CHAPTER 2 MATERIALS AND METHODS

2.1 INSTRUMENTATION

Table 2.1: List of instruments and equipment’s

Instrument Make & Model

HPLC system Shimadzu SPD-M20A, Prominence Diode array

detector, Software- LC-Solution, Japan
pH meter Equip-Tronics pH meter, EQ-610, Equip
Sonicator UCB40 spectra Lab, Mumbai
UV-Vis spectrophotometer V630 Jasco, Japan
Electronic balance CB-50 Contech, Navi Mumbai
Distillation Unit Lab Sil Instrument, Bangalore
Hot air oven 250BSS, Pathak, Mumbai
Constant temperature water Intex, FRNW-326
bath

2.2 CHEMICAL AND REAGENT

1) Acetonitrile (HPLC grade), Research Lab., Mumbai, India

2) Methanol (HPLC grade), Research Lab., Mumbai, India

3) Ammonium Acetate (AR grade), Research Lab., Mumbai, India

4) Hydrochloric acid (AR grade), Qualigens fine chemicals, Mahape, India

5) Sodium Hydroxide(AR grade), Research Lab., Mumbai, India

6) Hydrogen peroxide (LR grade) S.D. Fine-Chem Ltd., Mumbai, India

7) Glacial acetic acid (AR grade), Research Lab., Mumbai, India

Double Distilled Water (HPLC grade), double distilled, Lab sil Instrument,
Bangalore, India

2.2.1 CONSUMABLES

Column - Kromasil C18 (150 x 4.6 mm, 5 µm)

Filter paper - Pall India Pvt. Ltd. (0.22 µm)

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Methods
2.3 Characterization of drug by IR Spectroscopy
An accurately weighed drug (1 mg) was mixed with 100 mg KBr and kept in sample
holder. A spectrum was recorded in the range 400-4000 cm-1 .Wave numbers were
noted and correlated with functional groups present in the drug.

2.4 Preparation of analyte stock solution

An accurately weighed sample ( 10 mg ) of Tenofovir was transferred to a 10 mL
volumetric flask and dissolved in 5 mL methanol, it was shaken well. After drug was
dissolved, methanol was added upto the mark to make a solution of 1 mg mL -1. From
the stock solution, working dilutions of 10 - 100 µg mL -1 were made in 10mL
volumetric flasks by using methanol.

2.5 Selection of analytical wavelength

Stock solution was diluted by taking 0.1, 0.2, 0.3, 0.4, 0.5 mL to make solutions of 10-
50 µg/mL by using methanol and scanned over the range of 200-400 nm. ƛ max of
drug was noted.

2.6 Development and validation of RP-HPLC method for Tenofovir.

a) Screening and selection of mobile phase

Chromatographic separation studies were carried out on the working standard solution
of Tenofovir (100 μg mL-1). Initially,reversed phase analytical column (C18) was
tested by carrying out trials with methanol, water, acetonitrile, potassium dihydrogen
phosphate and ammonium acetate buffer in various proportions. When pure water
was tested, Tenofovir did not appear to dissolve completely and results in cloudy
solution. On the other hand, when 100% methanol was used, the compound dissolved
instantly. To optimize the chromatographic conditions, different combinations of
methanol– water (90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70), acetonitrile– water
(90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70), and methanol–ammonium acetate
(90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70) were tested.

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Based on the results obtained from screening studies and from literature survey,
Ammonium acetate buffer and methanol was used in various proportions, molarity
and pH to obtain the desired system suitability parameters.

b) Preparation of mobile phase ( 10 mM Ammonium acetate buffer & Methanol)

Ammonium acetate 0.038 g transferred to 500 mL Volumetric flask containing 250
mL double distilled water, degassing it and volume was made upto 500 mLwith
double distilled water. The pH of buffer was adjusted to 8.5 using Triethylamine. It
was filtered through 0.22 μ membrane filter paper and then sonicated on ultra sonic
bath for 10 min. Methanol was also filtered through 0.22 μ membrane filter paper and
then sonicated for 10 min.

c) SIAM Development:

