INTRODUCTION:

Lamivudine is a medication used in the management and treatment of HIV-1 and hepatitis B. It is in the nucleoside reverse transcriptase inhibitor (NRTI) class of medications used in combination with other drugs as antiviral treatment for human immunodeficiency virus type-1 (HIV-1) and as a monotherapy for hepatitis B virus (HBV). Lamivudine is globally important as first-line NRTI therapy; it is currently on the World Health Organization's (WHO) "List of Essential Medications."

Salicylic acid is one of the process related impurities (Lamivudine impurity C) present in the API of the lamivudine. It should be present within the limit specified in the monograph of Indian Pharmacopoeia-2018.

Literature survey revealed that few analytical methods for determination of lamivudine alone or in combinations including’ UPLC^{1-2 ,
23-25}, HPTLC^{3-6 , 16}, HPLC^7-11, and UV^13-14,
17-22 spectrophotometry.

MATERIALS AND METHODS:

The experimental requirements including analytical instruments, reagents chemicals, active pharmaceutical ingredients, excipients and formulation used are discussed below.

Analytical Instruments:

Jasco V-630 UV/Vis-spectrophotometer (Jasco Corporation, Tokyo, Japan). Camag Linomat 5 Applicator, TLC Scanner 4 controlled by Vision CATS software, version 3.1 (Camag, Muttenz, Switzerland). Merck TLC plates coated with silica gel 60F254 on aluminium sheet were used as stationary phase (Merck chemicals Ltd., Darmstadt, Germany).

API Lamivudine, Salicylic acid, Excipients like citric acid, sucrose, propylene glycol and methyl paraben was procured from Yarrow Chem Products., Mumbai, India and s d fine-chem limited, Mumbai, India.

Lamivir - 100mg and Lamivir - 150mg formulation was procured from Cipla Ltd., Sikkim, India.

EXPERIMENTAL METHODS:

Optimization of experimental conditions:

Selection of Solvent:

The drug lamivudine was found to be soluble in solvents like methanol, water and dimethyl sulfoxide, and the solution was found to be clear. The drug salicylic acid was found to be soluble in methanol, ethyl acetate, tetrahydrofuran, acetonitrile, and dimethyl sulfoxide. Both the drugs were completely soluble in methanol so it was chosen for the preparation and for dilution of the stock solution.

Selection of Stationary phase:

Silica gel of particle size 10-12μm and pore size of 60Å along with fluorescent F₂₅₄ indicator coated on the aluminium plates of thickness 200μm was selected with F₂₅₄ the indicator used for visualizing compounds that absorb UV light near 254nm which can be used for the detection of drug substance in the UV-visible region (200nm 800nm).

Selection of wavelength:

The solution lamivudine in the concentration of 10μg/ml was scanned in the UV region (200nm - 400nm) and its related substance (salicylic acid) was also scanned under same UV region (200nm - 400nm) with 10μg/ml concentration. The wavelength of measurement was selected based on the absorbance readings.

Selection of mobile phase:

Composition of mobile phase:

The mobile phase of different compositions of various solvents such as water, methanol, hexane, toluene, acetonitrile and ethanol was performed. The solvent system consisting of methanol: water: ethyl acetate shows a consistent and acceptable peak characteristic.

Ratio of mobile phase:

The three solvent system made up of methanol: water: ethyl acetate was varied in different ratios by increasing the volume of the polar solvents methanol and water. The composition and ratio of mobile phase was selected for the study according to the peak properties of both the compounds lamivudine and salicylic acid.

Selection of distance travelled by the solvent front:

The distance of the solvent front was varied between 7.5 to 9cm and the Rf value was calculated for the drug lamivudine and salicylic acid both as individual compounds and also as a mixture. The distance travelled by the solvent was selected depending upon the separation of both the drugs with the acceptable resolution.

Preparation of standard stock solutions:

The standard stock solution of lamivudine and salicylic acid was prepared using methanol as a solvent in the concentration of 1000µg/ml each.

Preparation of working standard solutions:

The drug solution lamivudine was diluted to get the concentration of 500µg/ml and the related substance salicylic acid solution was diluted to get the concentration of 50µg/ml.

