Stability indicating Method development and Validation of Lamivudine, Zidovudine and Nevirapinein Tablet dosage form

 

Shinde G. S.1*, Jadhav R. S.2, Vikhe D. N.2

1Department of Pharmaceutical Chemistry, Pravara Rural College of Pharmacy, Pravaranagar.

Tal - Rahata, Dist.- Ahmednagar.

2Department of Pharmacognosy, Pravara Rural College of Pharmacy, Pravaranagar.

Tal-Rahata, Dist.- Ahmednagar.

*Corresponding Author E-mail: shinde.ganesh904@gmail.com

 

ABSTRACT:

The RP-HPLC method for Lamivudine, Zidovudine and Nevirapine was developed using Inertsil ODS 3V C18 column (5µm, 150mm × 4.6mm) as stationary phase and 0.01M 1-octane Sulphonic acid: Methanol (60:40v/v, pH2.6) as mobile phase. The mobile phase was maintained at a flow rate of 1ml/min, run time 15 min and detection was carried out at 270nm.Lamivudine, Zidovudine and Nevirapine were found to be linear in the concentration range of 75-225µg/ml, 150-450µg/ml and 100-300µg/ml respectively. The result of % assay of marketed formulation was found as 101.93±0.1527, 100.86±0.2021and 94.2±0.1527 for Lamivudine, Zidovudine and Nevirapine respectively. Accuracy of the method was determined by performing recovery study and the result were found in the range of 100.9-101.4%, 100.1-100.7% and 99.0-99.9% for Lamivudine, Zidovudine and Nevirapine respectively. Percentage RSD of precision study of these drugs were found less than 2 percent which indicated good precision of the developed method. The proposed method was validated for linearity, accuracy, precision, and robustness. The proposed method is simple, rapid. precise and reproducible hence can be applied for routine quality control analysis of Lamivudine, Zidovudine and Nevirapine in pharmaceutical dosage form.

 

KEYWORDS: Lamivudine, Zidovudine, Nevirapine, RP-HPLC, Validation.

 

 


INTRODUCTION:

One of the deadliest and unmanageable chronic health catastrophes is HIV/AIDS. Fixed dose combinations (FDCs) form the main stay in clinical management of HIV-1 infection as they offer several advantages over single products with respect to storage, prescribing, dispensing, patient use, consumption and disease management.1

 

Lamivudine is chemically 1[(2R,5S)-2-(Hydroxy methyl)-1-3 oxathiolan-5yl] cytosine and Lamivudine is an analogue of cytosine. Polymerase2, Zidovudine is chemically 1-[(2R,4S,5S) azido(hydroxymethyl)tetrahydrofuran-2-yl]-5-methylprimidine-2,4(1H,3H0-dione and Nevirapine is chemically 11-cyclopropyl-4-methyl-5, 11-dihydro-6H-dipyrido [3, 2-b: 2’, 3’-e][1,4] diazepin-6-one and are reverse transcriptase (RT) inhibitors of human immune deficiency virus types (1 and 2) of  HIV reverse transcriptase and also the reverse transcriptase of hepatitis B3. Various pharmaceutical drugs are being validated using HPLC.4 Literature revealed that few methods have been reported for the individual estimation of Zidovudine and Lamivudine and in combination with other drugs5-9. The method developed is precise, simple and precise RP-HPLC method to determine Zudovudine and Lamivudine in pharmaceutical dosage forms.

 

MATERIALS AND METHOD:

Materials:

Lamivudine Zidovudine and nevirapine pure standards were received as gift samples from micro labs Pharmaceuticals banglore (India). All other reagents used were HPLC grade.

 

Apparatus:

The HPLC system was waters HPLC consisted with the pump-alliance 2690 seperation module, column Inertsil ODS-3V, C18, (150 × 4.6mm, 5µ), auto sampler, detector was waters 2489 and UV–Visible Detector, data processor Empower 2 (Water). Other instruments used were Melter balance AY 220, Eutech pH meter, Melter Ultrasonicatior and Millipore membrane filter.

