ACN. CurDD: 6.07% to
Mobile phase: Gradient Working solutions of 8.88%
elution ACN:THF: curcumin and IS were RSD Precision
H20 with 0.1% formic prepared by dilution Intraday:
acid (08 min: with MeOH, while for Curcumin:
35:20:45, 813 min: CurDD, ACN was 5.7111.5%
35:20:4550:40:10, and used. CurDD: 1.649.06%
1321 min: 50:40:10) Calibration standard Interday
Detection: Vis 425 nm and QC were prepared Curcumin:
by spiking the 7.2411.4%
appropriate working CurDD: 3.7718.7%
solutions into plasma
Samples (prepared in
low temperature
08 C)
50 mL
plasma 10 mL IS (so
the concentrations of
IS was 500 ng/mL).
After vortex-mixing
for 10 s, then 50 mL
of 10% sodium
dodecyl sulfate was
added, vortex-mixed
for 15 s.
Add 600 mL of
EtOA, vortex-mixed
90 s then centrifuged
5 min (12,000 rpm).
Supernatant was
taken, evaporated,
the residue was
dissolved in 80%
ACN, 50 mL was
injected.
Curcumin, Column (LCMS/ Mice Honokiol Standards: Stock Bias (RSD precision) 66
demethoxycurcumin MS): Metabolite tumor solutions were prepared Intrabatch:
(DMC), bis- identification: by dissolving each Curcumin: 1% to
demethoxycurcumin YMC Pack ODS analyte and IS in 5.5% (5.511.2%)
(BDMC) A C18 (250 4.6 mm, MeOH (500 mg/mL); DMC: 11.7% to
(administrated as 5 mm); guard column: working solutions were 14.3% (4.67.5%)
curcuminoid nano Agilent Zorbax Sb C18 prepared by mixing BDMC: 11.9% to
particle formulation) (12.5 4.6 mm, 5 mm) each of the stock 14.0% (3.34.8%)
Mobile phase: Solvent solutions Interbatch:
Continued
Table 3.12 Summary of bioanalysis's method of curcumin, related compounds, and its metabolitescont'd
Validation data
Biological of recovery/accuracy/
Chromatography sample/ Internal standard Preparation of standard, bias, precision,
Analytes (samples) conditions matrices (IS) sample extraction others results Reference
A: ACN, solvent B: Calibration standards Curcumin: 5.2% to
0.1% aqueous formic and QC samples were 5.1% (11.113.4%)
acid; gradient elution: prepared by spiking the DMC: 10.9% to
0 min: (A:B):5:95,v/v, appropriate working 13.2% (3.86.6%)
30 min linear gradient solutions into the tumor BDMC: 12.8% to
to 45:55, 50 min: 95:5, homogenate 14.1% (3.66.5%)
55 min 95:5 Samples:
Quantitative: Agilent
Zorbax SB C8 330 mL aliquot of
(150 4.6 mm, 5 mm); tumor
guard column: Agilent homogenate 20 mL
Zorbax Sb C18 IS (10 mg/mL)
(12.5 4.6 mm, 5 mm) vortex-mixed 30 s,
Mobile phase: 0 min: then extracted with
(A:B): 50:50, 6 min: 1400 mL ACN, then
85:15, 7 min: 95:5, vortex-mixing 1 min
8 min: 95:5 followed by
Detection: () m/z: ultrasonification 30 s.
Curcumin After centrifugation
(367.3149.2) for 10 min
DMC (337.1119.2) (9500 g), organic
BDMC (307.2119.1) layer was taken,
IS (265.2224.1) evaporated under
nitrogen, and the
residue was dissolved
in 200 mL MeOH
(vortex-mixing
1 min,
ultrasonification
30 s), 5 mL was
injected.
Curcumin Column (HPLC- Rat 2-(40 - Standard: Stock Accuracy, precision [67]
(administrated as PDA): Eclipse XDB plasma, hydroxy-benzene- solution: Curcumin intra- and interday are
curcumin-loaded Agilent C18 fecal, and azo) benzoic acid was dissolved in ACN within 15%
PLGA nano particle) (150 4.6 mm; 5 mm) urine (500 mg/mL); working
Mobile phase: ACN: solutions were prepared
10 mM monosodium by dilution of stock
phosphate pH 3.5, solution with 50%
adjusted by H3PO3 ACN; IS was dissolved
(40:60, v/v): in ACN (1.5 mg/mL)
Detection: 425 nm Calibration standards
and QC samples were
prepared by spiking
appropriate solutions
into blank plasma/
urine/fecal supernatant
Samples:
Plasma: 50 mL
plasma 100 mL IS
solution, vortex-mixed
then centrifuged
15 min (12,000 g);
Continued
Table 3.12 Summary of bioanalysis's method of curcumin, related compounds, and its metabolitescont'd
Validation data
Biological of recovery/accuracy/
Chromatography sample/ Internal standard Preparation of standard, bias, precision,
Analytes (samples) conditions matrices (IS) sample extraction others results Reference
20 mL was injected
Fecal: 50 mL fecal
supernatant 100 mL
IS solution, vortex-
mixed then centrifuged
15 min (12,000 g);
20 mL was injected
Urine: 50 mL urine
supernatant was
extracted with 1 mL
EtOAc by vortex-
mixed, then
centrifuged 5 min
(8000 rpm), after
drying with vacuum
centrifugation, the
residue was dissolved
100 mL 50% IS solution,
then 20 mL was injected
Curcumin, Curcumin- Column (LCMS/ Human Hesperitin Samples extraction:Accuracy (% RSD) [68]
O-glucoronide (COG) MS): Beta Basic C8 plasma Intraday:
(50 2.1 mm; 2 mm), 10 mL standard Curcumin: 91.3112%
guard column: Beta C8 solutions of (3.458.32%)
(10 2.1 mm; 2 mm) curcumin and COG COG: 82.7102%
Mobile phase: ACN: (2020,000 ng/mL), (3.129.73%)
H2 containing 0.1% IS (10 mg/mL) were Interday:
formic acid (50:50, v/v) added to 100 mL Curcumin: 99.5101%
Detection MRM: human plasma; (5.1812.7%)
Curcumin: m/z calibrations curve COG: 105109%
369177 containing curcumin (6.0811.3%)
COG: m/z 545369 and COG
IS: m/z 303177 (22000 ng/mL)
while QC samples
containing
2500 ng/mL.
The mixture was
vortex-mixed and
diluted with 0.15 M
phosphate saline
buffer (900 mL) then
extracted with
EtOAc, after the
organic layer was
taken and
evaporated, residue
was dissolve in
100 mL mobile
phase.
After centrifuged for
2 min (12,000 g),
25 mL of supernatant
was injected.
Continued
Table 3.12 Summary of bioanalysis's method of curcumin, related compounds, and its metabolitescont'd
Validation data
Biological of recovery/accuracy/
Chromatography sample/ Internal standard Preparation of standard, bias, precision,
Analytes (samples) conditions matrices (IS) sample extraction others results Reference
Curcumin Column (HPLC- Rat plasma Rhoda-mine 6G Standard: n/r n/r [69]
(administrated as Fluorescent): Plasma was extracted
amorphous curcumin) LiChrosphere C18 with ACN
(200 4.6, mm)
Mobile phase: 1% (w/v)
citric acid mono
hydrate pH 3: ACN:
THF (48:32:20)
Detection:
Fluorescent detector:
lex/lem 420/530 nm
Curcumin Column (HPLC-UV/ Human n/r Standard: n/r n/r [70]
Vis): Shim-Pack CLC serum Extraction method was
C18 (250 4.6 mm; performed using
5 mm) dispersive liquid-liquid
Mobile phase: microextraction in a
THF:0.1% citric acid cold column trapping
pH 3 (60:40) (CCT-DLLME)
Detection: n/r
Curcumin Column (HPLC-UV/ Mice n/r Standard: n/r n/r [71]
(administrated as free Vis): Hypersil ODS blood and Stomach tissue:
C18 (150 4.6 mm,
curcumin and 5 mm) stomach The freeze-dried
nanoencapsulation) Mobile phase: tissue tissue (1020 mg)
MeOH:3.6% aqueous was shaken with
acetic acid (73:27, v/v) 2 mL EtOAc
Detection: UV 425 nm overnight, centrifuge
8000 g (10 min),
filtered and dried
under N2,dissolved
in 1 mL MeOH,
then injected
(20 mL).
Blood:
200 mL
plasma 500 mL
EtOAc vortex-
mixed for 60 s and
sonicated for 60 s,
filtered and dried
under N2, dissolved
in 1 mL MeOH,
then injected
(20 mL).
Curcumin and Column (LCMS/ Human n/a Quantitative Intra- and interday [72]
metabolites, and MS): Phenomenex 3 m blood, measurements of accuracy and
13 other compounds C18(2) 100 nA urine, and curcumin metabolites precision < 15%
(administrated as (50 2 mm) feces were performed using
Continued
Table 3.12 Summary of bioanalysis's method of curcumin, related compounds, and its metabolitescont'd
Validation data
Biological of recovery/accuracy/
Chromatography sample/ Internal standard Preparation of standard, bias, precision,
Analytes (samples) conditions matrices (IS) sample extraction others results Reference
enriched bread; in Mobile phase: calibration curve of
microencapsulated A H2O:ACN:formic curcumin
curcumin) acid (94.9:5:0.1, v/v)
B ACN:formic acid Blood samples
(99.9:0.1, v/v); linear centrifuged
gradient:01 min: 4000 rpm (10 min,
440% B; 13 min: 4 C).
40100% B; 35 min: Urine samples were
100% B; 610 min: 4% treated with 0.005%
B butylated hydroxyl
Detection: MRM toluene (BHT).
(negative ion mode): Feces was diluted
m/zCurcumin: 1:10 (w/v) PBS
367 > 217 10 mM, containing
DeMC:337 > 217 0.005% BHT,
BDeMC: 307 > 217 vortex-mixed and
COG: 543 > 367 centrifuged
Curcumin sulfate: 4000 rpm (15 min,
447 > 367 4 C).
THCG: 547 > 135 Serum, urine and
Hexa hydrocurcumin fecal supernatant
(HHC): 373 > 179 were stored at
HHCG: 549 > 373 40 C before
analysis.
1 mL serum, 3 mL
urine and fecal were
extracted with 6 and
9 mL EtOAc,
respectively.
Supernatant were
dried under N2,
residue was dissolved
in 50 mL (MeOH:
H2O 70:30), 20 mL
was injected
into HPLC.
n/r: not reported; n/a: not available.
Desmethoxycurcumin demethoxycurcumin; Bis-desmethoxycurcumin bis-demethoxycurcumin.
198 Maria L.A.D. Lestari and Gunawan Indrayanto
scanning of excitation and emission from 300 to 700 nm; the analytical
wavelength for measurements was 538.5 nm. Recoveries were
97.3104.5% (RSD 1.32.45).
Detection and quantification of curcumin in mouse lung cell cultures by
matrix-assisted laser desorption ionization time mass spectrometry
(MALDI-TOFMS) was described by May et al. [116]. Matrices were
prepared using a-cyano-4-hydroxycinnamic acid (CHCA),
trihydroxyacetophenone, and dihydroxybenzoic acid, and it was shown that
CHCA was the superior matrix. MALDI analysis showed that cells take up
712% available curcumin.
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CHAPTER FOUR
Dasatinib
Hesham M. Korashy*, A.F.M. Motiur Rahman,
Mohammed Gabr Kassem
*Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Riyadh,
Saudi Arabia
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
Contents
1. Introduction 205
1.1 Nomenclature 206
1.2 Formula 206
1.3 Elemental analysis 207
1.4 Physical properties 207
1.5 Uses and applications 207
2. Methods of Preparation 207
3. Physical Properties 214
3.1 Spectroscopy 214
3.2 Mass spectrum 218
4. Methods of Analysis 220
4.1 Chromatographic methods 220
4.2 Colorimetric methods 228
4.3 UV spectrophotometric methods 229
5. Pharmacology 229
5.1 Pharmacokinetics 229
5.2 Pharmacodynamics 231
5.3 Toxicities 231
Acknowledgment 232
References 232
1. INTRODUCTION
Tyrosine kinase inhibitors (TKIs) are a class of small molecule drugs
that block the intracellular signals which drive proliferation in many malig-
nant cells by specifically inhibiting the kinase function of individual intracel-
lular pathways involved in receptor-mediated growth signaling [1].
Dasatinib (BMS-354825, Sprycel), is a thiazole-based ATP-competitive,
Profiles of Drug Substances, Excipients, and Related Methodology, Volume 39 # 2014 Elsevier Inc. 205
ISSN 1871-5125 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800173-8.00004-0
206 Hesham M. Korashy et al.
dual Src/Abl kinase inhibitor [2], approved for the treatment of imatinib-
resistant and imatinib-intolerant patients across all phases of chronic myelog-
enous leukemia (CML) [3].
1.1. Nomenclature
1.1.1 Systematical chemical names
N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-
2-methylpyrimidin-4-yl)amino)thiazole-5-carboxamide [4,5]
N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-
2-methyl-4-pyrimidinyl]amino]-1,3-thiazole-5-carboxamide [6,7]
N-(2-chloro-6-methylphenyl)-2-({6-[4-(2-hydroxyethyl)piperazin-1-yl]-
2-methylpyrimidin-4-yl}amino)-1,3-thiazole-5-carboxamide [8]
1.2. Formula
1.2.1 Empirical formula, molecular weight, obtained mass
and CAS number
Chemical formula: C22H26ClN7O2S, molecular weight: 488.0055, exact
mass: 487.1557, monoisotopic mass: 487.155721508, m/z: 487.7 [M H]
(obtained), m/z: 488.1635 [M H] [6], CAS no. 302962-49-8 [6,912]
O Cl
H S
N N
HO N
N H
N
N H3C
N
CH3
Dasatinib
1.2.3 SMILES
CC1]NC(NC2]NC]C(S2)C(]O)NC2]C(C)C]CC]C2Cl)]
CC(]N1)N1CCN(CCO)CC1 [13]
Dasatinib 207
1.2.4 InChI
1S/C22H26ClN7O2S/c1-14-4-3-5-16(23)20(14)28-21(32)17-13-24-22
(33-17)27-18-12-19(26-15(2)25-18)30-8-6-29(7-9-30)10-11-31/h3-5,12-
13,31H,6-11H2,1-2H3,(H,28,32)(H,24,25,26,27) [13]
1.4.2 Solubility
Water solubility: 0.0128 mg/mL
2. METHODS OF PREPARATION
In most of the reports [2,4,6,16,17], synthesis of dasatinib (1)
involves the reaction of N-(2-chloro-6-methylphenyl)-2-(6-chloro-2-
methylpyrimidin-4-ylamino)-1,3-thiazole-5-carboxamide 2 with 1-(2-
hydroxyethyl) piperazine by heating the mixture at 80 C refluxing in
208 Hesham M. Korashy et al.
Cl Cl
O OH O
H S H S
N N NH N
N HO N N
Cl H N H
N N
N H3C Heat N H3C
N N
2 1
CH3 CH3
Cl Cl HO HO N Cl
N NH N
N N DIEA N
N
4
CH3 CH3
3
Cl
O
H2N S Cl
N O
N H H S
HO N N
5 H3C N
N H
N
N H3C
N
K2CO3, Pd(OAc)2, BINAP 1
CH3
O
NCS
H2N Cl OH N NH2
OH 8
N NH N
N N DIEA N NaOH
N
CH3 2 7 CH3
6
S NH2
H3C O CH3
HO N NH N N HO N CH3
N
N H3C O CH3 N
N 10 N
N
N
CH3 11 HN N CH3
9 S H3C
O Heat
Cl O H OH
Cl Cl N
Cl O S N
Cl N
13 Cl N N
H2N CH2Cl2, pyridine N H N N
H
CH3 CH3 CH3 1
12 14 H3C
O Boc2 O O O O
S H H (COCl)2 H
H2N S N S S
N N
OEt DMAP Boc OEt NaOH Boc OH Boc Cl
N N N N
15 16 17 18
Cl Cl Cl
Cl Cl N N
H2N 12 O O
H S 3
CH3 N H2N S CH3
Boc N TFA N
TEA N H H
N
H3C
19 5 H3C
Cl HO Cl
O N O
H S H S
N NH HO N N
Cl N N
H N H
N N
N H3C Heat N H3C
N N
2 1
CH3 CH3
O
H3C
Cl Cl
O Cl
S N n-BuLi S 23
Cl Cl
C N
N O THF, 78 C H
N NaH, THF
H3C
20 21 22 H3C
Cl N CH3
Cl Cl
O O
N H S
Cl S N
25 Cl N TfOH, TFA,
N NH2
N N
N H3C
H3C N CH2Cl2
NaH, THF
24 CH3 O 26
H3CO CH3
Cl Cl
O OH O
H S H S
N N NH N
N HO N N
Cl N H
N H N
N H3C O N N H3C
N
2 1
CH3 , Heat CH3
O
Cl Cl
NBS Cl
O N N
H H S
Cl NH2 SCN O CH3 Cl N NH2 N 3
Cl N CH3
29 N H
N N O N N S N
N 2 H3C t-BuONa
6 NaOH 30
CH3 CH3 CH3
HO
Cl N
O
H S NH
HO N N
N N
N H Heat
N H3C
N
2
CH3
O
H
Cl Cl Cl Ph N N N
Cl O
31 Ph 33
Cl Ph S
H2N N
CH2Cl2, pyridine H EtOH
CH3 CH3
12 32
Cl Cl
N N
Cl Cl
O O 3
H S HCOOH S CH3
N H2N
Ph N N
N H N H NaOBut
Ph Ph THF
34 H3C 5 H3C
Cl OH
O Cl O H OH
H HN N N
N S S N
N N
Cl H N
N H N N N
N H3C DIPEA, EtOH
N
2 MW CH3 1
CH3 H3C
O HO O
H H S
N S N NH N
HO N OEt Hydrolysis
Cl OEt N
N N
N N
N N
36
35 CH3
CH3
O O
H S H S
N N N N
HO
N OH Protection PgO N OH (COCl)2
N N
N N
N 37 N
38
CH3 CH3
Cl
Cl
O
O H2N H S
H S PgO N N
N N 12 N N
PgO Cl H3C N H
N N
N N H3C
N
N Base 40
39 CH3
CH3
Cl
O
H S
Deprotection HO N N
N
N H
N
N H3C
N 1
CH3
Cl Cl N Cl
HO
N NH N Protection
N N HO N
N
3 4
CH3 CH3
O
H2N S O
CH3 H
N Cl O N S
PgO N PgO N
N 42 N OMe Hydrolysis
N N
N N
N
41 43
CH3 CH3
Cl
O O
H H S H2N
S N
PgO N N
OH (COCl)2
PgO N Cl 12
N N H3C
N N
N N
N N
44 CH3 45
CH3
Cl Cl
O O
H S H S
N N Deprotection HO N N
PgO N N
N H N N H
N
N H3C N H3C
N N 1
40
CH3 CH3
H
N
H Cl
N H N N H H N
Cl O S Cl O S N
N N
N N N N
NH DIEPA NH
2 CH3 Dioxane, reflux 12 h CH3
CH3 CH3
46
18
F
Br Cl
O H 18F
S N N
N N
Na2CO3, KI, CH3CN, H N
60 C, 4 h N N
CH3 1-18F
H3C
Darren R. Veach et al. [22] synthesized the 18F derivative of dasatinib from
the intermediate 2. Intermediate 2 was reacted with piperazine in the presence
of DIEPA in dioxane to give N-(2-chloro-6-methylphenyl)-2-(2-methyl-6-
(piperazin-1-yl)pyrimidin-4-ylamino) thiazole-5-carboxamide (46), which
was then reacted with 1-bromo-2-fluoro (18F) ethane to give 18F derivative
of dasatinib 1-18F (Scheme 4.10).
214 Hesham M. Korashy et al.
3. PHYSICAL PROPERTIES
3.1. Spectroscopy
3.1.1 Ultraviolet spectroscopy
The ultraviolet/visible (UV/VIS) absorption spectrum of dasatinib was
recorded for selecting the proper maximum absorption peak (lmax). Using
a UV/VIS spectrometer (Varian Cary 50 Conc UV/VIS spectrophotome-
ter), the absorption spectrum of dasatinib in ethanol was scanned from 200 to
400 nm. As shown in Figure 4.1, the lmax of dasatinib is located at 321 nm.
Chemical shifts were expressed in parts per million (ppm) (Table 4.2) with
respect to the tetramethylsilane signal for 1H and 13C NMR.
13
Figure 4.4 C NMR spectrum of dasatinib in DMSO-d6.
Dasatinib 217
Table 4.2 Comparative study of 1H NMR spectra for dasatinib (DMSO-d6) with literature
[6,14]
Chemical shift Chemical shift [14] Chemical shift [6]
Entry (500 MHz, DMSO-d6) (500 MHz, DMSO-d6) (400 MHz, DMSO-d6)
1 11.47 (s, 1H, NH) 11.48 (s, lH) 11.67 (br s, 1H)
(D2O exchangeable)
2 10.50 (br s, 1H)
3 9.88 (s, 1H, NH) 9.88 (s, lH) 9.96 (s, 1H)
(D2O exchangeable)
4 8.23 (s, 1H) 8.23 (s, lH) 8.27 (s, 1H)
5 7.40 (d, J 6.5 Hz, 7.257.30 (m, 3H) 7.40 (d, J 7.7 Hz, 1H)
1H, HAr)
6 7.29 (d, J 6.0 Hz, 7.28 (dd, J 6.6, 6.7 Hz, 1H)
1H, HAr)
7 7.27 (t, J 7.5 Hz, 7.25 (d, J 7.7 Hz, 1H)
1H, HAr)
8 6.06 (s, 1H) 6.05 (s, lH) 6.17 (s, 1H)
9 4.454.30 (m, 1H) 4.46 (s, lH) 4.33 (d, J 12.6 Hz, 2H)
10 3.52 (s, 4H) 3.523.56 (q, 6H) 3.79 (dd, J 5.0, 5.5 Hz, 2H)
11 3.453.35 (m, 1H) 3.60 (d, J 11.6 Hz, 2H)
12 2.50 (s, 3H) 2.492.51 (m, 4H) 3.38 (dd, J 12.1, 12.6 Hz, 2H)
13 2.50 (m, 2H) 3.223.19 (m, 2H)
14 2.42 (s, 4H) 2.412.45 (s, 5H) 3.133.07 (m, 2H)
15 2.25 (s, 3H) 2.24 (s, 3H) 2.45 (s, 3H)
16 1.06 (t, J 6.5 Hz, 2.24 (s, 3H)
1H, dOH)
3.453.35 (m, 1H), 2.50 (s, 3H), 2.50 (m, 2H), 2.42 (s, 4H), 2.25 (s, 3H),
and 1.06 (t, J 6.5 Hz, 1H, dOH) ppm.
13
3.1.3.2 C NMR spectrum
13
C NMR (DMSO-d6, 125 MHz): d 165.14, 162.56, 162.37, 159.91,
156.91, 140.80, 138.80, 133.51, 132.42, 128.99, 128.13, 126.98, 125.67,
82.59, 60.18, 58.50, 56.01, 52.71, 43.60, 25.55, and 18.28 ppm.
218 Hesham M. Korashy et al.
13
C NMR (DMSO-d6, 125 MHz): d 165.7, 162.8, 162.1, 160.4, 157.5,
141.2, 139.4, 133.8, 133.0, 129.6, 128.8, 127.6, 126.5, 84.0, 58.1, 55.2,
51.1 (2), 41.2 (2), 25.7, 18.8 [3].
13
C NMR (DMSO-d6, 125 MHz): d (ppm) 18.756, 26.034, 44.098,
53.186,58.997, 60.658, 83.098, 126.157, 127.458, 128.612, 129.474,
132.910, 134.002, 139.285, 141.286, 157.410, 160.393, 162.964,
165.629 [9].
Figure 4.5 Dasatinib shows m/z 487.3 [M(35Cl) H] molecular ion peak in
positive mode.
Figure 4.6 MS/MS spectrum of the m/z 487.3 dasatinib fragment in the
positive mode.
Dasatinib 219
Figure 4.7 MS/MS/MS spectrum of the dasatinib m/z 399.8 fragment in the
positive mode.
H3C
N
N OH
N N
CH3 N
H NH
N
S
O
Cl Exact mass: 487.1557
MS2
H3C H3C
H3C
N N
N N OH
N N N N N
N N N N
CH3 N CH3
H H NH NH
N NH N S
S S
O O
O Cl
Cl
Scheme 4.11 Possible MS/MS fragments of dasatinib for the m/z 487.3 fragment.
pattern interpretation of the drug substance. The reported m/z for dasatinib
(C22H26ClN7O2S) was ESI (M 1): 490.27 [5], 488.1635 [M H] [6],
and the obtained m/z 487.3 [M(35Cl) H], the reported m/z 488.9 is
[M(37Cl) H], while the calculated exact mass is 487.1557.
H3C
N CH3 N
N N
N N
CH3 S
N N
Cl
Exact mass: 310.0854 Exact mass: 285.0116
Cl
4. METHODS OF ANALYSIS
4.1. Chromatographic methods
4.1.1 High-performance liquid chromatography/mass spectrometry
In recent years, numerous laboratories have reported the use of high-
throughput bioanalytical procedures for the single and simultaneous
quantification of plasma concentrations of antileukemia drugs [2336].
High-performance liquid chromatography (HPLC) methods for the phar-
macokinetics (PK) of imatinib have been well investigated [2,3749]. Mea-
surement of TKIs plasma concentrations, in fact, is a reliable tool to perform
therapeutic drug monitoring (TDM); however, only the drugs fraction
reaching the intracellular compartment is expected to exert action. Then
a convenient correlation should be done, also, between clinical outcome
and intracellular drug levels reached in treated patients. As variability in drug
PK and inadequate patient compliance, also poor penetration of drugs into
body compartments, particularly in leukocytes or peripheral blood mono-
nuclear cell (PBMC), may contribute to the occurrence of subtherapeutic
drug level, leading to loss of treatment efficacy. The mechanisms by which
TKI drugs accumulate within cells remain generally unknown and very few
data are published to date [2,5052]. DAvolio et al. [53] have described a
new method using HPLC coupled with electrospray mass spectrometry
for the quantification of PBMC concentration of TKIs imatinib, dasatinib,
and nilotinib. A simple PBMC isolation and extraction procedure was
applied on 1014 mL of blood aliquots. Chromatographic separation of
drugs and Internal Standard (quinoxaline) was achieved with a gradient (ace-
tonitrile and water formic acid 0.05%) on a C18 reverse-phase analytical
Dasatinib 221
column with 25 min of analytical run, at a flow rate of 0.25 mL/min. Mean
intra- and interday precision for all compounds were 8.76% and 12.20%,
mean accuracy was 3.86%, and extraction recovery ranged within 79%
and 91%. Calibration curves ranged from 50.0 to 0.25 ng. The limit of quan-
tification was set at 0.25 ng for all the analyzed drugs. This method allows a
specific, sensitive, and reliable simultaneous intracellular determination of
the three TKIs imatinib, dasatinib, and nilotinib in a single chromatographic
run, useful for drugs estimation in PBMC of patients affected by CML.
Cl
O
H S
HO N N N
N H
N
N H3C
N
CH3
(A) Dasatinib
Cl Cl
O H O
H S 15 13 S
HN N HO N 13C 13 N C
N N
N H N 13C C 15
H
N N
N H3C N H3C
N N
CH3 CH3
(B) Metabolite M4 (D) Dasatinib stable-labeled internal standard
Cl Cl
O H O
O H S 15 13 S
HO N N HN 13 13 N C
N C C N
N H N 13C 15 H
N N
N H3C N H3C
N N
CH3 CH3
(C) Metabolite M5 (E) Metabolite M4 stable-labeled internal standard
Figure 4.8 Chemical structures of analytes and internal standards. The stable-labeled
internal standard for dasatinib was used to quantify dasatinib and M5 [54].
222 Hesham M. Korashy et al.
m/z 401 Cl
O
H S
HO N N
N
N H
N
N H3C
N
CH3
(A) Dasatinib
m/z 387 Cl
H
m/z 281O Cl
O O
H S 15 13 S
N 13 13 N C
HO N HN C C N
N N N 13C 15 H
N H N
N H3C N H3C
N N
CH3 CH3
(C) Metabolite M5 (E) Metabolite M4 stable-labeled internal standard
Figure 4.9 Arrows indicate the proposed fragmentation pathways leading to the prod-
uct ions monitored in the assay for dasatinib and its metabolites as well as stable-
labeled internal standard [54].
three core analytical runs. The lower limit of quantitation was evaluated by
spiking the three analytes into six unique human plasma lots at 1.00 ng/mL
followed by extraction and quantification. Specificity was assessed for poten-
tial matrix interferences in six lots of blank human plasma by extraction and
inspection of the resulting chromatograms for interfering peaks at the reten-
tion times of the three analytes and internal standards. Specificity was sim-
ilarly assessed for potential internal standard-derived interferences using six
lots of internal standard-spiked blank plasma. The recovery of dasatinib, M4,
M5, and the internal standards from human plasma during extraction was
determined at 50 and 800 ng/mL by comparing the response ratios in
human plasma samples spiked with the analytes prior to extraction with
those spiked postextraction. The matrix effect was determined at concentra-
tions of 50 and 800 ng/mL for dasatinib, M4, M5, and the internal standards
by dividing the analyte peak area responses in human plasma spiked
postextraction by the analyte responses spiked in reconstitution solution.
Capability of dilution was evaluated using QC samples that were prepared
at a concentration of 5000 ng/mL and subsequently diluted 10-fold with
blank human plasma prior to analysis.
Dasatinib 223
was in the range of 90.3106.5%, thanks to the use of stable isotopes as inter-
nal standard. There was no significant ion suppression observed at the respec-
tive TKI retention times. SPE was chosen because it allows the use of a single
protocol to quantify several TKIs that have a wide range of chemical prop-
erties: pKa values from 5 to 10, and log D values from 0 to 4.35 at pH 7 or
3.75 to 3.9 at pH 2 (benched using Marvin 5.0.0 software, www.
chemaxon.com). This could not be done with liquid/liquid extraction
because of the different pKa values and thus the need of different steps or
buffers to extract all analytes. The second reason for choosing SPE concerns
the matrix effect because it is considered as the Achilles heel of mass spec-
trometry, Taylor reporting that protein precipitation using an organic solvent
or dilute and shoot are the dirtiest sample preparation techniques and thus
produces the most matrix effects compared to solid phase extraction [58].
SPE coupled to UPLC/MS/MS remains the most effective sample prep-
aration to reduce matrix effect and specifically ion suppression [59]. Further-
more, the recent marketing of the SPE-plates (such as MCX used here)
makes the method presented herein ready-to-use for robotic automation.
This is less time-consuming and more compatible than SPE-cartridges that
often require an evaporation step or protein precipitation that must be
followed by centrifugation. By combining SPE and UPLC/MS/MS, this
method allowed great sensitivity, which is of particular importance for drugs
such as dasatinib and axitinib. These molecules have a short half-life (36 h)
and thus the trough concentration is low. EPARs data show that mean
trough concentration is of 1 ng/mL.
