www.ijpsonline.com
Hyderabad, for supplying gift samples of drotaverine
hydrochloride and paracetamol and the Principal, Dr.
D. Y. Patil College of Pharmacy, Pune, for providing
excellent research facilities.
REFERENCES
1.
2.
3.
4.
5.
Budavari S. The Merck Index. 13th ed. Whitehouse Station, NJ: Merck
and Co Inc; 2003.
Sweetman SC. Martindale, The Complete Drug reference. 33rd ed.
London: Pharmaceutical press; 2002.
Mahajan VK, Dahivelkar PP, Fursule RA, Shirkhedkar AA, Surana SJ.
Specrophotometric method for estimation of Drotaverine HCl. Indian
Drugs 2006;43:656-9.
Jameelunnisa B, Abdul R. Specrophotometric determination of
Drotaverine in tablets. Asian J Chem 2008;20:4173-84.
Bolaji O, Onyeji CO, Ogungbamila FO. A HPLC method is developed
for the determination of drotaverine HCl in human plasma and urine. J
Chromatography Biomed App 1993;622:93-7.
6.
Lalla KJ, Shah MU, Jain MB, Sharma AH. Modified HPLC method
for analysis of drotaverine in human plasma. J Pharm Biomed Anal
1993;11:385-8.
7. Indian Pharmacopoeia, Vol. 1, New Delhi: The Controller of
Publications; 1996. p. 554.
8. British Pharmacopoeia, Vol. 1, 4th ed. London: Her Majesty Stationary
Office; 2004. p. 36-8.
9. United States Pharmacopoeia, 26th ed. Rockville, MD: United States
Pharmacopoeial Convention; 2003. p. 16.
10. Chan CC. Analytical method validation and instrument performance
verification. Malden USA: Wiley Interscience; 2004. p. 16-22.
11. Validation of analytical procedure: methodology Q2B, ICH Harmonized
Tripartite Guidelines; 1996. p. 1-8.
Accepted 15 January 2010
Revised 30 September 2009
Received 5 May 2009
Indian J. Pharm. Sci., 2010, 72 (1): 133-136
Spectrophotometric and HPLC Methods for Simultaneous
Estimation of Amlodipine Besilate, Losartan Potassium
and Hydrochlorothiazide in Tablets
S. B. WANKHEDE*, K. C. RAKA, S. B. WADKAR AND S. S. CHITLANGE
Department of Pharmaceutical Chemistry, Padmashree Dr. D. Y. Patil Institute of Pharmaceutical Sciences and
Research, Sant Tukaram Nagar, Pimpri, Pune-018, India
Wankhede et al.: Simultaneous Estimation of Amlodipine, Losartan and Hydrochlorthiazide
Two UV-spectrophotometric and one reverse phase high performance liquid chromatography methods have been
developed for the simultaneous estimation of amlodipine besilate, losartan potassium and hydrochlorothiazide in
tablet dosage form. The first UV spectrophotometric method was a determination using the simultaneous equation
method at 236.5, 254 and 271 nm over the concentration range 5-25, 10-50 and 5-25 µg/ml for amlodipine
besilate, losartan potassium and hydrochlorothiazide, respectively. The second UV method was a determination
using the area under curve method at 231.5-241.5, 249-259 and 266-276 nm over the concentration range of
5-25, 5-25 and 10-50 µg/ml for amlodipine besilate, hydrochlorothiazide and losartan potassium, respectively.
In reverse phase high performance liquid chromatography analysis is carried out using 0.025 M phosphate
buffer (pH 3.7):acetonitrile (57:43 v/v) as the mobile phase and Kromasil C18 (4.6 mm i.d×250 mm) column as
stationery phase with detection wavelength of 232 nm linearity was obtained in the concentration range of 2-14,
20-140 and 5-40 µg/ml for amlodipine besilate, losartan potassium and hydrochlorothiazide, respectively. Both
UV-spectrophotometric and reverse phase high performance liquid chromatography methods were statistically
validated and can be used for analysis of combined dose tablet formulation containing amlodipine besilate, losartan
potassium and hydrochlorothiazide.