 

Method:

Preparation of Buffer:

2.16gm of 1-Octane sulphonic acid sodium salt was weighed and dissolved in 1000ml of Milli Q water and adjusted the pH to 2.6±0.05 with 1% orthophosphoric acid. The solution was filtered through 0.45 µ membrane filter and sonicated for 10min to degas.

 

Preparation of mobile phase:

Buffer and methanol was mixed in the ratio 60:40v/v and filtered through 0.45µ membrane filter and degassed by sonication.

 

Preparation of diluent:

Mixture of methanol and water in the ratio of 50:50 v/v was prepared and degassed.

 

Preparation of Standard Solution:

Standard Stock Solution of Lamivudine, Zidovudine, Nevirapine:

About 37.5mg of Lamivudine, 75.0mg of Zidovudine, 50.0mg of Nevirapine standard were weighed accurately into 25ml of volumetric flask.  20ml of methanol was added and sonicated for 5 minutes. After sonication, the volume was made up to the mark with same solvent to obtain final concentration of 1500μg/ml, 3000μg/ml, 2000 μg/ml of Lamivudine, Zidovudine, Nevirapine respectively. From the above solution 10ml was pipetted out in a 100 ml volumetric flask and the volume was made up to the mark with diluent to obtain final concentration of 150μg/ml, 300μg/ml, 200μg/ml of Lamivudine, Zidovudine, Nevirapine respectively.

 

Assay preparation of Tablet formulation :

20 tablets were accurately weighed and average weight was determined. Powdered tablet equivalent to 300mg of Lamivudine was transferred into a 200ml volumetric flask. 10ml of water was added and sonicated for 10 minutes to disperse the tablet powder. Then 130ml of methanol was added and sonicated for 25 minutes with intermittent shaking. The volume was made upto mark with methanol after cooling and mixed well, filtered through 0.45µ nylon filter. First 2ml of the filtrate was discarded and 5ml of this filtrate was diluted to 50 ml with diluent and mixed well to get concentration of Lamivudine as about 150µg/ml, Zidovudine 300µg/ml, Nevirapine 200µg/ml. The blank, standard and sample solutions were injected and result are given in table no 1.


 

Fig 1: Chromatogram of Combined Standard Solution of Lamivudine, Zidovudine and Nevirapine

 

Fig 2 : Chromatogram of Lamivudine, Zidovudine and Nevirapine Sample Solution.

 


Table 1: Analysis of Tablet formulation

Sr

no

parameters

Drug

Lamivudine

Zidovudine

Nevirapine

1

Label claim (%/tab)

150 mg

300 mg

200mg

2

Drug content (%)

101.9

100.8

94.3

3

% RSD

0.14

0.20

0.16

 

Table 2: Accuracy (Recovery studies)

Sr.no

Level of % Recovery

Weight Taken

(mg)

Mg

Recovered

% recovery

% mean recovery

Lamivudine

50

150.05

152.74

101.8

101.4

100

300.40

304.79

101.05

150

450.05

454.07

100.9

Zidovudine

50

300.20

305.18

101.7

100.7

100

599.50

604.81

100.9

150

899.00

899.54

100.1

 

Validation parameters:

1.    Accuracy:

The method was validated in accordance to ICH guidelines Recovery study was performed by standard addition method by adding the known amount of Lamivudine, Zidovudine Nevirapine (Reference standard) to the placebo at three different concentration levels i.e 50%, 100% and 150% of assay concentration and % recovery for all these drug were calculated.% Recoveries of Lamivudine, Zidovudine, and Nevirapine were found in the range of 100.9-101.4 %, 100.1-100.9 % and 99.0-99.99% respectively(RSD < 2) as in table no 2.

 

2. Precision:

The system precision was checked by using standard Lamivudine, Zidovudine and Nevirapine to ensure that the analytical system is precise.

 

a. Intra-day Precision:

Intra-day precision was determined by analyzing the combined standard solutions of Lamivudine, Zidovudine and Nevirapine (150, 300, 200 μg/ml) at three different time intervals on same day. The % RSD of precision study of these drugs was found to be less than 1 %.