A HPLC method coupled with electrospray mass spectrometry was
described for the quantification of plasma concentration of imatinib,
dasatinib, and nilotinib [26]. A simple protein precipitation extraction pro-
cedure was applied on 250 mL of plasma aliquots. Chromatographic separa-
tion of drugs and IS (quinoxaline) was achieved with a gradient (acetonitrile
and water formic acid 0.05%) on a C18 reverse-phase analytical column
with 20 min of analytical run, at a flow rate of 1 mL/min. Mean intra-
and interday precision for all compounds were 4.3% and 11.4%; mean
accuracy was 1.5%; extraction recovery ranged within 95% and 114%.
Calibration curves ranged from 10,000 to 62.5 ng/mL. The limit of quan-
tification was set at 78.1 ng/mL for imatinib and at 62.5 ng/mL for dasatinib
and nilotinib. This novel developed methodology allows a specific, sensi-
tive, and reliable simultaneous determination of the three TKIs imatinib,
dasatinib, and nilotinib in a single chromatographic run, useful for drugs
estimation in plasma of patients affected by CML.
226 Hesham M. Korashy et al.
Roche et al. [51] have developed an assay for the determination of cel-
lular levels of lapatinib and dasatinib based on LLE coupled to LC/MS/MS.
This method had been applied to cancer cell line models and used to exam-
ine potential mechanisms of PK resistance in cancer cell models. There are
significant differences in the overall cellular uptake of lapatinib and dasatinib;
the biological implications of this difference is unclear but clearly this has the
potential to impact efficacy. Cellular samples were extracted with a tert-butyl
methyl ether:acetonitrile (3:1, v/v):1 M ammonium format pH 3.5 (8:1, v/v)
mixture. Separation was achieved on a Hyperclone BDS C18
(150 mm 2.0 mm, 3 mm) column with isocratic elution using a mobile
phase of acetonitirile10 mM ammonium formate, pH 4 (54:46, v/v), at
a flow rate of 0.2 mL/min.
The TKIs were quantified using a triple quadrupole mass spectrometer
which was operated in multireaction-monitoring mode employing positive
electrospray ionization. The limits of detection and quantification for lapa-
tinib was determined to be 15 and 31 pg on column, respectively, whereas
for dasatinib was 3 and 15 pg on column, respectively.
Haouala et al. [56] described a sensitive LC/MS/MS method for the
simultaneous analysis in a small volume of plasma of the six major TKIs cur-
rently used: imatinib, nilotinib, dasatinib, sunitinib, sorafenib, and lapatinib.
This assay is notably applied for plasma levels monitoring of TKIs in some
specific clinical situations (toxicity, questionable compliance, managing
drug interactions, and less than optimal clinical response) where information
on drug plasma exposure may be useful for optimizing patient treatment
management. They developed a LC/MS/MS method requiring 100 mL
of plasma for the simultaneous determination of the six major TKIs currently
in use. Plasma is purified by protein precipitation and the supernatant is
diluted in ammonium formate 20 mM (pH 4.0) 1:2. Reverse-phase chro-
matographic separation of TKIs is obtained using a gradient elution of
20 mM ammonium formate (pH 2.2) and acetonitrile containing 1% formic
acid, followed by rinsing and reequilibration to the initial solvent composi-
tion up to 20 min. Analyte quantification, using matrix-matched calibration
samples, is performed by electrospray ionizationtriple quadrupole mass
spectrometry by selected reaction-monitoring detection using the positive
mode. The method was validated according to FDA recommendations,
including assessment of extraction yield, matrix effects variability
(<9.6%), overall process efficiency (87.1104.2%), as well as TKIs short-
and long-term stability in plasma. The method is precise (interday CV%:
1.39.4%), accurate (9.2 to 9.9%), and sensitive (lower limits of
Dasatinib 227
B over 2 min and was held at that composition for 1.5 min. The HPLC was
interfaced to a Finnigan LCQ Advantage ion-trap mass spectrometer oper-
ated in the positive ion electrospray and full tandem mass spectrometry
mode. The limit of quantitation for the purpose of this assay was 1 ng/mL.
5. PHARMACOLOGY
5.1. Pharmacokinetics
5.1.1 Absorption
A phase 1 trial study on 29 patients with advanced solid tumors receiving oral
dasatinib (20 mg/d) for two days showed a Cmax of 14 ng/mL with area
under the curve (AUC) of approximately 71 ng h/mL [64]. Dasatinib is rap-
idly absorbed after oral administration of 1.25, 2.5, or 5 mg/kg with a Tmax
of 1 h for all three oral doses, whereas the Cmax and the (AUC)024 seemed
dose dependent [60]. Oral bioavailability of dasatinib ranged from 14% in
mouse to 34% in the dog [37]. Upon oral administration, solubility of
dasatinib is dependent on pH; therefore, antacids, histamine H2-receptor
antagonists, or proton pump inhibitors such as famotidine or omeprazole
should be avoided. If antacid therapy is needed, it should be given at least
2 h before or 2 h after the dose of dasatinib [65]. In this regard, famotidine,
a H2-receptor antagonists, reduced dasatinib exposure by 61% [66]. In addi-
tion, it has been reported that oral administration of dasatinib with high-fat
containing meal increased the AUC by approximately 14%, which was not
clinically relevant. The maximum concentration of dasatinib is reached
between 0.5 and 6 h.
5.1.2 Distribution
Dasatinib extensively binds to human plasma proteins in vitro by approxi-
mately 96%. In leukemic patient, the calculated apparent volume of distri-
bution for dasatinib was 2502 L, implying that dasatinib is extensively
distributed in the extravascular space and tissues [37]. The elimination
half-life of dasatinib was approximately 35 h [37]. Although the brain
230 Hesham M. Korashy et al.
penetration of dasatinib is poor, with a CSF: plasma ratios ranging from 0.05
to 0.28, dasatinib appears to be more potent against CNS tumors than
imatinib. This could be a result of a much greater potency of dasatinib
and the large fraction of unbound drug [67].
5.1.3 Metabolism
In vitro studies demonstrate that multiple cytochrome P450 (CYP) isoforms
(e.g., CYP1A1, 1B1, and 3A4) are involved in metabolizing dasatinib [68].
Dasatinib is metabolized in humans markedly by CYP3A4 to active metab-
olites that represent around 5% of the parent compound, which is unlikely to
play a major role in the anticancer effect of dasatinib (Figures 4.8 and
4.9) [69]. Thus, coadministration of dasatinib with enzyme inducers (carba-
mazepine, dexamethasone, phenobarbital, phenytoin, and rifampicin) or
inhibitors (ketoconazole, macrolide antibacterials, HIV-protease inhibitors,
and nefazodone) may reduce or increase blood concentrations of dasatinib,
respectively. For example, it has been reported that coingestion of ketoco-
nazole with dasatinib resulted in a fivefold increase in dasatinib concentra-
tion in healthy volunteers, whereas rifampicin decreased dasatinib exposure
by 82% [70]. On the other hand, flavin-containing monooxygenase 3 and
phase II drug-metabolizing enzymes, such as UDP glucuronosyltransferase,
are also involved in metabolizing dasatinib [71]. Furthermore, in vitro studies
have demonstrated that dasatinib is a substrate of transporters, such as ATP-
Binding Cassette (ABC)B1 and ABCG2, but unlike nilotinib, not a potent
inhibitor of these transporters [72,73], in that, inhibition of ABCB1 trans-
porter by nilotinib increased the intracellular concentration of dasatinib in
CML cells [72]. In Caco-2 cells, the efflux ratio of dasatinib was approxi-
mately twofold, indicating that it may be a substrate for an intestinal efflux
transporter [37]. However, studies on P-gp knockout mice showed no dif-
ference in the amount of dasatinib remaining unabsorbed in the GIT as com-
pared to wild-type mice, suggesting that P-gp may not be responsible for
poor bioavailability of dasatinib [37].
5.1.4 Excretion
Dasatinib is mainly eliminated via the feces (85%), of which relatively small
amount of dasatinib is excreted unchanged as intact drug (19%) [70,74]. Uri-
nary excretion of dasatinib represents only 4%, of which <1% as unchanged
dasatinib. Dasatinib exhibited a biexponential and high clearance exceeding
the hepatic blood flow [60]. Although there are no data on the excretion of
Dasatinib 231
dasatinib into human milk, the manufacturer recommends that women who
are taking dasatinib do not breast-feed.
5.2. Pharmacodynamics
Dasatinib, a second-generation TKI, has been shown to be effective as an
anticancer drug in the treatment of patients with CML or Philadelphia
chromosome-positive (Ph) acute lymphoblastic leukemia who are resis-
tant or intolerant to imatinib. Dasatinib inhibits several multiple tyrosine
kinases, including BCR-ABL and SRC family kinases, which are indicated
for the treatment of adults with newly diagnosed chronic phase of CML.
Dasatinib exerts its anticancer effect through inhibiting several oncogenic
tyrosine kinases such as BCR-ABL, expressed by Ph stem cells, and is
directly involved in the pathogenesis of CML. Importantly, the ability of
dasatinib to inhibit 18 of the 19 BCR-ABL mutants that are resistant to
imatinib makes dasatinib more potent against BCR-ABL than imatinib
in vitro [75,76]. In this regard, dasatinib that is reported as being approxi-
mately 325-fold more active than imatinib in inhibiting wild-type ABL
kinase in vitro is active against a wide variety of imatinib-resistant BCR-
ABL mutants. However, the primary mechanism of resistance to dasatinib
is believed to be attributed to resistance of dasatinib to The T315I, a novel
BCR-ABL mutant clone [77,78]. Four-week treatment with dasatinib has been
shown to reduce BCR-ABL transcript by approximately 32% using real-time
polymerase chain reaction [79]. Dasatinib is also effective against multiple
myeloma cell lines in vitro, at clinically achievable concentrations, through
increased caspase-8 and caspase-12 activation and sensitized primary multi-
ple myeloma cells to other agents that activate caspase-9, such as dexameth-
asone and bortezomib [80]. Furthermore, dasatinib inhibited the viability of
both non-small cell lung cancer and head and neck squamous cell cancer cell
lines in vitro through apoptosis-dependent mechanism [81].
5.3. Toxicities
Most of the adverse effects associated with dasatinib therapy are mild to
moderate in severity and are usually reversible and manageable with appro-
priate intervention. Examples of these common side effects include cardiac
failure, hypertension, and coronary artery disease [10]. Dasatinib has the
potential to prolong the QT interval and thus should be given with caution
to patients with hypokalemia or hypomagnesemia. Pleural effusion is an
another common side effect associated with dasatinib; however, it can be
232 Hesham M. Korashy et al.
effectively managed with prompt delivery of supportive care and dose mod-
ification. Dasatinib also causes thrombocytopenia and bleeding, which may
be additive with antiplatelet and anticoagulant drugs [16].
ACKNOWLEDGMENT
This work was supported by the College of Pharmacy Research Center, King Saud
University.
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CHAPTER FIVE
Gefitinib
A.F.M. Motiur Rahman*, Hesham M. Korashy,
Mohammed Gabr Kassem*
*Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Riyadh,
Saudi Arabia
Contents
1. Introduction 239
1.1 Nomenclature 240
1.2 Formulae 240
1.3 Elemental analysis 241
1.4 Physical properties 241
1.5 Uses and applications 242
2. Methods of Preparation 242
3. Physical Properties 247
3.1 Spectroscopy 247
3.2 Mass spectrum 251
3.3 X-Ray powder diffraction pattern 253
4. Methods of Analysis 253
4.1 Chromatographic methods 253
5. Pharmacology 259
5.1 Pharmacokinetics 260
5.2 Toxicities 261
Acknowledgment 261
References 262
1. INTRODUCTION
The epidermal growth factor receptor (EGFR) protein tyrosine kinase
is one of the important kinases that play a fundamental role in signal trans-
duction pathways [1]. Many human cancers overexpress EGFR and the
related human epidermal growth factor receptor (HER-2). Compounds,
such as gefitinib (Iressa), that inhibit the kinase activity of EGFR and
Profiles of Drug Substances, Excipients, and Related Methodology, Volume 39 # 2014 Elsevier Inc. 239
ISSN 1871-5125 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800173-8.00005-2
240 A.F.M. Motiur Rahman et al.
HER-2 after binding of their cognate ligand, have been used as new ther-
apeutic antitumor agents [2,3].
Gefitinib, a potent and selective ATP-competitive inhibitor of EGFR
and HER-2 kinases, is the first EGFR-targeting agent launched as an anti-
cancer drug in Japan, Australia, and the United Sates for the treatment of
chemoresistant non-small cell lung cancer (NSCLC) patients [4,5]. In pre-
clinical studies, gefitinib has demonstrated antitumor activity against a vari-
ety of human cancer cell lines expressing EGFR, including lung, ovarian,
breast, and colon [68]. In human xenograft models, gefitinib (ZD1839)
in combination with standard cytotoxic agents resulted in both delayed
tumor growth and tumor regression, leading to enhanced survival [9].
Gefitinib is indicated as monotherapy for the treatment of patients with
locally advanced or metastatic NSCLC after failure of both platinum-based
and docetaxel chemotherapies.
1.1. Nomenclature
1.1.1 Systematical chemical names
N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-
4-ylpropoxy)quina-zolin-4-amine [10].
N-(3-chloro-4-fluorophenyl)-7-methoxy-6-[3-(morpholin-4-yl)
propoxy]quina-zolin-4-amine [11,12].
4-(30 -Chloro-40 -fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)
quinazoline [1315].
4-Quinazolinamine, N-(3-chloro-4fluorophenyl)-7-methoxy-6-(3-4-
morpholin) propoxy [16].
1.2. Formulae
1.2.1 Empirical formula, molecular weight, and CAS number
Gefitinib: C22H24ClFN4O3, average: 446.902, monoisotopic: 446.152096566,
CAS number: 184475-35-2 [10,11].
Gefitinib 241
O N
N
N O
O HN
F
Cl
Gefitinib
1.2.3 SMILES
COC1]CC2]C(C]C1OCCCN1CCOCC1)C(NC1]CC(Cl)]C
(F)C]C1)]NC]N2 [11,17].
1.2.4 InChI
1S/C22H24ClFN4O3/c1-29-20-13-19-16(12-21(20)31-8-2-5-28-6-9-
30-10-7-28)22(26-14-25-19)27-15-3-4-18(24)17(23)11-15/h3-4,11-
14H,2,5-10H2,1H3,(H,25,26,27) [11].
1.4.2 Solubility
Gefitinib can be defined as sparingly soluble at pH 1, but is practically insol-
uble above pH 7, with the solubility dropping sharply between pH 4 and
pH 6. In nonaqueous solvents, gefitinib is freely soluble in glacial acetic acid
and dimethyl sulfoxide, soluble in pyridine, sparingly soluble in tetrahydro-
furan, and slightly soluble in methanol, ethanol (99.5%), ethyl acetate, and
propan-2-ol and acetonitrile [16].
1.4.4 Stability
1.4.4.1 Storage and stability
Gefitinib is provided as a solid product and shipped at room temperature.
Store at 20 C. Solid product is stable 1 year at 20 C when properly
stored. Upon resuspension, gefitinib should be aliquoted and stored at
20 C, and one must avoid repeated freezethaw cycles. Resuspended
product is stable for 3 months at 20 C when properly stored.
1.4.4.2 Description
Gefitinib is a selective inhibitor of EGFR, a growth factor that plays a pivotal
role in the control of cell growth, apoptosis, and angiogenesis. EGFR acti-
vation stimulates many complex intracellular signaling pathways, primarily
the mitogen-activated protein kinases/extracellular signal-regulated kinases
and PI3K/AKT pathways [18,19]. Following EGFR activation, Src tyrosine
kinases and signal transducer and activator of transcription downstream sig-
naling have also been well documented. Recent studies demonstrated that
gefitinib can inhibit nucleotide oligomerization domain protein 2 (NOD2)-
induced cytokine release and nuclear factor kappa B (NF-kB) activation by
inhibiting receptor-interacting protein 2 tyrosine phosphorylation which is
critical for activation of NOD2 downstream signaling pathways [20].
2. METHODS OF PREPARATION
Synthesis of gefitinib (1) (Scheme 5.1) has been accomplished starting
from key intermediate 3 [13]. Reduction of the nitro group of compound 3
was carried out in aqueous medium using sodium dithionite at 50 C to
Gefitinib 243
O CN O CN
Na2S2O3 / H2O
N O NO2 N O NH2
50 C
O 2 O 3
DMF-DMA, AcOH,
toluene, 150 C
Cl
O N
F
N O CN
N O
H2N
O HN
N O N N
AcOH, 130 C
1 O 4
F
Cl
O O O
O
O Methansulfonic acid, HO O
NH NH Ac2O,
NH
L-methionine, reflux pyridine,
O N O N O N
DMAP
5 6 7
POCl3,
DEA
F H2N Cl O
Cl
O O
HN Cl F N
NH3/MeOH, . HCl
O
N i-PrOH O N
H 2O . HCl
O N
9 8
O
O N
F Cl N
N
4-(3-Chloropropyl)morpholine N O
HN Cl
O HN
HO K2CO3, DMF
N
10 1 F
O N
Cl
O O O 1. Oxidation
HO HNO3 HO X(CH2)3X X O 2. Reduction
O O NO2 O NO2
11 12 13
O O Cl
Niementowski POCl3, X O
X O X O
OH synthesis NH DEA N
HCONH2 O N O N
O NH2
14 15 16
Cl
F
O
H2N Cl O NH
NH O Cl
N O N O
Morpholine N F N
N K2CO3, DMF O N
O
17 1
(OHNH3)2SO4; O Base
O O
11 18 19
70%HNO3,
70%H2SO4, O O KOH,
CH3COOH N O CN Na2S2O4 N O CN t-amyl alcohol
O NO2 O NH2
2 3
HCOOH,
O O O O
HCOONH4 POCl3
N O N O
NH2 NH
O NH2 O N
20 21
Cl
H2N Cl
F
O Cl
N O F
N O NH
K2CO3, DMF N O
O N N
17 1
O N
O O Ac2O, AcOH,
HO 1-bromo-3-chloropropane, Cl O HNO3,
OCH3 OCH3
K2CO3, 60 C 05 C
O O
22 23
O O
Cl O Fe, AcOH, Cl O Formamidine acetate,
OCH3 OCH3
MeOH ethanol, reflux
O NO2 O NH2
24 25
O Cl H2N Cl
Cl O Cl O
NH SOCl2 N F
O N DMF reflux O N K2CO3, DMF
27
26
Cl Cl
F F
O
NH O NH
NH
Cl O N O
N N
28 KI, 50 C 1
O N O N
18
F-gefitinib was prepared in a three-step reaction sequence as depicted
in Scheme 5.6 [27]. Fluorination of 3-chloro-4-trimethyl ammonium-
nitrobenzene triflate provided 3-chloro-4-[18F]fluoro-nitrobenzene, which
was reduced to 3-chloro-4-[18F] fluoroaniline, and coupled to the
quinazoline precursor to yield [18F] gefitinib.
Deprotection of methyl group of gefitinib done for the radiosynthesis of
[ C] gefitinib obtained 31. The radiosynthesis was carried out using an
11
-
OTf K[18F]F /
18 18
NMe3 K CO -Kryptofix 222 F NaBH4, Pd/C F
2 3
MeCN MeOH
O2N Cl O2N Cl H 2N Cl
40 C, 25 min rt, 7 min 31
30
29
Cl
18
O Cl F
N O
N O NH
17 O N N O
N
1[18F]
IPA, 120 C, 15 min O N
18
Scheme 5.6 Synthesis of F-gefitinib (1) [27].
Cl Cl
F F
NaSCH2CH2SNa
O NH O NH
N O DMF, 140 C, 3h N O
N N
1 31
O N HO N
Cl
F
[11C]CH3I
O NH
NaH, 70 C, 5 min N O
N
H311C
O N
1[11C]
Scheme 5.7 Synthesis of 1 [ C] gefitinib ([11C]Iressa) [28].
11
Gefitinib 247
3. PHYSICAL PROPERTIES
3.1. Spectroscopy
3.1.1 Ultraviolet spectroscopy
Gefitinib ultraviolet/visible (UV/VIS)-absorption spectrum was recorded
for selecting the proper maximum absorption peak (lmax). The absorption
spectrum of gefitinib in ethanol was scanned from 200 to 400 nm, using
UV/VIS spectrometer (Varian Cary 50 Conc UV/VIS spectrophotometer).
As shown in Figure 5.1, the lmax of gefitinib is located at 331 nm.
1
2
4
F
l max = 278 (1),
O HN CI 280 (2), 291 (3),
N O 331 (4), 345 (5) nm
N
3
O N
4
Abs
3
2 5
0
300 350
Wavelength (nm)
Figure 5.1 UV spectra of gefitinib (1 104 M solution in ethanol).
248 A.F.M. Motiur Rahman et al.
O N
N
N O
O HN
F
Cl
7.19 (s, 1H, HAr.), 4.18 (d, J 5.5 Hz, 2H, ArOCH2), 3.94 (s, 3H, OCH3),
3.59 (s, 4H, O(CH2)2), 3.40 (s, 1H, NH), 2.502.40 (m, 6H, N(CH2)3), and
2.00 (m, 2H, CH2CH2CH2) ppm.
13
3.1.3.2 C NMR spectrum
Unreported 13C NMR data of gefitinib are given below: 13C-NMR
(DMSO-d6, 125 MHz): d 155.97, 154.48, 153.10 (2JCF 241.25 Hz),
152.56, 148.32, 146.75, 136.80, 123.44, 122.27 (4JCF 6.38 Hz), 118.72
(3JCF 18.25 Hz), 116.45 (3JCF 21.25 Hz), 108.75, 107.26, 102.49,
67.11, 66.15, 55.83, 54.93, 53.41, and 25.85 ppm.
Figure 5.3 1H NMR spectra of gefitinib in DMSO-d6.
13
Figure 5.4 C NMR Spectra of gefitinib in DMSO-d6.
Gefitinib 251
Figure 5.5 Gefitinib shows a m/z 447.0 molecular ion peak in positive mode.
252 A.F.M. Motiur Rahman et al.
Figure 5.6 MSMS spectra for the m/z 447.0 fragment in positive mode.
Figure 5.7 MSMSMS spectrum for the m/z 428.3 fragment in positive mode.
Gefitinib 253
Formula: C22H24ClNFN4O3
Formula weight: 446.91
Crystal system: triclinic
Space group: P1 Z 2
a 8.928 (1) A; a 97.12 (1)
b 9.717 (2) A; b 93.31 (1)
c 12.604 (2) A; g 101.47 (1)
V 1059.6 (3) A [30]
Dx 1.401 g/cm [30]
No. of reflections (I > 2.00 s (I)) 2766
ymax 68.25 with Cu Ka
R (I > 2.00 s (I)) 0.053
(D/s)max 0.000
(D/s)max 0.33 eA [30]
(D/s)min 0.29 eA [30]
Measurement: Rigaku RAXIS-RAPID
Program system: CrystalStructure 3.5.1 [31]
Structure determination : SIR92 [30]
Refinement: full-matrix
4. METHODS OF ANALYSIS
4.1. Chromatographic methods
4.1.1 High-performance liquid chromatography/mass spectrometry
[7,3134]
Several high-performance liquid chromatography/mass spectrometry
(LC/MS/MS) methods have been reported to determine gefitinib, either
as a single drug or in combination with other tyrosine kinase inhibitors
(TKIs) and/or its metabolite o-desmethyl gefitinib in plasma [31,3539].
Table 5.4 outlines recent methods published for the analysis of gefitinib
LC/MS/MS. Honeywell et al. [32] have reported the use of LC/MS/MS
techniques for the analysis of gefitinib. Utilizing a simple protein precipitation
with acetonitrile, a 20 mL sample volume of biological matrixes can be
254 A.F.M. Motiur Rahman et al.
(399.1 m/z; 283.1 m/z), and sorafenib (465.0 m/z; 251.9 m/z) at an ion voltage
of 3500 V. The accuracy, precision, and limit of quantification (LOQ) from
cell culture medium were as follows: gefitinib: 100.2 3.8%, 11.2 nM;
erlotinib: 101.6 3.7%, 12.7 nM; sunitinib: 100.8 4.3%, 12.6 nM;
sorafenib: 93.9 3.0%, 10.8 nM, respectively. This was reproducible for
plasma, whole blood, and serum. The method was observed to be linear
between the LOQ and 4000 ng/mL for each analyte. Effectiveness of the
method is illustrated with the analysis of samples from a cellular accumulation
investigation and from determination of steady-state concentrations in clini-
cally treated patients.
Zhao et al. [34] described a rapid and sensitive analytical method for the
determination of gefitinib concentrations in human plasma and mouse
plasma and tissue based on LC/MS/MS with electrospray positive ionization
after a single protein precipitation with acetonitrile. Sample preparation
involved a single protein precipitation step by the addition of 0.1 mL of
plasma or a 200 mg/mL tissue homogenate diluted 1/10 in human plasma
with 0.3 mL acetonitrile. Separation of the compounds of interest, including
the internal standard (d8)-gefitinib, was achieved on a Waters X-Terra
C18 (50 mm 2.1 mm i.d., 3.5 mm) analytical column using a mobile phase
consisting of acetonitrilewater (70:30, v/v) containing 0.1% formic acid
and isocratic flow at 0.15 mL/min for 3 min. The analytes were monitored
by tandem mass spectrometry with electrospray positive ionization. Linear
calibration curves were generated over the range of 11000 ng/mL for the
human plasma samples and 51000 ng/mL for mouse plasma and tissue sam-
ples with values for the coefficient of determination of greater than 0.99.
The values for both within- and between-day precision and accuracy were
well within the generally accepted criteria for analytical methods (less than
15%). This method was subsequently used to measure concentrations of
gefitinib in mice following administration of a single dose of 150 mg/kg
I.P. and in cancer patients receiving an oral daily dose of 250 mg.
The development of an on-column focusing gradient capillary LC
method coupled to tandem mass spectrometry (quadrupole-linear ion trap)
for the quantitative determination of gefitinib in blood plasma was described
by Guetens et al. [33]. Plasma samples (0.2 mL) were extracted with methyl
tert-butyl ether. The analytes of interest, ZD1839 and the internal standard
[(2)H8]ZD1839 (ZD1839-d8) were eluted on a 50 mm 1 mm, 5 mm par-
ticle size, capillary ODS Hypersil column using an aqueous ammonium ace-
tate gradient at 40 mL/min. Mass spectrometric detection was performed by
a Q-Trap tandem mass spectrometer with electrospray positive ionization
256 A.F.M. Motiur Rahman et al.
and monitored in the multiple reaction monitoring transitions 447 > 128
and 455 > 136, respectively. The LOQ of ZD1839 was 0.1 ng/mL. The
method proved to be robust, allowing quantification of ZD1839 with suf-
ficient precision, accuracy, and sensitivity.
Jones et al. [35] described a method for the quantitative determination of
gefitinib concentrations in treated healthy volunteers and patients with can-
cer that has been developed and validated. Plasma samples (0.5 mL) were
extracted, at basic pH, with methyl-t-butyl ether using deuterated gefitinib
as an internal standard. The extracts were chromatographed on an Inertsil
ODS3 column eluted with acetonitrile/ammonium acetate and gefitinib
and the internal standard quantified by mass spectrometric detection. The
method was validated with respect to linearity, selectivity, precision, accu-
racy, LOQ, recovery, and stability. The precision and accuracy of the assay
were good, and the LOQ was 0.5 ng/mL. The assay has been successfully
applied to a number of clinical and pharmacokinetic studies and been shown
to be robust and reliable during routine use.
A LC/MS/MS method was developed and validated by Wang et al. [36]
for the simultaneous quantification of gefitinib and its predominant metab-
olite, O-desmethyl gefitinib, in human plasma. Chromatographic separation
of analytes was achieved on an Alltima C18 analytical HPLC column
(150 mm 2.1 mm, 5 mm) using an isocratic elution mode with a mobile
phase comprised acetonitrile and 0.1% formic acid in water (30:70, v/v).
The flow rate was 300 mL/min. The chromatographic run time was
3 min. The column effluents were detected by API 4000 triple quadrupole
mass spectrometer using electrospray ionization (ESI) in positive mode.
Linearity was demonstrated in the range of 51000 ng/mL for gefitinib
and 5500 ng/mL for O-desmethyl gefitinib. The intra- and interday preci-
sions for gefitinib and O-desmethyl gefitinib were 10.8% and the accuracies
ranged from 89.7% to 104.7% for gefitinib and 100.4% to 106.0% for
O-desmethyl gefitinib. This method was used as a bioanalytical tool in a phase
I clinical trial to investigate the possible effect of hydroxychloroquine on the
pharmacokinetics of gefitinib. The results of this study enabled clinicians to
ascertain the safety of the combination therapy of hydroxychloroquine and
gefitinib in patients with advanced (Stage IIIBIV) NSCLC.
A quantitative LC/MS/MS method was developed and validated for the
tyrosine kinase inhibitors erlotinib, gefitinib, and imatinib in human plasma
by Chahbouni et al. [37]. Pretreatment of the samples was achieved by using
liquidliquid extraction using d8 imatinib as internal standard. Separation
was performed on a Waters Alliance 2795 LC system using an XBridge
Gefitinib 257
acid (30:70, v/v). The analytes were detected with a PE Sciex API 365 triple
quadrupole mass spectrometer using turbo ionspray source with positive
ionization. Ions monitored in the MRM mode were m/z 447.2 (precursor
ion) to m/z 127.8 (product ion) for gefitinib and m/z 455.2 (precursor ion)
to m/z 136.0 (product ion) for d8-ZD1839. The lower limit of quantitation
of gefitinib was 0.30 ng/mL (S/N 10), and results from a 5-day validation
study demonstrated acceptable within-day and between-day precision (CV
% values 6.0% and 5.2%, respectively) and accuracy (range 91.097.7%).
This method is now used to analyze plasma samples from pediatric pharma-
cokinetic studies of ZD1839, and the wide linear range (approximately 4 log
units) of this method provides a distinct advantage, as shown by the results of
a representative patient.
and sorafenib and from 0.1 to 200 ng/mL for dasatinib, axitinib, gefitinib
and sunitinib. Peaks of each compound (retention time from 0.76 to
2.51 min) were adequately separated. The mean relative extraction recovery
was in the range of 90.3106.5%. There was no significant ion suppression
observed at the respective TKI retention times. SPE was chosen because it
allows the use of a single protocol to quantify several TKIs that have a wide
range of chemical properties: pKa values from 5 to 10, and log D values from
0 to 4.35 at pH 7 or 3.75 to 3.9 at pH 2 (benched using Marvin 5.0.0
software, www.chemaxon.com). This could not be done with liquid-liquid
extraction because of the different pKa values and thus the need of different
steps or buffers to extract all analytes. The second reason for choosing SPE
concerns the matrix effect because it is considered as the Achilles heel of
the mass spectrometry, Taylor reporting that protein precipitation using an
organic solvent or dilute and shoot are the dirtiest sample preparation
techniques and thus produce the most matrix effects compared to solid phase
extraction [40].
SPE coupled to UPLC/MSMS remains the most effective sample prep-
aration to reduce matrix effect and specifically ion suppression [41]. Further-
more, the recent marketing of the SPE-plates (such as MCX used here)
makes the method presented herein ready-to use for robotic automation.