Key words: Amlodipine besilate, area under curve method, hydrochlorothiazide, losartan potassium, reverse phase
high performance liquid chromatography, simultaneous equation method, area under curve method
*Address for correspondence
E-mail: sagar2277@rediffmail.com
136
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January - February 2010
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Amlodipine besilate (AMLO), chemically is
[3-ethyl-5-methyl(4RS)-2-[(2-aminoethoxy)methyl]4-(2-chlorophenyl)-methyl-1-dihydropyridine-3,5dicarboxylate benzenesulfonate[1]. It is a long acting
calcium channel blocker used as an antihypertensive
agent. Losartan potassium (LOS), chemically, is
2-butyl-4-chloro-1-[p-(o-1H-tetrazol-5-ylphenyl)
benzyl]imidazole-5-methanol monopotassium [2]
salt. It is an angiotensin II receptor blocker and
chemically is used as an antihypertensive agent.
Hydrochlorothiazide (HCTZ), 6-chloro-3,4-dihydro2H-1,2,4-benzothiadiazine-7-sulfonamide[3] is used as
a diuretic. AMLO is official in BP, LOS is official
in IP and USP, whereas HCTZ is official in BP and
IP. These three drugs are marketed as combined
dose tablet formulation in the ratio of 05:12.5:50
mg (AMLO:HCTZ:LOS). Literature survey revealed
that a number of methods have been reported for
estimation of AMLO, LOS and HCTZ individually
or in combination with other drugs [4-18]. However,
there is no analytical method reported for the
simultaneous estimation of these drugs in a combined
dosage formulation. Present work describes rapid,
accurate, reproducible, and economical methods
for simultaneous estimation of these drugs in tablet
formulation. For UV-spectrophotometric method
double-beam Shimadzu UV/Vis spectrophotometer,
1700 Pharmaspec, with spectral bandwidth of 2
nm, wavelength accuracy of ±0.5 nm and a pair
of 1-cm matched quartz cells, was used. For high
performance liquid chromatographic method Merck
Hitachi with L-7100 double reciprocating pump,
L-7400 UV detector with Winchrom software for
data processing was used. Standard gift sample of
AMLO was received from Emcure Pharmaceuticals
Ltd, Pune, India, LOS as gift sample as from Cipla
Ltd, Patalganga, India and HCTZ from Unichem
Laboratories, Baddi, India. Combined dose tablet
formulation Trilopace, of Sun Pharmaceutical
Industries, Dadra, India, containing AMLO (5 mg),
LOS (12.5 mg) and HCTZ (50 mg) was purchased
from a local pharmacy Store. Methanol used for
UV-spectrophotometric method was of AR grade.
Acetonitrile (Universal Lab., Mumbai) and other
chemicals (Research Lab., Mumbai) used for
preparation of buffer solution in RP-HPLC were of
HPLC grade, were procured from the local market.
In the UV-spectrophotometric methods, simultaneous
equation method (method A), standard stock solutions
of AMLO (100 µg/ml), HCTZ (100 µg/ml) and
January - February 2010
LOS (100 µg/ml) were prepared in methanol. For
the selection of analytical wavelength solutions of
AMLO (2 µg/ml), HCTZ (5 µg/ml) and LOS (20
µg/ml) were prepared separately by appropriate
dilution of standard stock solution and scanned in
the spectrum mode from 200 to 400 nm. From the
overlain spectra of these drugs (fig. 1), wavelengths
236.5 nm (λmax of AMLO), 254 nm (λmax of LOS) and
271 nm (λmax of HCTZ) were selected for analysis.
The calibration curves for AMLO, HCTZ and LOS
were prepared in the concentration range of 5-25
µg/ml, 5-25 µg/ml and 10-50 µg/ml, respectively at
the selected wavelengths. Absorptivity values were
determined for AMLO, HCTZ and LOS and were
found to be 32.20/13.36/3.09, 10.86/19.07/61.14 and
37.24/26.55/12.74 at 236.5/254/271 nm, respectively.