 

b. Inter-day Precision:

Inter-day precision was determined by analyzing the combined standard solutions of Lamivudine, Zidovudine and Nevirapine (150, 300, 200 μg/ml) on three consecutive days. The % RSD of precision study of these drugs was found to be less than 1 %.


 

Table 3: Result of Intraday and Interday precision of Lamivudine (150μg/ml)

Conc. μg/ml

Time(hr.)

Mean Peak Area

n=6

Std. Deviation

n=6

%RSD

Time (days)

Mean Peak Area

n=6

SD

n=6

%

RSD

150

(Lamivudine)

0

4152892

3613.939

0.08%

1

4156201

5091.232

0.12%

2

4139386

3583.033

0.08%

2

4138035

3296.168

0.07%

4

4142319

4589.268

0.11%

3

4155800

8119.642

0.19%

     

Table 4: Result of Intraday and Interday precision of Zidovudine (300μg/ml)

Conc. μg/ml

Time

(hr.)

Mean Peak Area n=6

Std. Deviation

n=6

%RSD

Time

(days)

Mean Peak Area n=6

SD

n=6

% RSD

300

(zidovudine)

0

6636319

11496.57

0.17%

1

6641437

10733.47

0.16%

2

6485820

7281.153

0.11%

2

6485858

7267.965

0.11%

4

6536319

11496.57

0.17%

3

6536291

11534.14

0.17%

 

Table5:  Result of Intraday and Interday precision of Nevirapine (200μg/ml)

Conc. μg/ml

Time

(hr.)

Mean Peak Area n=6

Std. Deviation

n=6

%RSD

Time

(days)

Mean Peak Area n=6

SD

n=6

% RSD

200

Nevirapine

0

3192686

38231.68

1.19%

1

3184422

31298.39

0.98%

2

3154635

1728.371

0.054%

2

3153290

7658.574

0.24%

4

3260535

27990.07

0.85%

3

3245086

29443.96

0.90%

 


2. Linearity and Range:

The solutions for linearity were prepared in the concentrations as follows and chromatographic peak area was plotted against the concentration of the each drugs. From the data obtained, co-relation coefficient, slope and y-intercept were calculated (fig 3, 4, 5) .

 

Table 6: Results of linearity for RP-HPLC Method

Parameter

Lamivudine

Zidovudine

Nevirapine

Linearity and Range

75-225

150-450

100-300

Slope(m)

27974.69

21915.68

15950.85

Intercpt

-0.8280

-0.6870

-0.7489

Correction  3coefficient(R2)

0.9999

0.9999

 

3. Robustness:

Robustness was done by changing the column temperature (±5°C), flow rate (±10%), wavelength (±5 nm) . It was observed that % RSD of Peak Area was less than 2% for all the drugs in the conditions.( Table-7,8 and 9)

 

Fig  3: Linearity of Lamivudine

 

Fig 4: Linearity of zidovudine

 

Fig 5: Linearity of Nevirapine

 

Table 7:Variation in column Temperature

Column Oven Temperature

Analyte

Retention Time* (min)

Tailing Factor (T)

Resolution (R)

Theoretical Plates (N)

%RSD Peak Area

25°

Lamivudine

8.77

1.06

12.23

6038

1.15

Zidovudine

4.25

1.10

NA

3813

1.13

Nevirapine

10.87

1.08

4.01

5581

1.17

30°

lamivudine

7.856

1.05

11.42

5963

0.10

Zidovudine

4.027

1.10

NA

3990

0.10

Nevirapine

10.11

1.07

4.85

6365

0.08

35°

lamivudine

7.05

1.05

10.41

5671

0.43

Zidovudine

3.82

1.11

NA

4054

0.42

Nevirapine

9.44

1.08

5.64

6860

0.38

 

Table 8 : Variation in Flow Rate (ml/min)

Flow rate (ml/min)

Analyte

Retention Time* (min)

Tailing Factor (T)

Resolution (R)

Theoretical Plates (N)