This is less time-consuming and more compatible than SPE-cartridges that
often require an evaporation step, or protein precipitation that must be
followed by centrifugation. By combining SPE and UPLC/MSMS, this
method allowed great sensitivity, which is of particular importance for drugs
such as dasatinib and axitinib. These molecules have a short half-life (36 h)
and thus the trough concentration is low. EPARs data show that mean
trough concentration is of 1 ng/mL.
The method was validated for linearity, accuracy, precision, specificity, limit
of detection, LOQ, and robustness. Limit of detection and LOQ were found
to be 0.09 and 0.29 ppm, respectively, and recovery of gefitinib from tablet
formulation was found to be 99.16%.
In another method for the estimation of gefitinib in tablet dosage form
has been developed by Kumar et al. [43]. A Hypersil BDS RP C18,
250 4.6 mm, 5 mm particle size, with mobile phase consisting of 0.02 M
dipotassium hydrogen orthophosphate and methanol in the ratio of 10:90
v/v was used. The flow rate was 1.0 mL/min and the effluents were mon-
itored at 246 nm. The retention time was 3.7 min. The detector response
was linear in the concentration of 25300 g/mL. The respective linear
regression equation being Y 94342.26x 77672.7. The limit of detection
and LOQ was 0.125 and 0.15 mg/mL respectively. The percentage recovery
of gefitinib was 99.5%.
A degradation pathway for gefitinib is established as per ICH recommen-
dations by validation and stability indicating reverse-phase liquid chromato-
graphic method, which was developed by Madireddy Venkataramanna
et al. [44], gefitinib is subjected to stress conditions of acid, base, oxidation,
thermal, and photolysis. Significant degradation is observed in acid and base
stress conditions. Two impurities are studied, among which one impurity is
found to be the prominent degradant. The stress samples are assayed against a
qualified reference standard, and the mass balance is found close to 99.5%.
Efficient chromatographic separation is achieved on a Agilent make XDB-
C18, 50 4.6 mm with 1.8 mm particles stationary phase with simple mobile
phase combination delivered in gradient mode, and quantification is carried at
250 nm at a flow rate of 0.5 mL/min. In the developed RPLC method, the
resolution between gefitinib and the potential impurities is found to be greater
than 5.0. Regression analysis shows an r value (correlation coefficient) of
greater than 0.998 for gefitinib and the two potential impurities. This method
is capable to detect the impurities of gefitinib at a level of 0.01% with respect to
test concentration of 0.5 mg/mL for a 4-mL injection volume.
5. PHARMACOLOGY
Gefitinib is a small-molecule (446.9 Da) chemotherapeutic agent that
specifically inhibits EGFR-tyrosine kinase for the treatment of NSCLC [45].
Gefitinib competitively and reversibly binds to the ATP-binding sites of
EGFR, causing downregulation of EGFR phosphorylation and subsequent
downstream signaling molecules [45,46]. This in turn strongly increases
260 A.F.M. Motiur Rahman et al.
5.1. Pharmacokinetics
5.1.1 Absorption
Gefitinib is usually administered as a once-daily oral tablet. After oral admin-
istration, gefitinib is absorbed slowly with a bioavailability of approximately
60% in human [47]. However, this bioavailability is not altered dramatically
by food or any other gastrointestinal factors [48].
5.1.2 Distribution
The relationship between the pharmacokinetics and long-term antitumor
activity of gefitinib in patients with EGFR mutation-positive lung adeno-
carcinoma has been reported. In that, lung cancer patients on gefitinib
who showed partial response exhibited a lower Cmax (278 ng/mL) than that
of patients with stable disease 588 ng/mL) [49]. Conversely, a significant
negative correlation was found between the area under the plasma
concentrationtime curve (AUC)024 of gefitinib and longer survival [49].
After oral administration, gefitinib is widely distributed throughout the
body. Following IV dosing (5 mg/kg) of gefitinib, a plasma elimination
half-life of 714 h was reported in rats and dogs using HPLCMS assay, with
an apparent volume of distribution of 1400 L [50]. Gefitinib is highly bound
to human plasma albumin and a1-acid glycoprotein by 90%. In human, the
maximum elimination half-life reaches 48 h and the steady state plasma
concentrations are achieved within 10 days. Following administration of
[14C]-gefitinib, concentrations of radioactivity in plasma exceeded gefitinib
throughout the profile, indicating the presence of circulating metabo-
lites [51]. In comparison with erlotinib, gefitinib has a longer elimination
half-life and a larger tissue distribution [52].
Gefitinib 261
5.1.3 Metabolism
Gefitinib is metabolized extensively in the liver by cytochrome P450
enzymes, primarily by CYP3A4 and to a lesser extent by CYP3A5 and
CYP2D6 [53]. Five metabolites were identified in human plasma, among
which only O-desmethyl gefitinib has a similar EGFR-TK activity to
gefitinib [51]. Gefitinib has also been implicated as an inhibitor of CYP2C19
and CYP2D6 activity, but weak inhibitor of CYP2C9, CYP3A4, and
CYP1A2. Therefore, gefitinib may inhibit the metabolism of
coadministered drugs that are substrates of CYP2C19 and CYP2D6. At
the highest concentration studied (5000 ng/mL), gefitinib inhibited
CYP2C19 by 24% and CYP2D6 by 43% [53]. After administration of
gefitinib 5 mg/kg to rats and dogs, five metabolites were detected, but at
levels much lower than the parent drug [50]. In clinical studies,
coadministration of gefitinib and rifampicin reduced gefitinib maximum
concentration and AUC by approximately 65% and 83%, respectively.
5.1.4 Excretion
In human, gefitinib is mainly eliminated hepatically, with total plasma clear-
ance of 595 mL/min after intravenous administration, in which the excre-
tion is predominantly via the feces (86%) [47]. However in rats and dogs, the
plasma clearance was reported approximately 25 and 16 mL/min/kg for
both male rats and dogs, respectively [50].
5.2. Toxicities
In phase I clinical trials, gefitinib was found to be well tolerated, with clinical
efficacy observed well below the maximum tolerated dose. Most of the
adverse effects associated with gefitinib therapy are mild to moderate in
severity and are usually reversible and manageable with appropriate inter-
vention. Examples of these common side effects include diarrhea, dry skin,
acneiform rash, and nausea and vomiting [54]. Rare cases (about 1%) of
interstitial lung disease such as pneumonia or inflammation were also
reported. Serious side effects have been reported with the use of gefitinib
including: allergic reactions, difficulty of breathing, swelling of the lips,
tongue, and elevated liver enzymes [55].
ACKNOWLEDGMENT
This work was supported by the College of Pharmacy Research Center, King Saud
University.
262 A.F.M. Motiur Rahman et al.
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[52] D. Leveque, Pharmacokinetics of gefitinib and erlotinib, Lancet Oncol. 12 (2011) 1093.
[53] M. Scheffler, P. Di Gion, O. Doroshyenko, J. Wolf, U. Fuhr, Clinical pharmacokinetics
of tyrosine kinase inhibitors: focus on 4-anilinoquinazolines, Clin. Pharmacokinet.
50 (2011) 371403.
[54] X. Li, T.M. Kamenecka, M.D. Cameron, Bioactivation of the epidermal growth factor
receptor inhibitor gefitinib: implications for pulmonary and hepatic toxicities, Chem.
Res. Toxicol. 22 (2009) 17361742.
[55] R. Kumasaka, N. Nakamura, K. Shirato, H. Osawa, S. Takanashi, Y. Hasegawa,
H. Yamabe, M. Nakamura, M. Tamura, K. Okumura, Side effects of therapy: case
1. Nephrotic syndrome associated with gefitinib therapy, J. Clin. Oncol. 22 (2004)
25042505.
CHAPTER SIX
Imatinib Mesylate
Badraddin M.H. Al-Hadiya*, Ahmed H.H. Bakheit*,
Ahmed A. Abd-Elgalil
*Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
Research Center, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
Contents
1. Background 266
2. Description 266
2.1 Nomenclature 266
2.2 Formulae 267
2.3 Elemental analysis 267
2.4 Appearance 267
3. Methods of Preparation of Imatinib 267
4. Physical Characteristics 270
4.1 Optical activity 270
4.2 Ionization constant 270
4.3 Solubility characteristics 271
4.4 Particle morphology 271
4.5 Crystallographic properties 271
4.6 Hygroscopicity 272
4.7 Thermal methods of analysis 272
4.8 Spectroscopy 275
4.9 Mass spectrometry 277
5. Methods of Analysis 279
5.1 Compendial methods of analysis 279
5.2 Electrochemical methods of analysis 282
5.3 Spectroscopic methods of analysis 283
5.4 Chromatographic methods of analysis 286
5.5 Determination in body fluids and tissues 288
6. Stability 288
7. Clinical Applications 291
7.1 Pharmacodynamics (an overview) 291
7.2 Mechanism of action 291
7.3 Clinical uses 291
7.4 Mechanisms of drug resistance 292
7.5 Pharmacokinetics 293
7.6 Toxicity 294
References 294
Profiles of Drug Substances, Excipients, and Related Methodology, Volume 39 # 2014 Elsevier Inc. 265
ISSN 1871-5125 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800173-8.00006-4
266 Badraddin M.H. Al-Hadiya et al.
1. BACKGROUND
Imatinib was developed in the late 1990s by biochemist Nicholas
Lydon, a former researcher for Novartis, and oncologist Brian Druker of
Oregon Health & Science University. Other major contributions to
imatinib development were made by Carlo Gambacorti-Passerini, a physi-
cian scientist and hematologist at University of MilanoBicocca, Italy, John
Goldman at Hammersmith Hospital in London, UK, and later on by Charles
Sawyers of Memorial Sloan Kettering Cancer Center. Druker led the clin-
ical trials confirming its efficacy in myelogenous leukemia (CML) [1].
Imatinib was developed by rational drug design. After the Philadelphia
chromosome mutation and hyperactive bcrabl protein were discovered,
the investigators screened chemical libraries to find a drug that would
inhibit that protein. With high-throughput screening, they identified
2-phenylaminopyrimidine. This lead compound was then tested and mod-
ified by the introduction of methyl and benzamide groups to give it
enhanced binding properties, resulting in imatinib [2].
Gleevec received FDA approval in May 2001. Druker, Lydon, and Saw-
yers received the Lasker-DeBakey Clinical Medical Research Award in
2009 for converting a fatal cancer into a manageable chronic condition.
Gleevec also holds the record for the drug with the fastest approval time by
the FDA. Novartis filed international patent applications, was the manufac-
turer of the drug and marketed it under the name Gleevec.
2. DESCRIPTION
2.1. Nomenclature
2.1.1 Systematic chemical names
a-(4-Methyl-1-piperazinyl)-30 -{[4-(3-pyridyl)-2-pyrimidinyl]amino}-
p-tolu-p-toluidide methanesulfonate [3].
N-(4-methyl-3-{[4-(pyridin-3-yl) pyrimidin-2-yl] amino} phenyl)
4-[(4-methylpiperazin-1-yl)methyl]benzamide [4].
4-(4-Methylpiperazin-1-ylmethyl)-N-[4-methyl-3-[[4-(pyridin-3-yl)
pyrimidin-2-yl]amino]phenyl]benzamide [5].
H N
N N N N
N O
O
HO S O
N
CH3
2.4. Appearance
White-to-off-white, odorless, crystalline powder [7,8].
H
NH2 N NH2
HNO3, ethanol, cyanamide H
NH N N
reflux, 21 h
NO2 NO2 HNO3 Isopropanol, NaOH N
2 O 4 O reflux, 12 h NO2
(1) Na, toluene, methanol
0 C, 75 C, 45 min N
N
(2) 3-Acetylpyridine, ethyl formate N N 6
toluene, 25 C, 16 h
3 (3) Dimethylamine, toluene 5 N
acetic acid, 1 h
COCI H
H N N
N N N
N
O H N
N
1-Methylpiperazine
methanol Br
N
Pt/C, 5 bar, H2
80 C, 20 h Trimethylaluminum
COOMe 70% COOMe toluene, Ar
10 HN
+ 40 C, 30 min
9
75% O 12
Br +
NH2
N N
NH2 N
11
13
Sodium tert.-butylate, rac-BINAP, Pd2(dba)3CHCl3
N
Ar, xylene, reflux, 5 h
H
72% N N N
HN
N O
N N NH2
reflux, 20 h reflux, 12 h
92.6% 85.5%
N N N DMEDA, Cul, K2CO3
a b 13
3 5 Br dioxane, 100 C, 20 h
82.0%
Br2, Fe, 80 C, 1.5 h
O2N O2N
89.5%
d
14 c 15
H H
N N N N
N2H4.H2O/FeCl3 N
N
H
CH3OH, reflux, 68 h NH2 N N
NO2 83.8% Cl
N N
N 6 7 THF, TEA
e
HN
0 C, 3 h
OH Cl 93.0%
N O
g
SOCl2, CH2Cl2 18
reflux, 5 h
87.2% 1-Methylpiperazine reflux, 3 h
COCl 91.4%
O OH f
16 17 h
N
H
N N N
HN
N O
4. PHYSICAL CHARACTERISTICS
4.1. Optical activity
Imatinib mesylate does not have chiral centers, and it is not optically
active.
a copper Ka radiation. The pattern obtained is shown in Figure 6.2, and the
data of scattering angle (degrees 2y) and the relative intensities (I/Imax) were
displayed in Table 6.1 [17,18].
4.6. Hygroscopicity
The compound is nonhygroscopic [19].
Table 6.1 An a2 crystalline form of imatinib mesylate which has the XRPD
characteristics given below
Angle (2u) D value () Intensity (%)
4.841 18.24057 33.6
10.410 8.49070 100.0
11.194 7.89775 14.2
11.856 7.45827 19.9
12.881 6.86709 6.8
13.819 6.40328 12.9
14.860 5.95663 67.
16.439 5.38788 32.4
17.049 5.19665 5.6
17.623 5.02870 58.6
18.052 4.9100 61.6
18.567 4.77491 98.8
19.032 4.65925 70.2
19.772 4.48657 15.3
21.236 4.18055 60.8
21.582 4.11431 59.4
22.594 3.93217 19.7
23.137 3.84112 21.8
23.696 3.75172 25.0
24.851 3.57993 58.6
26.250 3.39226 9.1
27.341 3.25932 18.7
28.475 3.13204 42.4
31.896 2.80347 9.0
32.533 2.75005 6.6
43.447 2.08117 6.4
274 Badraddin M.H. Al-Hadiya et al.
4.7.4 Boiling point, enthalpy of vapor, flash point, and vapor pressure
The calculated value of the boiling point of imatinib mesylate under a pressure of
760 mmHg was 754.9 C. The calculated value of enthalpy of vapor was
115.42 kJ/mol. The calculated value of flash point was found to be 410.3 C,
andthevaporpressurewascalculatedtobe6.03 1024 mmHgat25 C[21,22].
Imatinib Mesylate 275
4.8. Spectroscopy
4.8.1 UV/vis spectroscopy
The UV absorption spectra of imatinib mesylate in different solvent systems,
buffer phases (pH 113), are shown in Figure 6.5. The figures were recorded
using a V-570 double beam UVvis spectrophotometer (Jasco, Japan) in
10 mm matched quartz cells. Data acquisition and analysis were performed
using Spectramanager workstation (Jasco, Japan). Absorption spectra were
recorded from 190 to 800 nm at a speed of 400 nm/min with 0.2 nm data
interval, using medium response and 1 nm bandwidth. The formulation
matrix has shown a UV absorbance spectrum with minimum change at
A
2.5
2
Abs
-1
200 250 300 350 400
Wavelength (nm)
B
2.3
2
-0.1
190 400 600 800
Wavelength (nm)
Figure 6.5 (A) UVvisible absorption spectrum of imatinib mesylate (A) in 50:50 v/v
methanol:phosphate buffer solution (PBS) (125 mg/mL) and (B) in buffer and
unbuffered media (pH 113).
276 Badraddin M.H. Al-Hadiya et al.
Table 6.2 Assignments for the resonance bands observed in the 1H NMR spectrum of
imatinib
(n)
CH N
(m) (h) (e) (d)
HC C NH O CH CH
(g)
C N C CH C C C CH2
(l) (b)
CH C H3C C (c)
C NH CH CH N CH2
(i) (o) (f) (e) (d)
N CH CH CH H2C CH2
(p) (q) (b) (b)
CH CH
(k) CH2 N
(j)
(b)
CH3
(a)
Product ion spectra of imatinib with the TSQ-MS and the LTQ-MS are
shown in Figures 6.10 and 6.11, respectively. The major peaks in the spec-
trum occur at m/z 394, 379, 377, 351, 290, 264, 247, and 222. The base
peak appeared at m/z 394. The first steps of imatinib fragmentation are
drawn in Figure 6.4 and are single-bond cleavages. But only one major frag-
ment at m/z 394, which corresponds to the neutral loss of methylpiperazine,
is observed [33].
Proposed structures of fragment ions formed by collision-induced disso-
ciation of the imatinib ion, [M H] at m/z 494, with ESIC-TSQ-MS2 and
ESIC-TQ-MS23 product scan acquisitions were given in the Figure 6.13AC.
Fragment ions of imatinib-d8 (d8, m/z 502) are also given where
necessary.
5. METHODS OF ANALYSIS
5.1. Compendial methods of analysis
5.1.1 Identification [8]
Imatinib mesylate was determined by infrared absorption spectrophotome-
try. The spectrum was compared with that of imatinib mesylate RS, or with
a qualified reference spectrum of imatinib mesylate.
280 Badraddin M.H. Al-Hadiya et al.
Table 6.3 Assignments for the resonance bands observed in the 13C NMR spectrum of
imatinib
O
NH
(g) (i) (j) (k)
(h)
394
100
90
80
70
Relative abundance
60
50 222 247
378
40
174 379
30 185
189 264
119 364
20 111 194 366 494
105 204 217 290 351
10 99 131 158 238
58 70 274 Loss of water:476
0
50 100 150 200 250 300 350 400 450 500
m/z
Figure 6.10 MS product ion spectrum of [M H] imatinib ion (m/z 494) with
ESI-TSQ-MS (triple stage quadrupole MS).
282 Badraddin M.H. Al-Hadiya et al.
377
100
90 100 394
Relative abundance
80
222 351
70 50 494
Precursor ion
Relative abundance
30
no 217 247
20 Precursor ion
105 = Low mass cut-off
10 of linear ion trap MS 204
185199 367
131 237
274 394
0
120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
m/z
Figure 6.11 MS/MS (A, insert) and MS3 (B) product ion spectra of [M H] imatinib ion:
m/z 494 ! product ions and m/z 494 ! 394 ! productions, respectively, on the ESIC-
LTQ-MS (linear ion trap MS). MS2 product ion at m/z 394 is the base peak and corre-
sponds to imatinib with the loss of methylpiperazine (100 amu). A product ion with
the methylpiperazine ring is observed at m/z 217 (A) (see also Figure 6.12).
Figure 6.12 First step of imatinib fragmentation. Proposed mechanism and MS frag-
ment structures of imatinib obtained in LTQ-MS spectra. Single-bond cleavage with
charge migration is drawn and is dependent on the initial HC positions in the imatinib
structure.
A
N N
+
189 N N N N N N
See below H H
+ HN
364 C23 H18N5 N 378 C23 H18N6 N
N
+
N N N N
H
N
+ N
N
476 C29H30N7, (1.3%) LTQ/TSQ
N
d8 = 484 C29H22D8N7
189 C12 H17N2, (0.5%) LTQ, (5%) TSQ
d8 = 197 C12H9D8N2
+
H
O N
N N N N
H H
N
N
+
B H
O N
N N N N
H H
N
N
70 C4H8N
.
N
+
O N +
+
O
75 C4H3D5N, 74 C4H4D4N
N N N
+
202 C12H14N2O 189 C12H17N2 174 C11H12NO NH
210 C12H6D8N2O 197 C12H9D8N2 180 C11H6D6NO
58 C3H8N
N N
62 C3H4D4N, 61 C3H5D3N
N
N + +
N N
+ CH2 CH2
Figure 6.13cont'd
Imatinib Mesylate 285
C H
+
O N
N N N N
N H H
N
O N
N N N
+ H H
H2C N
394 C24H20N5O, (100%) LTQ/TSQ
O N O N
H
N N N +
N N N
H H H H
+ N
N
379 C23H17N5O 290 C16H12N5O
O N N
+ H
N N N H3N N N
H H H
+ N N
264 C15H14N5
378 C23H16N5O
N
H
+ N N
H
N
247 C15H11N4
+
C N
H
HC
N N
H
N
222 C13H10N4
N N
+ or + N
N H N H2C +
H H N N
N N H
158 C9H8N3 N
185 C10H9N4
+
CH +
+ HC
HN HN
or +
N N HN
105 C6H5N2 N N
104 C7H6N
131 C8H7N2
Figure 6.14 Electropherograms of human plasma spiked with 1.00 mg/mL of IMAT and
1.25 mg/mL of LID (IS) (A) and human blank plasma (B). (1) IMAT and (2) LID (IS). Elec-
trophoretic conditions: uncoated fused-silica capillary, 46.5 cm total length, 38.0 cm
effective length, 50 mm i.d., 30 kV of voltage, 351 C capillary temperature, detection
at 200 nm, hydrodynamic injection at a pressure of 50 mbar for 20 s.
Imatinib Mesylate 287
analysis takes about 5 min. A linear response over the 0.430.0 mg/L con-
centration range was investigated for two compounds. A dilution of the sam-
ple was the only step necessary before the electrophoresis analysis. Detection
limits of 0.1 mg/L for Gleevec and its metabolite (S/N 3) were obtained.
The developed method is easy, rapid, and sensitive and has been applied to
determine Gleevec and its main metabolite in clinical urine samples [37].
tandem mass spectrometry were worked out [32,39]. Rapid and sensitive
HPLC methods were developed to estimate imatinib in human plasma using
UV detectors [40]. Ivanovic et al. [41] have reported the HPLC method for
simultaneous determination of imatinib and its main metabolite
N-desmethyl-imatinib in pharmaceutical dosage forms (Table 6.4).
6. STABILITY
A literature survey revealed few stability-indicating analytical methods
for the quantification of imatinib based on HPLC [47], reverse phase (RP)
ultra-performance liquid chromatography [48], and HPTLC [38]. There
have been some RP-HPLC methods for the estimation of imatinib and
its impurities in Glivec capsules [41,49].
Preliminary investigations have established the main degradation path-
ways, namely, oxidation to N-oxide under oxidative stress conditions. Deg-
radation was not observed after storage at high temperatures (100 C).
Imatinib Mesylate 289
Stability study was carried out for the formulation by exposing it to dif-
ferent temperatures, 0 C, ambient temperature, and 40 C for 3 months.
The sample was analyzed for drug content at the regular intervals. No
remarkable change was found in the drug content of formulation. This indi-
cates that the drug was stable at the above optimized formulation [15].
Stability studies have been performed under The International Confer-
ence on Harmonization of Technical Requirements for Registration of
Pharmaceuticals for Human Use (ICH Q1A) conditions and other experi-
mental conditions have been reported. No apparent change of the drug sub-
stance quality was observed in these studies. A retest period of 2 years at
25 C is proposed when the substance is stored in tight packing protected
from light.
Szczepek et al. [50] studied the decomposition of imatinib mesylate
(ImM) under hydrolytic (neutral, acidic, alkaline), oxidative, and photolytic
conditions. They were found that the imatinib mesylate is effectively
290 Badraddin M.H. Al-Hadiya et al.
N N
H H H N
N N NH2 N N N N
N
N N N O
HOOC
2 3 4
O N
N
H H N N N N
N N N N H H
N
N O
5
-
O
N N +
H H N H H N
+
N N N N N N N N
-
O
N O N O
6 7
- -
O O
N + N +
H H N H H N
+ +
N N N N N N N N
O OH
N - N O
O -
CH3SO3
8 8a
Figure 6.17 Structure of degradation products obtained under oxidation conditions
(compounds 6, 7, 8, and 8a).
Imatinib Mesylate 291
7. CLINICAL APPLICATIONS
7.1. Pharmacodynamics (an overview)
Imatinib was identified through high-throughput screening against the
Breakpoint cluster regionAbelson proto-oncogene kinase (BCRABL)
kinase, then it considered as a potent and selective inhibitor of the protein
tyrosine kinase BcrAbl, platelet-derived growth factor receptors
(PDGFRa and PDGFRb), and KIT. The lead compound of this series, a
2-phenylaminopyrimidine, had low potency and poor specificity, inhibiting
both serine/threonine and tyrosine kinases. The addition of a 30 -pyridyl
group at the 30 position of the pyrimidine enhanced its potency. Imatinib
mesylate was the first molecularly targeted protein kinase inhibitor to receive
FDA approval. It targets the BCRABL tyrosine kinase, which underlies
CML. A single molecular event, in this case the 9:22 translocation, leads
to expression of the ABL fused to BCR, yielding a constitutively activated
protein kinase, BCRABL, and then the malignant phenotype.
Imatinib clinical and molecular remissions were found in more than 90%
of CML patients in the chronic phase of disease. Imatinib effectively treats
other tumors that carry related tyrosine kinase mutations, including GI stro-
mal tumors (driven by protein encoded by kit gene (c-KIT) mutation), and
hypereosinophilia syndrome, chronic myelomonocytic leukemia, and
dermatofibrosarcoma protuberans (all driven by mutations that activate
the PDGFR) [51].
7.5. Pharmacokinetics
Imatinib is well absorbed after oral administration and reaches maximal
plasma concentrations within 24 h. The elimination t1/2 of imatinib and
that of its major active metabolite, the N-desmethyl derivative, are
18 and 40 h, respectively. Mean imatinib area under the curve (AUC)
increases proportionally with increasing dose in the range 251000 mg.
Food does not change the pharmacokinetic profile of imatinib. Doses more
than 300 mg/day achieve trough levels of 1 mM, which correspond to in vitro
levels required to kill BCRABL-expressing cells. Inhibition of the BCR
ABL tyrosine kinase in white blood cells from patients with CML reaches a
maximum in the dose range of 250750 mg/day. Nonrandomized studies
suggest that response may be restored in a minority of resistant patients with
doses of 600 or 800 mg/day, as opposed to the standard 400 mg/day. In the
treatment of GIST, higher doses (600 mg/day) may improve response
rates [51].
Imatinib is approximately 95% bound to human plasma proteins, mainly
albumin and a1-acid glycoprotein. The drug is eliminated predominantly
via the bile in the form of metabolites, one of which (CGP 74588) shows
comparable pharmacological activity to the parent drug. The fecal to urinary
excretion ratio is approximately 5:1 [52].
Imatinib is metabolized mainly by the cytochrome P450 (CYP) 3A4 or
CYP3A5; enzymes CYPs 1A2, 2D6, 2C9, and 2C19 play minor roles in its
metabolism. Accordingly, imatinib can competitively inhibit the metabo-
lism of drugs that are CYP3A4 or CYP3A5 substrates. Interactions may
occur between imatinib and inhibitors or inducers of these enzymes, lead-
ing to changes in the plasma concentration of imatinib as well as
coadministered drugs [52]. As examples of such interactions, single dose
of ketoconazole, an inhibitor of CYP3A4, increases the maximal imatinib
concentration in plasma and its plasma AUC by 26% and 40%, respectively.
Coadministration of imatinib and rifampin, an inducer of CYP3A4, lowers
the plasma imatinib AUC by 70%. Likewise, imatinib, as a competitive
294 Badraddin M.H. Al-Hadiya et al.
7.6. Toxicity
Imatinib causes GI distress (diarrhea, nausea, and vomiting), but these symp-
toms usually are easily controlled; also the drug promotes fluid retention,
which may lead to dependent edema and periorbital swelling. Significant
myelosuppression occurs infrequently but may require transfusion support,
dose reduction, or discontinuation of the drug. Drug intake can be associ-
ated with hepatotoxicity. Most nonhematological adverse reactions are self-
limited and respond to dose adjustments. After the adverse reactions, such as
edema, myelosuppression, or GI symptoms, have resolved, the drug may be
reinitiated and titrated back to effective doses [53].
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296 Badraddin M.H. Al-Hadiya et al.
Moxifloxacin Hydrochloride
Mahmoud M.H. Al Omari*, Deema S. Jaafari*, Khaldoun A. Al-Souod,
Adnan A. Badwan*
*The Jordanian Pharmaceutical Manufacturing Co., PO Box 94, Naor, Jordan
Contents
1. Description 300
1.1 Nomenclature 300
1.2 Formulae 301
1.3 Elemental analysis 301
1.4 Appearance 302
2. Methods of Preparation 302
2.1 Method I 302
2.2 Method II 303
2.3 Method III 304
2.4 Method IV 305
2.5 Method V 307
2.6 Method VI 309
2.7 Method VII 309
2.8 Method VIII 309
2.9 Method IX 309
2.10 Method X 310
2.11 Method XI 311
2.12 Method XII 312
2.13 Method XIII 313
3. Physical Characteristics 316
3.1 Ionization constants 316
3.2 Solubility characteristics 316
3.3 Partition coefficients 318
3.4 Optical activity 318
3.5 Polymorphism 319
3.6 Particle morphology 325
3.7 Hygroscopicity 325
3.8 X-ray powder diffraction pattern 327
3.9 Thermal analysis 329
3.10 Spectroscopy 332
3.11 Mass spectrometry 340
Profiles of Drug Substances, Excipients, and Related Methodology, Volume 39 # 2014 Elsevier Inc. 299
ISSN 1871-5125 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800173-8.00007-6
300 Mahmoud M.H. Al Omari et al.
1. DESCRIPTION
1.1. Nomenclature
1.1.1 Systematic chemical names
1-Cyclopropyl-6-fluoro-8-methoxy-7-[(4aS,7aS)-octahydro-6H-
pyrrolo[3,4-b]pyridin-6-yl]-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid hydrochloride [1].
1-Cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-[(4aS,7aS)-
octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl]-4-oxo-3-quinolinecarboxylic
monohydrochloride [2].
(4aS-cis)-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-
[-octahydro-6H-pyrrolo [3,4-b] pyridin-6-yl]-4-oxo-3-quinolinecarboxylic
monohydrochloride [2].
1-cyclopropyl-7-[(S,S)-2,8-diazabicyclo[4.3.0]non-8-yl]-6-fluoro-8-
methoxy-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid hydrochloride [3].
7-[(4aS,7aS)-octahydro-1H-pyrrolo[3,4-b]pyridin-6-yl]-1-cyclopropyl-
6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid [4].
Moxifloxacin Hydrochloride 301
1.2. Formulae
1.2.1 Empirical formula, molecular weight, CAS number [2,5]
Moxifloxacin C21H24FN3O4 401.4 [151 09609-2]
Moxifloxacin HCl C21H25ClFN3O4 437.9 [186826-86-8]
H OCH3
1.4. Appearance
Light yellow or yellow powder or crystals, slightly hygroscopic [1].
Slightly yellow to yellow powder or crystals [9].
