INTRODUCTION:

Human immunodeficiency virus (HIV), the causative agent of acquired immunodeficiency syndrome (AIDS), develops resistance rapidly.^1,2 Therapeutic strategy regimens require the combination of antiretroviral drugs to inhibit HIV.^3,4 The combination of two nucleoside reverse transcriptase inhibitors (NRTIs) such as lamivudine (3TC) and stavudine (D4T) and a non-nucleoside reverse transcriptase inhibitor (NNRTI) such as nevirapine (NVP) was the first line regimen in HIV therapy.^5,6 Chemical structures of these drugs are shown in Figure 1. A number of analytical methods have been developed for analyzing these drugs individually or in combination with other drugs in various formulations and biological fluids.^7-12

However there is no method available for the simultaneous determination of these three drugs. Therefore, an attempt was made to develop a new, rapid and sensitive method for the simultaneous determination of 3TC, D4T and NVP. To access the reproducibility and wide applicability of the developed method, it was validated as per ICH norm, which is mandatory also.^{13, 14}

MATERIALS AND METHODS:

Instrumentation:

Liquid chromatographic system from Shimadzu comprising of manual injector, double reciprocating plunger pump LC10 ATvp for constant flow and constant pressure delivery and Photodiode array detector SPD-M10 Avp connected to software Class M10A for controlling the instrumentation as well as processing the data generated was used.

Chemicals and reagents:

Lamivudine, stavudine and nevirapine pure drugs were kindly supplied as gift samples by Ranbaxy Lab, Dewas India. Concentrated orthophosphoric acid, methanol were of HPLC grade supplied by Merck Ltd., India. Triple distilled water was generated in house. Tablets, Triamune–30 of Cipla Limited India containing Lamivudine, Nevirapine and Stavudine in ratio of 150 mg: 200mg: 30mg respectively were purchased from local market.

Chromatographic condition:

An Inertsil-ODS-3 (C-18) Column (5 µm, 250mm x 4.60mm) was used as the stationary phase. Two mobile phase components: mobile phase A comprising of 0.1% OPA and mobile phase B (methanol). The mobile phases were filtered and then degassed by sonication prior to use. A gradient elution was programmed as described in Table 1. The optimum wavelength of 254nm was suitable for measuring absorbance of all analytes. The injection volume was 20ml at a flow rate of 1.0 ml/min.

Fig-1 Chemical structures of (A) Lamivudine (B) Stavudine (C) Nevirapine

Table 1 Mobile phase program for gradient elution with a constant flow rate of 1.0 mL/min

Time (Min)	Solvent A (%)	Solvent B (%)
0.01	60	40
6.00	40	60
13.0	60	40
15.0	Stop

Note: Solvent A = 0.1% OPA, Solvent B = Methanol.

Standard preparation:

Standard stock solution:

Standard stock solutions of 2000, 1500, 300 µg ml^-1 of NVP, 3TC and D4T were prepared in methanol respectively.

Working standard solution:

Working standard solutions were prepared by taking dilutions ranging from 40-200, 30-150, 6-30 mg/ml for NVP, 3TC and D4T respectively. These solutions were used to calculate the linear dynamic range and for the relative quantification of the tablets. Samples in triplicates were made for each concentration. Peak areas were plotted against corresponding concentrations to obtain the calibration curves.

Sample preparation:

Twenty tablets of Triamune – 30 of Cipla Limited India containing 3TC, NVP and D4T 150 mg: 200mg: 30mg respectively were weighed and crushed to fine powder. Powder equivalent to 150mg lamivudine was weighed and dissolved in 100 ml methanol, sonicated for 10 min and filtered through whatmann filter paper No. 42, finally different concentrations of tablet sample were prepared by serial dilution technique.

RESULTS AND DISCUSSION:

Chromatography:

Initially reverse phase LC separation was tried to develop using different organic solvents in combination isocratically in which the drugs were resolve poorly. Thereafter, taking into consideration the system suitability parameter like RT, tailing factor, No. of theoretical plates, in gradient flow the mobile phase comprising of 0.1% OPA and methanol was found suitable in gradient elution which was programmed as described in Table 1. The gradient systems run in the flow rate of 1 mL/min. To analyze these three drugs detection were tried at various wavelengths ranging from 230nm to 280nm. The optimum wavelength of 254 nm was suitable for measuring absorbance of all analytes.

Under optimized conditions, 3TC and D4T, NVP showed good selectivity, resolution with retention time 2.6, 4.8 and 10.09 min respectively.

System suitability:

System suitability parameters such as number of theoretical plates, HETP and peak tailing are determined. The results obtained are shown in Table 2. The number of theoretical plates for NVP, 3TC and D4T were 22354, 2391 and 5044 respectively.

Table 2 System suitability

Serial No.	Parameters	3TC	D4T	NVP
1	No. of Theoretical plates	2391	5044	22354
2	HETP	0.051	0.055	0.151
3	Tailing factor	1.32	1.24	1.15

Linearity:

NVP, 3TC and D4T showed a linearity of response between 40-200, 30-150, 6-30 mg/ml respectively. The linearity was represented by a linear regression equation as follows.