%RSD Peak Area

0.9ml/min

lamivudine

8.6

1.05

11.56

6007

0.36

Zidovudine

4.450

1.11

NA

4132

0.36

Nevirapine

11.14

1.09

4.88

6675

0.43

1.0ml/min

lamivudine

7.856

1.05

11.42

5963

0.10

Zidovudine

4.027

1.10

NA

3990

0.10

Nevirapine

10.11

1.07

4.85

6365

0.08

1.1ml/min

lamivudine

7.159

1.05

11.08

5658

0.17

Zidovudine

3.665

1.09

NA

3736

0.18

Nevirapine

9.21

1.07

4.69

5884

0.14  

 

Table 9 : Variation in Wavelength (nm)

Wavelength

(nm)

Analyte

Retention Time (min)

Tailing Factor (T)

Resolution (R)

Theoretical Plates (N)

%RSD Peak Area

265nm

lamivudine

7.85

1.06

11.32

5983

0.40

Zidovudine

4.02

1.11

NA

3949

0.40

Nevirapine

10.12

1.09

4.80

6009

0.41

270nm

lamivudine

7.856

1.05

11.42

5963

0.10

Zidovudine

4.027

1.10

NA

3990

0.10

Nevirapine

10.11

1.07

4.85

6365

0.08

275nm

lamivudine

7.85

1.06

11.27

5745

0.05

Zidovudine

4.03

1.11

NA

3937

0.05

Nevirapine

10.12

1.08

4.87

6494

0.06

 


1.    System Suitability Parameters:

The system suitability tests   are carried out to evaluate the reproducibility of the system for the analysis to be performed. The results of system suitability tests are given in Table 10, showing that the parameters are within the suitable range.

 

Table 10 : System Suitability Parameters:

Analyte

Retention Time* (min)

Tailing Factor* (T)

Theoretical Plates* (N)

Resolution* (R)

lamivudine

8.016

1.05

2795

8.45

Zidovudine

4.059

1.18

2428

-

Nevirapine

10.274

1.16

3087

3.36

Required limits

--

T < 2

N > 2000

R >2

 

Forced Degradation Studies:

Forced degradation studies were carried out on the sample preparations of LAMI, ZIDO and NEVI tablets 150:300:200mg and the degradation was evaluated by calculating the percentage degradation of LAMI, ZIDO and NEVI in comparison with unstressed sample preparation. The Acid, alakali, Peroxide Degradation are the stress conditions which were followed for forced degradation studies;

 

1.Acid Stressed Degradation:

Accurately weighed 2046.10mg of tablet powder was transferred into 200ml volumetric flask, 5ml 5N Hydrochloric acid was added and heated for 1 hour on a water bath at 80°C. The solution was cooled and neutralized with 5ml 5 N sodium hydroxide, 10ml water was added, sonicated for 10 minutes, then 130ml methanol was added, sonicated for 25 minutes with intermittent shaking. The volume was made upto mark with methanol, mixed well, filtered through 0.45µ nylon filter. The 5ml of this solution was diluted to 50ml diluent and analysed to record chromatogram as shown in figure no 5

 

2.Alkali Stress Degradation:

Accurately weighed 2048.10mg of tablet powder was transferred into 200ml volumetric flask, 5 ml 5N sodium hydroxide was added and heated for 1 hour on a water bath at 80°C. The solution was cooled and neutralized with 5 N hydrochloric acid, 10ml water was added and sonicated for 10 minutes, then 130ml methanol was added, sonicated for 25 minutes with intermittent shaking. The volume was made up to mark with methanol, mixed well, filtered through 0.45µ nylon filter. The 5ml of this solution was diluted to 50ml diluent and analysed to recorded chromatogram as shown in figure no 6

 

3.Peroxide Stress Degradation:

Accurately weighed 2046.20mg of tablet powder and transferred into 200ml volumetric flask, 5ml 6% H2O2 was added and heated for 1 hour on a water bath at 80°C, 10ml of water was added and sonicated for 10 minutes, then 130ml methanol was added and sonicated for 25 minutes with intermittent shaking. The volume was made upto mark with methanol, mixed well, filtered through 0.45µ nylon filter. The 5ml of this solution was diluted to 50ml diluent and analysed to recorded chromatogram as shown in figure no 7

 

Table 11: Result of Peak Purity of Drug in Acid Stressed Degradation

Sr. No.