2. METHODS OF PREPARATION
2.1. Method I [1012]
l-Cyclopropyl-6,7-difluoro-8-methoxy-l,4-dihydro-4-oxo-3-quinoline-
carboxylic acid (I) is condensed with cis [S,S]-2,8-diazabicyclo[4.3.0]non-
ane (II) under reflux in a mixture of acetonitrile and dimethylformamide in
the presence of 1,4-diazabicyclo [2.2.2]octane (DABCO) as a catalyst to
get moxifloxacin base. The later compound is dissolved in HCl solution
by means of heat, then concentrated, cooled, and precipitated by ethanol
to give moxifloxacin HCl (Scheme 7.1). For further purification, it is dis-
solved in hot water, HCl is added, cooled and the crystalline product is
filtered, washed well with ethanol, and then dried.
To prepare compound (I), 3,4,6-trifluoro-5-methoxybenzoyl chloride (1)
is reacted with diethyl malonate (2) in a mixture of absolute ethanol and tol-
uene in the presence of magnesium ethoxide as a base to give diethyl (3,4,6-
trifluoro-5-methoxybenzoyl)-malonate as a crude product, followed by
partial hydrolysis and decarboxylation with aqueous p-toluenesulphonic
acid to give ethyl (3,4,6-trifluoro-5-methoxybenzoyl)-acetate (3). The later
compound is heated under reflux with triethyl orthoformate in acetic
anhydride to produce ethyl 2-(3,4,6-trifluoro-5-methoxybenzoyl)-3-
ethoxy-acrylate (4) as an oil, which is further condensed with cyclopropylamine
Moxifloxacin Hydrochloride 303
O O O O
H H
F N F
OH DABCO H H OH
N
+ NH
F N N N
H
OCH3 H OCH3
I II Moxifloxacin base
O O O O
F F
H H OH HCl H H OH
N N
N N . HCl
N N
H OCH3 H OCH3
O O O
F C2H5O OC2H5 F
Cl (1) Mg(OC2H5)2 OC2H5
+ +
O O (2) H /H2O
F F F F
OCH3 OCH3
1 2 3
O O O O
F F
OC2H5 Ac2O OC2H5
+ CH(OC2H5)3
F F F F OC2H5
OCH3 OCH3
3 4
O O O
O
NH2
F F
OC2H5 C2H5OH OC2H5
+
F F OC2H5 F F NH
OCH3 OCH3
4 5 6
O O O O
F F
OC2H5 (1) K2CO3, DMF OH
+
(2) H
F F NH F N
OCH3 OCH3
6 I
O O
O C N
N CH3CN
N-CH2-C6H5 + N-CH2-C6H5
N(CH3)2
O O
1 2 3
O O
H
N N
H2/Ru-C or Pd-C
N-CH2-C6H5 N-CH2-C6H5
O O
3 4
O
H H
N N
LiAlH4 or NaBH4/BF3.(C2H5)2O
N-CH2-C6H5 N-CH2-C6H5
O
4 5 (Racemic)
(1) L-(+)-tartaric acid
H (2) Crystalization/recrystallize/NaOH, H H
N or mother liquor/NaOH/D-()-tartaric acid N
(3) NaOH
N-CH2-C6H5 N-CH2-C6H5
5 (Racemic) 6
H H H H
N N
H2/Pd-C
N-CH2-C6H5 NH
H
H
6 II
Scheme 7.3 Preparation of cis [S,S]-2,8-diazabicyclo[4.3.0]nonane (II).
O O O O
F (CH3)3COK or F
H H OH C2H5(CH3)2COK H H OH
N N
THF/CH3OH
N N N N
H F H OCH3
1 Moxifloxacin base
O O O O
F F
H H OH (1) HCl/H2O H H OH
N N
(2) Cryst H2O/C2H5OH . HCl . H O
N N N N
2
H OCH3 H OCH3
O O R2 O O
H
F N R2 F
NRR 1 DBU H NRR1
NH N
F N N N
H
OCH3 H OCH3
1 2 3
O O O O
R2 F F
H NRR1 NaOH H H OH
N N
N N N N
H OCH3 H OCH3
3 Moxifloxacin base
O O O O
F F
H H OH H H OH
N HCl N
N N . HCl . H O
N N 2
H OCH3 H OCH3
Scheme 7.5 Preparation of moxifloxacin HCl monohydrate. Where R and R1 are hydro-
gen or selected from C1 to C5 linear or branches chain alkyl group, R2 is hydrogen, trityl,
silyl, or COOR3 group, where R3 is phenyl, ethyl, or butyl analogs.
O O O
F CN F
OCH3
F N F N
OCH3 OCH3
2 H+ 3
RR1NH
O O
F
NRR1
F N (1) Triethylorthoformate/Ac2O
DCC/HOBT/RR1NH
OCH3 or DMF-DMS/NaOCH3
or SOCl2/RR1NH (2) Cyclopropylamine
1 (3) K2CO3
O O O O
F F
OH NRR1
F N F F
OCH3 OCH3
4 5
H CPh3
H H
N N
(1) Ph3CCl, (C2H5)3N, CH2Cl2
N-CH2-C6H5 NH
(2) H2/Pd-C
H
H
1 2
Scheme 7.7 Preparation of N-substituted diazabicyclononane (2).
H3CCOO OOCCH3
B
O O O O
F F
OC2H5 H3BO3 O
(CH3CO)2O
F N F N
OCH3 OCH3
1 2
H3CCOO OOCCH3 OOCCH3
H3CCOO
B B
O O O O
H H
F N F
O (C2H5)3N H H O
+ NH N
CH3CN
F N N N
H
OCH3 OCH3
H
2 3 4
H3CCOO OOCCH3
B
O O O O
F F
H H O CH3OH H H OH
N N
HCl . HCl
N N N
N
OCH3 H OCH3
H
O O O O
F F
H H OH H H OH
N C2H5OH N
. HCl HCl . HCl. H O
N N N N 2
H OCH3 H OCH3
F F F
F
B B
O O Boc O O
F H
N Boc F
O (C2H5)3N H O
+ NH N
F Organic solvent
N N N
H
OCH3 OCH3
H
1 2 3
F F
B
O O O O
Boc F Boc F
H O Reflux H OH
N N
N N N N
H OCH3 H OCH3
3 4
O O O O
Boc F F
H OH H H OH
N HCl N
. HCl
N N N N
H OCH3 H OCH3
4 Moxifloxacin HCl
are demonstrated by its simple procedures, high product yield and purity,
low cost, high selectivity, and mild reaction conditions.
F3CCOO OOCCF3
B
O O O O
F F
OC2H5 B2O3 O
(CF3CO)2O
F N F N
OCH3 OCH3
1 2
F3CCOO OOCCF3 OOCCF3
F3CCOO
B B
O O O O
H H
F N F
O H H O
+ NH N
F N N N
H
OCH3 OCH3
H
2 3 4
F3CCOO OOCCF3
B
O O O O
F F
H H O (1) NaOH H H OH
N N
(2) CH3COOH
N N N
N
OCH3 H OCH 3
H
4 Moxifloxacin base
OCH3
Mg
O O O O
F F
OH Mg(OCH3)2, 30 C O
DMF
F N F N
OCH3 OCH3
1 2
OCH3 OCH3
Mg Mg
O O O O
H H
F N F
O 4550 C, 16 h H H O
+ NH N
F N N N
H
OCH3 OCH3
H
2 3 4
OCH3
Mg
O O O O
F F
H H O HClaq. H H OH
N N
. HCl
N N N N
H OCH3 H OCH3
4 Moxifloxacin HCl
O O O
H
N N N
OH CH3OH, HCl OCH3 Pd/C, H2 OCH3
OH OCH3 OCH3
O O O
1 2 3
O CH3 O CH3 O CH3
O O O O
H
N N N N
OCH3 Ac2O, C7H8 OCH3 CAL-B, H2O OCH3 OCH3
OCH3
TEA, DMAP; 25 C OCH3 25 C OCH3 + OH
O O O O
3 4 5 6
O CH3 O CH3
O O O
HHCl
N N N
Ac2O, C7H8
OCH3 HCl, Reflux OH
OCH3 OH O
O O O
5 7 8
O CH3 O CH3
O O O
H
N N N
Benzylamine, C7H8 HCl, Reflux
O N-CH2-C6H5 N-CH2-C6H5
O O O
8 9 10
O
H H H H
N N N
LiAlH4, THF (1) Pd/C, H2;
N-CH2-C6H5 N-CH2-C6H5 NH
(2) Distillation
H
O
10 11 II
Scheme 7.12 Preparation of cis [S,S]-2,8-diazabicyclo[4.3.0]nonane (II).
O Cl O
H H H
N N
R NaDCC
N-CH2-C6H5 N-CH2-C6H5
S C7H8, H2O, 20 C
H O H O
1 2
Cl O
H O
N H
N
TEA, 20 C
N-CH2-C6H5 N-CH2-C6H5
H O O
2
3 (95.4%)
O O
H H H
N N
Pd/C, H2, 50 C
N-CH2-C6H5 N-CH2-C6H5
Butyl acetate
H
O O
3 4 (99%)
O O
H H H H
N N
Resolution S
N-CH2-C6H5 N-CH2-C6H5
R
H H
O O
4 5
produce [R]-3-methoxycarbonylmethlene-5-phenyl-3,4,5,6-tetrahydro-2H-
1,4-oxazin-2-one (3), followed by reaction with acryloyl chloride in THF
to give [4S]-1,6-dioxo-4-phenyl-1,3,4,6,7,8-hexahydropyrido-[2,1-c][1,4]
oxazine-9-carboxylic acid methyl ester (4). The later lactam (4) is hydrogenated
with Pd/C in a mixture of ethanol and ethylacetate (10:1) to give
[4S-4a,9a,9aa]-1,6-dioxo-4-phenyloctahydropyrido [2,1-c][1,4] oxazine-9-
carboxylic acid methyl ester (5). Amidation of compound (5) with benzyl
amine in toluene produces [2R,3S]-methyl-2-(benzylcarbamoyl)-1-([R]-2-
hydroxy-1-phenylethyl)-6-oxopiperidine-3-carboxylate (6), which is then
hydrolyzed by NaOH in methanol, and then acidified with HCl to produce
compound (7). The later compound is reacted with acetic anhydride in the pre-
sence of lithium acetate to produce [R]-2-([4aR,7aS]-6-benzyl-2,5,7-tri-
oxooctahydro-1H-pyrrolo[3,4-b] pyridin-1-yl)-2-phenylethyl acetate (8),
followed by hydrogenation with lithium aluminum hydride in THF to produce
compound (9). Further hydrogenation with PD/C in methanol is carried out to
yield cis [S,S]-2,8-diazabicyclo[4.3.0]nonane (II) (Scheme 7.14).
Moxifloxacin Hydrochloride 315
OH
NH2 CO2CH3
O O Cl O O
MeOH O
+ THF/Reflux CO2CH3
Ph N Ph N
H
CO2CH3 CO2CH3 O
1 2 3 4
Bn
O O O O HO HN O
Pd/C, H2 H BnNH2
CO2CH3 CO2CH3 CO2CH3
Ph N EtOH/EtOAc Ph N C7H8/TEA Ph N
O O O
4 5 6
Bn Bn
HO HN O HO HN O AcO O
H NBn
(1) NaOH/MeOH Ac2O/LiAc
CO2CH3 CO2H O
Ph N (2) HCl Ph N 100 C Ph N
H
O O O
6 7 8
AcO O HO H H
H NBn H NBn N
LiAlH4 Pd/C, H2
N O N
NH
Ph THF Ph MeOH
H H
O H
8 9 II
3. PHYSICAL CHARACTERISTICS
3.1. Ionization constants
Moxifloxacin is an amphoteric compound, which contains a secondary alkyl
amine, two tertiary arylamines, and a carboxylic acid (Figure 7.1).
The ionization constants (pKa1 and pKa2) were measured by means of
potentiometry and spectrophotometry [27]. The obtained two pKa values
were 6.25 and 9.29 for carboxylic acid and secondary amine, respectively.
The low acidic character of moxifloxacin was explained by the formation
of intramolecular hydrogen bond between the carboxyl and keto groups
in the quinoline ring. Nearly similar values (pKa1 6.4 and pKa2 9.5) were
also reported [28].
Tertiary amine
Secondary amine (weak base)
(basic; pKa2 = 9.5)
O O
F
H H OH
N
N N
H OCH3
Carboxylic acid
(acidic; pKa1 = 6.4)
Figure 7.1 The chemical structure of moxifloxacin showing the various functional
groups with their ionization constants (pKas).
Moxifloxacin Hydrochloride 317
35 5
Water
30 Ethanol
2-Propoanol 4
25 Acetone
Solubility (mg/ml)
Solubility (mg/ml)
3
20
15
2
10
1
5
0 0
15 20 25 30 35 40 45 50 55
Temperature (C)
Figure 7.2 The solubility of moxifloxacin HCl in water and different organic solvents as a
function of temperature, left y-axis corresponds to water.
60
50
40
Solubility (mg/ml)
30
20
10
0
0 1 2 3 4 5 6 7 8
pHeq
Table 7.2 The pH solubility (S) data of moxifloxacin HCl and the calculated
dose/solubility (D/S)
pHeq S (mg/ml) D/S (ml)
1.2 6.8 59
2.1 31.3 13
3.0 31.0 13
3.9 31.3 13
5.4 26.0 15
6.4 37.6 11
6.9 58.0 7
The highest single dose (D) is 400 mg.
Moxifloxacin Hydrochloride 319
O O O O
F F
H H OH H H OH
N N
N . HCl N . HCl
N N
H OCH3 H OCH3
O O O O
F F
H H H H OH
OH N
N
N . HCl N N . HCl
N
OCH3 H OCH3
H
3.5. Polymorphism
Moxifloxacin HCl exists in different forms as shown in Table 7.4. The anhy-
drous form can be converted to the monohydrate when exposed to water, a
mixture of water, and ethanol [35] or a controlled humidification environ-
ment at 60% or 80% RH [36].
The amorphous form can be prepared by dissolving crystalline
moxifloxacin HCl in a solvent (e.g., methanol) and then spray dried [37].
Table 7.4 The list of different forms of moxifloxacin HCl with their preparations and methods of characterization
Form Preparation Characterization Reference
Form I (anhydrous Moxifloxacin hydrochloride or/and its hydrates are suspended XRPD, FT-IR, DSC, [15]
crystalline form of in a suitable polar solvent, the pH is adjusted to 78 with microscope
moxifloxacin base) NaOH or NH4OH at 2530 C, the reaction mixture is
extracted with suitable chloro or ester solvents, the solvent is
completely distilled under reduced pressure at below 60 C,
the reaction mixture is cooled to 2535 C, the crude is
treated with a suitable organic solvent like keto solvent, the
product is isolated by filtration and optionally washed with
water, and then dried to get the form
Form I (anhydrous The anhydrous form is the only crystal modification known in XRPD, DSC, TGA, 13C [35]
crystalline) the prior art [1012] NMR, Raman, FT-IR,
microscope
Form II (monohydrate The anhydrous form is suspended and stirred in aqueous media
crystalline) until hydration or dissolved in ethanol either with or without
water and distilled off and dried under humid condition to get
prism and needle crystals
Hydrated crystalline Crystalline anhydrous or monohydrate forms of moxifioxacin XRPD [36]
HCl, or mixtures is placed into a controlled humidification
environment at 60% or 80% RH and 30 C for 18 and 16 h,
respectively
Amorphous Crystalline moxifloxacin HCl is dissolved in a solvent (e.g., XRPD, FT-IR [37]
methanol), subjected to spray drying and further dried
Form A (crystalline) Moxifloxacin is suspended in organic solvents (e.g., XRPD, FT-IR, DSC [38]
methanol), treated with dry hydrogen chloride, dried under
control conditions to get the crystalline Form A of
moxifloxacin HCl with moisture content below 2%
Form X (anhydrous Moxifloxacin HCl is azeotropically refluxed in hydrocarbon XRPD [39]
crystalline) solvents, cooled, filtered, and dried
Form Y (anhydrous Moxifloxacin is dissolved in alcoholic solvents by heat and
crystalline) adjusting the pH to 7.58.5 using with aqueous alkaline
solution. The pH is adjusted to below 0.5 with aqueous HCl at
<15 C and the reaction mass is maintained for 3060 min at
<15 C, followed by drying
Form III (anhydrous Moxifloxacin HCl is azeotropically refluxed in lower XRPD, 13C NMR, FT-IR, [40]
crystalline) branched or chained acid esters or an aliphatic hydrocarbon TGA, DSC
solvent or aromatic hydrocarbons. The reaction mixture is
cooled with stirring till the solid mass crystallizes, followed by
drying
New crystalline form Moxifloxacin HCl is dissolved in a mixture of methanol/water XRPD, FT-IR [41]
by heating at the reflux temperature; acetone is added and the
solution is heated at 4045 C, cooled to 1525 C, filtered,
and dried
Continued
Table 7.4 The list of different forms of moxifloxacin HCl with their preparations and methods of characterizationcont'd
Form Preparation Characterization Reference
Crystalline form of Moxifloxacin or it HCl salt is suspended in water and pH XRPD, 13C NMR, FT-IR, [42]
moxifloxacin base was adjusted to >11 with NaOH. The basified solution is TGA, DSC
washed with toluene and then pH adjusted to 8.08.2 with
HCl. The reaction mixture is extracted with methylene
chloride and the organic layer is dried under reduced pressure.
The solid thus obtained is recrystallized from acetonitrile
Form A (hydrated Moxifloxacin HCl (anhydrous or monohydrate) is suspended XRPD, 13C NMR, FT-IR, [43]
crystalline) in a solvent selected from alcohols and polyols or mixtures, in TGA, DSC
which the resulting mixture has an overall water content of
between 0.01% and 2.5% by weight, refluxed, cooled, and
then the form is isolated
Form B (anhydrous The same procedure of Form A, but the isolated form is
crystalline) reslurried at reflux in a solvent selected from alcohols and
polyols or mixtures, in which the resulting mixture has an
overall water content of between 0.01% and 2.5% by weight
and then the form is isolated
13
Form IV (anhydrous Moxifloxacin base is dissolved (or its HCl salt is suspended) in XRPD, C NMR, FT-IR [44]
crystalline) an organic solvent, cooled, saturated with HCl (not required
in case of the HCl salt), maintained for 2 h, the solvent is
removed under vacuum and the residue is resuspended in the
same solvent, cooled to a temperature to 0 and 10 C for
13 h, the formed crystals are separated by filtration, wash and
dry the obtained product until a constant weight
Form a1 (hydrated Moxifloxacin HCl is suspended in a mixture of water and 37% XRPD, DSC [45]
crystalline) HCl, dissolved by heating at 100 C, cooled to 20 C and the
attained solid material was filtered, washed with water, and
dried under reduced pressure at 95 C to afford the form
having a water content of 3.64%
Form a2 (hydrated Moxifloxacin HCl is suspended in N-methyl-2-pyrrolidinone
crystalline) and water, heated to 130 C, cooled to 20 C. The
obtained solid is filtered, washed with acetone, and dried
under reduced pressure at 60 C to afford the form having a
water content of 4.02%
Form IV (monohydrate Moxifloxacin HCl is suspended in methanol and water and the XRPD [46]
crystalline) pH is adjusted to 1.02.0 with concentrated HCl at 25 C, and
cooled to 5 C. The solid obtained is collected by filtration
and the solid is dried at 6065 C
Form b (monohydrate Moxifloxacin base is suspended in methanol and water. HCl and XRPD, DSC [47]
crystalline) EDTA are added, heated to 3438 C for 1 h, cooled to 05 C
for 1 h, filtered, and washed with chilled methanol. The solid
Form g (anhydrous
again is heated in methanol and water to 55 C to get a clear
crystalline)
solution, cooled slowly to 4045 C (2025 C for Form g) and
HCl is added (not required for Form g), cooled slowly to 05 C
and maintained for 1 h, filtered, washed with chilled methanol,
and water thrice (only methanol for Form g) and dried at
5055 C for 2 h (8085 C for 12 h for Form g), sieved, and
dried further under vacuum at 4555 C for 30 h (8085 C for
36 h for Form g)
Continued
Table 7.4 The list of different forms of moxifloxacin HCl with their preparations and methods of characterizationcont'd
Form Preparation Characterization Reference
Form F (anhydrous Moxifloxacin HCl is suspended in water and ethanol, heated XRPD, FT-IR, DSC, TGA [48]
crystalline) at 7585 C under stirring, filtered, and washed with
ethanol. Ethanol is added dropwise at 7080 C to the solid,
cooled slowly to 2030 C, and crystallized for 120 min,
filtered and washed with absolute ethanol. Absolute ethanol
and HCl are added to the wet crystal, stirred at 4050 C for
1.52 h, cooled to 1020 C, stirred for 23 h, washed
with absolute ethanol, and dried under vacuum to water
content 1.0%
Hydrate crystalline Anhydrous moxifloxacin or/and moxifloxacin hydrate is XRPD [49]
dissolved in organic solventwater mixture by heat at
50150 C, the pH is adjusted with HCl to pH 12, stirred for
560 min, and cooled for crystallization. The organic solvent is
one or more of acetone, dioxane, and ethyl acetate
Form C (crystalline) Moxifloxacin HCl is stirred with methanol and triethyl amine at XRPD, FT-IR, DSC, [50]
2530 C. The reaction mass is concentrated partially. Raman
Further methanol is added and the pH is adjusted to 1.02.0 using
HCl gas dissolved in methanol at 2025 C. The content is
cooled to 05 C and maintained at 05 C for 2 h. The
resulting solid is filtered, washed with chilled methanol, and
dried under vacuum at 8090 C
Moxifloxacin Hydrochloride 325
Different forms of moxifloxacin HCl hydrate and its anhydrous form can
be obtained from different aqueous and organic solvents under controlled
experimental conditions (Table 7.4). They are characterized by distinctive
X-ray powder diffraction (XRPD) patterns, 13C-nuclear magnetic reso-
nance (13C NMR), differential scanning calorimetry (DSC), Fourier trans-
form infrared (FT-IR), Raman spectroscopy, thermal gravimetric analysis
(TGA), and microscope.
3.7. Hygroscopicity
Anhydrous moxifloxacin HCl is hygroscopic and absorbs water under
adverse storage conditions and handling [35].
A B C
Figure 7.5 The photographs of microscopic moxifloxacin HCl monohydrate (A) prism,
(B) needle, and (C) oval shapes.
326 Mahmoud M.H. Al Omari et al.
Figure 7.6 The DSC thermograms of anhydrous moxifloxacin HCl after incubation at (a)
initial, (b) 25 C/25% RH/12 h, (c) 25 C/52% RH/1 h, (d) 25 C/95% RH/1 h, (e) 40 C/75%
RH/3 weeks, and (f ) Ph. Euro. RS.
Moxifloxacin Hydrochloride 327
The maximum water uptake (<0.1%) was attained after 3 days of incubation
at 40 C/75% RH.
Figure 7.7 The XRPD patterns of (A) anhydrous moxifloxacin HCl (Form I), (B)
moxifloxacin HCl monohydrate (Form II), and (C) amorphous moxifloxacin HCl.
328 Mahmoud M.H. Al Omari et al.
Table 7.5 The crystallographic results from the X-ray powder diffraction patterns of
anhydrous moxifloxacin HCl (Form I) and moxifloxacin HCl monohydrate (Form II)
Anhydrous moxifloxacin HCl (Form I) Moxifloxacin HCl monohydrate (Form II)
Relative Relative
Scattering angle intensity intensity
(degrees 2u) d-Spacing () (%) (degrees 2u) d-Spacing () (%)
5.8 15.2373 53.8 5.8 15.2373 23.5
8.6 10.2816 71.2 8.5 10.4023 39.2
10.3 8.5881 50.0 10.1 8.7577 37.3
11.6 7.6284 9.6 13.4 6.6075 33.3
13.6 6.5108 42.3 14.5 6.1086 80.4
14.5 6.1086 100.0 14.8 5.9855 7.8
15.0 5.9061 7.7 15.6 5.6803 39.2
15.8 5.6088 32.7 17.0 5.2155 9.8
17.3 5.1257 17.3 17.4 5.0965 21.6
17.5 5.0676 34.6 17.5 5.0676 9.8
18.3 4.8478 9.6 17.9 4.9552 15.7
18.9 4.6953 5.8 18.6 4.7703 15.7
19.3 4.5988 5.8 19.6 4.5291 9.8
19.6 4.5291 11.5 20.4 4.3533 31.4
20.6 4.3115 25.0 17.4 5.0965 21.6
21.5 4.1330 3.8 22.7 3.9171 3.9
24.2 3.6776 17.3 23.0 3.8667 2.0
24.7 3.6043 15.4 23.6 3.7698 21.6
25.0 3.5617 19.2 24.1 3.6927 21.6
26.3 3.3886 5.8 24.5 3.6333 3.9
27.0 3.3023 61.5 26.5 3.3634 7.8
27.4 3.2550 59.6 26.7 3.3387 94.1
27.8 3.2090 44.2 27.0 3.3023 5.9
29.4 3.0379 38.5 27.3 3.2667 49.0
29.7 3.0079 3.8 27.5 3.2434 100.0
30.0 2.9785 5.8 27.8 3.2090 3.9
Moxifloxacin Hydrochloride 329
Table 7.5 The crystallographic results from the X-ray powder diffraction patterns of
anhydrous moxifloxacin HCl (Form I) and moxifloxacin HCl monohydrate
(Form II)cont'd
Anhydrous moxifloxacin HCl (Form I) Moxifloxacin HCl monohydrate (Form II)
Relative Relative
Scattering angle intensity intensity
(degrees 2u) d-Spacing () (%) (degrees 2u) d-Spacing () (%)
30.3 2.9497 3.8 22.7 3.9171 3.9
31.3 2.8577 7.7 28.9 3.0893 3.9
31.8 2.8139 5.8 29.2 3.0583 45.1
29.7 3.0079 11.8
31.4 2.8488 9.8
28.9 3.0893 3.9
34.2 2.6217 3.9
35.1 2.5566 3.9
generated from the XRPD patterns of both forms are listed in Table 7.5. The
interplaner d-spacing is calculated from Bragg equation (2dsin y nl),
where l (1.5418 A ) is the wavelength of the X-ray (Cu Ka radiator).
In this study, the monohydrate form (Form II) is characterized with a band
at 2y 26.7 (Figure 7.7B).
The XRPD pattern of the amorphous form of moxifloxacin HCl pre-
pared by spray drying technique is shown in Figure 7.7C [37]. The plain
halo shape of the pattern proves the amorphous nature when the drug is
spray dried.
In addition to these three forms and as mentioned previously in
Section 3.5, there are other crystalline forms of anhydrous and hydrate
moxifloxacin HCl that can be clearly characterized using this technique.
Figure 7.8 The DSC thermograms of (A) anhydrous moxifloxacin HCl (Form I) and (B)
moxifloxacin HCl monohydrate (Form II).
3.10. Spectroscopy
3.10.1 UV/VIS spectroscopy
The UV/VIS absorption spectra of moxifloxacin HCl monohydrate in dif-
ferent solvents were recorded using the Beckman Coulter DU-650 spectro-
photometer [30]. Figure 7.10 shows the UV/VIS absorption spectrum of
moxifloxacin HCl monohydrate (0.0043 mg/ml as anhydrous) in methanol.
The two maxima, recorded at 232 and 295 nm, are apparently due to the
p ! p* electronic transitions in the aromatic ring. The longest wavelength
Moxifloxacin Hydrochloride 333
Figure 7.9 The TGA thermograms of (A) anhydrous moxifloxacin HCl (Form I) and (B)
moxifloxacin HCl monohydrate (Form II).
0.6
0.5
0.4
Absorbance
0.3
0.2
0.1
0
220 240 260 280 300 320 340 360 380 400
Wavelength (nm)
Figure 7.10 The UV/VIS absorption spectrum of moxifloxacin HCl (0.0043 mg/ml as
anhydrous) in methanol.
Table 7.8 The UV/VIS absorption data of moxifloxacin HCl in different solvents
Solvent lmax (nm) A (1%, 1 cm) (gm/100 ml)1cm1
Methanol 232 255
295 1100
334 280
358 250
358 250
Water 245 300
288 995
338 385
0.1 N HCl 217 340
295 1050
335 260
360 235
0.1 N NaOH 242 305
291 1055
339 335
355 285
Moxifloxacin Hydrochloride 335
Figure 7.11 The FT-IR absorption spectrum of moxifloxacin HCl monohydrate (KBr disc).
Table 7.9 The assignments of the FT-IR absorption bands of moxifloxacin HCl
monohydrate
Wavenumber (cm1) Assignment
35303472 n(OdH); H2O; COOH
31563056 n(CdH); aromatic
29762800 n(CdH); aliphatic
27332422 n(NH2 )
1709 n(C]O); COOH
16201520 n(C]O); phenyl breathing
14541358 dCH; deformations of CH2
13231256 db(dCH2)
1184 n(CdO)
1166 n(CdN)
1111 n(CdC)
1049 dr(dCH2)
991802 dCH-bend; phenyl
772721 db(COO)
691424 Ring deformation
n, stretching; db, bending.
336 Mahmoud M.H. Al Omari et al.
Figure 7.12 The FT-IR absorption spectrum of (A) anhydrous (Form I) and (B) mono-
hydrate (Form II) of moxifloxacin HCl (KBr disc).
Figure 7.13 The Raman absorption spectrum of (A) anhydrous (Form I) and (B) mono-
hydrate (Form II) of moxifloxacin HCl.
Table 7.10 The assignments of some of the Raman absorption bands of moxifloxacin
HCl monohydrate
Wavenumber (cm1) Assignment
31503050 n(OdH, NdH)
3120 n(](CdH))
30002800 n(CdH)
1711 n(C]O)
1619 n(C]C)
1433 db(CH2)
13761352 n(Quinolone ring)
n, stretching; db, bending.
13
3.10.3.2 C NMR spectrum
The 13C NMR spectrum of moxifloxacin HCl (water content 22.5%) was
obtained using the BRUKER-AV-500 spectrometer [54]. The sample was
dissolved in DMSO-d6 and all resonance bands were referenced to the tetra-
methylsilane (TMS) internal standard. The 13C NMR, DEPT, HMQC, and
HMBC spectrum of moxifloxacin HCl are shown in Figures 7.167.19. Their
corresponding assignments are given in Table 7.12.
13
3.10.3.3 Solid C NMR spectrum
13
The solid C NMR spectra of anhydrous moxifloxacin HCl (Form I) and
moxifloxacin HCl monohydrate (Form II) were recorded using the BRUKER
SML300spectrometer[35].Thesolid 13CNMRspectraareshowninFigure7.20
and the assignments of the solid 13C NMR bands are given in Table 7.13. The 13C
NMR spectra show the significant difference between the two forms due to the
presence of a sharp peak at 168.1 ppm for the monohydrate form (Form II) in the
range180160 ppm(Figure7.20).Suchdifferenceinthespectramaybeattributed
to existing inter-hydrogen bonding between water and the carboxylic group.
340 Mahmoud M.H. Al Omari et al.
H2 H1 F
H O
J IH
H H H P
B
K1 N N H C . HCl
H N G
O1 H
K2 H
O
N H H
H
H H H3 C E1 F1
H H O2 D
L1 M2 H H
H H
L2 M1 E2 F2
Chemical shift
1
(ppm) Number of proton Multiplicity Assignment H1H COSY
15.11 1a s C /
10.28 1a s, br P /
a
9.07 1 s, br J /
8.65 1 s B /
7.63 1 d A /
4.16 1 m G GE, GF
4.08, 3.65 2 dd H1, H2 H1I, H2I
3.91, 3.76 2 m O O1N, O2N
3.88 1 m I IH, IN
3.61 3 s D /
3.18, 2.92 2 m K1 , K 2 K1L, K2L
2.67 1 m N NI
1.84, 1.70 2 m M1, M2 M1N, M2N
1.751.80 2 m L1, L2 L1K, L2K
1.021.14 2 m F1, F2 F1G, F2G
0.89, 1.21 2 m E1, E2 E1G, E2GE1G, E2G
a
Disappear by increase of water content.
d, doublet; s; singlet; m, multiplet; dd, doublet doublet; br, broad.