Mixture solution containing lamivudine and salicylic acid was also prepared in same proportion of 500µg/ml of lamivudine and 50µg/ml of salicylic acid. All the dilutions were carried out using methanol as a solvent.

VALIDATION OF THE METHOD¹⁵:

The validation of the developed method was carried out in terms of specificity, linearity, inter and intraday precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), and stability studies as per ICH guidelines.

Specificity:

The specificity of the method was determined by performing the peak purity test for the drugs. Peak purity of drugs was assessed by comparing their UV spectra at three points, viz. peak start (s), peak area (m) and peak end (e) positions of the spots.

Linearity and range:

The linearity of response was assessed by applying different volumes from the working standard solutions of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 and 6μl spotted on TLC plate to obtain linear concentration range of 500-3000 ng/band of Lamivudine and 50- 300ng/band of Salicylic acid. The spots were developed and evaluated densitometrically using CAMAG HPTLC system. Peak areas were noted for each spot and they were plotted against their concentrations to get a linear graph. The calibration graph was statistically evaluated for regression equation and correlation coefficient.

Precision:

Precision of the method was studied by

· Intra-day precision

· Inter-day precision

Intra-day precision:

The spotting volume of 4μl was injected from the drug solution containing 500μg/ml of Lamivudine and 50μg/ml of Salicylic acid. The concentration of Lamivudine and Salicylic acid was 2000ng/band and 200 ng/band. The studies were carried out at 3 different time intervals on the same day with 6 determinations at each time. The %RSD for values obtained were calculated.

Inter-day precision:

The spotting volume of 4μl was injected from the drug solution containing 500μg/ml of Lamivudine and 50 μg/ml of Salicylic acid. The concentration of Lamivudine and Salicylic acid was 2000ng/band and 200ng/band. The studies were carried out on 3 consecutive days with 6 determinations at each time. The % RSD for values obtained were calculated.

Accuracy:

The oral dosage of Lamivir- 100mg and Lamivir- 150 mg tablets were studied for the accuracy. The tablet powder equivalent to 10mg was weighed and transfer into the 10ml volumetric flask. The solvent methanol was added and the drug was extracted by sonication for 5 mins and the final volume was made up. The formulation solutions were double filtered using Whatmann filter paper. The first 2ml was discarded and the remaining solutions were used for the accuracy studies.

50% Recovery:

5ml of 1000μg/ml solution of tablet solution and 2.5ml of 1000μg/ml of standard solution of lamivudine was pipetted into 10ml volumetric flask to made up the volume to give concentration of 500μg/ml of assay solution and 250μg/ml of standard drug lamivudine.

100% Recovery:

5ml of 1000μg/ml solution of tablet solution and 5ml of 1000μg/ml of standard solution of lamivudine was pipetted into 10 ml volumetric flask to made up the volume to give concentration of 500μg/ml of assay solution and 500μg/ml of standard drug lamivudine.

Limit of detection (LOD) and limit of quantification (LOQ):

The LOD and LOQ of lamivudine and salicylic acid were determined by using the equation; LOD = 3.3 x σ/S and LOQ = 10 x σ/S, where σ is the standard deviation of the intercept and S is the slope of the calibration curve.

Robustness and Ruggedness:

Intentional variations in the chamber saturation time by ±2mins, mobile phase ratio ±0.1ml and detection wavelength by ±1nm were carried out for lamivudine. The peak area and the R_f value were evaluated for notable changes. The ruggedness was carried out by 2 different analysts under same experimental conditions. The chromatograms were checked for the similar results.

Stability:

Stability of solution:

Freshly prepared solution of Lamivudine in methanol was diluted to 10mcg/ml and the peak area and detector responses were noted. The same solutions were checked at regular intervals under two conditions for stability. At room temperature, the study was carried out and the solutions were checked till 5 days. The stability of the solutions under refrigeration condition was also checked for continuous 1 week.

Stability of plate:

The pure drug solution of lamivudine was prepared and spotted on the plate. The plates were developed using the fixed experimental conditions and detected for its response. After detection, the plate was preserved and scanned at different time intervals any notable changes in the peak area were compared with the initial values. This study was carried out for 48hrs.