Using these absorptivity values following Eqns.
were developed for determining concentration of
AMLO, HCTZ and LOS in tablet sample solution.
A 1 = 32.20C AMLO +10.86C HCTZ +37.24C LOS (1) ,A =
13.36C AMLO +19.07C HCTZ +26.55C LOS (2) and A 3 =
3.09CAMLO+61.14CHCTZ+12.74CLOS (3), where A1, A2
and A3 are absorbance of the tablet sample solution
at 236.5, 254 and 271 nm, respectively. CAMLO is the
concentration of AMLO, CHCTZ is the concentration of
the HCTZ, and CLOS is the concentration of the LOS.
For estimating AMLO, HCTZ and LOS in tablet
formulation, twenty tablets were weighed and average
weight was calculated. The tablets were crushed to
obtain fine powder. Tablet powder equivalent to 75
mg of LOS was transferred to 50.0 ml volumetric
flask added 30 ml of methanol, sonicated for 20
min and volume was made up to the mark with
methanol. The solution was then filtered through
Whatmann filter paper No. 41. The filtrate was
appropriately diluted with the same solvent to obtain
Fig. 1: Overlain spectra of AMLO, HCTZ and LOS.
AMLO is amlodipine besilate, LOS is losartan potassium and HCTZ
is hydrochlorothiazide
Indian Journal of Pharmaceutical Sciences
137
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final concentration within Beer Lambert’s range for
each drug. Absorbance of diluted sample solution was
measured at selected wavelengths. The concentration
of drugs was determined by using the Eqns 1, 2 and
3. Results of analysis of tablet formulation mentioned
in Table 1.
In the area under curve method (method B),
preparation of standard stock solution was same as
mentioned in method A. From the overlain spectra
of drugs (fig. 1) wavelengths range 231.5-241.5 nm
(AMLO), 266-276 nm (HCTZ) and 249-259 nm
(LOS) were selected for the analysis. The calibration
curves for AMLO, HCTZ and LOS were prepared
in the concentration range as mentioned in method
A at the selected wavelength range. Absorptivity
values were determined for AMLO, HCTZ and
LOS and were found to be 313.28/374.37/30.96,
153.25/200.57/581.14 and 374.37/264.49/123.96
at 231.5-241.5/266-276/249-259 nm, respectively.
Using these absorptivity values following equations
were developed for determining concentration of
AMLO, HCTZ and LOS in tablet sample solution.
A 1 = 313.28C AMLO +153.25C HCTZ +374.37C LOS (4),
A 2 = 374.37C AMLO +200.57C HCTZ +264.49C LOS (5)
and A3= 30.96CAMLO+581.14CHCTZ +123.96CLOS (6),
where A 1 , A 2 and A 3 are area under curve of the
sample at 231.5-241.5, 266-276 and 249-259 nm,
respectively, CAMLO is the concentration of AMLO,
CHCTZ is the concentration of the HCTZ, and CLOS is
the concentration of the LOS. Preparation of sample
solution for analysis of tablet formulation was same
as described under method A. The concentrations of
AMLO, HCTZ and LOS were determined by using
the Eqns. 4, 5 and 6. Result of analysis of tablet
formulation are mentioned in Table 1.