Table 7.12 The 13C NMR spectral assignments for moxifloxacin HCl
O O
1
F 2 8 6
H H 7 10 OH
N 15
17
16 . HCl
N 3 9 N 5
18 20 4
19 21 OCH3 12
H 11
14 13
Figure 7.20 The solid 13C NMR spectrum of moxifloxacin (A) anhydrous (Form I) and (B)
monohydrate (Form II) of moxifloxacin HCl.
while the fragment ion at m/z 145 is probably formed by the migration of
a fluorine atom to nitrogen followed by cleavage of the NdC bond.
The driving force for such migration appears to be due to the loss of a
stable neutral molecule with a triple bond in the aromatic ring (substituted
benzyne). The mechanism involving loss of benzyne is expected to be
initiated with the abstraction of orthohydrogen to the fluorine by the
nitrogen atom [55]. Furthermore, fragment ion at m/z 364 is formed by
loss of HF leading to the formation of the four-membered ring
azetidine [56].
Moxifloxacin Hydrochloride 345
Table 7.13 The solid 13C NMR spectral assignments for moxifloxacin HCl monohydrate
O O
1
F 2 8 6
H H 7 10 OH
N 15
17
16 . HCl
N 3 9 N 5
18 20 4
19 21 OCH3 12
H 11
14 13
4. METHODS OF ANALYSIS
4.1. Compendial methods
4.1.1 Moxifloxacin HCl drug substance
Moxifloxacin HCl is a drug substance listed in the European pharmacopeia
(Ph. Euro.) [1], United States pharmacopeia-national formulary (USP-NF)
[2] and United States pharmacopeia-medicines compendium (USP-MC)
[57]. Table 7.15 shows its summary of specifications and methods of analysis.
346 Mahmoud M.H. Al Omari et al.
1.2 384.1721
1.0
169.1131
Counts 10-3
0.6
332.1773 358.1921
108.0449 145.1131 181.1338
0.4 374.1880
232.0769 276.0663 321.1589
402.1821
0.2 110.0615 249.1037
0
80 120 160 200 240 280 320 360 400
m/z
Figure 7.21 The mass spectrum of moxifloxacin HCl monohydrate.
H OCH3
H OCH3
H OCH3
Continued
Table 7.14 The mass spectral fragmentation for moxifloxacin HCl monohydratecont'd
Fragment
m/z Relative abundance (%) Formula Structure
364.16 6.3 [C21H25FN3O4] [H2O HF] O O+
NH H
H N
N
OCH3
F
H H
N
N N
H OCH3
N N
OCH3
341.17 10.1 [C21H25FN3O4][CO2 NH3] H+
O
F
H
CH2
N N
H OCH3
N
N
H OCH3
H N
N
OCH3
Continued
Table 7.14 The mass spectral fragmentation for moxifloxacin HCl monohydratecont'd
Fragment
m/z Relative abundance (%) Formula Structure
301.14 10.1 [C21H25FN3O4] [CO2 C3H7N] O H+
N N
OCH3
F
OH
H2N N
OCH3
+
N
OCH3
249.10 15.2 [C21H25FN3O4][CO2 C3H7NC4H4] O H+
H2N N
OCH3
+
N
OCH3
H OCH3
H
Continued
Table 7.14 The mass spectral fragmentation for moxifloxacin HCl monohydratecont'd
Fragment
m/z Relative abundance (%) Formula Structure
145.11 31.6 [C7H14FN2] H H H+
N
NF
H
110.06 11.4 [C6H8NO] O H+
H2N
OH
Table 7.15 The summary of the compendial methods of moxifloxacin HCl
Test Eur. Ph. USP-NF USP-MC
Definition 1-Cyclopropyl-6-fluoro-8-methoxy-7-[(4aS,7aS)-octahydro-6H-pyrrolo[3,4-b]
pyridine-6-yl]-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride
(4aS-cis)-1-cyclopropyl-6-fluoro-1,4-
dihydro-8-methoxy-7-(octahydro-6H-pyrrolo [3,4-b]
pyridin-6-yl)-4-oxo-3-quinolinecarboxylic acid,
monohydrochloride
Moxifloxacin HCl contains NLT 98.0% and NMT 102.0% of moxifloxacin HCl
(C21H24FN3O4 HCl), calculated on the anhydrous basis.
Characters Light yellow or yellow powder Slightly yellow to yellow powder Slightly yellow to yellow,
or crystals, slightly hygroscopic. or crystals. Soluble in 0.1 N crystalline powder
Sparingly soluble in water, NaOH, sparingly soluble in
slightly soluble in ethanol (96%), water, and in methanol; slightly
practically insoluble in acetone soluble in 0.1 N HCl, in
dimethylformamide, and in
alcohol; practically insoluble in
methylene chloride, in acetone,
in ethyl acetate, and in toluene;
insoluble in tert-butyl methyl
ether and n-heptane
Identification A Specific optical rotation (see Test IR Absorption <197K >
below)
Continued
Table 7.15 The summary of the compendial methods of moxifloxacin HClcont'd
Test Eur. Ph. USP-NF USP-MC
Identification B IR absorption The RT of the major peak in the Identification testsgeneral,
spectrophotometry (2.2.24) chromatogram of the assay chloride < 191 >
preparation corresponds to that
in the chromatogram of the
standard preparation, as obtained
in the assay
Identification C To 10 mg/ml in H2O, add 1 ml To a solution (1 in 160), add
of dilute HNO3, mix, stand for diluted HNO3, and filter. The
5 min and filter. The filtrate gives filtrate meets the requirements of
reaction (a) of chlorides (2.3.1) the tests for Chloride <191 >
Appearance of solution Not more opalescent than RS II
(2.2.1) and not more intensely
colored than RS GY2 (2.2.2,
Method II). If intended for use in
the manufacture of parenteral
preparations, the solution is clear
(2.2.1) and not more intensely
colored than RS GY2 (2.2.2,
Method II). 50 mg/ml in dilute
NaOH solution
pH 3.94.6, 2 mg/ml in CO2-free H2O [(2.2.3) and <791 >]
Specific optical rotation 125 to 138 (anhydrous substance), 10 mg/ml in CAN:H2O
(1:1 v/v) [(2.2.7) and <781S >]
Related substance/related compounds/organic impurities (protect solutions from light) [(2.2.29) and < 621 >]
Diluent 0.50 g of TBAHSO4 and 1.0 g 0.50 g of TBAHSO4 and 1.0 g MeOH
KH2PO4 in about 500 ml of KH2PO4 in H2O, add 2 ml of
H2O. Add 2 ml of H3PO4 and H3PO4 and 20 mg of anhydrous
50 mg of anhydrous Na2SO3, Na2SO3, then dilute to
then dilute to 1000.0 ml with 1000.0 ml with H2O
H2O
Mobile phase (v/v) MeOH:A solution containing MeOH:A solution containing MeOH: A solution A: 0.1% DEA
0.5 g/l TBAHSO4, 1.0 g/l 0.5 g/l TBAHSO4, 1.0 g/l in H2O, pH of 2.3 with TFA
KH2PO4 and 3.4 g/l H3PO4 KH2PO4 and 2 ml/l H3PO4 (40:60).
(28:72) (7:18)
Test solution (a) 1.0 mg/ml in diluent 0.1 mg/ml in diluents, prepare 1.0 mg/ml in MeOH
from stock solution (5 mg/ml)
Test solution (b) 0.1 mg/ml in diluent, prepare 0.1 mg/ml in MeOH, prepare
from test solution (a) from test solution (a)
Reference solution 0.1 mg/ml moxifloxacin HCl in 0.1 mg/ml of moxifloxacin HCl
diluent in MeOH
Reference solution (a) 0.1 mg/ml moxifloxacin HCl in 0.002 mg/ml moxifloxacin HCl 1 mg/ml each of moxifloxacin
diluent in diluent HCl, impurities A, B, C, D, and
E in MeOH
Continued
Table 7.15 The summary of the compendial methods of moxifloxacin HClcont'd
Test Eur. Ph. USP-NF USP-MC
Reference solution (b) 1 mg/ml moxifloxacin for peak 0.1 mg/ml and 0.001 mg/ml of 1 mg/ml of moxifloxacin HCl
identification CRS (containing moxifloxacin HCl and impurity and impurity A in MeOH
impurities A, B, C, D, and E) in A in diluent, respectively
diluent
Reference solution (c) 0.001 mg/ml in diluent, prepare 0.05 mg/ml [Note: Store the
from test solution (b) solution under refrigeration and
protected from light], prepare
from reference solution (a).
Column End-capped phenylsilyl silica gel Packing L11 (5 mm, Packing L11 (5 mm,
for chromatography R (5 mm, 250 4.0 mm) 150 4.6 mm)
250 4.6 mm)
Detection UV: 293 nm 200700 nm [calculations should
be made at an isobestic point of
moxifloxacin and impurity
spectra or at 293 nm if an
isobestic point is not available]
MS source: ES scan ( and ),
source temperature: 90 C,
desolvation temperature: 400 C
Injection (ml) 10 25 10
Flow rate (ml/min.) 1.3 0.9 1.0
Temperature ( C) 45 Oven: 40, autosampler: 5
Run time 2.5 the RT of moxifloxacin 2 the RT of moxifloxacin
a
Identification of impurities Reference to moxifloxacin Reference to moxifloxacin
(rRT) Impurity A: about 1.1 Impurity A: about 1.15
Impurity B about 1.3 Impurity B about 1.32
Impurity C about 1.4 Impurity C about 1.48
Impurity D about 1.6 Impurity D about 1.71
Impurity E about 1.7 Impurity E about 1.83
System suitability Resolution: 1.5 between Resolution: 1.5 between Resolution: 1.5 between
moxifloxacin and impurity A; moxifloxacin and impurity A, moxifloxacin and impurity A,
The chromatogram obtained is use reference solution (b) use reference solution (b)
similar to the chromatogram Column efficiency: 4000 Relative standard deviation:
supplied with moxifloxacin for theoretical plates 1.0%, use reference solution
peak identification CRS, use Tailing factor: 2.0
reference solution (b) Relative standard deviation:
2.0%, use reference solution (a)
Signal-to-noise ratio: 10,
use reference solution (c)
Limits Correction factors: for the Correction factors: for the Any individual impurity: 0.1%
calculation of content, multiply calculation of content, multiply Total: 2.0%
the peak areas of the following the peak areas of the following
impurities by the corresponding impurities by the corresponding
correction factor: impurity correction factor: impurity
B 1.4; impurity E 3.5. B 0.71; impurity E 0.29.
Continued
Table 7.15 The summary of the compendial methods of moxifloxacin HClcont'd
Test Eur. Ph. USP-NF USP-MC
Impurities A, B, C, D, E: 0.1% Impurities A, B, C, D, E: 0.1%
(each) (each)
Unspecified impurities: 0.10% Unspecified impurities: 0.1%
Total: 0.3% Total: 0.5%
Disregard limit: 0.05%
Assay As described in the test for related As described in the test for related As described in the test for related
substances with the following substances with the following substances with the following
modification: modification: modification:
Injection: Test solution (b) Injection: Test solution (a) and Injection: Test solution (a) and
and reference solution (a). reference solution reference solution, detector: at
293 nm
To confirm the absence of
co-eluting known and unknown
impurity, substitute the Detector
under related substances
List of impurities Specified impurities: A, B, C, D, O O
E. R
Impurity A (6,8-difluro): H H OH
N
R R0 F
Impurity B (6,8-dimethoxy): N N
R R0 OCH3 R'
H
Impurity C (8-ethoxy): R F,
R0 OC2H5
Impurity D (6-
methoxy-8-fluoro): R OCH3,
R0 F
Impurity E (8-hydroxy): R F,
R0 OH
Water 4.5%, use 0.200 g [(2.5.12) and <921 >], method Ia
Anhydrous: 0.5%.
Monohydrate: 4.0%
[<921 >, method I]
Sulfated ash 0.1%, use 1.0 g in a platinum crucible [(2.4.14) and <281 >]
Sulfate A 0.6-g portion shows no more
sulfate than corresponds to
0.25 ml of 0.020 N sulfuric acid
(0.04%) [<221 >]
Elemental impurities Proceed as directed in the general
chapter.[< 232 >]
Residual solvents Proceed as directed in the general
chapter [< 467 >]
Microbial enumeration tests The total aerobic microbial
and Tests for specified count: 1000 cfu/g
microorganisms The total combined molds and
yeasts count: 100 cfu/g [<61 >
and < 62 >]
Continued
Table 7.15 The summary of the compendial methods of moxifloxacin HClcont'd
Test Eur. Ph. USP-NF USP-MC
Storage In an airtight container, In tight, light-resistant
protected from light containers. Store at room
temperature
Labeling The label states, where
applicable, that the substance is
suitable for use in the
manufacture of parenteral
preparations
a
Use the chromatogram supplied with moxifloxacin for peak identification CRS and the chromatogram obtained with reference solution (b) to identify the peaks due to
impurities A, B, C, D, and E.
ACN, acetonitrile; TBA, tetrabutylammonium; RT, retention time; rRT, relative retention time; DEA, diethylamine; TFA, trifluoroacetic acid.
Table 7.16 The summary of the compendial methods of moxifloxacin HCl ophthalmic solution
Test USP-NF USP-MC
Definition Moxifloxacin ophthalmic solution is a sterile, self-preserved aqueous solution of Moxifloxacin Moxifloxacin ophthalmic solution
HCl. It contains NLT 90.0% and NMT110.0% of the labeled amount of moxifloxacin contains an amount of
(C21H24FN3O4) moxifloxacin HCl equivalent to
NLT 95.0% and NMT 105.0% of
the labeled amount of
moxifloxacin (C21H24FN3O4)
Identification The retention time of the major peak in the chromatogram of the assay preparation corresponds to The response of moxifloxacin
that in the chromatogram of the standard preparation, as obtained in the Assay from the sample solution
corresponds to that of the standard
solution, as obtained in the assay
Uniformity of dosage units Meets the requirements [<905 >]
Sterility It meets the requirements when tested as directed for Membrane Filtration under Test for Sterility of the product to be examined
[< 71>]
pH 6.37.3 [<791 >]
Osmolality 260320 mOsmol/kg [<785 >]
Related compounds/Organic impurities [< 621 >]
Test 1: Early-eluting related compounds (relative retention time less than 1.8) [protect solutions
from light. Analyze the Test solution immediately after preparation]
Buffer solution/Blank 0.5 g of TBAHSO4 and 1.0 g of KH2PO4 in 1000 ml of H2O, add 2 ml of H3PO4
solution
Continued
Table 7.16 The summary of the compendial methods of moxifloxacin HCl ophthalmic solutioncont'd
Test USP-NF USP-MC
Mobile phase (v/v)/Flow Time (min) Flow rate (ml/ Buffer solution MeOH Elution MeOH: A solution A: 0.1% DEA
rate (ml/min) min) in H2O, pH of 2.3 with TFA
(40:60)/1.0
030 0.5 69 31 Isocratic
3031 0.5 69 ! 60 31 ! 40 Linear
gradient
3136 0.9 60 40 Isocratic
3136 0.9 60 40 Isocratic
3637 0.9 60 ! 69 40 ! 31 Linear
gradient
3742 0.5 69 31 Equilibration
Resolution solution 0.1 mg/ml moxifloxacin HCl and 0.001 mg/ml impurity A in buffer solution 1 mg/ml each of moxifloxacin
HCl and impurity A in MeOH
Reference solution 0.002 mg/ml moxifloxacin HCl in buffer solution 1 mg/ml each of moxifloxacin
HCl, impurity A, impurity B,
impurity C, impurity D, and
impurity E in MeOH
Sensitivity solution 0.05 mg/ml moxifloxacin HCl, prepared from reference solution [store under refrigeration and
protected from light]
Test solution A volume of solution equivalent to 0.1 mg/ml moxifloxacin in buffer solution A volume of solution equivalent
to 1 mg/ml moxifloxacin in
MeOH
Column Packing L11 (5 mm, 250 4.0 mm) Packing L11 (5 mm,
150 4.6 mm)
Detection UV: 293 nm 200700 nm [calculations should
be made at an isobestic point of
moxifloxacin and impurity spectra
or at 293 nm if an isobestic point is
not available]
MS source: ES scan ( and ),
source temperature: 90 C,
desolvation temperature: 400 C
Temperature ( C) 45 Column oven: 40, autosampler: 5
System suitability Resolution: 2.0 between moxifloxacin and impurity A, use resolution solution Resolution: 1.5 between
Column efficiency: 4000 theoretical plates moxifloxacin and impurity A, use
Tailing factor: 2.0 resolution solution
Relative standard deviation: 2.0%, use reference solution Relative standard deviation:
Signal-to-noise ratio: 10, use sensitivity solution 1.0%, use reference solution
Limits Related compound F rRT Limit (%) Any individual impurity: 0.20%
Specified unknown 1.0 0.3 0.2
impurity #1
Decarboxy 0.13 0.4 0.3
Specified unknown 1.0 0.9 0.3
impurity #2
Any specified and identified 1.0 1.0
impurity
Other single impurities 1.0 0.1
a
Impurity A 1.1
b
8-Hydroxy (impurity E)
Continued
Table 7.16 The summary of the compendial methods of moxifloxacin HCl ophthalmic solutioncont'd
Test USP-NF USP-MC
R F F
H H OH H H OH
N N
N N H2N N
N N
R' OCH3
H OCH3
Specified impurities: A, B, C, D, E. 7-Amino-1-cyclopropyl-6-fluoro-8-
1-Cyclopropyl-6-fluoro-8-
Impurity A: R R0 F methoxy-7-[(4aS,7aS)-
methoxy-4-oxo-1,4-dihydroquinoline-
Impurity B: R R0 OCH3 octahydro-pyrrolo[3,4-b]pyridin-
3-carboxylic acid (specified impurity #4)
Impurity C: R F, R0 OC2H5 6-yl]-1H-quinolin-4-one
Impurity D: R OCH3, R0 F (decarboxy)
Impurity E (8-hydroxy): R F, R0 OH
Continued
Table 7.16 The summary of the compendial methods of moxifloxacin HCl ophthalmic solutioncont'd
Test USP-NF USP-MC
Impurity C (8-ethoxy):
R F, R0 OC2H5
Impurity D (6-
methoxy-8-fluoro):
R OCH3, R0 F
Impurity E (8-hydroxy):
R F, R0 OH
Elemental Proceed as directed in the chapter [<232 >]
impurities
Residual Proceed as directed in the chapter [< 467 >]
solvents
Assay [< 621 >]
Mobile phase MeOH: A solution A: 0.1% DEA in H2O, pH of 2.3 with TFA
(v/v) (40:60).
Resolution 1 mg/ml each of moxifloxacin HCl and impurity A in MeOH
solution
Reference 0.1 mg/ml moxifloxacin HCl in MeOH
solution
Test solution 0.1 mg/ml moxifloxacin in MeOH, prepare from test solution
obtained from organic impurities test
Column Packing L11 (5 mm, 150 4.6 mm)
Temperature Column oven: 40 C, autosampler: 5 C
( C)
flow rate 1.0
(ml/min)
Detection UV: 293 [to confirm the absence of co-eluting known and
(nm) unknown impurity peaks, substitute the detector from the test for
Organic Impurities].
Continued
370 Mahmoud M.H. Al Omari et al.
Impurity C (8-ethoxy):
R F, R0 OC2H5
Impurity D (6-
methoxy-8-fluoro):
R OCH3, R0 F
Impurity E (8-hydroxy):
R F, R0 OH
Elemental Proceed as directed in the chapter [<232 >]
impurities
Residual Proceed as directed in the chapter [< 467 >]
solvents
Assay [< 621 >]
Mobile phase MeOH: A solution A: 0.1% DEA in H2O, pH of 2.3 with TFA
(v/v) (40:60).
Resolution 1 mg/ml each of moxifloxacin HCl and impurity A in MeOH
solution
Reference 0.1 mg/ml moxifloxacin HCl in MeOH
solution
Test solution 0.1 mg/ml moxifloxacin in MeOH, prepare from test solution
obtained from organic impurities test.
Column Packing L11 (5 mm, 150 4.6 mm)
Continued
372 Mahmoud M.H. Al Omari et al.
Impurity C (8-
ethoxy): R F,
R0 OC2H5
Impurity D (6-
methoxy-8-fluoro):
R OCH3, R0 F
Impurity E (8-
hydroxy): R F,
R0 OH
Assay (protect solutions from light) [<621 >]
Diluent 0.5 g/l of TBAHSO4 and 1.0 g/l KH2PO4 in 0.3 M H3PO4
Continued
374 Mahmoud M.H. Al Omari et al.
4.3.2 Polarography
Differential pulse polarographic (DPP) method for moxifloxacin HCl determi-
nation was developed and validated [66]. This method was applied for the
determination of trace amounts of moxifloxacin in pharmaceutics, serum,
and urine. Moxifloxacin showed a well-defined irreversible cathodic peak at
Moxifloxacin Hydrochloride 377
Table 7.20 The UV/visible parameters of the methods used for the determination of
moxifloxacin
Material Mode l (nm) Solvent Reference
Tablets and bulk First derivative 280.4 0.3 N HCl [69]
material in the 303.4
presence of its acid
degrades
(decarboxylated)
Human plasma Second Peak to peak Citrate-PO4 [70]
derivative amplitude in buffer
Peak to peak a l range (pH 7.2) and
amplitude in a 335345 SDS
wavelength (12.0 mN)
range 335345
Bulk material, tablets, Zero order 296 0.1 N HCl [71]
i.v. infusions, eye 289 PO4 buffer
drops, and polymeric (pH 7.4)
nanoparticles
Tablets Zero order 296 0.1 N HCl [72]
Bulk material and Zero order 295 0.1 N HCl [73]
tablets
Tablets Zero order 295 H2O [74]
Bulk material and Zero order 294.4 0.01 N HCl [75]
tablets
Bulk material and AUC zero 279.0296.4 H2O [76]
ophthalmic solution order 289.4305.6
AUC first
derivative
Bulk material, tablets, Zero order 293 H2O [77]
and eye drops First derivative 282 (max.)
First derivative 302 (min.)
Tablets (combination Zero order 286 and 295 0.1 N HCl [78]
with cefixime) (simultaneous 279 and 295
equation)
Zero order
(ratio-Q
analysis)
Eye drops Zero order 265 and 294 Methanolic [79]
(combination with (simultaneous 276.66 and 0.1 N HCl
bromefenac Na) equation) 301.71
Moxifloxacin Hydrochloride 379
Table 7.20 The UV/visible parameters of the methods used for the determination of
moxifloxacincont'd
Material Mode l (nm) Solvent Reference
Zero order 293.22 and
(ratio-Q 301.71
analysis)
First derivative
Eye drops Zero order 268 and 289 H2O [80]
(combination with (simultaneous
bromefenac Na) equation)
Pharmaceutical First derivative 283 (max.) H3PO4 [81]
formulations First derivative 304 (min.)
Ophthalmic solution First derivative 243.30 and H2O [82]
(combination with Second 261.90
dexamethasone Na derivative 266 and 241
PO4)
Eye drops First derivative 247 and 288 H2O/MeOH [83]
(combination with (ratio-Q
prednisolone acetate) analysis)
Tablets (combination Zero order 287.0 and H2O [84]
with cefixime) First derivative 317.9
(ratio-Q 269.6 and
analysis) 359.3
Tablets (combination Zero order 275 and 295 0.1 N HCl [85]
with cefixime) (ratio-Q
analysis)
AUC, area under curve; SDS, sodium dodecyl sulfate.
4.4.2 Colorimetry
Three different methods (AC) were used for the determination of
moxifloxacin in tablets based upon the formation of its complexes with alkaloidal
380 Mahmoud M.H. Al Omari et al.
4.4.3 Fluorimetry
Determination of moxifloxacin by fluorimetric technique was reported by
different workers (Table 7.21). The methods were based on the use of fluo-
rescence enhancer (e.g., sodium dodecyl sulfate), Eu (III) with sodium
dodecyl benzene sulfonate as enhancer or derivative formation with
4-chloro-7-nitrobenzofurazan. The extent of the linear range and detection
Moxifloxacin Hydrochloride 381
Table 7.21 The fluorimetric parameters of the method used for the determination of
moxifloxacin
Fluorescence lexc lem Range DL
Material agent Solvent (nm) (nm) (ng/ml) (ng/ml) Reference
Tablets None PO4 287 465 30300 10 [93]
buffer
(pH 8.3)
Human SDS AC 294 503 30300 15
serum and enhancer buffer
urine (pH 4.0)
Tablets NBD-Cl EAC 464 537 33.51000 10 [94]
Tablets, i.v. SDS H2 O 294 494 1320 0.5 [95]
infusions, enhancer
eye drops,
human
urine, and
plasma
Tablets, Eu (III) with Tri 373 614 0.027.3 0.003 [96]
human SDBS HCl
serum and buffer
urine (pH 9.2)
DL, detection limit; SDS, sodium dodecyl sulfate; NBD-Cl, 4-chloro-7-nitrobenzofurazan; SDBS,
sodium dodecyl benzene sulfonate; AC, acetate; EAC, ethylacetate.
limit depends upon the fluorescence probe formed, pH of solution, and sol-
vent type. The use of Eu (III) with sodium dodecyl benzene sulfonate as
fluorescence enhancer gave the highest sensitivity in comparison with the
other methods (Table 7.21).
It was found that the presence of Y (III) enhances intensity of fluores-
cence of moxifloxacin [97]. As a result, a novel method for the determina-
tion of moxifloxacin in urine was developed. The linearity range and
detection limit were reported to lie within 4.0400 and 0.34 ng/ml,
respectively.
In a BrittonRobinson buffer medium of pH 6.50, an indirect method
for the determination of moxifloxacin in tablets by the fluorescence
quenching of rhodamine B-acridine system in the presence of sodium dode-
cyl sulfate was proposed [98]. The method was found to be linear in the
range of 1.0010.0 mg/ml with a detection limit of 0.039 mg/ml. Further-
more, it was found that the effective energy transfer could occur between
congo red and calcein in the BrittonRobinson buffer (pH 6), which
quenched the fluorescence of calcein. The fluorescence of calcein
382 Mahmoud M.H. Al Omari et al.
4.4.4 Chemiluminometry
Determination of moxifloxacin in tablets by a flow injection method includ-
ing chemiluminescence detection was reported [100]. The proposed
method is based on the luminescent properties of the system Ce (IV)
sulphitemoxifloxacin and the addition of Eu (III), a trivalent lanthanide
ion, as emission sensitizer. The effect of acidity of Eu (III) and Ce (IV) solu-
tions, concentration of Ce (IV) and sulfate, and the flow rate on the chemi-
luminescence emission were considered to attain the optimal experiment
variables. The linearity range and detection limit are 0.22.0 and
0.035 mg/ml, respectively.
A batch type chemiluminescence method was used to determine
moxifloxacin [101]. The method is based on the enhancement of chemilu-
minescence emission of tris(2,2-bipyridyl) Ru (II)Ce(IV) system. Under
the optimum experimental conditions, the linear range and detection limit
are 0.440.0 and 0.12 mg/ml, respectively. The same tris(2,2-bipyridyl)
ruthenium(II)Ce(IV) system was studied by flow injection method
[102]. The concentration of moxifloxacin was held in the range of
0.0440.0 mg/ml with a detection limit of 0.012 mg/ml. The influence of
potential interfering substances was also studied. The proposed method
was successfully applied for the determination of moxifloxacin in pharma-
ceutical preparations.
Table 7.22 The HPTLC parameters of the method used for the determination of
moxifloxacin
Linearity DL
l range (mg/
Material Mobile phase (v/v) (nm) (mg/spot) spot) Reference
Tablets and bulk 0.3 M 290 0.11.4 [69]
material in the presence NH4AC:25%
of its acid degrades NH3:n-PrOH
(decarboxylated) (1:1:8)
Tablets n-BuOH:MeOH: 295 0.4001.0 0.01 [103]
NH3 (4:4:2)
Polymeric n-PrOH: 298 0.10.8 0.004 [104]
nanoparticles and bulk EtOH:6 M NH3
material in the presence (4:1:2)
of its degrades
Tablets MC:MeOH: 25% 292 0.0090.054 [105]
NH3:ACN
(10:10:5:10)
Tablets and bulk MC:EtOH: 294 0.061.5 0.012 [106]
material in the presence toluene:n-BuOH:
of its acid degrades 25% NH3:H2O
(6:6:2:3:1.8:0.3)
Eye drops with MC:ACN: 260 0.64.2 [107]
bromfenac sodium MeOH:NH3
(2.5:2.5:2.0:1.0)
Stationary phase used: Precoated silica gel G60F254. DL, detection limit; AC, acetate; MC, methylene
chloride; ACN, acetonitrile.
384 Mahmoud M.H. Al Omari et al.
5. STABILITY
5.1. Solid-state stability
Forced degradation of moxifloxacin HCl in solid state was investigated by
using densitometric TLC method [104]. It was found that heating
moxifloxacin HCl at 100 C for 8 h leads to a decrease in percentage recov-
ery to 91% with the detection of three degrades.
Stability of moxifloxacin HCl in its tablet dosage form after incubation at
40 C/75% RH and 50 C/75% RH for 6 months was investigated using
HPLC/UV detection [143]. The stability data for two products (Avelox
and Staxin) proved that the drug is stable without any significant decrease
in percentage assay after 6 months of incubation (percentage assay range:
99.8100.0% at 40 C/75% RH).
Forced degradation of moxifloxacin HCl in the solid state was monitored
by HPLC/UV detection [146]. It was found that the drug is stable when
exposed to daylight and to thermal stress at 60 C for 30 days.
Following the photodegradation by UPLC/MS detection, moxifloxacin
HCl in its solid state was found to be not stable when exposed to a UV lamp
(365 nm) in a watch glass for 48 h as one photodegrade was detected [165].
The effect of heat, humidity, and light on the racemization of
moxifloxacin HCl in its solid state was studied by capillary electrophore-
sis [34]. Stress degradation studies were carried out at 40 C/75% RH,
60 C for 12 weeks, and in a stability light chamber (provides ICH-required
illumination levels with 4-week exposure) at 25 C/40% RH. Results rev-
ealed that racemization was not a significant degradation pathway of the drug
under the investigated conditions.
The effect of heat, humidity, and light on the stability of moxifloxacin
HCl in its pure solid state was investigated by HPLC/UV detection
Moxifloxacin Hydrochloride 399
Table 7.26 The stability data for different tablet formulations of moxifloxacin HCl at
40 C/75% RH in close container
Assay Organic impurities
95.0105.0% Any individual 0.2%
Month Month
Formula 0 1 2 3 0 1 2 3
F-1 101.9 101.8 101.7 0.03 0.02 0.02
F-2 102.9 103.7 103.5 0.03 0.02 0.02
F-3 102.2 102.9 103.4 0.03 0.02 0.02
F-4 102.9 99.3 100.1 101.3 0.02 0.02 0.04 0.04
Figure 7.22 Comparative dissolution profiles for different formulations with Avelox
tablets (Bayer product).