Application of the validated HPTLC method:

Tablet formulation:

Tablet formulation used for the study is Lamivir-100mg and Lamivir-150mg.Twenty individual tablets of Lamivudine were weighed accurately and average weight was calculated. Tablet powder equivalent to 10 mg of Lamivudine was weighed and added to a 10ml volumetric flask. To this tablet powder, methanol was added and the drug was extracted by sonication for 5 mins and the final volume was made up. The formulation solutions were double filtered using Whatmann filter paper. The first 2ml was discarded and the remaining solutions were used for the assay study of lamivudine tablet formulation. The filtrate was further diluted to give a concentration of 500μg/ml with methanol. The solution was analyzed according to the established procedure. The content was calculated for both the formulations.

Oral solution:

Oral solution of lamivudine was prepared in-house according to the composition given in the product monograph of Epivir oral solution with the dosage of 10 mg/ml and the total volume for each container is 240ml.

Excipients mentioned were sucrose, propylene glycol, methyl paraben, citric acid, sodium citrate, artificial strawberry essence and artificial flavouring banana based on the above formula the oral solution with selected excipients such as citric acid, methyl paraben, propylene glycol and sucrose were weighed and spotted individually. The spots were checked under fixed conditions, for any interference peak. The mixture of excipients was prepared and added to 10mg of lamivudine and the solutions were prepared and used for the analysis of lamivudine.

Identification of Salicylic acid in oral dosage forms:

Tablet formulation used for the study is Lamivir-100mg and Lamivir-150mg. 20 individual tablets of Lamivudine were weighed accurately and average weight was calculated. Tablet powder equivalent to 10mg of Lamivudine was weighed and added to a 10ml volumetric flask. To this tablet powder, methanol was added and the drug was extracted by sonication for 5 mins and the final volume was made up. The formulation solutions were double filtered using Whatmann filter paper. The first 2ml was discarded and the remaining solutions were used for the study. The filtrate was further diluted to give a concentration of 500μg/ml with methanol. The solution was analysed for the presence of salicylic acid.

Oral solution was prepared according to the established procedure given in the product monograph of Epivir oral solution. The oral solution containing lamivudine in the concentration of 500μg/ml excipients were spotted and the bands were studied for the peaks and evaluated.

RESULTS AND DISCUSSION:

The drug was found to be completely soluble in methanol which results in a clear solution with no particle precipitation. The solution of lamivudine with 10 µg/ml concentration in methanol was scanned which shows a smooth spectrum with absorption at 272nm is given in fig 1. The solution of salicylic acid in methanol prepared at 10µg/ml concentration scanned in UV region showed the absorbance peaks as given in the fig 1 with maximum absorbance at 233nm.

Based on the solubility of selected drug lamivudine and its related substance the work was carried out using methanol as a common solvent.

Selection of Stationary phase:

The activated plates were immediately used for the selected study in two different dimensions of 10x10cm and 20x10cm for the method development, validation and application of study.

Selection of wavelength:

The spectral scan of lamivudine was found to show absorbance peaks at 205nm, and 272nm with acceptable absorbance at selected wavelength of 233nm. The related substance salicylic acid categorized as impurity C according to the monograph in Indian Pharmacopoeia was found to have notable absorbance at three different wavelengths of 205nm, 233nm and 302nm. It was found that salicylic acid had acceptable absorbance at 233nm which was fixed as a wavelength of measurement and shown in fig 1.

Fig 1: Overlay UV spectrum of Lamivudine and Salicylic acid

The wavelength of 233nm was selected for the study from the scanned plate in the HPTLC (CAMAG) scanner, as both drugs gave good detection response with an acceptable peak area and peak shape having a resolution more than 2.

Selection of mobile phase optimization:

The mobile phase was selected based on the acceptable peak characteristics of lamivudine and salicylic acid such as the retardation factor, resolution between both the peaks, tailing factor, asymmetric factor, peak area and peak purity and scanned spectra in the mixture solutions. The chemical properties of selected drug lamivudine were found to have a basic pKa value based on which the solvent water was included in the mobile phase composition, whereas the impurity C (salicylic acid) was found to have an acidic nature with the value of 2.98. So, when both lamivudine and salicylic acid was put as mixture and spotted, the R_f value of Lamivudine and Salicylic acid was found to be 0.76±0.03 and 0.65 ±0.02.