In the reverse phase high performance liquid
chromatography (method C), following optimum
conditions were established for quantitative analysis
of AMLO, HCTZ and LOS in tablet formulation
by trial and error. Mobile phase: a mixture of
phosphate buffer (0.025M, pH-3.7 adjusted with
ortho phosphoric acid) and acetonitrile in the ratio of
(57:43 v/v), column: Kromasil C18 (4.6 mm i.d×250
mm), flow rate: 1.0 ml/min for 6.3 min then 1.3 ml/
min from 6.3 min onwards, detection wavelength: 232
nm, temperature: room temperature. Sample solution
of AMLO, HCTZ and LOS 6 µg/ml, 15 µg/ml and
60 µg/ml, respectively were prepared in mobile phase
and chromatographed under optimum chromatographic
conditions. The proposed chromatographic conditions
were found suitable for effective separation and
quantitation of AMLO (RT-5.12 min), HCTZ (RT-
TABLE 1: ANALYSIS OF TABLET FORMULATION
Method
A
B
C
Component
AMLO
HCTZ
LOS
AMLO
HCTZ
LOS
AMLO
HCTZ
LOS
Label Claim (mg/tab)
05
12.5
50
05
12.5
50
05
12.5
50
Amount Found
5.02
12.51
50.05
5.03
12.52
49.98
4.97
12.498
50.05
% Label Claim*
100.40
100.08
100.10
100.60
100.16
99.96
100.21
99.98
99.35
SD
0.8042
0.1824
0.1189
0.8672
0.1237
0.0738
0.8810
1.0073
0.9165
CV
0.8012
0.1823
0.1188
0.8620
0.1235
0.0738
0.8791
1.0075
0.9225
AMLO is amlodipine besilate, LOS is losartan potassium and HCTZ is hydrochlorothiazide *Average of six determinations, SD - Standard Deviation, CV- Coefficient
of Variation.
TABLE 2: RESULTS OF RECOVERY STUDIES
Methods
A
B
C
Level of %
recovery
80
100
120
80
100
120
80
100
120
AMLO
99.78
101.16
100.05
99.35
99.51
99.33
100.37
100.71
99.47
% Recovery*
HCTZ
100.75
100.74
100.45
100.14
100.11
99.92
100.51
100.58
99.85
LOS
99.81
99.68
99.91
99.97
100.05
100.05
100.40
100.07
99.73
Standard Deviation
AMLO
HCTZ
LOS
0.5869
0.2758
0.0070
0.9122
0.3536
0.2192
0.2192
0.0212
0.0566
0.2121
0.0778
0.1768
0.0919
0.0212
0.0707
0.1061
0.0989
0.0070
0.5515
0.2051
0.4243
0.0212
0.3182
0.1485
0.2263
0.0070
0.2828
Relative standard deviation
AMLO
HCTZ
LOS
0.5882
0.2737
0.0070
0.3510
0.2199
0.9017
0.0567
0.2191
0.0211
0.1780
0.2118
0.0778
0.0710
0.0918
0.0212
0.1068
0.0989
0.0070
0.2041
0.4226
0.5495
0.0211
0.3164
0.1484
0.2275
0.0070
0.2836
AMLO is amlodipine besilate, LOS is losartan potassium and HCTZ is hydrochlorothiazide, *Average of three determinations, SD - Standard Deviation,
CV- Coefficient of Variation.
138
Indian Journal of Pharmaceutical Sciences
January - February 2010
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Fig. 2: Typical chromatogram of standard solution of AMLO, HCTZ
and LOS.
AMLO is amlodipine besilate, LOS is losartan potassium and HCTZ
is hydrochlorothiazide
3.42 min) and LOS (8.02 min) with resolution of
2.32 (between HCTZ and AMLO) and 7.91 (between
AMLO and LOS), tailing factor- 1.5 for AMLO,
1.33 for HCTZ and 1.05 for LOS. The calibration
curves (mean peak area Vs concentration) for AMLO,
HCTZ and LOS were prepared in the concentration
range of 2-14 µg/ml, 5-40 µg/ml and 20-140 µg/ml,
respectively at 232 nm. For the estimation of these
drugs in the tablet formulations, twenty tablets were
weighed and average weight was calculated. The
tablets were crushed to obtain fine powder. Tablet
powder equivalent to 50 mg of LOS was transferred
to 50.0 ml volumetric flask added 30 ml mobile phase
and ultrasonicated for 20 min, volume was then made
up to the mark with mobile phase. The solution was
then filtered through a Whatmann filter paper No.
41. The filtrate was appropriately diluted with the
mobile phase to obtain final concentration within
linearity range for each drug in the ratio 60:15:6 μg/
ml LOS: HCTZ: AMLO, respectively. The contents
were mixed thoroughly and filtered through a 0.2 μ
filter. Twenty microlitres of solution was injected and
chromatographed under optimum chromatographic
condition. A typical chromatogram of AMLO, LOS
and HCTZ is shown in (fig. 2). The concentration of
AMLO, HCTZ and LOS in tablet sample solution was
determined by comparing the peak area of the sample
with that of standard at 232 nm. Results of analysis
tablet formulation are shown in Table 1.