Figure 7.23 XRPD of moxifloxacin HCl (A) Form II, (B) Form W, and (C) and (D) Form
W incubated at 40 C/75% RH for 1 and 2 months, respectively, in open container. Form
II (Bayer hydrate form prepared by recrystallization from water/ethanol system).
Moxifloxacin Hydrochloride 403
Figure 7.24 XRPD of (A) moxifloxacin HCl Form II (Bayer form), (B) Avelox tablets, and
(C) formula 4 (F-4) after grinding. Form II (Bayer hydrate form prepared by recrystalliza-
tion from water/ethanol system).
404 Mahmoud M.H. Al Omari et al.
Table 7.28 The stability data for different tablet formulations of moxifloxacin HCl at
40 C/75% RH packed in different packaging materials
Assay Organic impurities
95.0105.0% Any individual 0.2%
Month Month
Product 0 3 6 0 3 6
Avelox (Al/Al) 103.0 98.1 0.02 0.04
Avelox (PP/Al) 101.0 101.3 0.00 0.00
F-5 (Al/Al) 98.8 98.7 99.0 0.00 0.01 0.01
F-5 (PVDC/Al) 98.8 100.8 101.2 0.00 0.00 0.02
F, formula; PP, polypropylene, PVDC, polyvinylidene chloride.
Figure 7.25 pH stability profile of moxifloxacin HCl at 50 and 80 C for 1 and 3 days,
respectively.
110 C, the decomposition was found to be faster and followed the order at
48 h: Cu (II) (79.35%) > Fe (III) (63.97%) > Al (III) (62.42%) > Zn (II)
(58.62%) > Drug alone (52.04%). Moxifloxacin HCl was found to undergo
thermal degradation through the loss of the functional carboxyl (decarbox-
ylation) and hydrolysis of 2,8-diazabicyclo[4.3.0]non-8-yl groups
(Figure 7.26).
Stability of moxifloxacin HCl in solution was investigated by HPLC/
fluorescence detection [108]. It was found that the drug in organic solvent
stored at room temperature and at 37 C for 22 days is stable when protected
from light (brown glass or in dark). The obtained percentage recoveries were
97.1% and 99.3%, respectively. For the case of exposure to daylight in a
white glass at room temperature, the drug showed a significant decrease
in percentage recovery to 39.8%.
Photodegradation of moxifloxacin HCl in water by sunlight was mon-
itored by HPLC/MS detection as a function of time [121]. Samples were
kept under environmental conditions (open air, protected from rain). After
136 h, the drug content decreased by at least 50% of the initial concentra-
tion. Moxifloxacin N-oxide, formed by photooxidation of the alicyclic ring,
was detected by MS analysis.
The degradation of moxifloxacin was studied by HPLC/UV detection
under different stress conditions (0.1 N HCl, water, and 0.1 N NaOH) at
Moxifloxacin Hydrochloride 407
O
F
H H
N
N N
H OCH3
Decarboxylation
O O
H H F
OH
N
N N
H OCH3
Moxifloxacin
Figure 7.26 Acid degradation pathways of moxifloxacin HCl at 110 C in the presence
of Cu (II) ions.
O O
F
OH
H2N N
OCH3
O O
F
H H OH
N
N N
H OCH 3
Moxifloxacin
O O O O
F F
H H OH H OH
O N NH
HO
N N N N
borate buffer (pH 9.0) and in water (unbuffered) sparged with O2 for 20 min
and stored at 50 C for 12 weeks. The unbuffered sample in water was stored
in a stability light chamber at 25 C/40% RH for 12 weeks. Results revealed
that racemization was not a significant degradation pathway of the drug sub-
stance in solution.
Moreover, moxifloxacin HCl in aqueous solution showed instability
when exposed to 245 nm UV lamp [173]. The extent of photodegradation
(30%, 54%, or 83%) was achieved using various flow rate and retention coil
lengths of the photochemical reaction unit and monitoring by HPLC/fluo-
rescence detection.
Forced degradation of moxifloxacin in 0.5 N HCl and 0.5 N NaOH at
70 C for 5 days and in 3% H2O2 at ambient temperature for 3 days was
studied by HPLC/UV detection [176]. Significant degradation was
observed by oxidative stress (83.4%) and by basic (94.6%) conditions; no
degradation was observed under acidic (99.2%) condition.
412 Mahmoud M.H. Al Omari et al.
6. PHARMACOLOGY
6.1. Uses, applications, and pertinent history
6.1.1 Systemic use
Moxifloxacin HCl is a fluoroquinolone antibacterial indicated for treating
infections in adults caused by designated, susceptible bacteria. It was initially
approved in a tablet form by the EMA in June 1999 [185] and by the
USFDA in December 1999 [186].
Moxifloxacin HCl tablets and intravenous are indicated in patients aged
18 years and older for the treatment of the following bacterial infections if
they are caused by bacteria susceptible to moxifloxacin:
Acute bacterial sinusitis
Acute exacerbations of chronic bronchitis.
Community-acquired pneumonia [187,188].
Uncomplicated and complicated skin and skin structure infections.
Complicated intraabdominal infections, including polymicrobial infec-
tions such as abscess. [188].
The intravenous administration is recommended when it offers a route of
administration advantageous to the patient (e.g., severe infection or the
patient cannot tolerate the oral dosage form) [3,188].
Moxifloxacin HCl tablets are also indicated for the treatment of mild to
moderate pelvic inflammatory disease (i.e., infections of the female upper
416 Mahmoud M.H. Al Omari et al.
6.2. Absorption
6.2.1 Oral
Moxifloxacin is readily absorbed from the gastrointestinal tract after oral
administration. It is not subject to significant presystemic biotransformation
(first-pass effect). In consequence, the absolute bioavailability is almost
complete (
90%) [191].
Peak plasma concentrations (Cmax) and area under the plasma
concentrationtime curve (AUC) increased linearly with dose after admin-
istration of single oral moxifloxacin doses of 50800 mg. After the
recommended dose of 40 0 mg, a mean Cmax of 2.5 mg/l was reached in
1.5 h (tmax) and AUC was 26.9 mg/l h [192].
Moxifloxacin has no clinically relevant interactions with food [191].
Concomitant intake of dairy products slightly delayed the rate, but not
the extent, of absorption of the drug [192] Changes in gastric pH by pre-
treatment with ranitidine had no influence on absorption. In common with
the fluoroquinolones, moxifloxacin is likely to form nonabsorbable com-
plexes in the presence of multivalent cations. Accordingly, its absorption
is impaired by the concomitant administration of Maalox and iron
supplements [191].
Moxifloxacin pharmacokinetics are linear and dose-proportional with
repeated oral doses of up to 600 mg once daily over 10 days. Plasma con-
centrations rose readily after oral doses, achieving and maintaining appropri-
ately high levels over 24 h. Stable steady-state conditions are reached within
23 days with no indication of clinically relevant accumulation [193].
Moxifloxacin Hydrochloride 417
6.2.2 Intravenous
Moxifloxacin pharmacokinetics are linear and dose-proportional in the
range of 100400 mg single dose [193]. Intravenous administration of
moxifloxacin 400 mg produced a Cmax of 3.62 mg/l and AUC of
34.6 mg h/l [192].
6.2.3 Topical
Following the administration of bilateral topical ocular doses of
moxifloxacin 0.5% ophthalmic solution 3 a day in healthy subjects, mean
steady-state Cmax (2.7 ng/ml) and estimated daily exposure AUC
(45 ng h/ml) values were 1600 and 1000 lower than the respective values
reported after therapeutic 400 mg doses of moxifloxacin [190].
Conjunctival tissue levels of moxifloxacin rose to peak levels within
1530 min after topical administration of moxifloxacin 0.5% ophthalmic
solution. Conjunctival tissues Cmax was 24.1 mg/g. The AUC03 was
27.1 (mg h)/g [194].
6.3. Distribution
Moxifloxacin is approximately 3050% bound to serum proteins, indepen-
dent of drug concentration. The volume of distribution ranges from 1.7 to
2.7 l/kg. It is widely distributed throughout the body, with tissue concen-
trations often exceeding plasma concentrations. The rates of elimination of
moxifloxacin from tissues generally parallel the elimination from plasma
[188]. Moxifloxacin reaches higher concentrations in saliva, capillary blood
[195], skin blister fluid, epithelial lining fluid, bronchial biopsies, and max-
illary sinus mucosa than in plasma. Similar results were noted in anterior
ethmoid mucosa and nasal polyp tissue [192]. Free drug concentrations
in subcutaneous tissue and skeletal muscle are comparable to those of
the unbound drug in plasma. Very high concentrations are achieved in
the alveolar macrophages and bronchial mucosa [195]. Moxifloxacin was
also detected in nasal and bronchial secretions and abdominal tissues and
fluids following oral or intravenous administration of 400 mg [188]. The
418 Mahmoud M.H. Al Omari et al.
high volume of distribution, low protein binding, and rapid early distribu-
tion phase into target tissues are indicative of optimal distribution proper-
ties allowing for effective treatment of the indicated infections [195]. In
animal studies, orally administered moxifloxacin penetrated across the pla-
cental barrier and into breast milk in rats, and achieved good penetration
into CSF in rabbits, particularly in those with experimental
meningitis [192].
6.4. Metabolism
Moxifloxacin does not appear to be metabolized by the cytochrome P-450
pathway. It is metabolized to inactive metabolites via conjugation to a sulfo-
(M1) and a glucuronide (M2) derivative in principal (Figure 7.29)
[55,192,196,197].
6.5. Elimination
Up to 96% and 98%, respectively, of the oral and intravenous dose of
moxifloxacin was recovered as either parent compound (
45%), metabolite
M1 (
38%), or metabolite M2 (
14%). About 20% of the administered
dose is recovered unchanged in urine. Urinary excretion is independent
of the dose and route of administration. Renal clearance is lower than
O O
H F
H OH
N
N N
H OCH3
Moxifloxacin base
Urine: 1922%
Faeces: 26%
HO
O O O O HO OH
HO3S H O
F F COOH
H OH H O
N N
N N N N
H OCH3 H OCH3
Sulfo-compound Acyl-glucuronide
Urine: 2.5% Urine: 14%
Faeces: 35% Faeces: 0%
Figure 7.29 The metabolic pattern of the main metabolites of moxifloxacin with their
recovery in humans.
Moxifloxacin Hydrochloride 419
REFERENCES
[1] European Pharmacopeia, seventh ed., Council of Europe, Strasbourg, Moxifloxacin
Hydrochloride Monograph, 2011, pp. 25312532.
[2] United States Pharmacopeia 35/National Formulary 30 (USP 35/NF 29), Moxifloxacin
Hydrochloride Monograph, vol. 3, USP Convention. INC, Maryland, 2012,
pp. 39593960.
[3] Avelox Product Monograph, Bayer Site, http://www.bayer.ca/files/AVELOX-PM-
ENG-20JAN2012-150618.pdf, accessed date March 2013.
[4] Drug Bank Site, Moxifloxacin Monograph, http://www.drugbank.ca/drugs/
DB00218, accessed date March 2013.
[5] Food and Drugs Administration (FDA) Site, Vigamox: Chemistry Review, http://
www.accessdata.fda.gov/drugsatfda_docs/nda/2003/21-598_Vigamox_chemr.PDF,
accessed date March 2013.
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CHAPTER EIGHT
Pravastatin Sodium
Abdullah A. Al-Badr, Gamal A.E. Mostafa
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457,
Riyadh, Kingdom of Saudi Arabia
Contents
1. Description 434
1.1 Nomenclature 434
1.2 Formulae 435
1.3 Elemental analysis 435
1.4 Solubility 435
1.5 Appearance 436
1.6 Partition coefficient 436
2. Uses and Applications 436
3. Methods of Preparation 436
4. Physical Characteristics 447
4.1 X-ray powder diffraction 447
4.2 Thermal methods of analysis 448
4.3 Ultraviolet spectroscopy 448
4.4 Vibrational spectroscopy 448
4.5 Nuclear magnetic resonance spectrometry 452
4.6 Mass spectrometry 454
5. Methods of Analysis 456
5.1 Compendial methods 456
5.2 Spectrophotometric methods 474
5.3 Electrochemical methods 476
5.4 Chromatographic methods 477
5.5 Immunoassay methods 497
6. Pharmacokinetics 499
7. Metabolism 501
8. Stability 505
9. Reviews 506
Acknowledgments 506
References 506
Profiles of Drug Substances, Excipients, and Related Methodology, Volume 39 # 2014 Elsevier Inc. 433
ISSN 1871-5125 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800173-8.00008-8
434 Abdullah A. Al-Badr and Gamal A.E. Mostafa
1. DESCRIPTION
1.1. Nomenclature
1.1.1 Systematic chemical names
3b-Hydroxycompactin sodium.
bR,dR,1S,2S,6S,8S,8aR)-1,2,6,7,8,8a-Hexahydro-b,d,6-trihydroxy-2-
methyl-8-[(2S)-2-methyl-1-oxobutoxy]-1-naphthaleneheptanoic acid.
Sodium (+)-(3R,5R)-3,5-dihydroxy-7-[(1S,2S,6S,8S,8aR)-6-hydroxy-
2-methyl-8-[(S)-2-methylbutyryloxy]-1,2,6,7,8,8a-hexahydro-1-
naphthyl]heptanoate.
Sodium(+)(3R,5R)-3,5-dihydroxy-7-{(1S,2S,6S,8S,8aR)-6-hydroxy-
2-methyl-8-[(S)-2-methybutyryloxy]-1,2,6,7,8,8a-hexahydro-1-
naphthyl}heptanoate.
1-Naphthalene heptanoic acid, 1,2,6,7,8,8a-hexahydro-b,d,6-trihydroxy-
2-methyl-8-(2-methyl-1-oxobutoxy)-, monosodium salt, [1S-[1a(bS*,-
dS*)-2a,6a,8b(R*),8aa]].
[1S-[1a(bS*,dS*),2a,6a,8b-(R*),8aa]]-1,2,6,7,8,8a-Hexahydro-b,d,6-
trihydroxy-2-methyl-8-(2-methyl,1-oxobutoxy)-1-naphthalene heptanoic
acid monosodium salt.
(3R,5R)-7-[(1S,2S,6S,8S,8aR)]-1,2,6,7,8,8a-hexahydro-6-hydroxy-2-
methyl-8-[(S)-2-methylbutyryloxy-1-naphthyl]-3,5-
dihydroxyheptanoate.
Sodium (+)-(3R,5R)-3,5-dihydroxy-7-{(10 S,20 S,60 S,80 S,80 aR)-60 -
hydroxy-20 -methyl-80 -[(S)-2-methylbutyryloxy]-10 ,20 -60 -70 -80 ,80 a-
hexahydro-10 -naphthyl}heptanoate.
Sodium (+)(bR,dR,1S,2S,6S,8S,8aR)-1,2,6,7,8,8a-hexahydro-b,d,6,8-
tetrahydroxy-2-methyl-1-naphthalene heptanoate, 8[(2S)-2-
methylbutyrate].
1,2,6,7,8,8a-Hexahydro-6-hydroxy-2-methyl-8-((2-methylbutyryloxy)-
1-naphthyl)-3,5-dihydroxynaphthoate [13].
1.2. Formulae
1.2.1 Empirical formula, molecular weight, CAS-number [1]
Pravastatin
C23H36O7 425.5 [81093-37-0]
Pravastatin sodium
C23H35O7 Na 446.52 [81131-70-6]
O O
C OH C ONa
OH OH
HO HO
O O
O O
H H
CH3 CH3
HO HO
1.4. Solubility
Pravastatin is freely soluble in water and methanol, relatively insoluble in
chloroform, ether, acetone and acetonitrile [13].
436 Abdullah A. Al-Badr and Gamal A.E. Mostafa
1.5. Appearance
Pravastatin sodium is a hygroscopic white to off-white crystalline powder.
Pravastatin lactone is colorless plate-like crystal [13].
3. METHODS OF PREPARATION
Terahara et al. [4,5] isolated pravastatin as a metabolite of compactin
through enzymatic hydroxylation by means of microorganisms.
Daniewski et al. [6] described the following total synthesis of pravastatin:
1-Methyl-4-methylenecyclohexane 1 reacted with methyl glyoxylate 2
to form an inseparable mixture of two diastereomers 3 and 4 in 68:32 ratio in
72% yield. Without separation, the mixture 3 and 4 was carried through
benzylation of the hydroxyl group (80%), hydrolysis and iodolactonization
to 5 and 6 (82% yield) followed by dehydroiodination with 1,4-diazabicyclo
[2,2,2]-octane (DABCO) in DMSO (99%) to give 7 and 8 as a chromato-
graphically separable mixture. The lactone 7 was reduced to the lactol 9
followed by Wittig methylenation to give 10 (75% yield for the two steps).
Heating compound 10 with the dimethyl acetal of N,N-dimethylacetamide
produced the amide 11 in 92% yield. Hydroboration and oxidation
provided the aldehyde 12. While the intramolecular ene reaction of 12 with
dimethyl aluminum chloride produced the desired ene product 13
Pravastatin Sodium 437
9
O CH3
O CH3 O CH3
C N
CH3 C N O C N
CH3 CH3 Cl
CH3 C H
CH3 CH3 Al
HO CH2 H3C CH3
OBn OBn OBn
11 11a 12
O CH3 O CH3 O
(CH3)3Si (CH3)3Si
C N C N C H
HO CH3 O CH3 O
H H H
CH3 CH3 CH3
silyation Li in
liq. NH3
OBn OBn OH
13 14 15
O
(CH3)3Si
CH OCH3 CH OCH3
CH OCH3 HO HC H3C O HC
O HC H
H H CH3
CH3 CH3
H3C C O
OH OH
O
16 17 18
O
O O H O
O
H3C CH2 C H O
O
H (CH3)3 Si H3C O
CH3 H
OCH3 CH3
20
H3C C O TiCl4
H3C C O
O
19 O
21
O OCH3 HO OCH3
H H H
O O
O O
H3C O H3C O
H H
CH3 CH3
reduction
H3C C O H3C C O
O 22 O 23
HO HO
H H
CH3 CH3
disilylation epoxidation
HO tBuPh2Si O
23a 24
tBuPh2Si O O tBuPh2Si O O
H H
O O
O O
O O O
H H
CH3 F3C S O Si(CH3)3 CH3
O dehydration
tBuPh2Si O tBuPh2Si O
O OH
27 28
O OH
tBuPh2Si O O HO
O
O O OH
C Bu
O O
H Bu N Bu F H
Burgess CH3 CH3
Bu
reagent
tBuPh2Si O HO
29 30
Pravastatin
Li N O
O O CH2
14
CO2 (COCl)2
CH3 CH2 CH2 MgBr CH3 CH2 CH2 C O Na CH3 CH2 CH2 C Cl +
14 14
1 2 3 4
O O O O
C CH2 C
CH3 CH2 CH2 14
N O CH3 CH 14
N O
CH3
CH2 CH2 1. LiOH/H2O2 O
THF (1) NaHMDS 2. HCl C
14
O Na
LiCl (2) CH3I 3. NaOH
CH3
5 6 7
O OH 14 O
H H
(COCl)2 CH3 DMAP CH3 CH3 TBAF
14 Cl +
CH2 Cl 2 Acetic acid
CH3
8 9 10
O O
HO O HO
NH 4
O OH
O O
CH3 14 O CH 3 14 O
H H
CH3 CH3 Mucor CH3 CH3
hiemalis
HO
11 12
14
[ C]-Pravastatin
O
C ONa O
HO HO
C ONa
H H H
H
O HO O HO
O O
H H
CH 3 CH 3
Cytochrome + Isomer
P450 HO
stirred for 60 min, and the product is filtered, washed, and dried to afford
about 2.8 g of pravastatin sodium salt as a dry powder.
Lee et al. [11] prepared pravastatin from new saccharothrix isolates by
fermentation of saccharothrix (1848 h) under the presence of compactin
(12 g). The process of production of pravastatin using saccharothrix isolates
is provided.
Abe [12] provided a DNA sequence of a gene cluster from Penicillium
citrinum. The gene cluster can be used for the biosynthesis of pravastatin pre-
cursor, compactin.
Kumar et al. [13] prepared pure pravastatin by microbial hydroxylation of
compactin. The microorganism is species of the genus Streptomyces, such as
Streptomyces carbophilus. The conditions capable of converting compactin to
pravastatin include the fermentation production medium containing glucose
at a concentration level of about 1525 g/l, Soya bean meal, cotton seed
meal, corn steep liquor, sodium chloride, and calcium carbonate at a certain
concentration levels. The conditions also include the maintenance of tem-
perature at about 1850 C and pH from 5 to 10.
Jekkel et al. [14] used a new microbial process: Mortierella maculata for the
preparation of pravastatin sodium from compactin sodium.
Mei et al. [15] used Micropolyspora roseoalba for preparation of pravastatin.
M. roseoalba is useful for manufacturing pravastatin sodium at low cost.
The physiological and morphological characteristics of M. roseoalba
were given.
Keri and Melczer [16] described a method for synthesizing and purifying
pravastatin. This work encompasses methods of synthesizing pravastation
comprising less than about 0.1% by weight pravastation C comprising
(a) purification of compactin containing compactin C until the amount of
compactin C is less than about 0.16% by weight, and (b) synthesizing prav-
astatin using compactin from (a). The work also encompasses compactin
prepared according to the crystallization process and the pharmaceutical for-
mulations comprising thereof.
Klaassen et al. [17] used Recombinant Escherichia coli cells and compactin
hydrolases for preparation of the pharmaceutically active b-variant of
pravastatin. Amycolatopsis orientalis is capable of converting compactin into
pravastatin with 100% efficiency. The gene (cmpH) encoding the cyto-
chrome P450 enzyme capable of hydroxylating compactin to pravastatin
was isolated from A. orientalis gene library and cloned into E. coli.
A number of synthetic genes encoding derivatives of compactin hydroxylase
were tested in E. coli and shown to improve the compactin into pravastatin
Pravastatin Sodium 443
mixture of hydroxy ester and lactone 13. Lactone 13 was heated at 210 C
with 1-methylnaphthalene to introduce the double bond 14. The resulting
olefinic lactone 14 was reduced with lithium aluminum hydride to give the
diol 15. The primary alcohol of the diol 15 was protected as the pivalate ester
to afford 16 and the secondary alcohol as the triisopropyl silane (TIPS) ether
to produce the silyl ether 17. Removal of the pivaloyl group with lithium
aluminum hydride gives 18 and treatment of 18 with Swern oxidation gave
the aldehyde 19.
Treatment of the aldehyde 19 with potassium carbonate in methanol pro-
vided an equilibrium mixture of isomeric aldehyde 20 along with the recov-
ered aldehyde 19. These two aldehydes 19 and 20 were separated by medium
pressure liquid chromatography enabling easy recycling. Elongation of the
two carbon atom unit from the aldehyde 20 was performed by Horner
Emmons reaction leading to the ab-unsaturated ester 21. The double bond
of 21 was reduced with magnesium in methanol to give the methyl ester 22
which was reduced with lithium aluminum hydride to the alcohol 23.
Deprotection of tert-butyldimethylsilyl group of the alcohol 23 gives the diol
24 quantitatively. The diene moiety was introduced by bromination
dehydrobromination sequence. The diol 24 was treated with a solution of
bromine in chloroform to give the dibromide 25. After selective protection
of the primary alcohol of the dibromide 25, the silyl ether 26 was produced.
Treatment of 26 with diazabicycloundecene (DBU) furnished the diene 27.
Esterification of 27 with (S)-2-methyl butanoic anhydride provided the ester
28 which was deprotected to give hydroxy ester 29 quantitatively. Swern oxi-
dation of 29 proceeded to give the aldehyde 30. Aldol condensation of the
dianion of methyl acetoacetate proceeded to give the aldol product 31 which
were an inseparable mixture of epimers. The carbonyl group in 31 was
reduced by sodium borohydride in the presence of diethyl methoxy borane
to afford the diol 32 quantitatively. Treatment of the diol 32 with HF
pyridine complex in acetonitrile gave the desired product, compactin 33.
Enzymatic hydroxylation of (+)-compactin (ML-236B) afforded pravastatin
lactone 34 which is finally converted to pravastatin.
O
O OH OAc
(1)
O , Lipase Amano AK. K2CO3,
ClSO3H, (CH2OH)2 Vinyl acetate/hexane CH3OH
(2) NaBH4, CeCl3,
CH3OH O O O O
OEt
1 2 3
Pravastatin Sodium 445
OH OH TBDMSO
9 S S
10 11
O O
O CO2 Me O O
H H H
NaBH 4 , 1-Methylna-
15 15
CH 3 OH phthalene, LiAlH4
60 C 10
210 C 10
ether
H O S CH3 H O H
S CH3
12 S 13 S 14
O CH3
OH
OH OH CH 2 O C C CH3
H
Pivalayl CH3
15
chloride, TIPSOTf
10 pyridine (C2 H 5 ) 3 N, CH 2 Cl 2
H H
15 16
O CH3
TIPSO CH 2 O C C CH3 TIPSO CH 2 OH
H H
CH3
LiAlH 4 / (COCl) 2 , DMSO
ether (C 2 H 5 )3 N, CH2Cl2
H H
17 18
COO CH3
H O H O
TIPSO TIPSO TIPSO
H H H 3 CO O
H
8 K 2 CO3 , 8 P
CH 3 OH H 3 CO CH2 COOCH 3 Mg, CH3 OH,
NaH, HMPA, THF 0 C
H H H
19 20 21
446 Abdullah A. Al-Badr and Gamal A.E. Mostafa
OH OH
COO CH 3
TIPSO TIPSO HO
H H H
OH OTBDMS
HO HO
H H
TBDMSOTf,
2,6-dimethyl- DBU, HMPA,
Br
pyridine, CH 2 Cl 2 60 C
H Br Br
H Br
25 26
OTBDMS O OTBDMS
HO O
H H
(S)-methylbutanoic
anhydride, DMAP, TBAF,
CH2 Cl 2 THF
27 28
OH H O
O O BuLi, NaH,
O O OH OH
H H
OCH 3
(COCl) 2 , DMSO, THF, 0 C
(C2 H 5 )3 N, CH 2 Cl 2
29 30
COOH COOCH 3
O HO
HO HO
O O
O O
H H
(C2 H 5 ) 2 BOCH3 HFpyridine
NaBH 4 , CH3OH complex,
78 C CH 3 CN
31 32
Pravastatin Sodium 447
O OH O OH
O O
O O
O O
H
Enzymatic
hydroxylation HO
33 34
(+)-Compactin Pravastatin
ML-236B lactone
COOH
HO
HO
O
O
H
HO
Pravastatin
4. PHYSICAL CHARACTERISTICS
4.1. X-ray powder diffraction
X-ray powder diffraction (XRD) pattern of pravastatin sodium was per-
formed using a Simmon XRD-5000 diffractometer (Figure 8.1).
Table 8.1 shows the values for the scattering angles ( 2y), the interplanar
d-spacing (A), and the relative intensities (%) observed for the major diffrac-
tion peaks of the pure sample of pravastatin sodium drug substance.
Keri et al. [22] prepared a new crystalline form of pravastatin sodium and
studied the X-ray powder diffraction data, the differential scanning calorim-
etry and thermogravimetric analysis, and other experiments for the
drug form.
Table 8.2 Vibrational assignments for pravastatin sodium infrared absorption bands
Frequency (cm1) Assignments
3352 OdH stretch
2965, 2881 CH3 and CH2 stretch
1725 C]O stretch
1578 C]C stretch
1400, 1329 CH2 bending
1187 CH bending
1015 CdO stretch
865 CH bending
Clarke [3] reported the following: Pravastatin sodium salt (KBr disk).
Principal peaks at wavenumbers 1727, 1579, and 1187 cm1.
15
1.00
5.921
6.0
0.99 5.910
5.892
14
1.00 5.534
5.5
13
1.02 5.390
12
5.0
4.930
11
6.021
6.002
5.921
10
5.910
5.892
4.5
4.324 5.534
0.98
9
5.390
4.930
4.094 4.324
0.97
8
4.094
4.0
3.718
3.718 3.711
3.711 3.703
7
0.98 3.703 2.520
2.520 2.506
2.506 2.494
3.5
1.00
6
2.494 0.99 2.443
0.25 2.443 1.00 2.421
2.421 1.02 2.388
5
2.388 2.379
2.379 2.374
3.0
2.374 0.98 2.358
2.358 0.97 4 2.349
2.349 0.98 2.292
2.292 0.25 2.276
2.276 2.262
3
4.92 2.262
2.5
1.712
1.712 4.92 1.704
1.704 1.00 1.689
2
2.0
1
5.94
0
1.616 1.611
1.611 1.591
1.5
2.96 1.591 1.580
1.580 1.571
-1
1.483 1.481
1.0
1.454 1.423
1.423 1.407
1.407 1.393
ppm
ppm
454 Abdullah A. Al-Badr and Gamal A.E. Mostafa
2 .520
2 .506
2 .494
2 .443
2 .421
2 .388
2 .379
2 .374
2 .358
2 .349
2 .292
2 .276
2 .262
1.712
1.704
1.689
1.684
1.669
1.654
1.641
1.626
1.616
1.611
1.591
1.580
1.571
1.562
1.553
1.483
1.481
1.469
1.454
1.423
1.407
1.393
1.257
1.250
1.148
1.134
0.947
0.933
0.918
2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 ppm
4.92
1.00
5.94
2.96
1.05
2.97
5.95
Bacher et al. [23] reported the complete assignments of the 1H and 13C
NMR data of pravastatin derivatives.
HO 14
O 13
3
2 C 12
4 1 O 11
H
5 9 CH 3
2 1 10 8
3
5 7
HO 4 6
Assignment hydrogen
Chemical shift d (ppm) Multiplicity Number of protons at carbon number
0.920.95 m 6H 40 , 18 2 CH3
1.14 d J7 3H 50 CH3
1.251.26 m 1H 8
1.391.42 m 2H 11
1.451.48 m 1H 9
1.551.75 m 6H 12, 14, 17
2.262.29 m 1H 10
2.352.44 m 4H 2, 30
2.492.52 m 1H 20
3.703.72 m 1H 13
4.09 m 1H 15
4.32 s 1H 3
5.39 s 1H 1
5.53 s 1H 4
5.895.92 m 1H 7
6.01 d J 9.9 1H 6
5. METHODS OF ANALYSIS
5.1. Compendial methods
5.1.1 British pharmacopoeia methods [26]
Identification
A. Specific optical rotation, this test should be carried out as described in
the general procedure (2.2.7) (see Tests).
Pravastatin Sodium 457
180.536
177.987
136.793
136.511
128.637
127.407
71.653
71.041
69.285
65.386
49.567
49.397
49.227
49.056
48.886
48.716
48.545
45.520
45.178
43.004
38.808
38.337
37.065
35.629
32.288
27.877
25.353
17.40.
13.94.
12.27.
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
Liquid Carry out this test according to the general procedure (2.2.29).
chromatography
Solvent mixture Mix 9 volumes of methanol R with 11 volumes of water R.