The solubility of Tenofovir was studied in order to determine the proper ratio of
solvents used as mobile phase to the drug substance for analysis. Referring to
chemical structure, the compound is a base and is able to accept proton (s), therefore
polarity of the dissolved solvent will affect the solubility. The composition pH and
flow rate of the mobile phase were changed to optimize the separation conditions.
Increasing the organic modifier content resulted in a decrease in the retention time
(RT) of the drug. The ternary mixture was proved to be the appropriate among all
combinations, with a better defined and well resolved peak, free from tailing. In order
to get sharp peak and base line separation of the components, a number of
experiments were carried out by varying the pH of solvents in mobile phase and its
flow rate. The pH of the mobile phase will greatly affect its retention time as it
interacts with the stationary phase. The effect of pH on analyte elution was related to
the degree of ionization. Reducing the pH resulted in a shorter RT because of
ionization of its basic site. A pH of 8.5 was regarded as optimum because at this pH
the analyte peak was sharp and well-defined. Finally a mixture of Methanol:
Ammonium acetate buffer (60:40 % v/v) was at pH-8.5 adjusted with Triethylamine
is proved to be the best suitable mobile phase among all the tested combinations. The
chromatographic peaks obtained were better defined and resolved and almost free
from tailing.

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A minimum flow rate and minimum run time results the less usage of solvents. Flow
rate of the mobile phase was tested from 0.6-1.5 mL/min for optimum separation and
it was found that 1.0 mL/min flow rate was ideal for the successful elution of the
analyte.

2.7. Reagents preparation:

2.7.1 Preparation of 0.1N sodium hydroxide solution
Sodium hydroxide pellets (0.4 g) were dissolved in 50 mL of double distilled water in
100 mL volumetric flask, the volume was made up to 100 mL with water to get 0.1 N
sodium hydroxide solution. Other strengths such as 0.01 N and 0.001 N were prepared
by diluting the 0.1 N NaOH appropriately with water.

2.7.2 Preparation of 0. 1N hydrochloric acid solution

Hydrochloric acid (0.1N) was prepared by diluting 0.85 ml of concentrated
hydrochloric acid solution to 100 mL with double distilled water by using 100 ml
volumetric flask. Other strengths (0.01 N, 0.005 N) were prepared by diluting 0.1N
HCl with appropriate quantity of water.

2.7.3 Preparation of 15 % (v/v) hydrogen peroxide solution

Hydrogen peroxide (15%) was prepared separately by diluting 50 mL of 30% of
hydrogen peroxide to 100 mL with distilled water by using 100 mL volumetric flask.
Other strength (3%) was prepared similarly by diluting 30% H2O2.

2.8 Forced degradation studies:

Forced degradation studies were carried out to achieve 5-30 % degradation of the
drug. In present study, drug was exposed to Acid, Alkali, Oxidation Stress for
different time interval (0-24 h). The results were obtained by comparing samples
subjected to stress treatment with appropriate controls (Std. untreated). The %
degradation in each case was noted.

2.8.1 Hydrolytic Degradation

The hydrolytic degradations were carried out in acidic, alkaline and neutral
conditions. Samples were prepared by taking 1 mL of stock solution of drug (1000

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CHAPTER 2 MATERIALS AND METHODS

μg/mL) and 1mL of hydrolytic agent (0.1N HCl/ 0.01N HCl/ 0.005 N HCl / 0.1N
NaOH/ 0.01N NaOH/ 0.001N NaOH / water) in 10 mL volumetric flask. For few
conditions, samples were heated on constant temperature water bath at 80 0C for
definite time intervals. After required exposure, samples were neutralized by using
equal strength of acid or alkali which ever was required. Finally volume was made up
to the mark by using Methanol and subjected for HPLC analysis.

2.8.2 Oxidative Degradation

Oxidative degradation was carried out by using hydrogen peroxide. Samples were
prepared by taking 1 mL of stock solution of drug (1000 μg/mL) and 1ml of hydrogen
peroxide (3% or 15% or 30%) in 10 mL volumetric flask. After required exposure,
samples were diluted up to the mark by using Methanol and subjected for HPLC
analysis.

2.8.3 Photo Degradation

Photo degradation of drug was carried out in solid phase, drug (10 mg) was kept in
sealed ampoule and exposed to ICH recommended dose of light ( 1.2 million lux h
and 200 W h/m2 ) in photo stability chamber. After definite exposure time solution of
drug sample (100µg/mL) was prepared by using methanol and subjected to HPLC
analysis.