Fig 2: Chromatogram of 1000 ng/band and 100 ng/band of Lamivudine and Salicylic acid

Selection of chamber saturation:

The saturation of the chamber used for the plate development affects the R_fvalue. The chamber saturation of 10 to 30 min was tried to avoid edge effects and fluctuations in R_fvalue. The suitable saturation time of 20 min was fixed for the study.

Selection of distance travelled by the solvent front:

After carrying out the trails between the distance of 7.5 cm to 9cm for height of 10cm plate, the solvent front of 9cm was selected for the work.

METHOD VALIDATION:

Specificity:

The ability of the analytical method to measure the analyte was carried out by testing the peak purity for the drug lamivudine and the related substance in the selected range of linearity. The peak purity for both the compounds lamivudine and salicylic acid was found to be more than 0.999. Hence the method is specific for the drug.

Linearity and range:

Different volumes of working standard solutions (1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 and 6μl) were spotted with the help of Linomat V automatic sample applicator on the TLC plates. The plates were developed in 10 x10 twin trough chamber saturated with the fixed mobile phase system and scanned using CAMAG TLC scanner 3. The peak areas of Lamivudine and Salicylic acid were noted. Rf value of lamivudine was found to be 0.76±0.03 and for related substance was found to be separate at 0.65± 0.02 respectively.

The linear regression data showed a good linear relationship over a concentration range of 500-3000 ng/band of Lamivudine and 50-300ng/band of Salicylic acid. The calibration data are shown in Table 1 and 2. The slope, intercept and correlation coefficient values calculated with linear graph are presented in Table 3. The calibration graph of Lamivudine and Salicylic acid was shown in Fig 3-4.

Table 1: Calibration data for Lamivudine

Concentration (ng/band)	Peak area (AU)
500	178
750	256
1000	356
1250	443
1500	531
1750	599
2000	694
2250	761
2500	851
2750	938
3000	1002

Fig 3: Calibration graph of Lamivudine

Table 2: Calibration data for Salicylic acid

Concentration (ng/band)	Peak area (AU)
50	30
75	32
100	37
125	40
150	42
175	49
200	50
225	51
250	57
275	60
300	67

Fig 4: Calibration graph of Salicylic acid

Table 3: Regression data of Lamivudine and Salicylic acid

Regression data	Lamivudine	Salicylic acid
Slope	0.321	0.1407
Intercept	19.691	22.191
Correlation coefficient	0.9989	0.9826
Regression equation	y= 0.3321x + 19.691	y=0.1407x + 22.191

Precision:

Intraday precision:

The intraday precision studies were carried out using the solution of drug mixture in the concentration of 2000:200ng/band of Lamivudine and Salicylic acid. The intraday precision studied on the same day at three-time intervals were statistically evaluated for the %RSD value was calculated.

Inter day precision:

The inter day precision studies were carried out using the solution of drug mixture in the concentration of 2000:200ng/band of Lamivudine and Salicylic acid. The intraday precision studied on the 3 different days at three-time intervals were statistically evaluated for the % RSD value was calculated for mean determinations.

Accuracy:

The recovery of the standard drug added to the assay solution at two different levels of 50% and 100% were studied for the tablet formulation in two dosage forms of 100mg and 150mg. The % recovery for Lamivudine at the two different levels was calculated. The % RSD for the three determinations at each level was found to be below 2.

Limit of detection (LOD) and limit of quantification (LOQ):

The detectable limit based on the slope of the curve and repeated measurement of standard solutions are calculated. The quantifiable limit for the drug lamivudine and the impurity C was found to be below the linearity range.

Robustness and ruggedness:

The robustness studies carried out with slight modifications in the chamber saturation time, mobile phase composition and detection wavelength. The changes in the peak area for the positive and negative variations in all the selected conditions were calculated and the values obtained were found to be robust. The method was found to be rugged with the results obtain from the two analysts under similar experimental conditions.