Test solution (a) Dissolve 0.1 g of pravastatin sodium in the solvent mixture and
dilute to 100 ml with the solvent mixture.
Test solution (b) Dilute 10 ml of test solution (a) to 100 ml with the solvent
mixture.
Reference solution Dissolve 5 mg of pravastatin sodium and 5 mg of pravastatin
(a) impurity A CRS in the solvent mixture and dilute to 50 ml with
the solvent mixture.
Reference solution Dilute 2 ml of test solution (a) to 100 ml with the solvent
(b) mixture. Dilute 1.010 ml of the solvent mixture.
Reference solution Dissolve 12.4 mg of pravastatin 1,1,3,3-tetramethylbutylamine
(c) CRS in the solvent mixture and dilute to 100 ml with the
solvent mixture.
Column:
size: l 0.15 m, 4.6 mm
stationary Octadecylsilyl silica gel for chromatography R (5 mm)
phase:
temperature: 25 C
Mobile phase: Glacial acetic acid R, triethylamine R, methanol R, water R
(1:1:450:550 V/V/V/V).
Flow rate: 1.3 ml/min.
Detection: Spectrophotometer at 238 nm.
Injection: 10 ml; inject test solution (a) and reference solutions (a) and (b).
Run time: 2.5 times the retention time of pravastatin.
Relative retention with reference to pravastatin (retention time about 21 min):
impurity B about 0.2; impurity A about 0.6; impurity C about 2.1.
Pravastatin Sodium 459
Assay
Liquid chromatography Carry out this test according to the general
procedure (2.2.29) as described in the test for related substances.
Injection: Test solution (b) and reference solution (c).
Calculate the percentage content of C23H35Na07 using the chromato-
gram obtained with reference solution (c) and the declared content of
pravastatin in pravastatin 1,1,3,3-tetramethylbutylamine CRS.
460 Abdullah A. Al-Badr and Gamal A.E. Mostafa
136.80
128.64
127.40
72.01
71.63
71.04
69.26
67.08
65.39
49.35
49.17
48.88
45.53
45.18
43.00
38.80
38.33
37.06
35.62
32.30
27.88
25.35
12.28
A COOH
HO
H
HO
O
H
H3C O
H H H H
CH3 CH3
H
HO
H
6-Epipravastatin
Pravastatin Sodium 461
136.80
136.76
128.64
127.40
72.02
71.66
71.63
71.04
69.29
69.25
65.39
65.33
49.35
49.17
48.88
45.53
45.51
45.18
43.02
42.99
38.80
38.35
38.32
37.06
35.65
35.61
32.30
32.27
27.88
25.39
25.35
17.40
13.96
13.93
12.28
200 180 160 140 120 100 80 60 40 20 0 ppm
13
Figure 8.11 The DEPT 135 C NMR spectrum of pravastatin sodium in CD3OD.
A. (3R,5R)-3,5-dihydroxy-7-[(1S,2S,6R,8S,8aR)-6-hydroxy-2-methyl-8-
[[(2S)-2-methylbutanoyl]oxy]-1,2,6,7,8,8a-hexahydronaphthalen-1-yl]
heptanoic acid. (60 -epipravastatin),
B COOH
H
HO
HO
HO O
H H
H 3C O
H H H
H3C H CH3
H
HO
H
3-Hydroxypravastatin
Figure 8.12 The HSQC NMR experiment of pravastatin sodium in CD3OD.
HO 14
O 13
3
2 C 12
4 1 O 11
H
5 9
CH 3
2 1 10 8
3
5 7
HO 4 6
Chemical shift d (ppm) Assignment Chemical shift d (ppm) Assignment
12.27 C4 45.52 C16
13.94 C18 65.39 C3
17.40 C5 69.29 C1
25.35 C11 71.04 C15
27.88 C30 71.65 C13
32.29 C8 127.4 C4
35.63 C12 128.64 C6
37.07 C2 136.51 C5
38.34 C9 136.79 C7
38.83 C10 177.99 C10
43.00 C20 180.54 C17
45.18 C14
303.4 32 C18H35O4 HO
C
O
HO
322.3 42 C18H26O5 HO
C
O
OH
CH3
HO
HO
HO
O
O
H
HO
B. (3R,5R)-3,5-dihydroxy-7-[(1S,2S,6S,8S,8aR)-6-hydroxy-8-[[(2S,3R)-
3-hydroxy-2-methylbutanoyl]oxy]-2-methyl-1,2,6,7,8,8a-
hexahydronaphthalen-l-yl]heptanoic acid. (300 -hydroxy pravastatin),
C COOH
HO
H
HO
O
H
H3 C
O
H3 C H H H
H CH3
H
HO
H
Pravastatin Sodium 465
C. (3R,5R)-3,5-dihydroxy-7-[(1S,2S,6S,8S,8aR)-6-hydroxy-2-methyl-
8-[[(2S)-2-methylpentanoyl]oxy]-1,2,6,7,8,8a-hexahydronaphthalen-l-
yl]heptanoic acid.
D O OH
H
O
O
H
H3C O
H H H
H
CH 3 CH 3
HO H
Pravastatin lactone
D. (1S,3S,7S,8S,8aR)-3-hydroxy-8-[2-[(2R,4R)-4-hydroxy-6-oxotetra-
hydro-2H-pyran-2-yl]ethyl]-7-methyl-I,2,3,7,8,8a-hexahydronaph-
thalen-l-yl (2S)-2-methylbutanoate (pravastatin lactone).
in which 446.51 and 553.78 are the molecular weights of pravastatin sodium
and pravastatin 1,1,3,3-tetramethylbutylamine, respectively; C is the con-
centration, in mg/ml, of pravastatin 1,1,3,3-tetramethylbutylamine in the
Standard solution; V is the volume, in ml, of the Test solution; W is the weight,
in mg, of pravastatin sodium taken to prepare the Test solution; ri is the peak
response for each impurity obtained from the Test solution; and rs is the prav-
astatin peak response obtained from the Standard solution, in addition to not
exceeding the limits for each impurity specified in Table 8.6 not more than
0.1% of any other individual impurity is found, and not more than 0.6% of
total impurities is found.
Assay
Solution APrepare a 0.08 M phosphoric acid solution, adjust with a 25%
sodium hydroxide solution to a pH of 5, mix, filter, and degas.
Solution BUse acetonitrile.
Mobile phaseUse variable mixtures of Solution A and Solution B as
directed for Chromatographic system. Make adjustments if necessary (see Sys-
tem Suitability under Chromatography h621i).
Standard preparationDissolve an accurately weighed quantity of USP
Pravastatin 1,1,3,3-Tetramethylbutylamine RS in methanol, and dilute
quantitatively, and stepwise if necessary, with methanol to obtain a solution
having a known concentration of about 0.25 mg of pravastatin 1,1,3,3-
tetramethylbutylamine per ml.
System suitability preparationDissolve accurately weighed quantities
of USP Pravastatin 1,1,3,3-Tetramethylbutylamine RS and USP
Pravastatin-Related Compound A RS in methanol to obtain a solution con-
taining about 0.25 mg of USP Pravastatin 1,1,3,3-Tetramethylbutylamine
RS and 0.001 mg of USP Pravastatin-Related Compound A RS per ml.
Assay preparationTransfer about 20 mg of pravastatin sodium, accu-
rately weighed, to a 100-ml volumetric flask, dissolve in and dilute with
methanol to volume, and mix.
Chromatographic system (see Chromatography h621i)The liquid chro-
matograph is equipped with a 238-nm detector and a 4-mm 10-cm col-
umn that contains 3-mm packing L1. The flow rate is about 1 ml/min. The
chromatograph is programmed as follows.
470 Abdullah A. Al-Badr and Gamal A.E. Mostafa
Identification
A: The retention time of the major peak in the chromatogram of the
Assay preparation corresponds to that in the chromatogram of the Standard
preparation, as obtained in the Assay.
B: Ultraviolet Absorption Carry out this test as directed in the general
procedure h197UiFinely powder a number of tablets and extract with
water a portion equivalent to about 10 mg of pravastatin sodium. The
UV absorption spectrum of a solution of pravastatin sodium in water
containing about 10 mg/ml exhibits maxima at the same wavelength
as that of a similar solution of USP Pravastatin Sodium RS, concomi-
tantly measured between 220 and 340 nm.
Dissolution Carry out this test as directed in the general procedure h711i
Medium: water; 900 ml.
Apparatus 2: 50 rpm.
Time: 30 min.
ProcedureDetermine the amount of C23H35NaO7 dissolved by employing
UV absorption at the wavelength of maximum absorbance at about 238 nm
on filtered portions of the solution under test, suitably diluted with Medium,
if necessary, in comparison with Standard solution having a known concen-
tration of USP Pravastatin 1,1,3,3-Tetramethylbutylamine RS in the same
Medium. [Note: To express the concentration of the Standard solution as
pravastatin sodium, use the conversion factor of (446.51/553.78), in which
446.51 and 553.78 are the molecular weights of pravastatin sodium and
pravastatin 1,1,3,3-tetramethylbutylamine, respectively.]
TolerancesNot less than 80% (Q) of the labeled amount of
C23H35NaO7 is dissolved in 30 min.
Uniformity of dosage units Carry the test as directed in the general
procedure h905i: meet the requirements.
Related compounds
Mobile Phase and Chromatographic systemProceed as directed in the Assay.
Test solutionUse the Assay preparation, prepared as directed in the Assay.
[Note: Use this solution within 24 h of preparation.]
ProcedureInject a volume (about 20 ml) of the Test solution into the
chromatograph, record the chromatograms for up to 4 times the retention
time of the pravastatin peak, identify the impurities listed in Table 8.7, and
measure the peak responses. Calculate the percentage of each impurity in the
portion of tablets taken by the formula:
100ri =rs
472 Abdullah A. Al-Badr and Gamal A.E. Mostafa
in which ri is the peak response of the individual impurity, and rs is the sum of
the responses of all the peaks obtained from the Test solution. In addition to
not exceeding the limits of each impurity in Table 8.7, not more than 0.2%
of any unspecified individual impurity is found and not more than 3% of
total impurities are found. Disregard the peak due to pravastatin-related
compound B that elutes at the relative retention time of about 0.7 and
the peak due to 300 -hydroxy pravastatin at the relative retention time of
about 0.3, as these impurities are controlled in the drug substance mono-
graph. Disregard any impurity that is less than 0.05%.
Assay[Note: The Standard preparation, Assay stock preparation, and
Assay preparation can be stored for up to 7 days at room temperature.]
Mobile phasePrepare a filtered and degassed mixture of methanol,
water, glacial acetic acid, and triethylamine (500:500:1:1). Make adjust-
ments if necessary (see System Suitability under Chromatography h621i).
Diluent 1Transfer 16.4 g of anhydrous sodium acetate into a 2000-ml
volumetric flask. Add 1600 ml of water, adjust with glacial acetic acid to a
pH of 5.6, dilute with water to volume, and mix.
Diluent 2Prepare a mixture of Diluent 1 and methanol (80:20).
Standard preparationTransfer an accurately weighed quantity of USP
Pravastatin 1,1,3,3-Tetramethylbutylamine RS to a suitable volumetric flask
and dissolve in Diluent 1 using sonication to obtain a solution having a
known concentration of about 0.6 mg of pravastatin 1,1,3,3-
tetramethylbutylamine per ml. Dilute 5.0 ml of this solution with Diluent
2 to 25 ml and mix.
Pravastatin Sodium 473
100446:51=553:78CVD=NL rU =rs
in which 100 is the conversion factor to percentage; 446.51 and 553.78 are
the molecular weights of pravastatin sodium and pravastatin 1,1,3,3-
tetramethylbutylamine, respectively; C is the concentration, in mg/ml, of
474 Abdullah A. Al-Badr and Gamal A.E. Mostafa
analytes were detected and quantified with the selected reaction monitoring
mode of a triple quadrupole mass spectrometer. The assay was validated in a
3.47100 ng/ml concentration range for pravastatin, 1.32200 ng/ml for
30 a-isopravastatin, and 0.5215 ng/ml for 60 -epipravastatin using only
plasma for calibration. For plasma samples, subjected to full validation,
within- and between-day precisions were 17% (918% at the lower limit
of quantitation level) and accuracies were between 91% and 103%. For tissue
homogenates, subjected to partial validation, within- and between-day pre-
cisions were 212% (619% at the lower limit of quantitation level) and
accuracies were between 87% and 113% (81% and 113% at the lower limit
of quantitation level). Drug and metabolites were shown to be chemically
stable under most relevant analytical conditions. Finally, the assay was
applied for a pilot study in mice. After intravenous administration of the
drug, all isomeric compounds were found in plasma; however, in liver
and kidney homogenate, only the parent drug showed levels exceeding
the lower limit of quantitation.
Abdullah [68] developed an RP-HPLC method for the determination of
pravastatin and two other statins. The method involves the use of a
15 cm 4.6 mm of Zorbax Extend C18 column (5 mm) and different chro-
matographic conditions for the separation of the drugs. Linearity range was
1060 mg/ml for pravastatin. The method was used for the determination of
the drugs in spiked human plasma samples.
Sultana et al. [69] developed a validated RP-HPLC method for the
simultaneous determination of pravastatin, lisinopril, and other statins in
active pharmaceutical ingredients, formulations, and human serum.
A Purospher star C18 (5 mm, 25 cm 4.6 mm) column was used with
mobile phase consisting of acetonitrile:water (60:40, pH 3) with flow rate
of 1 ml/min, and the quantitative evaluation was performed at 225 nm.
The retention time was 2 min for lisinopril and 3.1 min for pravastatin. Suit-
ability of this method for the quantitative determination of the drugs was
proved by validation in accordance with the requirements laid down by
International Conference on Harmonization guidelines. The method was
applied to the determination of the drugs in active pharmaceutical ingredi-
ents and in pharmaceutical preparations, with high percentage of recovery,
good accuracy, and precision.
Sampath et al. [70] developed and validated an ultra-flow liquid
chromatography-tandem mass spectrometric method for the estimation of
pravastatin in human plasma. Pravastatin and omeprazole (internal standard)
were extracted from human plasma using a solid-phase extraction procedure
Pravastatin Sodium 487
monitoring mode using target ions at m/z 423.4 (pravastatin and 30 a-iso-
pravastatin) and m/z 319.2 (internal standard). The blank plasma did not
interfere with the determination of the analytes. The linear concentration
ranges of the calibration curves for pravastatin and 30 a-isopravastatin were
both 1.25200 ng/ml. The limits of quantitation of pravastatin and 30 a-
isopravastatin were both 1.25 ng/ml. The extraction recovery was more
than 80%. The method is suitable for pharmacokinetics study of the drug
and its main metabolite.
Vlckova et al. [78] reported a simple and reproducible ultra HPLC-
tandem mass spectrometric method for the determination of pravastatin
and its lactone in rat plasma and urine using deuterium-labeled internal
standard for the quantification. Separation of the analytes was performed
on BEH C18 analytical column (5 cm 2.1 mm, 1.7 mm) using gradient
elution by mobile phase consisting of acetonitrile and 1 mM ammonium
acetate at pH 4. Run time was 2 min. Quantification of the analytes was
performed using the selected reaction monitoring experiment in
electrospray ionization negative ion mode for pravastatin and in electrospray
ionization positive-ion mode for pravastatin lactone. Sample treatment con-
sisted of a protein precipitation by acetonitrile and microextraction by
packed sorbent for rat plasma. Simple microextraction by packed sorbent
procedure was sufficient for rat urine. Microextraction by packed sorbent
was implemented using the C8 sorbent inserted into a microvolume syringe,
an eVol hand-held automated analytical syringe, and a small volume of sam-
ple (50 ml). The analytes were eluted by 100 ml of the mixture of acet-
onitrile:0.01 M ammonium acetate, pH 4.5 (90:10). The method was
validated and demonstrated good linearity in the range of 5500 nmol/l
for plasma and urine samples. Recovery was within 97109% for plasma
samples and 92101% for urine samples. The method was applied for the
measurement of pharmacokinetic plots of pravastatin and its lactone in rat
plasma and urine samples.
Li et al. [79] determined pravastatin in rat muscle by RP-HPLC. Prav-
astatin was extracted from muscle homogenate with solid-phase extraction
and analyzed by RP-HPLC with a UV detector. The lowest detection limit
of the drug was 0.4 ng and the quantitation limit was 5 ng/g of muscles. The
linearity range was 5100 ng/g of muscle and the solid-phase extraction
recovery was 52.66%. The precision of the method was 61.8%. The method
is sensitive with good precision to assay pravastatin in rat muscle.
A pharmacokinetic application to determine the drug in muscle has been
practiced.
490 Abdullah A. Al-Badr and Gamal A.E. Mostafa
the statins were detected in an untreated sewage sample at 4117 ng/l and in
a treated sewage sample at 159 ng/l.
Kawabata and Urasaki [88] developed a liquid chromatography-APCI
tandem mass spectrometric method for the quantitative determination of
pravastatin and its main metabolite (R-416) in human plasma with a fully
automated online solid-phase extraction system. The method employed
the direct online injection of human plasma into the Prospekt-2 system
for extraction of the analytes followed by column-switching introduction
of analytes to the LC/APCI-MS/MS system. The use of online solid-phase
extraction system resulted in reducing the sample preparation time and
decreasing endogenous interfering substances in the extract. The lower limit
of quantification for the assay was 0.1 ng/ml for pravastatin and its metab-
olite R-416 on a 100 ml plasma sample. The calibration curves were linear
for concentrations ranging from 0.1 to 100 ng/ml for both analytes. The
intra- and interassay precision was less than 4%. The method provided an
automated sample analysis in a total cycle time of 6 min, allowing analysis
of many plasma samples to be conducted with increased throughput and
improved robustness.
Jain et al. [89] described a rapid, specific, and sensitive LC-MS/MS assay
method using solid-phase extraction for the determination of pravastatin, in
human plasma. The plasma filtrate obtained after solid-phase extraction,
using a polymer base, a hydrophiliclipophilic balance cartridge, was sub-
mitted directly to short-column liquid chromatography-tandem mass spec-
trometric (LC-MS/MS) assay, with negligible matrix effect on the analysis.
For validation of the method, the recovery of the free analytes was compared
with that from an optimized extraction method, and the analyte stability was
examined under conditions mimicking the sample storage, handling, and
analysis procedures. The extraction procedure yielded extremely clean
extracts with a recovery of 107.44% and 98.93% for pravastatin and internal
standard, respectively. The intra- and interassay precisions for the samples at
the lower limit of quantitation were 3.30% and 7.31%, respectively. The cal-
ibration curves were linear for the dynamic range 0.5200 ng/ml with cor-
relation coefficient r
0.9988. The intra- and interassay accuracy ranged
from 95.87% to 112.40%. The method is simple and reliable with a total
run time of 3 min. This validated method was applied to the pharmacoki-
netic study in human volunteers receiving a single oral dose of 40 mg imme-
diate release formulation.
Deng et al. [90] developed a liquid chromatography and tandem mass
spectrometry (LC-MS/MS) method for determining pravastatin or
494 Abdullah A. Al-Badr and Gamal A.E. Mostafa
Pilli et al. [95] developed and validated a simple, rapid, and sensitive liq-
uid chromatography/tandem mass spectrometry method for the quantita-
tion of pravastatin in human plasma. Topiramate was used as the internal
standard. The analytes were extracted from human plasma samples by liq-
uidliquid extraction. The reconstituted samples were chromatographed
on a C18 column by using a 90:10 mixture of acetonitrile and 5 mM ammo-
nium acetate as the mobile phase at a flow rate of 1 ml/min. The method is
applicable to clinical studies.
were incubated in the wells at 4 C for 2024 h. After washing with buffer
A, 200 ml 0.01% tetramethylbenzidine in 50 mM acetate/citrate buffer of
pH 5.5 containing 3% DMSO and 0.002% hydrogen peroxide was added
for 30 min, followed by 0.05 M sulfuric acid, and the absorbance was mea-
sured at 450 nm. The antisera were obtained using bovine serum albumin
conjugates of the b-alanine derivative of pravastatin (for method A) or
the S-deoxy derivative of pravastatin for method B. At optimal antise-
rum/labeled antigen dilutions, pravastatin could be determined in the range
of 5500 pg/well with an IC50 of 36130 pg/well. The detection limit for
method A was 500 pg/ml pravastatin, and the relative standard deviation at
5 ng/ml was 4.5%.
Darwish et al. [102] developed and validated a new highly sensitive EIA
for the determination of pravastatin in human plasma samples. Pravastatin
was coupled to keyhole limpet hemocyanin and bovine serum albumin via
its terminal carboxylic acid group by carbodiimide reagent. Pravastatin
keyhole limpet hemocyanin conjugate was used as an immunogen for raising
anti-pravastatin polyclonal antibody in rabbits. The generated anti-
pravastatin antibody recognized pravastatin with high affinity and selectivity.
Pravastatinbovine serum albumin conjugate was immobilized onto
microwell plates and used as a solid phase. The assay involved a competitive
binding reaction between pravastatin, in plasma sample, and the
immobilized pravastatinbovine serum albumin for the binding sites on a
limited amount of the anti-pravastatin antibody. The anti-pravastatin anti-
body bound to the plate wells was quantified with horseradish peroxidase-
labeled anti-immunoglobulin second anti-rabbit IgG antibody and 3,30 ,5,
50 -tetramethylbenzidine as a substrate for the peroxidase enzyme. The con-
centration of pravastatin in the sample was quantified by its ability to inhibit
the binding of the anti-pravastatin antibody to the immobilized pravastatin
bovine serum albumin and subsequently the color development in the assay
wells. The conditions of the enzyme immune assay method were investi-
gated, and the optimum conditions were employed in the determination
of pravastatin in plasma samples. The assay limit of detection was 0.2 ng/ml,
and the effective working range at relative standard deviation of 5% was
0.520 ng/ml. The mean analytical recovery of pravastatin from spiked
plasma was 100.9 2.98%. The precision of the assay was satisfactory; rel-
ative standard deviation was 2.613.70% and 3.964.17% for intra- and
interassay precision, respectively. The analytical procedure is convenient,
and one can analyze approximately 200 samples per working day, facilitating
the processing of large-number batch of samples. The enzyme immune assay
Pravastatin Sodium 499
method has a great value in the routine analysis of pravastatin in plasma sam-
ples for its therapeutic monitoring and pharmacokinetic studies.
6. PHARMACOKINETICS
In a two-way crossover study, eight healthy male subjects each
received an intravenous and an oral dose of [14C]-pravastatin sodium.
The oral absorption of [14C] activity from pravastatin sodium was about
34%, and the oral bioavailability was about 18%, suggesting first-pass metab-
olism of the drug. After intravenous dose, the recovery of radioactivity aver-
aged 60% and 34% in urine and feces, respectively. Corresponding values
were 20% (urine) and 71% (feces) for oral dose [103].
Biotransformation profile of pravastatin in pooled human urine, plasma,
and feces from healthy male volunteers given single oral dose or single intra-
venous doses of [14C]-pravastatin were determined by HPLC. The drug-
related component in urine, plasma, and feces corresponds to the intact prav-
astatin in the pooled urine samples. Pravastatin constituted 29% and 69% of
the radioactivity after the oral and the intravenous doses, respectively [104].
The disposition and metabolism of pravastatin sodium in rats, dogs, and
monkeys have been studied using [14C]-labeled compound. Absorption was
about 70% in rat and 50% in dogs. Tissue distribution examined by both
whole-body autoradiography, and radiography measurement demonstrated
that the drug was selectively taken up by the liver and excreted via bile
mainly in unchanged form [105].
The effect of oral contraceptive steroids on the pharmacokinetics of
pravastatin in young women was evaluated. Normal healthy male and female
were subjected to receive a single dose of pravastatin after an overnight fast.
The pharmacokinetic profiles of pravastatin and SQ-31906 in young and
elderly subjects of men and women differed little. Concomitant administra-
tion of oral contraceptives in young women did not affect the pharmacoki-
netics of pravastatin and SQ-31906. No difference was detected between the
disposition of the parent drug or its metabolite in men and women [106].
Pravastatin differs from other statins because of its greater hydrophilicity
due to the hydroxy group attached to its decalin ring. The hydrophilic
nature of pravastatin accounts or its minimal penetration into the intracel-
lular space of nonhepatic tissues, including an apparent inability to cross the
bloodbrain barrier. The drug is well tolerated and is rapidly absorbed and
excreted. Pravastatin does not accumulate in plasma even with repeated
administration. The drug is taken up into the liver by an active transport
500 Abdullah A. Al-Badr and Gamal A.E. Mostafa
carrier system. The hepatic excretion ratio is high (0.66). Pravastatin and
metabolites are cleared through both hepatic and renal routes. The drug
is 50% protein bound [107].
The single dose and steady-state pharmacokinetics of pravastatin and its
two metabolites, SQ-31906 and SQ-31945, were evaluated in hemodialysis
patients. No statistical differences in the pharmacokinetics of pravastatin or
SQ-31906 were evident when comparing the first and last days of oral dos-
ing with pravastatin. The pharmacokinetic parameters of pravastatin and
SQ-31906 were similar to those of healthy volunteers. SQ-31945, the inac-
tive polar metabolite, accumulated in dialysis patients. Pravastatin can be
administered in the usual dosages to subjects with renal failure on hemodi-
alysis and no change of dosing is necessary [108].
Many of the in vivo and in vitro human and animal studies suggest that an
active transport mechanism is involved in the pharmacokinetics of prava-
statin. The drug is rapidly absorbed from the upper part of the small intestine,
via proton-coupled carrier-mediated transport, and then taken up by the
liver by a sodium-independent bile acid transporter [109].
Co-administration of pravastatin with gemfibrozil leads to the inhibition
of human organic anion transporter 3-mediated pravastatin transport and the
metabolites in humans. It causes a decrease in renal clearance of the drug by
about 40% in healthy volunteers. An uptake study was undertaken of prav-
astatin using human organic anion transporter-expressing S2 cells. Human
organic anion transporter 3 and human organic anion transporter 4 trans-
ported pravastatin in a saturatable manner with MichaelisMenten constant
of 27.7 and 257 mM, respectively. Human organic anion transporters 1 and 2
did not transport pravastatin [110].
Pharmacokinetic properties of pravastatin in Mexicans were studied in
health adult volunteers. To evaluate the pharmacokinetic properties of prava-
statin in healthy Mexican mestizos volunteers and to compare them with those
in white and Japanese populations described in the literature. Twenty-four sub-
jects (15 women, 9 men; mean age, 30.6 years) participated in the study. The
mean (SD) Cmax was 9.5 (2.4) ng/ml; Tmax, 0.8 (0.3) h; AUC01, 35.7 (19.7)
ng/ml; tl/2, 2.7 (1.1) h; and mean residence time, 3.1 (1.1) h. One volunteer
(4%) had an AUC value that differed substantially from the rest of the study
population, producing a bimodal distribution of the pharmacokinetic param-
eters. No adverse events were observed or reported during the trial [111].
Studies compared the multiple-dose pharmacokinetic interaction pro-
files of pravastatin when co-administered with four inhibitors of cytochrome
P450-3A4 isoenzymes in healthy subjects was studied. Compared with
Pravastatin Sodium 501
7. METABOLISM
Iwabuchi et al. [113] reported that, in rat, pravastatin was metabolized
to two metabolites. After incubation of one of the metabolites with rat hepa-
tocytes, a glutathione conjugate was found. Metabolic pathways for prava-
statin were proposed.
Everett et al. [104] studied the biotranformation profiles pravastatin in
pooled urine, plasma, and feces from healthy males given single 19.2 mg oral
or 9.9 mg intravenous doses of [14C]-pravastatin and determined them by
HPLC. The main drug-related component in urine, plasma, and feces cor-
responded to intact pravastatin. Twelve metabolites were isolated and iden-
tified as unchanged pravastatin 1, 3a-iso-pravastatin 2, 6-epi-pravastatin 3,
desacyl pravastatin 4, pravastatin glucuronide 5, 5,6-epoxy 3a-iso-
pravastatin 6, 30 (S)-OH-pravastatin 7, unknown conjugate of metabolite
9, tetranor of 30 (S)-OH-pravastatin 8, tetranor analog of 30 (S)-
OH-pravastatin 9, 3-keto-5,6-dihydroxy derivative of pravastatin 10,
7-OH-3a-iso-pravastatin 11, and triol metabolite of pravastatin 12.
Figure 8.15 shows the biotransformation pathways of pravastatin in human.
Muramatsu et al. [114] studied the metabolic fate of pravastatin sodium in
isolated rat hepatocytes. Two polar metabolites were isolated and identified
as a glutathione conjugate and a dehydrodiol. Both metabolites were formed
via an epoxide which has been identified as the 40 b,50 b epoxide on the
decalin moiety. Formation of the glutathione conjugate was enzymic, while
the dehydrodiol was formed by nonenzymic hydrolysis of the epoxide
accompanied by the intramolecular migration of the double bond.
Nakamura et al. [115] determined the structures, including stereochem-
istry, of the two major metabolites of pravastatin sodium in an isolated rat
hepatocyte system, 4-a-glutathione conjugates, and 30 ,50 -dehydroiol by
one- and two-dimensional NMR spectroscopy. The structures of two syn-
thetic pravastatin epoxides, possible precursors of the metabolites, were
established. One of the synthetic epoxides, 40 a beta, 50 beta epoxide, was
converted to the pravastatin metabolite, 40 a-glutathione conjugate by a
rat liver cytosol system, and is proposed as the common metabolic interme-
diate between pravastatin sodium and the two metabolites.
H H H H
HO CO2- Na+ HO CO2- Na+ HO CO2- Na+ HO CO2- Na+
OH OH OH O OH
H O H O H
H
H3 C O
H3 C O H3C O
H H CH3 H
CH3 CH3 CH3 CH3 CH3 CH3
oxidation oxidation HO
HO OH OH OH
O
Desacyl-PV (4) 5,6-epoxy-3- 3 -iso-PV (2) 7-OH-3-iso-PV (11)
iso-PV (6)
da CO2- Na+
1 xid
t io HO
w- -O
n
b
H OH
HO CO2- Na+ O H H
Ox HO CO2- Na+
OH H3 C O ida
OH O CO2- Na+ O H H tio
CH3 CH3 n OH
O H
H3 C O H3 C O
H H
CH3 CH3 HO 2 C CH3 CH3 HO H3C O
OH H
Conjugate of 3(S)- O OH CH3 CH3
OH-PV Tetranor (8)
HO HO O
Triol (12)
HO O
3(S)-OH-PV HO OH OH
Tetranor (9)
Komai et al. [105] studied the disposition and the metabolism of prava-
statin in rat, dogs and monkeys using [14C]-labeled compound. The extent
of absorption was approximately 70% in rats and 50% in dogs. Tissue distri-
bution examined by both whole-body autoradiography and radioactivity
measurement demonstrated that the drug was selectively taken up by the
liver, a target organ of the drug, and excreted via bile mainly in unchanged
form. Since pravastatin excreted by the bile was reabsorbed, the inter-
ohepatic circulation maintained the presence of unchanged pravastatin in
the target organ. The profiles of metabolites of pravastatin were studied
in various tissues and excreta, and the main metabolic pathway of pravastatin
sodium is shown in Figure 8.16.