2.8.4 Thermal Degradation

Solid drug sample 10 mg sealed in glass ampoules and exposed to dry heat in hot air
oven for 110ºC for definite time interval and another was kept as control at Room
temperature. After required exposure two separate solutions were prepared by
weighing appropriate amount of samples exposed to thermal stress and control to
produce concentration of 100μg/mL. The samples were diluted up to the mark with
Methanol and injected separately into HPLC.

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2.9 Optimization of SIAM:

(A) Forced Degradation Studies:
The stress studies were optimised to get % degradation in desired range by using
Design Expert software 10 in which 32 factorial design was applied by considering
strength and time as independent factors and % degradation as response.

Table 2.2 : Stress conditions for % degradation

Sr.No Stress Strength (N) Time (h)
.
1. Acid (HCl) 0.1, 0.01, 0.005 1, 7, 24 h
2. Alkali (NaOH) 0.1, 0.01, 0.001 1, 7, 24 h
3. Oxidation (H2O2) 3%, 15%, 30% 1, 7, 24 h

32 full factorial design indicated following trials.

Following trials were suggested by the software in different stress conditions
(acid, alkali, oxidation)

1) Acid Stress (HCl)

Table 2.3: Trials of stress studies in acidic medium (HCl) suggested by software
Run Factor 1 : Strength(N) Factor 2 : Time (h)

1 0.005 7

2 0.01 24

3 0.01 7

4 0.1 7

5 0.005 24

6 0.01 1

7 0.005 1

8 0.1 24

9 0.1 1

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2) Alkali Stress (NaOH)

Table 2.4 : Trials of stress studies in Alkali medium (NaOH) suggested by

software

Run Factor 1 : Strength(N) Factor 2 : Time (h)

1 0.001 24
2 0.001 7
3 0.1 7
4 0.1 24
5 0.01 7
6 0.01 1
7 0.001 1
8 0.1 1
9 0.01 24

3) Oxidation (H2O2)
Table 2.5: Trials of stress studies in oxidising maedium (H2O2) suggested by
software
Run Factor 1 : Strength(N) Factor 2 : Time (h)

1 15 1
2 30 24
3 3 7
4 30 1
5 3 1
6 15 24
7 30 7
8 15 7
9 3 24

After performing above trials, data of % degradation was obtained and inserted in the
designsoftware 32 factorial design. A List of solutions was suggested by the software.
Suggested solutions were further practically performed.
2.10 Method Validation

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2.10.1 Linearity and Range

Dilutions were made from the standard stock solution of Tenofovir ( 1 mg/mL ) using
Methanol to prepare solutions of concentration in range of 20-120 µg/mL. All
solutions were made in triplicate, samples of each concentration were injected and
peak areas were noted. The correlation graph was constructed by plotting the peak
areas obtained at the optimum wavelength of detection versus concentration of
drug.The linearity was evaluated by linear regression analysis,line equation and r 2
value was noted.

2.10.2 Precision
a) Intermediate precision (Inter-day precision)
Precision of the method was evaluated by taking samples of concentration
20-120 µg/mL. Samples were injected in triplicate on multiple days in same
laboratory. In each case peak area was noted; Standard deviation (SD) was calculated
to determine % RSD .

%RSD = SD *100
Mean

b) Repeatability (Intra-day precision)

For Intra-day precision, sets of three replicates of drug concentrations (ranging 20-120
µg/mL) were analyzed on the same day in same laboratory. Precision of the method
was evaluated in the similar manner as above, by calculating % RSD.

2.10.3. Recovery
Recovery of the method was determined by employing Standard addition method.
The standard of known concentration was spiked in degradants mixture (acid, alkali,
photo,neutral, oxidation degradants) and percent recovery of drug was calculated.
Three different concentrations were used as 80, 100 and 120 µg mL -1. Three
replicates of each concentration level were analysed.

2.10.4. Limit of Detection (LOD)

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LOD was calculated by using following formula:

3.3 σ
LOD = σ = Standard deviation of the Y intercept
S S = Slope of the calibration curve

2.10.5. Limit of Quantification (LOQ)

LOQ was calculated by using following formula:
10 σ
LOQ = σ = Standard deviation of the Y intercept
S S = Slope of the calibration curve

2.10.6. Robustness
For Robustness, selected parameters were pH of buffer, flow rate and wavelength.
Each selected parameter was varied by traditional One Factor At a Time approach
while other parameters were kept at constant level. Wavelength was varied at 258
and 260 nm, flow rate at 0.9 and 1.1 mL min-1,and pH of buffer at 8.4 and 8.6.

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