Stability study:

Stability study of the solution:

The stability of drug solution of lamivudine and salicylic acid were checked continuously at room temperature and refrigeration. The solution was found to be stable for 4 days at room temperature and 7 days in the refrigeration.

Stability study of the developed plate:

When the developed chromatographic plate was exposed to atmosphere, the analytes are likely to decompose. Hence it was necessary to conduct stability studies of the plate. Stability of the analytes on the plates was studied in 48 hours. The developed plate was found to be stable for up to 24 hours.

Application of the validated HPTLC method:

Tablet formulation:

The solution prepared with two different dosage forms of tablet formulation where the chromatogram and the peaks were evaluated. Both the dosage forms were found to have % label claim within the limits. The selected concentration of the solution for both the dosage forms were found to show peaks of impurity C which was calculated in comparison to the standard solutions of salicylic acid in the linear range. The salicylic acid was found to be within the limits specify of the peak response not more than the reference of salicylic acid.

Fig 5: Chromatogram of Lamivir – 100mg tablet formulation

Fig 6: Chromatogram of Lamivir – 150mg tablet formulation

Oral solution:

Oral solution is prepared as per the composition mentioned in the product monograph of Epivir oral solution containing 10 mg/ml lamivudine solution was diluted using methanol and spotted. The peak area of the drug lamivudine was compared with the peak area of the standard drug lamivudine and calculated. The % of drug found was equivalent to the concentration of standard drug lamivudine. The excipients studied according to the formula given in the oral solution of lamivudine product had no interference at the R_f value of drug. There was no peak found for salicylic acid in the chromatogram of oral solution.

Fig 7: Chromatogram of oral solution Lamivudine

Identification of Salicylic acid in oral dosage forms:

The tablet formulation in two dosage forms of 100mg and 150 mg studied for its % label claim was identified for the presence of impurity C detected as a separate spot at the R_f value of the standard salicylic acid. The peak area of the identified salicylic acid was calculated and quantified. It was found that the presence of salicylic acid identified in the both dosage forms are within the limits specified in the Pharmacopoeia and it is given in table 4.

Table 4: Quantification of Salicylic acid in oral dosage form (Lamivir- 100 mg and Lamivir- 150 mg)

Tablet formulation	Salicylic acid		Observation
Tablet formulation	Peak area	Concentration of salicylic acid obtained from linearity of reference salicylic acid	Observation
Lamivir- 100 mg	37	0.10 µg	Within the limit
Lamivir- 150 mg	44	0.16 µg	Within the limit
Oral solution	-	-	Not detected

CONCLUSION:

Lamivudine is a reverse transcriptase inhibitor used to treat HIV and Hepatitis B infection which is rapidly absorbed after oral administration and peak serum level is 1.5±0.05µg/ml with less than 26% plasma protein binding and eliminated unchanged in the urine with excretion in the human breast milk also. The presence of salicylic acid as a related compound is reported to decreases the excretion rate of lamivudine which can result in higher serum level. The salicylic acid has 90% plasma protein binding and it extensively metabolized with half-life of 2 to 4.5 hours and 10% excreted unchanged in the urine.

Salicylic acid is categorized as lamivudine impurity C which is chemically 2- hydroxybenzene carboxylic acid recommended to be present within the limits according to the monograph. The specification for salicylic acid in the drug substance lamivudine by HPLC method is not more than 0.1% peak area response in comparison to the reference standard Salicylic acid of concentration 0.5 µg/ml. For the presence of lamivudine impurity C (Salicylic acid) above the specified level can affect the pharmacokinetic of the drug lamivudine.

The literature survey shows no reported HPTLC method for the individual drug. Our study establishes a validated method to know the concentration of Salicylic acid in the bulk drug and different kinds of formulations in a sensitive level. So, this method can be used for the routine analysis of API lamivudine and in oral dosage form.

ACKNOWLEDGEMENT:

The authors are thankful to the M/S SNR Sons charitable trust for providing the necessary facilities to carry out the work.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

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Received on 04.12.2023 Modified on 25.04.2024

Asian J. Pharm. Res. 2024; 14(3):213-220.

DOI: 10.52711/2231-5691.2024.00034