80%, 100% and 120%. The results of recovery
studies, expressed as % recovery, are mentioned
in Table 2. Robustness of the proposed methods
was studied by analyzing tablet formulation under
varied conditions like different analysts, on different
days and different times on same day. The standard
deviation for analysis under different conditions
was below 2% indicating robustness of method.
Ruggedness of the reverse phase high performance
liquid chromatography method was studied by
deliberately changing the method parameters viz.
change in mobile phase composition (±1 ml) and
change in flow rate (±0.1 ml/min). Reverse phase
high performance liquid chromatography method was
found to withstand these variations as there was no
significant change in retention time, tailing factor and
resolution.
Based on the results obtained, it is found that the
proposed UV-Spectrophotometric and reverse phase
high performance liquid chromatography methods are
accurate, precise, reproducible economical and can be
employed for routine analysis of AMLO, LOS and
HCTZ in combined dose tablet formulation.
ACKNOWLEDGEMENTS
The authors thank Dr. Avinash D. Deshpande,
Director of Pharmacy, Pad. Dr. D. Y. Patil Institute of
Pharmaceutical Sciences and Research, Pimpri, Pune
for providing necessary facilities. The authors also
thank Emcure Pharmaceuticals Ltd., Pune; Unichem
laboratories, Baddi; and Cipla Ltd, Patalganga for
providing gift samples of drugs.
REFERENCES
1.
2.
3.
4.
5.
Accuracy of the proposed UV-spectrophotometry and
reverse phase high performance liquid chromatography
method was studied by performing recovery studies
by standard addition method at three different levels
January - February 2010
6.
7.
British Pharmacopoeia, Vol. 1, London: Her Majesty's Stationary Office;
2008. p.137, 1080.
The United States Pharmacopoeia Drug Information, Vol. 1, 18th ed.
Maryland: The United States Pharmacopoeia Convention; 1998. p.
1893-6.
Indian Pharmacopoeia, New Delhi: Government of India, The
Controller of Publication; 2007. p. 1194-5.
Erk N. Application of first derivative UV-spectrophotometry and
ratio derivatives pectrophotometry for simultaneous determination of
Candesartan cilexetil and Hydrochlorothiazide. Pharmazie 2003;58:796800.
Charles J, Brault J, Boyer S, Langlois C, Cabrero MS, Dubost L.
Simultaneous determination of Irbesartan and Hydrochlorothiazide in
tablets by derivative spectrophotometry. Anal Lett 2003;36:2485-95.
Prabhakar AH, Giridhar R. A rapid colorimetric method for the
determination of Losartan potassium in bulk and in synthetic mixture
for solid dosage form. J Pharm Biomed Anal 2002;27:861-6.
Satana E, Altinay S, Goger NG, Ozkan SA, Senturk Z. Simultaneous
Indian Journal of Pharmaceutical Sciences
139
www.ijpsonline.com
8.
9.
10.
11.
12.
13.
determination of Valsartan and Hydrochlorothiazide in tablets by first
order UV spectrophotometry and Liquid chromatography. J Pharm
Biomed Anal 2001;25:1009-13.
Lande NR, Shektar BM, Kadam SS, Dhaneshwar SR. Simultaneous
spectrophotometric estimation of Losartan potassium and
Hydrochlorothiazide in tablet dasage form. Indian drugs 2000;37:57781.
Dinc E, Ustundag O. A new application of chemometric technique to
HPLC data for the simultaneous analysis of two compound mixture. J
Liq Chrmatogr Relat Technol 2005;28:2179-94.
Ulu ST, Saglik S. Comparison of UV and Second derivative
spectrophotometric and HPLC methods for the determination of
Losartan in tablets. Turk J Pharm Sci 2004;1:165-75.
Dinc E, Ustundag O. Application of multivariate calibration
techniques to HPLC data for quantitative analysis of binary mixture
of Hydrochlorothiazide and Losartan in tablet. Chromatographia
2005;61:237-44.
