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METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF LAMIVUDINE AND ZIDOVUDINE IN TABLET BY REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Objective: The objective of the study was to develop and validate reverse-phase high-performance liquid chromatography (RP-HPLC) method and apply method to tablet dosage form. 
Methods: A simple, rapid, economical, precise, and accurate RP-HPLC method for simultaneous estimation of lamivudine and zidovudine in their combined dosage form has been developed. 
Results: A RP-HPLC method was developed for the simultaneous estimation of lamivudine and zidovudine. In their combined dosage form has been developed. The separation was achieved by LC-C18 column (150 mm ×4.6 mm, 5 μm) and water: methanol (65:35v/v) as mobile phase, at a flow rate of 0.8 ml/min. Detection was carried out at 272 nm. Retention time of lamivudine and zidovudine was found to be 3.007 min and 4.647, respectively. The method has been validated for linearity, accuracy, and precision. The assay method was found to be linear from 50% to 150% for lamivudine and zidovudine. 
Conclusion: Developed method was found to be accurate, precise, and rapid for simultaneous estimation of lamivudine and zidovudine in their combined dosage form.


System suitability parameters
System suitability tests were performed to verify that the resolution and repeatability of the system were adequate for the analysis intended. The parameters monitored for system suitability include retention time, theoretical plate number, peak area, tailing factor, and resolution. The repeatability of these parameters was checked by injecting 3 times the test solution of lamivudine 10 μg/mL and zidovudine 25 μg/mL. The results shown in Table 1 were within acceptable limits.

Specificity
Specificity of method can be termed as absence of any interference at retention times of samples. Specificity was performed by injecting blank and standard preparations. Chromatograms were recorded and retention times from sample and standard preparations were compared for the identification of analytes.

Linearity and range
A series of standard solutions 5-15 μg/ml of lamivudine and 15-35 μg/ml of zidovudine were prepared. An aliquot of 20 μl of each solution was injected 3 times for each standard solutions and peak area was observed. Plot of average peak area versus the concentration is plotted and from this, the correlation coefficient and regression equation were generated. The calibration data of lamivudine and zidovudine are given in Table 2a and b, while Fig. 3a and b represents linearity graphs of three drugs, respectively.

Precision
The method was validated in terms of intraday and interday precision. The solution containing lamivudine -10 μg/ml and zidovudine -25 μg/ml was injected 6 times for repeatability study. Interday and intraday study was performed by injecting 5, 10, and 15 μg/ml of lamivudine and 15, 25, and 35 μg/ml of zidovudine solutions 3 times for each aliquot. The %RSD for precision study was found to be <2%, as shown in Table 3a-c.

Accuracy
Accuracy was determined by calculating recovery of lamivudine and zidovudine by the standard addition method. Known amounts of standard solutions of lamivudine (5, 10, and 15 μg/ml) and zidovudine (15, 25, and 35 μg/ml) were added to a pre-quantified test solutions         of lamivudine (10 μg/ml) and zidovudine (25 μg/ml). Each solution was injected in triplicate and the recovery was calculated by measuring peak areas. Results obtained are shown in Table 4a and b.

Limit of detection (LOD) and limit of quantification (LOQ)
LOD and LOQ for SC and EV were calculated as suggested by ICH guidelines using equations LOD =3.3 σ/s and LOQ =10 σ/s, respectively. Where, σ is the SD of the response and S is the slope of the calibration curve.

Robustness
The robustness study was performed to evaluate the influence of small but deliberate variation in the chromatographic condition. The robustness was checked by making two small changes. The mobile ration was changed by ±2 ml and flow rate was changed by ±0.02 ml/min and pH was changed by ±0.2. After each changes, sample solution was injected and system suitability parameters were observed. The results are shown in Table 5.

System suitability study
The detection was carried out in the ultraviolet region at 272 nm. The different composition of mobile phase was testing and the composition giving retention time of 3.007 min for lamivudine and 4.660 min for zidovudine with good resolution and theoretical plates was selected that optimized mobile phase was water: methanol (65:35v/v). A chromatogram of the mixture in optimized conditions is shown in Fig. 4 and the system suitability parameters are shown in Table 1. Patel et al.

Specificity
The method was found to be specific as there was no interference observed in any of the parameters under observation.

Linearity and range
The linearity of lamivudine and zidovudine was found between 5-15 μg/ml and 15-35 μg/ml, respectively. The results are shown in Table 2a and b.

Precision
The %RSD for repeatability study for lamivudine and zidovudine was found to be 0.355 and 0.202, respectively. The interday and intraday study also shows %RSD value for lamivudine and zidovudine within the acceptable limit. Results for precision study are shown in Table 3a-c.

Accuracy
Accuracy of the method was confirmed by recovery study at three levels (50%, 100%, and 150%) of standard addition. Percentage recovery for lamivudine was found to be 99.72-99.82% and zidovudine was found to be 99.68-99.74%, as shown in Table 4a and b.

LOD and LOQ
The LOD was found to be 0.285 μg/ml for lamivudine and 0.225 μg/ml for zidovudine, while the LOQ was found to be 0.889 μg/ml for lamivudine and 0.723 μg/ml for zidovudine.

Robustness
The typical variations studied under this parameter were mobile phase composition, pH, and flow rate. Overall %RSD was found to be <2% for all the variations which indicates that the proposed method is robust. Robustness data are shown in Table 5.

Analysis of marketed formulation by proposed method
Applicability of the proposed method was tested by analyzing the commercially available marketed formulation. The percentage of lamivudine and zidovudine was found to be 100.02% for lamivudine and 99.86% for zidovudine, respectively.

CONCLUSION
From the above discussion, it can be concluded that the proposed method is specific, precise, and accurate. Results are in good agreement with label claim which indicates that there is no interference of excipients. Therefore, the proposed method can be used for routine analysis of lamivudine and zidovudine in combined tablet formulation.