The Development and Validation of RP-HPLC Method for Lamivudine, Dolutegravir, and Tenfovir in Human Plasma

Abstract

The present work aimed to develop a simple, rapid, and accurate method for the estimation of Dolutegravir, Lamivudine and Tenofovir disoproxil fumarate in human plasma, as per US-FDA guidelines. Moreover and the present method is the first for the estimation of this combination in a biological matrix. For the estimation of Lamivudine, Dolutegravir, and Tenfovir disoproxil fumarate in human plasma using the Cobicistat as internal standard by RP-HPLC (Reverse Phase-High Performance Liquid Chromatographic) technique, a straightforward, precise, and accurate method has been developed. The pure drug samples of Dolutegravir, Lamivudine and Tenofovir in were purchased from Selleck chem LLC supplied by Pro lab marketing. HPLC grade Acetonitrile, HPLC grade Methanol and all other chemicals were obtained from Merck chemical division, Mumbai. The Chromatographic separation was achieved on discovery C18, 250 mm x 4.6 mm, 5\(\mu\), Column at 30⁰C temperature.

The separation was achieved employing a mobile phase consists of 0.1% v/v Ortho phosphoric acid and Acetonitrile taken in the ratio of 65:35 (v/v). The flow rate was maintained at 1.0 ml/min, detection wave length was 277 nm. Retention time of Lamivudine, Dolutegravir and Tenfovir were found to be 2.994 min, 4.350 min and 5.688 min respectively. The peaks were found to be free of interference. The method was validated over a dynamic linear range of 60-2400 ng/ml, 190-7600 ng/ml, and 18-720 ng/ml for Lamivudine, Dolutegravir and Tenfovir respectively, with a Correlation coefficient of 0.998. The precision and accuracy of samples of six replicate measurements at lower limits of quantification level were within the limits. The analytes were found to be stable in human plasma at -28°C for 37 days. The determination of Lamivudine, Dolutegravir, and Tenfovirin in human plasma is appropriate due to the stability, sensitivity, specificity, and reproducibility of this method. According to US-Food and Drug Administration guidelines, the reported method was validated, and it was discovered to be substantially within the acceptable range.