Hertzog DL, McCafferty JF, Fang XG, Tyrell RJ, Reed RA.
Development and validation of stability indicating HPLC method for
simultaneous determination of Losartan Potassium, Hydrochlorothiazide
and their degradation product. J Pharm Biomed Anal 2002;30:747-60.
Sivakumar T, Venkatesan P, Manavalan R, Valliappan K. Development
of a HPLC method for the simultaneous determination of Losartan
potassium and Atenolol in tablets, Indian J Pharm Sci 2007;69:154-57.
14. Vora DN, Kadav AA. Development and validation of a simultaneous
HPLC method for estimation of bisoprolol fumarate and amlodipine
besilate from tablets. Indian J Pharm Sci 2008;70:542-46.
15. Sahu R, Patel VB. Simultaneous spectrophotometric determination of
amlodipine besilate and atorvastatin calcium in binary mixture, Indian
J Pharm Sci 2007;69:110-1.
16. Naidu KR, Kale UN, Shingare MS. Stability indicating RP-HPLC
method for simulatneus determination of amlodipine and benzapril
hydrochloride from their combination drug product. J Pharm Biomed
Anal 2005;39:147-55.
17. Gohil K, Trivedi P, Molvi KI. Spectrophotometric analysis of
amlodipine besilate in bulk and in tablet dosage forms. Indian J Pharma
Sci 2005;67:376-8.
18. Topale PR, Gaikwad NJ, Tajane MR. Simultaneous UVspectrophotometric estimation of losarten potassium and amlodipine in
tablet. Indian Drugs 2003;40:119-21.
Accepted 17 January 2010
Revised 14 October 2009
Received 18 July 2009
Indian J. Pharm. Sci., 2010, 72 (1): 136-140
Antioxidant Activities of Some Cameroonian Plants
Extracts Used in the Treatment of Intestinal and
Infectious Diseases
J. MOMENI*, W. P. DJIALEU NTCHATCHOUA1, FADIMATOU, M. T. AKAM2 AND M. B. NGASSOUM1
Department of Chemistry, Faculty of Science, University of Ngaoundere, P.O. Box 454, Ngaoundere, Cameroon;
1
Department of Applied Chemistry, National Advanced School of Agro-Industrial Sciences (ENSAI), University of
Ngaoundere, P.O. Box 455, Ngaoundere, Cameroon; 2Department of Chemistry, Faculty of Science, University of
Buea, P. O. Box 63 Buea, Cameroon
Momeni et al.: Antioxidant Activities of Some Cameroonian Plants Extracts
Antioxidant activity test using two different methods namely 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azinobis(3ethylbenzothialozinesulfonate) diammonium salt free radical scavenging test has been carried out on three
Cameroonian plant extracts used in the treatment of intestinal and infectious diseases: Pittosporum mannii Hook
f. (Pittosporaceae), Vepris heterophylla R. Letouzey (Rutaceae) and Ricinodendron heudelotii (Baill) Pierre ex Pax
(Euphorbiaceae). Results of this study in the 2,2-diphenyl-1-picrylhydrazyl scavenging test show that the ethyl
acetate extract of P. mannii and the methanol extract of V. heterophylla exhibit high free radical scavenging activities
with IC50 values of 177.74 and 204.69 µg/ml, respectively while the methanol/dichloromethane (1+1) extract of R.
heudelotii showed weak free radical scavenging activities as compared to Trolox (939.19 µg/ml) used as standard.
In the same manner, 2,2′-azinobis(3-ethylbenzothialozinesulfonate) diammonium salt radical scavenging test of
these extracts was in accordance of the result of 2,2-diphenyl-1-picrylhydrazyl test. The antioxidant properties of
these extracts probably explain partly, the use of these plants in traditional medicine for the treatment of infectious
diseases and inflammations.
Key words: Antioxidant activity, DPPH and ABTS radical, Pittosporum mannii, Ricinodendron heudelotii, Vepris
heterophylla
*Address for correspondence
E-mail: momenj_j@yahoo.com
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