Kitazawa et al. [116] stated that a major metabolite R-416 of pravastatin
sodium and a minor metabolite R-418 were produced in rat liver cytosol in
the presence of adenosine-30 -phosphate-50 -phosphosulfate as a cofactor.
The reactions were inhibited by the inhibitors for sulfotransferase, and
18
OH was introduced to the 30 -a and 60 -a positions of R-416 and
R-418, respectively, by incubation with H18 2 O. Pravastatin was, therefore,
metabolically activated by sulfation at the 60 -b-hydroxy group by sul-
fotransferase, followed by nucleophilic attack of hydroxy anions at 3-a or
6-a positions, to give R-416 or R-418, respectively (Figure 8.17).
Muramatsu et al. [117] studied the metabolism of pravastatin sodium by
3a-hydroxysteroid dehydrogenase. When incubated with isolated rat hepa-
tocytes, pravastatin sodium yields a small amount of a metabolite in addition
to two metabolites. The metabolite was found to be formed by at first being
enzymatically dehydrogenated to 60 -keto intermediate (R-195) through
spontaneous deesterification with accompanying aromatization. The
pravastatin-60 -b-hydroxydehydrogenase activity was localized in cytosolic
fraction and required NADP, preferentially over NAD, as a cofactor.
The formation of R-195 by rat liver cytosol was strongly inhibited by indo-
methacin. The results and high substrate specificity of purified pravastatin-
60 -b-hydroxy dehydrogenase toward 3a-hydroxysteroids suggested that the
enzyme is identical to 3a-hydroxysteroid dehydrogenase.
Jacobsen et al. [118] compared the intestinal metabolism of lovastatin and
pravastatin in vitro. Incubation of pravastatin with human small intestinal
microsomes resulted in the generation of 30 a,50 b,60 b-trihydroxy pravastatin
and hydroxy pravastatin, as in the liver, pravastatin was metabolized in the
small intestine by sulfation and subsequent degradation to its main metabo-
lite 30 a-iso-pravastatin.
504 Abdullah A. Al-Badr and Gamal A.E. Mostafa
OH OH
HOOC HOOC
HO HO
O O
H H
H3 C O H3 C O
H H
CH3 CH3 CH3 CH3
Isomerization
OH HO
Isomerization
COOH
OH O OH
NaOOC
O
HO O
O O
H3 C O H H
H
CH3 CH3 H3 C O H3 C O
H H
CH3 CH3 CH3 CH3
HO -Oxidation Lactonization
M-9 HO HO OH
COOH
Pravastatin sodium M-3 (R-414)
H
CH3 Oxidation
M-8 OH
HOOC
HO
O
H
H3 C O
H
CH3 CH3
O
Major pathway HO
Minor pathway
Conjugation Hydration
Proposed metabolite
OH OH
HOOC HOOC
HO HO
O O
H H
H3 C O H3 C O
H H
CH3 CH3 CH3 CH3
HO HO
COONa COOH
OH OH
O O
O O
H H
CH3 CH3 CH3 CH3
8
PAPS O H2 SO 4
6
3 sulfotransterases
HO HO S O
O OH
Pravastatin
sodium (I)
HO
HO COOH
COOH
OH
OH O
O
O
O H
H CH3 CH3
CH3 CH3 +
HO
OH
R-416 R-418
Figure 8.17 Plausible metabolic pathway of pravastatin sodium to R-416 and R-418
[116].
8. STABILITY
Deng et al. [120] found that pravastatin was unstable under the simu-
lated stomach conditions. Brain-Isasi et al. [63] reported that degradation of
pravastatin is dramatically influenced on both pH and temperature and that
stability of pravastatin increases with the increase of pH. Lotfy et al. [121]
studied the stability of pravastatin sodium under acidic hydrolytic condi-
tions. Polagani et al. [122] conducted a stability test to evaluate analyte sta-
bility in stock solutions and in plasma samples under different conditions.
Stock solutions were found to be stable for 23 days at 28 C. Pilli
et al. [95] studied the stability of pravastatin in human plasma and found that
the drug is stable for 30 days at 28 C. Bastarda et al. [53] studied the chem-
ical stability of pravastatin in aqueous media at pH between 1 and 7. Due to
the presence of a highly sensitive diene alcohol and 3-hydroxy-8-lactone,
pravastatin showed very characteristic chemical behavior. In the acidic
media of pH 13, pravastatin undergoes isomerization of 6a-hydroxy group
in the hexahydronaphthalone ring forming a mixture of 6a-, 6b-, 3a-, and
3b-hydroxyl isomers. Each isomer may also undergo lactonization of the
hydroxy acid chain to form the corresponding lactone. Thus in acidic media
pravastatin sodium could convert to the following seven different
506 Abdullah A. Al-Badr and Gamal A.E. Mostafa
9. REVIEWS
Serizawa [123] presented a review on two-stage manufacturing prav-
astatin by fermentation. It comprises the manufacturing of (+)-compactin,
ML-236B, fermentation of pravastatin with microorganisms, and future
perspective.
Arai et al. [124] presented a review on the production, chemistry,
enzyme inhibition, and animal studies, metabolic studies, of pravastatin.
Erturk et al. [125] presented a review on the analytical methods for the
quantitative determinations of pravastatin and four other statins in biological
fluids. Almost all the assays reviewed are based on HPLC or gas chromatog-
raphy. Some purification steps (liquidliquid extraction, solid-phase extrac-
tion, etc.) have been used before they are submitted to separation of
chromatographic procedures and are detected by various detection method
such as ultraviolet, fluorescence, and mass spectrometry. The review showed
that most methods may be used for quantitative determination of statin in
plasma and are suitable for therapeutic monitoring of these drugs.
Nirogi et al. [126] presented a review on chromatographymass spec-
trometry methods for the quantitation of pravastatin and six other statins
in biological samples. HPLC in combination with tandem mass spectrom-
etry is the analytical technique of choice for the quantification of these com-
pounds in biological samples. This review envisages that most of the
methods used for quantification of pravastatin and the other statins are in
plasma and they are suitable for therapeutic drug monitoring of these drugs.
ACKNOWLEDGMENTS
The authors wish to thank Mr. Tanvir Ahmed Butt, Pharmaceutical Chemistry Department,
College of Pharmacy, King Saud University for his secretarial assistance in preparing this
profile.
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510 Abdullah A. Al-Badr and Gamal A.E. Mostafa
Vardenafil Dihydrochloride
Abdelkader E. Ashour*, A.F.M. Motiur Rahman,
Mohammed G. Kassem
*Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Riyadh,
Saudi Arabia
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
Contents
1. Introduction 515
1.1 Nomenclature 516
1.2 Formula 516
1.3 Physical properties 517
2. Methods of Preparation 518
3. Physical Properties 522
3.1 Spectroscopy 522
3.2 Nuclear magnetic resonance spectrometry 523
3.3 Mass spectrum 526
4. Methods of Analysis 528
4.1 Chromatographic methods 528
4.2 Voltammetric method 535
4.3 Immunoassay methods 535
5. Pharmacology of Vardenafil 536
5.1 Pharmacodynamics 536
5.2 Pharmacokinetics 539
Acknowledgment 540
References 540
1. INTRODUCTION
Vardenafil (Levitra) is a potent and highly selective inhibitor of
cGMP-specific phosphodiesterase type 5 (PDE-5), the most prominent
PDE in the penile corpus cavernosum. It has been proven to be safe and
effective treatment for erectile dysfunction (ED). This action can result in
smooth muscle relaxation that is needed for a penile erection [1]. Vardenafil
is 510 times more potent than sildenafil, the classic PDE-5 inhibitor [2].
The drug is generally well tolerated, with a favorable safety profile. Its use
Profiles of Drug Substances, Excipients, and Related Methodology, Volume 39 # 2014 Elsevier Inc. 515
ISSN 1871-5125 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800173-8.00009-X
516 Abdelkader E. Ashour et al.
1.1. Nomenclature
1.1.1 Systematical chemical names
2-(2-Ethoxy-5-((4-ethylpiperazin-1-yl)sulfonyl)phenyl)-5-methyl-
7-propylimidazo[5,1-f][1,2,4]triazin-4(1H)-one (free base) [8].
2-[2-Ethoxy-5-(4-ethylpiperazine-1-sulfonyl)phenyl]-5-methyl-7-
propyl-1H,4H-imidazo[4,3-f][1,2,4]triazin-4-one (IUPAC, free base) [9].
2-{5-Ethoxy-2-[(4-ethyl-1-piperazinyl)sulfonyl]phenyl}-5-methyl-7-
propylimidazo[5,1-f][1,2,4]triazin-4(1H)-one (free base) [10].
2-{2-Ethoxy-5-[(4-ethyl-1-piperazinyl)sulfonyl]phenyl}-5-methyl-7-
propylimidazo[5,1-f][1,2,4]triazin-4(1H)-one dihydrochloride (ACD/
IUPAC name) [11].
2-{2-Ethoxy-5-[(4-ethylpiperazin-1-yl)sulfonyl]phenyl}-5-methyl-7-
propylimidazo[5,1-f][1,2,4]triazin-4(1H)-one dihydrochloride [11].
1.2. Formula
1.2.1 Empirical formula, molecular weight, obtained mass,
and CAS number
Chemical formula: C23H32N6O4S.2HCl; C23H34Cl2N6O4S
Molecular weight: 561.53
Monoisotopic mass: 560.17395 Da, molecular weight: 488.60, mon-
oisotopic mass: 488.220581 Da (free base), exact mass: 488.22, exact mass:
488.22, m/z 488.9 [M H] (obtained),
CAS number 224789-15-5, CAS number 224785-90-4 (free base) [9,12].
Vardenafil Dihydrochloride 517
1.2.3 SMILES
Cl.Cl.O]C2\N]C(/Nn1c(nc(c12)C)CCC)c3cc(ccc3OCC)S(]O)(]O)
N4CCN(CC)CC4 [11].
CCCC1]NC(C)]C2N1NC(]NC2]O)C1]C(OCC)C]CC(]C1)
S(]O)(]O)N1CCN(CC)CC1 (free base) [13].
1.2.4 InChI
InChI1S/C23H32N6O4S.2ClH/c1-5-8-20-24-16(4)21-23(30)25-22
(26-29(20)21)18-15-17(9-10-19(18)33-7-3)34(31,32)28-13-11-27(6-2)
12-14-28;/h9-10,15H,5-8,11-14H2,1-4H3,(H,25,26,30);2*1H [11].
InChI1S/C23H32N6O4S/c1-5-8-20-24-16(4)21-23(30)25-22(26-29
(20)21)18-15-17(9-10-19(18)33-7-3)34(31,32)28-13-11-27(6-2)12-14-28/
h9-10,15H,5-8,11-14H2,1-4H3,(H,25,26,30) (free base) [13].
1.3.2 Solubility
Water solubility: 0.11 mg/mL (HCl salt) [13].
1.3.4 Stability
30 C/75% relative humidity [15].
2. METHODS OF PREPARATION
Vardenafil dihydrochloride (1) was first synthesized by Ulrich
Niewohner et al. [17], then Mao et al. [18], and recently Nowakowski
et al. [19]. Alkylation of 2-hydrozybenzonitrile with ethyl bromide,
followed by addition of ammonia to the nitrile functionality using
AlMeClNH2, generated in situ by addition of ammonium chloride with
trimethyl-aluminum, to give amidine 3. On the other hand, D,L-alanine
was acylated with butyryl chloride to provide 7, which underwent a
DakinWest reaction with ethyl oxalyl chloride to furnish the intermediate
a-oxoamino ester 9 (Scheme 9.1).
O 70 C
O O
OEt
NH2 Et3N, TMSCl Cl
Pr NH Pr NH O
OH 040 C O 8
H3C OH H3C OEt
O H3C Pyridine, DMAP
O O
5 O THF, reflux
Pr Cl 6 7 9
10 C to RT
71 %
O O
O CH3
HN HN
HN NH N N
POCl3 N ClSO3H ClO2S N
N N N
N O
ClCH2CH2Cl 0 C to RT O
O
O CH3 reflux 91 %
10 28 % 11 12
O
.2HCl O
N 3 eq N
HN N
NH N O N 1M HCl in Et2O O HN
S N N N
N S N
O N
CH2Cl2, 0 C to RT Et2O, CH2Cl2, O
66 % O 99 % O
13 1
Vardenafil dihydrochloride
Scheme 9.1 Synthesis of vardenafil dihydrochloride [17,20,21].
Vardenafil Dihydrochloride 519
O O
O CH3
HN HN
HN NH N N
POCl3 N conc. H2SO4 HO3S N
N N N
N O
or 80 %
O O
O CH3 CH3COCl
11 17
10
O
.2HCl O
N Conc. HCl
1) SOCl2, DMF HN N
N O N Acetone, H2O O HN
S N 89 % N N
N S N
O N
N O
2) O
NH O
13 1
Vardenafil dihydrochloride
Scheme 9.2 Large scale synthesis of vardenafil dihydrochloride.
520 Abdelkader E. Ashour et al.
was reacted with N-ethylpiperazine in the same pot to give vardenafil (13) in
93 % yield. The vardenafil dihydrochloride (1) salt of vardenafil (13) was
prepared using concentrated HCl in acetone and water.
Recently, in 2009, Yongjun Mao et al. [18] presented an improved
synthetic route (Scheme 9.3) for the synthesis of vardenafil (13). In this
scheme, compound 5 was treated with chlorosulfonic acid to obtain sul-
fonyl chloride substituted product 18, which was then treated with
1-ethylpiperazine to provide 19, followed by POCl3 treatment to give
the benzonitrile 20.
The benzamidine 21 was afforded by the reaction of 20 with
lithium hexamethyldisilazane. Then benzamidine 21 was treated with
hydrazine hydrate in ethanol to convert the benzamidine 21 to the
benzamidrazone 22. Compound 22 [19] was then reacted with 9 to afford
the intermediate 23 which was directly transformed into the title
vardenafil (13).
For the synthesis of vardenafil (13), imidazole 26 was prepared according
to the reported procedure [2325]. Aminolysis of imidazole 26, followed by
OEt O
OEt O OEt O N
NH2
ClSO3H NH POCl3
NH2 NH2
CH2Cl2, < 20 C RT, 86 % S O 8090 C, 85 %
14 N
SO2Cl 18 O
N 19
Pr NH O O O
N O N
H3C OEt O HN O HN
N N POCl3, N N
O 9 S N H S N
N N
O O
EtOH, reflux 70 C
O 84% O
23 13
Vardenafil
Scheme 9.3 Improved synthetic route for the synthesis of vardenafil
dihydrochloride [21].
Vardenafil Dihydrochloride 521
OH OH O O
EtI, 14C AlMe3, NH2. HCl
I 14C 14C
K14CN N K2CO3 N NH4Cl NH2NH2.H2O
NH
CuI
O
O CH3 O
H NH O
O N NH2 HN NH HN
14C H3C OEt 14C
N
14C N POCl3 N
NH 9 N O N
O
EtOH O CH3 O
4 (14C) 10 (14C) 11 (14C)
O O
N
i) N
HN NH O HN
14C N N 14C N
ClO2S N S N
ClSO3H N EtiPr2N N .2HCl
O
O ii) HCl O
12 (14C) 1 (14C)
Vardenafil dihydrochloride (14C)
Scheme 9.4 Synthesis of [14C]-labeled vardenafil hydrochloride [27].
O O O
HN HN N
N N
HO3S N ClO2S N 27 NH
N SOCl2 N
O O
17 26
3
BuLi + H2 3H
O 3H O
O 1) TMEDA
N N
2) AlBr3 O HN
O HN N N .XHCl
N N S N
S
N
N LiAl3H4 N
O O
O O
28 1 (3H)
Vardenafil dihydrochloride (3H)
3
Scheme 9.5 Synthesis of [ H]-labeled vardenafil dihydrochloride [28].
two steps. In the first step, sulfonic acid 17 was treated with thionyl chloride
to obtain 26. Compound 26 was then treated with N-acetylpiperazine (27)
and gave 28. Reduction of carbonyl group of compound 28 was then per-
formed using [3H]-labeled lithium aluminum hydride to obtain [3H]-labeled
vardenafil dihydrochloride (1[3H]).
3. PHYSICAL PROPERTIES
3.1. Spectroscopy
3.1.1 Ultraviolet spectroscopy
The ultraviolet/visible (UV/VIS) absorption spectrum of vardenafil
dihydrochloride was recorded for selecting the proper maximum absorption
peak (lmax). The absorption spectrum of the compound in ethanol was
scanned from 200 to 800 nm, using a UV/VIS spectrometer (Varian Cary
50 UV/VIS spectrophotometer). As shown in Figure 9.1, the lmax of
vardenafil dihydrochloride is located at 270 nm.
O
0.4 N
O HN
N N
l max S N : 2HCl
Abs N
270 nm O
O
0.2
0.0
400
Wavelength (nm)
Figure 9.1 UV spectrum of vardenafil dihydrochloride (5.8 105 M solution in ethanol).
Figure 9.2 Infrared spectroscopy (IR) spectra of vardenafil dihydrochloride in KBr pellet.
1281.50, 1156.50, 950.28, 719.82, and 583.54 cm1. Assignments for the
major IR absorption bands are provided in Table 9.1.
Table 9.1 Infrared spectroscopic data for vardenafil dihydrochloride in KBr plate
Entry Bond Absorption peaks (lmax) (cm1) Appearance
1 CdN 719 Strong
2 CdO 1156 Strong
3 dCdH, HC]CH (aryl) 1600, 1491, 1333 Strong
4 C]O 1724 Strong
5 CH2, CdH (alkyl) 2970 Strong
6 NdH 3421 Broad
8 . 056
7 . 490
7 . 474
4 . 907
4 . 346
4 . 334
3 . 970
3 . 946
3 . 697
3 . 674
3 . 333
3 . 266
3 . 244
3 . 228
2 . 974
2 . 951
2 . 928
2 . 783
1 . 950
1 . 937
1 . 485
1 . 474
1 . 399
1 . 387
1 . 103
1 . 090
8 . 090
8 . 074
O
N
O HN
N N
S N
N
O
O
16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 -3 ppm
2 . 00
1 . 00
2 . 08
1 . 99
2 . 03
6 . 45
2 . 06
3 . 03
1 . 99
5 . 98
3 . 07
13
3.2.3 C NMR spectrum of vardenafil dihydrochloride
155 . 160
152 . 667
144 . 266
134 . 738
132 . 327
131 . 765
128 . 326
121 . 174
117 . 243
114 . 856
162 . 443
66 . 933
53 . 249
51 . 899
49 . 561
49 . 390
49 . 220
49 . 050
48 . 879
48 . 709
48 . 539
44 . 631
26 . 758
21 . 154
14 . 748
13 . 929
10 . 758
9 . 507
O
N
O HN
N N
S N
N
O
O
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0
13
Figure 9.5 C NMR spectra of vardenafil dihydrochloride in CD3OD.
Figure 9.6 Vardenafil dihydrochloride shows a m/z 88.9 [M H] molecular ion peak
in positive mode.
O
N
O HN
N N
S N
N
O
O
Exact mass: 488.2206
m/z 388.9
MS2
O O
N
NH2
N O N H
N O HN
N O HN
N
S N S N S N
N N N
O O H2O
O O O
m/z 461.9 m/z 419.7 m/z 377.7
O O
N
NH2
N O HN HN
N
S N
NH HS N N
O N N
O O
O
m/z 339.7 m/z 329.7 m/z 312.8
N N O
O O N S
N N S
S S O
O
O O O
O
O O
m/z 298.6 m/z 283.6 m/z 228.5 m/z 168.7
Scheme 9.6 MS2 scan for the vardenafil dihydrochloride at m/z 488.9.
The MS/MS/MS scan of the fragment m/z 377.9 gave number of peaks
including m/z 350.9, 312.7, 398.7, 280.6, 268.5, and 209.7. The fragments
are shown in Scheme 9.7.
4. METHODS OF ANALYSIS
4.1. Chromatographic methods
Literature surveys have revealed several chromatographic methods for de-
termination of vardenafil in bulk drug and biological samples published
by Kumar et al. [30], Subba Rao et al. [31], Carlucci et al. [32]
Zhang et al. [33], Gratz et al. [34], Zou et al. [35], Di et al. [36] Bartosova
et al. [37], Cheng et al. [38], Ku et al. [39], Lake et al. [40], Zhu
et al. [41], Zhang et al. [42], and Man et al. [43].
Vardenafil Dihydrochloride 529
O HN
N
S N
N
H2O
O
m/z 377.7
MS3
O O O
NH2 HN HN
O N N N
N N N
S N N N
N N N
HO
O O O O
O O
O NH
O HN HN
N S
S N N NH
N N
HO
O
O O
m/z 209.7
m/z 280.6 m/z 268.5
Scheme 9.7 Possible fragments for the MS/MS/MS scan of vardenafil dihydrochloride.
by Zou et al. [35] to screen for the presence of synthetic PDE-5 inhibitors,
including sildenafil, vardenafil, tadalafil, homosildenafil, acetildenafil, and
hydroxyhomosildenafil. The methods were applied to premarket samples
submitted to the Health Sciences Authority of Singapore for testing. One
sample was in the form of capsules while six other samples were premixed
bulk powder samples for dietary supplements to be repackaged or formu-
lated into the final dosage forms (usually capsules). Identification of
PDE-5 inhibitors was achieved by comparing individual peak retention
times (tR), UV spectra, and mass spectra with those of reference standards.
The seven samples were found to contain at least one of the following com-
pounds: sildenafil, vardenafil, hydroxyhomosildenafil, homosildenafil, and
acetildenafil. The five compounds were simultaneously determined by
LCESIMS/MS in multiple reactions monitoring (MRM) scan mode.
The method has been validated for accuracy, precision, linearity, and
sensitivity.
5. PHARMACOLOGY OF VARDENAFIL
5.1. Pharmacodynamics
5.1.1 An overview
Vardenafil (VAR) is a safe and well-tolerated treatment of men with ED. It is
a highly potent and selective inhibitor of PDE-5, the most prevalent phos-
phodiesterase in the human penile corpus cavernosum, and thus increases
intracellular cGMP levels in the cavernosum tissue of the penis [7]. It was
the first second-generation PDE-5 inhibitor that received marketing
approval in the United States. Unlike the other PDE-5 inhibitors, sildenafil
and tadalafil, VAR was developed from the outset specifically for use as an
erectogenic agent [46]. It is 510 times more potent than sildenafil, the clas-
sic PDE-5 inhibitor [2]. VAR has been shown to be efficacious in the treat-
ment of ED in the doses of 10 and 20 mg taken on demand, prior to
intercourse [47]. For most patients, the recommended starting dose is
10 mg, which according to US labeling information should be taken
approximately 60 min prior to sexual activity [46]. Adverse effects associated
with VAR are not severe, mostly dose dependent and tend to decrease with
time. These include headache, flushing, dyspepsia, and rhinitis [8,48].
active PDE involved in the termination of cGMP signaling in the penile cor-
pus cavernosum, the erectile tissue in the penis. This, in turn, potentiates
endogenous increases in cGMP levels in the corpus cavernosum and the ves-
sels supplying it, thus increasing dilatation of the corporeal sinusoids all-
owing more blood flow, which induces an erection. Interestingly, this
occurs only in the presence of nitric oxide release with sexual arousal. Sexual
stimulation causes the release of nitric oxide (NO) from neurons and endo-
thelial cells in the corpus cavernosum. NO, in turn, activates the enzyme
guanylyl cyclase, with the resultant conversion of guanosine triphosphate
to cGMP. This results in activation of cGMP-dependent protein kinase,
phosphorylation of several proteins and reduction of intracellular calcium
levels, and a consequent smooth muscle relaxation and an increased arterial
blood flow leading to enlargement of the corpus cavernosum. Because of the
increased tumescence, veins are compressed between the corpus
cavernosum and the tunica albuginea, leading to an erection [8,4953].
Notably, VAR has no effect on NO release and is, thus, ineffective in caus-
ing erection in the absence of sexual arousal [54].
5.1.3 Efficacy and safety of VAR for the treatment of erectile dysfunction
5.1.3.1 Efficacy of VAR for the treatment of erectile dysfunction
VAR has been reported to be highly effective in the treatment of ED in the
broad population at doses of 10 and 20 mg taken in an on-demand fash-
ion [55]. Efficacy and tolerability of VAR have been frequently reported.
Rosen et al. [56] have reported that VAR was clearly efficacious in treating
patients with mixed ED etiologies, achieving a high response rate with sig-
nificant improvement in the scores measuring sexual function and satisfac-
tion. The same study has also shown that VAR was safe and well tolerated
with few patients reporting adverse events. These data have been supported
by the recent findings of Tan et al. [57], Rosen et al. [58], and Porst et al. [59]
who demonstrated that VAR was a highly effective and very well-tolerated
treatment for patients with ED. Several clinical studies have shown that
VAR is effective in men with ED originating from various underlying
organic causes and severities, including traditionally difficult-to-treat men
with diabetes or a history of radical prostatectomy [6062]. Further, Porst
et al. [60] have confirmed the efficacy of VAR in ED regardless of organic,
psychogenic, or mixed causes, the baseline severity of the condition and
patient age.
With regard to potency, VAR has been shown to be the most potent and
specific of the commercially available PDE-5 inhibitors [54]. Potency can be
538 Abdelkader E. Ashour et al.
Nitrate preparations are commonly prescribed for the prevention and treat-
ment of angina pectoris, and the actions of these drugs are terminated by
cGMP hydrolysis in blood vessels [64]. Thus, PDE-5 inhibitors, including
VAR, could increase the effects of the nitrates, and this may result in severe
vasodilatation and hypotension. In fact, the patient information sheet for
VAR states that in 18 healthy subjects pretreated with VAR 20 mg, there
was an additional reduction in blood pressure and increase in heart rate with
nitroglycerin administration, and it is recommended that nitrate preparations
not be taken until at least 24 h after VAR, due to significant, potentially life-
threatening hypotension [64,70,71]. Due to its effect on Q-T interval, VAR
is also not recommended in patients taking type 1A (such as quinidine and
procainamide) or type 3 antiarrhythmics (such as amiodarone and sotalol) or
in patients with congenitally prolonged Q-T syndrome [70,72]. In addition,
a-adrenergic receptor blockers, such as doxazosin, should only be combined
with PDE-5 inhibitors, including VAR, with special caution and close mon-
itoring of blood pressure [71].
5.2. Pharmacokinetics
5.2.1 An overview
In addition to pharmacodynamic properties discussed above, pharmacoki-
netic properties of VAR (ingestion/food interaction, movement in the cir-
culation, tissue uptake, elimination) have great impact on efficacy. In this
context, Klotz et al. [73] have studied the pharmacokinetic and pharmaco-
dynamic properties of VAR in 21 ED patients. The results showed that sin-
gle doses of 10 and 20 mg VAR led to a rapid rise in the plasma
concentrations of VAR, with a tmax (the time required to achieve maximum
plasma concentration) of 0.9 and 0.7 h and a mean Cmax (the maximum
plasma concentration) of 9.1 and 20.9 ng/mL, respectively. In the post-
absorptive phase, the concentrations declined with an average half-life of
4.2 and 3.9 h, respectively. VAR is extensively metabolized by CYP3A4
and to a small extent by CYP3A5 and CYP2C isoforms into more than
14 metabolites. The major metabolite, N-desethyl VAR (M1), is pharma-
cologically active. It has 28% of VARs potency for PDE-5 inhibition
and its contribution to the overall VAR activity is 7%. The elimination
half-life of VAR and its major metabolite M1 is about 45 h and indepen-
dent of the dose. VAR is primarily excreted as metabolites in the feces and to
a small extent in urine. Only 1% of the administered VAR dose is excreted
into urine in an unchanged form [8,46].
540 Abdelkader E. Ashour et al.
ACKNOWLEDGMENT
This work was supported by a grant from the National Plan of Science, Technology, and
Innovation (Grant No. 10-MED1188-02) King Saud University, Riyadh, Saudi Arabia.
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Vardenafil Dihydrochloride 543
R T
Ranitidine, 15, 533 Tadalafil, 36, 287
Reserpine, 4, 384; 5, 557; 13, 737 Talc, 23, 517
Riboflavin, 19, 429 Teniposide, 19, 575
Rifampin, 5, 467 Tenoxicam, 22, 431
Risperidone, 37, 313 Terazosin, 20, 693
Rocuronium bromide, 35, 285 Terbutaline sulfate, 19, 601
Rutin, 12, 623 Terfenadine, 19, 627
Terpin hydrate, 14, 273
Testolactone, 5, 533
S Testosterone enanthate, 4, 452
Saccharin, 13, 487 Tetracaine hydrochloride, 18, 379
Salbutamol, 10, 665 Tetracycline hydrochloride, 13, 597
Salicylamide, 13, 521 Theophylline, 4, 466
Salicylic acid, 23, 427 Thiabendazole, 16, 611
Scopolamine hydrobromide, 19, 477 Thiamine hydrochloride, 18, 413
Secobarbital sodium, 1, 343 Thiamphenicol, 22, 461
Sertraline hydrochloride, 24, 443 Thiopental sodium, 21, 535
Sertraline lactate, 30, 185 Thioridazine, 18, 459
Sildenafil citrate, 27, 339 Thioridazine hydrochloride, 18, 459
Silver sulfadiazine, 13, 553 Thiostrepton, 7, 423
Simvastatin, 22, 359 Thiothixene, 18, 527
Sodium nitroprusside, 6, 487; 15, 781 Ticlopidine hydrochloride, 21, 573
Sodium valproate, 32, 209 Timolol maleate, 16, 641
Solasodine, 24, 487 Titanium dioxide, 21, 659
Sorbitol, 26, 459 Tobramycin, 24, 579
Sotalol, 21, 501 a-Tocopheryl acetate, 3, 111
Spironolactone, 4, 431; 29, 261 Tolazamide, 22, 489
Starch, 24, 523 Tolbutamide, 3, 513; 5, 557; 13, 719
Streptomycin, 16, 507 Tolnaftate, 23, 549
Strychnine, 15, 563 Tramadol hydrochloride, 38, 463
Succinycholine chloride, 10, 691 Tranylcypromine sulfate, 25, 501
Sucralose, 38, 423 Trazodone hydrochloride, 16, 693
Sulfacetamide, 23, 477 Triamcinolone, 1, 367; 2, 571; 4, 521;
Sulfadiazine, 11, 523 11, 593
Sulfadoxine, 17, 571 Triamcinolone acetonide, 1, 397; 2, 571;
Sulfamethazine, 7, 401 4, 521; 7, 501; 11, 615
Sulfamethoxazole, 2, 467; 4, 521 Triamcinolone diacetate, 1, 423;
Sulfasalazine, 5, 515 11, 651
Sulfathiazole, 22, 389 Triamcinolone hexacetonide, 6, 579
Sulfisoxazole, 2, 487 Triamterene, 23, 579
Sulfoxone sodium, 19, 553 Triclobisonium chloride, 2, 507
Sulindac, 13, 573 Trifluoperazine hydrochloride, 9, 543
Sulphamerazine, 6, 515 Triflupromazine hydrochloride, 2, 523;
Sulpiride, 17, 607 4, 521; 5, 557
Sunitinib malate, 37, 363 Trimethaphan camsylate, 3, 545
Cumulative Index 551