Name of Drug

Peak Purity Result

Purity Angle

Purity Threshold

Conclusion

1

Lamivudine

0.048

0.214

Passed

2

Zidovudine

0.217

0.232

Passed

3

Nevirapine

0.079

0.223

Passed

 

Table 12 Result of Peak Purity of Drug in Alkali Stressed Degradation

Sr.

No.

Name of Drug

Peak Purity Result

Purity Angle

Purity Threshold

Conclusion

1

Lamivudine

0.049

0.230

Passed

2

Zidovudine

0.063

0.234

Passed

3

Nevirapine

0.056

0.230

Passed

 

 

Table 13: Result of Peak Purity of Drug in Peroxide Stressed Degradation

Sr.

No.

Name of Drug

Peak Purity Result

Purity Angle

Purity Threshold

Conclusion

1

Lamivudine

0.057

0.209

Passed

2

Zidovudine

0.120

0.233

Passed

3

Nevirapine

0.069

0.230

Passed

 

Table 14: Result of Stress Study for Lamivudine

Standard Area

4210116

4227007

4214823

4190111

4200608

Average Area

4208533

 

 

 

%RSD

0.3%

 

 

 

Test No.

Unstressed

Acid Stress

Alkali Stress

Peroxide Stress

Avg. Wt. taken (mg)

1019.91

Wt. taken (mg)

2046.00

2046.50

2048.10

2046.20

Area (Inj.1)

4286040

3842747

3820584

3106146

Area (Inj.2)

4278296

3873847

3824608

3075875

Avg. Area

4282168

3858297

3822596

3091011

% RSD

0.1

0.6

0.1

0.7

Assay(mg/tab)

151.20

136.20

134.84

109.13

Assay (%)

100.8

90.8

89.9

72.8

% Degradation

NA

9.9

10.8

27.8

 


 

Fig 6:Chromatogram of Combined Tablet Solution of Lamivudine, Zidovudine and Nevirepine in Acid Stress Degradation

 

 

Fig 7: Chromatogram of Combined Tablet Solution of Lamivudine, Zidovudine and Nevirepinein Alkali Stress Degradation

 

 

Fig 8: Chromatogram of Combined Tablet Solution of Lamivudine, Zidovudine and Nevirepine in Peroxide Stress Degradation


 

 

Table 15: Result of Stress Study for Zidovudine

Standard Area

6618136

6622718

6605308

6589964

6589023

Average Area

6605030

 

 

 

%RSD

0.2%

 

 

 

Test No.

Unstressed

Acid Stress

Alkali Stress

Peroxide Stress

Avg. Wt. taken (mg)

1019.91

Wt. taken (mg)

2046.00

2046.50

2048.10

2046.20

Area (Inj.1)

6670294

5971907

6231620

6203838

Area (Inj.2)

6675162

6013361

6234864

6144817

Avg. Area

6672728

5992634

6233242

6174328

% RSD

0.1

0.5

0.0

0.7

Assay(mg/tab)

302.26

271.39

282.06

279.66

Assay (%)

100.8

90.5

94.0

93.2

% Degradation

NA

10.2

6.7

7.5

 

Table 16: Result of Stress Study for Nevirapine

Standard Area

3161136

3154240

3164981

3141171

3147091

Average Area

3153724

 

 

 

%RSD

0.3%

 

 

 

Test No.

Unstressed

Acid Stress

Alkali Stress

Peroxide Stress

Avg. Wt. taken (mg)

1019.91

Wt. taken (mg)

2046.00

2046.50

2048.10

2046.20

Area (Inj.1)

2997526

2101374

2844387

2949797

Area (Inj.2)

2997226

2115665

2845496

2928474

Avg. Area

2997376

2108520

2844942

2939136

% RSD

0.0

0.5

0.0

0.5

Assay(mg/tab)

190.84

134.21

180.94

187.11

Assay (%)

95.4

67.1

90.5

93.6

% Degradation

NA

29.7

5.1

1.9

 

