INTRODUCTION:

Lamivudine (3TC). Chemically lamivudine is 4-amino-1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2-dihydropyrimidin-2-one. Lamivudine is a synthetic drug and phosphorylated intracellularly to its active 5'-triphosphate metabolite, lamivudine triphosphate. This nucleoside analogue is incorporated into viral DNA by HIV reverse transcriptase and HBV polymerase, resulting in DNA chain termination. It can be used in HIV-1 and hepatitis B (HBV).

Stavudine (d4T) is a dideoxynucleoside analog. IUPAC name of stavudine is 1-[(2R, 5S)-5-(hydroxymethyl)-2,5-dihydrofuran-2-yl]-5-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione. Stavudine acts by an inhibiting the activity of HIV-1 reverse transcriptase (RT) both by competing with the natural substrate dGTP and by its incorporation into viral DNA.

Nevirapine (NEV) is a potent, non-nucleoside reverse transcriptase inhibitor used in combination with nucleoside analogues for treatment of HIV infection and AIDS. IUPAC Name of Nevirapine is 2-cyclopropyl-7-methyl-2,4,9,15-tetraazatricyclo[9.4.0.0^{3,8}]pentadeca-1(15),3,5,7,11,13-hexaen-10-one. Nevirapine acts binds directly to an enzyme i.e. reverse transcriptase (RT) and blocks the RNA-dependent and DNA-dependent DNA polymerase activities by causing a disruption of the enzyme's catalytic site. The structure of lamivudine, stavudine and nevirapine are given in the Fig 1(a), (b) and (c).^1-4

(a) Lamivudine

(b) Stavudine (c) Nevirapine

Fig 1

The litrerature survey^5-13 reveals that the analytical methods like UV, HPLC, UPLC and HPTLC for determination of these drugs individually and other combination in pharmaceuticals and biological preparations. In the present investigation an attempt was made to develop a simple, new, accurate, sensitive and economical HPLC method for the simultaneous estimation of lamivudine, stavudine and nevirapine in pure sample and tablet dosage forms. The proposed method has been validated as per ICH guidelines¹⁴.

MATERIALS AND METHODS:

All analytical works were performed on HPLC Shimadzu LC 2010 CHT series equipped with quaternary constant flow pump, auto injector, SPDM 10 A VP Shimadzu Photodiode Array Detector and LC Solution Version 1.22 SP1 Software, Phenomenex Gemini C₆ Phenyl column (250mm × 4.6mm, 5µ) forms the stationary phase. A calibrated single pan balance i.e. Sartorius CP 225 D, Velp scientific vortex mixer, pH meter of LAB INDIA, Enertech Fast Clean Ultrasonic cleaner, were also used during the analysis. All chemicals and reagents used were of AR/HPLC grade and HPLC water was prepared from Milli-Q in the lab. The reference standards of lamivudine, stavudine and nevirapine were collected from Cipla Laboratory. The tablet was purchased from the local market.

Preparation of mobile phase:

The mobile phase was prepared by mixing 500ml of 0.02M NH₄H₂PO₄ [pH adjusted to 2.5±0.1 with formic acid (98%)] buffer with 500ml of methanol. The mobile phase was sonicated for 10 mins and filtered through 0.45µ membrane filter paper.

Preparation of mixed standard stock solution:

It was prepared by weighing accurate quantity of Lamivudine 75mg, Stavudine 15mg, and Nevirapine 100mg and the same was dissolved in small volume of mobile phase and sonicated for 10mins in a 50ml of volumetric flask and finally made up to mark with mobile phase to get 1500, 300 and 2000µg/ml of Lamivudine, Stavudine and Nevirapine respectively.

Optimized Chromatographic conditions:

RP-HPLC analysis was performed by isocratic elution with flow rate of 1ml/min. The mobile phase consisting of 0.02 M ammonium dihydrogen phosphate buffer having pH 2.5 was adjusted with formic acid (98%) and methanol were used in the ratio of (50:50) to obtain well-resolved peaks of Lamivudine, Stavudine and Nevirapine as shown in the Fig. 2. Injection volumes of 20µl laboratory mixture of standard solutions were injected into the column. The detection wavelength was set at 264nm. The chromatographic run time was set at 25 minutes.

Fig. 2: Representative chromatogram of laboratory mixture of standard Lamivudine, Stavudine, and Nevirapine.

Preparation of Standard Calibration Curves for Lamivudine and Stavudine and Nevirapine: From the standard stock solution 0.2ml, 0.4ml, 0.6ml and 0.8ml, and 1ml were diluted to 10 ml with mobile phase to give final concentrations (30, 60, 90, 120, 150µg/ml) of Lamivudine, (6, 12, 18, 24, 30µg/ml) of Stavudine and (40, 80, 120, 160, 200µg/ml) of Nevirapine. 20µl of the working standard solutions were injected into the column (n=5). Peak areas were recorded for all the peaks. The plots of peak area of series of the respective concentration of 3TC, d4T, NEV were found to be linear in the range of 30-150, 6-30, and 40-200µg/ml, respectively. The linearity data of Lamivudine, Stavudine, and Nevirapine are given in Table 1.