Y (NVP) = 43920.06 conc. + 93306.13 (r²=0.9996)

Y (3TC) = 25989.09 conc. + 59765.08 (r²=0.9998)

Y (D4T) = 46161.09 conc. + 2954.65 (r²=0.9996)

Accuracy:

Recovery studies were performed to validate the accuracy of developed method. To preanalyzed sample solution, a definite concentration of standard drug was added and recovery was studied. These results are summarized in Table 3.

Precision:

Repeatability:

Five dilutions in three replicates were analyzed in same day for repeatability and results were found within acceptable limits (RSD ‹ 2) as shown in Table 4.

Intermediate Precision:

Five dilutions in three replicates were analyzed on two different days and by two analysts for day to day and analyst to analyst variation. The RSD values for all results were fall within acceptable limits (RSD ‹ 2) as shown in Table 4.

Robustness:

As per ICH norms, small, but deliberate variations in concentration of the mobile phase were made to check the method’s capacity to remain unaffected. The ratio of mobile phase was changed from, Methanol : 0.1% OPA in 40:60 V/V to Methanol : 0.05% OPA, Methanol : 0.15% OPA in 40:60 V/V to 40:60 V/V in gradient flow and Methanol : 0.1% OPA in 45:55 V/V to 45:55 V/V and 35:65 V/V to 35:65 V/V in gradient flow found robust as RSD is found less than 2.0% shown in Table 5.

Table 3 Results of recovery experiments

Conc. of drug in preanalyzed samples (mg/ml)			Std. drug sol. Added (mg/ml)			*Recovered amount (**mg/ml)			%Recovered
3TC	D4T	NVP	3TC	D4T	NVP	3TC	D4T	NVP	3TC	D4T	NVP
15.86	2.93	19.87	16.79	2.98	20.30	16.65	3.21	21.38	99.17	107.7	105.4
31.72	5.86	39.75	33.58	5.96	40.60	34.5	6.42	43.82	102.74	107.7	107.9
47.58	8.79	59.62	50.36	8.95	60.90	50.82	9.56	65.12	100.92	106.8	106.9
								Mean	100.94	107.4	106.73
								S.D	1.78	0.51	1.32
								%RSD	1.76	0.47	1.23

* Mean of three reading

Table 4 Results of precision

Validation Parameter	% Mean*			S.D.			% R.S.D.
	3TC	D4T	NVP	3TC	D4T	NVP	3TC	D4T	NVP
Repeatability	100.5	99.94	99.32	1.07	1.93	1.25	1.06	1.93	1.26
Intermediate precision
Day to Day	100.07	100.25	100.05	0.61	0.44	1.03	0.61	0.44	1.03
Analyst to Analyst	100.07	100.25	100.24	0.09	0.09	0.54	0.08	0.09	0.54

* Mean of fifteen determinations (3 replicates at 5 concentration level)

Fig 2 Representative Chromatogram of Lamivudine, Stavudine and Nevirapine

Table 5 Results of robustness

Parameters	Lamivudine	Stavudine	Nevirapine
Change in Concentration of Mobile Phase
%Mean*	99.51	105.37	102.84
S.D.	0.23	1.11	0.17
% R.S.D.	0.23	1.05	0.17
Change in Ratio of Mobile Phase
%Mean*	100.36	106.02	103.35
S.D.	0.31	0.01	0.31
% R.S.D.	0.31	0.00	0.30

* Mean of six determinations

Stability of sample solution:

The sample solution injected after 12 hr did not show any appreciable change. Results are shown in Table 6.

Table 6 Stability data of 3TC, D4T and NVP

AUC ±%RSD
Hours	3TC 30mg/ml	D4T 6mg/ml	NVP 40mg/ml
0	859299±0.31	286482±0.53	1894716±1.25
6	859198±0.42	285178±0.68	1861761±2.23
12	859429±0.35	285489±0.93	1938030±2.25

Tablet Analysis :

Content of NVP, 3TC and D4T found in the tablets by the proposed method are shown in Table 7. The low values of R.S.D. indicate that the method is precise and accurate.

Table 7 Results of the HPLC analysis for tablets

Serial. No.	Parameters	Triamune–30
		3TC	D4T	NVP
1	% Mean*	101.02	99.26	99.26
2	S.D	1.21	0.97	0.97
3	% R.S.D	1.20	0.98	0.98
4	SEσ	0.49	0.39	0.39

* Mean of fifteen determinations (3 replicates at 5 concentration level)

CONCLUSIONS:

RP-HPLC method was developed and validated for simultaneous estimation of NVP, 3TC and D4T in tablet dosage form. Proposed method is fast, accurate, precise and sensitive; hence it can be employed for routine quality control of tablets containing these three drugs in industries.

REFERENCES:

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