RESULT AND DISCUSSION:

The scope of the present work is to expand the optimization of the chromatographic conditions, to develop RP-HPLC method for the estimation of drugs in selected multi-component dosage forms. The developed method was also validated. Accuracy of the method was determined by performing recovery study and the result were found in the range of 100.9-101.4%, 100.1-100.7% and 99.0-99.9% for Lamivudine, Zidovudine and Nevirapine respectively. Percentage RSD of precision study of these drugs were found less than 2 percent which indicated good precision of the developed method. The proposed method was validated for linearity, accuracy, precision, and robustness Formulation was exposed to different stress condition such as Acidic, Alkali and Oxidative. The amount of degradation of lamivudine was found in the range of 10-30% in all the stress condition. While Zidovudine and Nevirapine were not showed significant degradation except in Acidic condition. As the purity angle of the peaks was less than the purity threshold so peak purity was compiled for all the drugs in all the stressed condition.

 

CONCLUSION:

The method gives good resolution for both the drugs with a short analysis time. Percentage recovery shows that the method is free from interference of the excipient used in the formulation. Proposed study describes an HPLC method for the estimation of Lamivudine, Zidovudine and Nevirapine in pharmaceutical dosage form. The developed method was also applied to find the percent degradation of these drug in acid, alkali and oxidative stress conditions. This method can be used for routine quantitative analysis of these drugs after different storage conditions.

 

REFERENCE:

1.     Mahua Sarkar, Sateesh Khandavilli, Ramesh Panchagnula, Development and validation of RPHPLC and ultraviolet spectrophotometric methods of analysis for the quantitative estimation of antiretroviral drugs in pharmaceutical dosage forms. Journal of Chromatography B 830, 349–354 (2006).

2.     USP-NF, The Official Compendia of Standards, published by The United States Pharmacopoeia Convention, City Press, Baltimore, US, 2009, Vol III, pp 3072-3.

3.     Indian Pharmacopoeia 2007, Ministry of Health and Family welfare, Controller of Publications, Delhi.

4.     Shinde Ganesh S, Development and Validation of RP HPLC Method for Simultaneous estimation of Meclizine Hydrochloride and Caffeine in Bulk and Tablet Dosage Form. EJPMR, 2019, 6(6):433-441

5.     Vibhuti Kabra, Vivek Agrahari, Chandrabose Karthikeyan, Piyush Trivedi, Simultaneous quantitative determination of zidovudine and nevirapine in human Plasma Using Isocratic, Reverse Phase High Performance Liquid Chromatography, TJPR, 2009; 8(1): 79-86.

6.     Prasada Rao C H, Channabasavaraj K P, Lakshmi Aswini G, Development and validation of RP-HPLC method for the estimation of Nevirapine in bulk drug and tablets, J Pharma Sci and Res, 2009; 1(2): 78-82.

7.     Anand Babu K, Jaykar B, Analytical method development and validation for simultaneous estimation of Zidovudine, Lamivudine and Nevirapine tablets by RP-HPLC, International Journal of Pharmaceutical Research and Development, 2011; 3(7): 9-14

8.     Venkatesh P and Rajesh Kumar R. Sepectrophotometric determination of nevirapine in pharmaceuticals after derivatization with 2,4-dinitro phenlyhydrazine. Asian J. Pharm. Health Sci., 2011; 1 (1): 33-4.

9.     Rohini P, Madhu Sudhana Reddy I, Gupta A, Lokeswara Babu V Sudharani G, Method development and validation for estimation of Nevirapine from tablets by RP-HPLC, Int. J Pharma, 2011; 1(1): 29-33.

10.   Purnima D Hamrapurkar, Mitesh D Phale, Nitul shah, Quantitative estimation of Nevirapine by high performance thin layer chromatography, JPRHC, 2009; 1(2):197-216.

 

 

 

Received on 09.04.2022        Modified on 05.05.2022

Accepted on 20.05.2022   ©AandV Publications All Right Reserved

Res.  J. Pharma. Dosage Forms and Tech.2022; 14(3):238-244.

DOI: 10.52711/0975-4377.2022.00039