Analysis of the marketed formulation:

Twenty Tablets were weighed accurately and crushed to a fine powder. An accurately weighed quantity of powder equivalent to 150mg of Lamivudine, 30 mg of Stavudine, and 200 mg of Nevirapine was dissolved in 75ml of mobile phase and sonicated for 15mins in a 100 ml volumetric flask and then the volume was made up to mark with the mobile phase and then filtered through a 0.45 µ membrane filter paper. From this above solution, 1 ml was transferred into 10 ml of volumetric flask and diluted up to the mark with the mobile phase. 20µl of these solutions were injected into the HPLC system. The concentration of drug present in tablet dosage form was computed from the calibration curve. The results of tablet analysis are given in Table 2. The chromatogram of the tablet sample is shown in Fig. 3.

Fig. 3: Representative chromatogram of tablet sample containing 3TC, d4T and NEV.

Method Validation:

The proposed method was validated according to ICH guidelines Q2 B.

System Suitability: System suitability test was carried out on freshly prepared standard stock solution of Lamivudine, Stavudine and Nevirapine (90, 18, 120 µg/ml), and the results of parameters were obtained by five replicate injections. The system suitability results are given in Table 3.

Specificity:

The peak purity of 3TC, d4T, and NEV was assessed by comparing the retention time of standard 3TC, d4T, and NEV. A good correlation was also found between the retention time of standard and sample of 3TC, d4T, and NEV.

Linearity:

Linearity was studied by preparing different concentration levels. The linearity ranges for Lamivudine, Stavudine and Nevirapine were found to be linear in the range of 30-150, 6-30 and 40-200µg/ml respectively. The linear regression equations for 3TC, d4T, and NEV were found to be Y = 182450.300 + 39412.77x, Y = 18110.4762 + 55791.5238 x and Y = 78,495.00 + 35,041.1550 x with Co-efficient of correlation (r2) 0.9991, 0.9992, 0.9996 respectively.

Precision:

The precision study was performed to find out intra-day and inter-day (three days) variations in the estimation of 3TC, d4T, and NEV of different concentrations, with the proposed method. Percentage relative standard deviation (%RSD) was found to be less than 1% within a day and day-to-day variations, which proves that method is precise.

Accuracy:

Standard addition method was used to perform an accuracy study. A known amount of standard 3TC, d4T, and NEV corresponding to 80%, 100%, and 120% of the label claim was added to the pre-analyzed sample of tablet dosage form separately. The recovery studies were carried out four times, at each level of recovery. The results of studies along with its evaluation are given in Table 3.

Stability study:

Stability study of reagents, mobile phase, standard, and sample solutions were studied for 48hrs at 1hr intervals and compared with freshly prepared solutions, which was found to be stable. The results are found to %RSD i.e. 0.28%, 0.37%, and 0.56% for Lamivudine, Stavudine, and Nevirapine respectively.

Table 3: Recovery studies of 3TC, d4T and NEV.

Analyte	Level of % Recovery	Pure drug added (µg/ml)	Formulation (µg/ml)	C.I.	% RSD	% SE	t
3TC	80%	48	60	100.104±0.169	0.105	0.053	1.956
	100%	60		100.037±0.175	0.109	0.055	0.681
	120%	72		100.003±0.174	0.108	0.054	0.068
d4T	80%	9.6	12	99.766± 1.221	0.769	0.383	0.610
	100%	12		100.250±0.742	0.464	0.233	1.072
	120%	14.4		100.399±0.994	0.622	0.312	1.278
NEV	80%	64	80	100.027±0.146	0.091	0.046	0.591
	100%	80		99.984± 0.085	0.053	0.026	0.577
	120%	96		100.039±0.227	0.143	0.071	0.547

SD: Standard deviation, % SE: Percent standard error, C.I.: Confidence Interval within which true value may be found at 95% confidence level = R ± ts/√n, R: Mean percent result of analysis of Recovery study (n = 4). Theoretical‘t’ values at 95% confidence level for n - 1 degrees of freedom t (0.05, 3) = 3.182.

Robustness:

By introducing small but deliberate changes in the mobile phase pH (±0.5), mobile phase composition (±2%), detection wavelength (±5.0nm), and flow rate (±1%) robustness of the described method were studied. The robustness of the method was assessed for 3 different levels of a mixture of concentration of the calibration plot (60, 12, 80µg/ml), (90, 18, 120µg/ml), and (120, 24, 160µg/ml). All the robustness parameters were less than 1.5% RSD.

Sensitivity:

The LOD and LOQ were separately estimated by using a standard calibration curve. The Lower limit of detection of Lamivudine, Stavudine, and Nevirapine was found to be 0.0622, 0.0125, and 0.0578µg/ml respectively. The Lower limit of quantitation of Lamivudine, Stavudine, and Nevirapine was found to be 0.1984, 0.1157, and 0.1021µg/ml respectively.

Forced degradation studies:

Forced degradation studies were performed to evaluate the stability-indicating properties and specificity of the method. Intentional degradation was carried out by exposing 25.25, 25.20, and 25.62mg of Lamivudine, Stavudine, and Nevirapine in 25ml volumetric flasks containing 0.1N HCl for 3 hours and ultrasonicate 1hr. Then aliquot 5ml was transferred into another 25ml volumetric flask and made up to the mark with mobile phase to obtain the below-mentioned concentration of 3TC and d4T and NEV (Table 4). Basic Hydrolysis degradation was carried out by exposing 25.25, 25.94, and 25.39mg of Lamivudine, Stavudine, and Nevirapine in a 25ml volumetric flask containing 0.1N NaOH for 3hours and ultrasonicate 1hr. Then aliquot 5ml was transferred into another 25ml volumetric flask and made up to the mark with mobile phase to obtain the below-mentioned concentration of 3TC and d4T and NEV (Table 4).

Acid, base, and oxidative degradation chromatograms of 3TC, d4T, and NEV are shown in Fig. 4, 5, and 6 respectively.

Table 4: Forced degradation study of 3TC, d4T, and NEV.

SL. No.	Agent	Analyte	Initial Concen tration (µg/ml)	Stressed Concen tration (µg/ml)	% Degradation
1	0.1 M HCl	3TC	202	177.26	12.24
		d4T	201.6	184.3	8.58
		NEV	204.96	182	11.2
2	0.1 M NaOH	3TC	202	178	11.88
		d4T	207.52	185	10.85
		NEV	203.12	181	10.89
3	3% H2O2	3TC	202	141	30.19
		d4T	201.12	143	28.89
		NEV	204.4	167	18.29

%degradation = [(initial concentration - stressed concentration)/initial concentration] x 100

Fig. 4: Acid (0.1N HCl) degradation chromatogram of Lamivudine, Stavudine and Nevirapine

Fig. 5: Base (0.1 M NaOH) degradation chromatogram of Lamivudine, Stavudine and Nevirapine

Fig. 6: Oxidative (3% H₂O₂) degradation chromatogram of Lamivudine, Stavudine and Nevirapine

RESULTS AND DISCUSSION:

To develop a precise, accurate, and suitable RP-HPLC method for the simultaneous estimation of 3TC, d4T, and NEV, different mobile phases and stationary phases were employed and the proposed chromatographic conditions were observed appropriate for the quantitative determination. In order to get sharp peaks and baseline separation of the components, a number of experiments were conducted by varying the composition of the mobile phase (buffer and methanol in the ratios of 70:30, 60:40, 40:60, 50:50, and 45:55) at a different flow rate (0.5, 0.8, 0.6 and 1ml/min) and at different wavelengths. A mobile phase comprising of 50:50 v/v preparation of buffer (pH 2.5) and methanol, at a flow rate 1ml/min and a Phenomenex Gemini C6 Phenyl column and an injection volume of 20µl and 25^OC temperature for the RP-HPLC system was found to be the best for analysis. The modalities adopted in the newly developed method were successfully validated as per analytical procedures laid down in routine. The proposed method was validated by preliminary analysis of the reference samples and by recovery studies. The percentages of average recoveries were observed in the range of 98 to 102%. The results of the analysis of average recoveries obtained in each instance were compared with the theoretical value of 100 percent by means of the Student’s‘t’ test. As the calculated‘t’ values were less than theoretical ‘t’ values (Table 3), it was concluded that the results of recoveries obtained in agreement with 100 percent for each analyte were accurate. Additional peaks were not seen in the chromatogram indicates non-interference of the common excipients used in the tablets. Stress studies were performed in 3TC, AZT, and NEV by exposing the drug to 0.1 N HCl, 0.1 N NaOH, 3% H₂O₂for three hours and ultra-sonication for one hour, and it was found that the drugs were reasonably degraded (≤ 30%) in acidic, basic and oxidative medium which is acceptable as per ICH guidelines. All the robustness parameters were less than 1.5% RSD. All the system suitability parameters were obeying the limits I.P. Some results are rejected by employing the Q test statistical equation. This demonstrates that the developed HPLC method is a new, simple, linear, accurate, sensitive, and reproducible, robust, and stability-indicating assay. So this method can be easily used for the routine quality control of bulk and tablet dosage forms.

ACKNOWLEDGMENTS:

The authors are thankful to the Dr. Tripathy of Startech Pvt Ltd, Hyderabad for providing the necessary facilities to carry out this research work.

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Received on 08.11.2020 Modified on 28.03.2021

Research J. Pharm.and Tech 2022; 15(2):541-545.

DOI: 10.52711/0974-360X.2022.00087

Concentration of Lamivudine (µg/ml)	Peak area	Concentration of Stavudine (µg/ml)	Peak area	Concentration of Nevirapine (µg/ml)	Peak area
30	1298758	6	350978	40	1542937
60	2622861	12	711448	80	2921035
90	3764544	18	1041605	120	4299133
120	4879462	24	1345174	160	5677230
150	6082373	30	1680695	200	7055328

Analyte	Label claim (mg/tab)	Amount found (mg/tab)	C.I.	%RSD	t
Lamivudine	150	150.152	100.371±0.480	0.745	1.110
Stavudine	30	30.072	100.008±0.921	0.578	0.028
Nevirapine	200	200.125	100.054±0.745	0.602	0.941