Selective, Sensitive and High-throughput Simultaneous Method Development of Zidovudine, Lamivudine and Nevirapine in Human Plasma Using 96 well Plate Solid Phase Extraction and Liquid Chromatography Coupled with Tandem Mass Spectrometry

A high throughput, sensitive, selective, and rugged liquid chromatography coupled with mass spectrometry (LC-MS/MS) method for the quantification of Zidovudine, Lamivudine and Nevirapine, in human plasma was developed and validated.  The analytes were extracted from plasma by solid phase extraction technique using Waters Oasis HLB 96 well plate 30 µm (30 mg). Isocratic  elution  of  Zidovudine, Lamivudine and Nevirapine  were  achieved  in  3 min  using  ACE, C18, 4.6 X 150mm, 5µ column having a mobile phase of 0.1% formic acid: Methanol 30: 70 v/v. The flow rate was 0.7 mL/min at a column temperature of 35 ± 5°C. Electron spray ionization technique in positive mode was selected to improve the selectivity and sensitivity required for this application. The retention times of Zidovudine, Lamivudine and Nevirapine were 1.5, 1.2, and 1.7 min, respectively. The method was validated for linearity, precision, accuracy, specificity, sensitivity, matrix effect, dilution integrity, ruggedness, reinjection reproducibility, and stability. The assay produced linear calibration curves over the concentration range for Zidovudine, Lamivudine and Nevirapine in the ranges of 5 to 1500, 5 to  1500 and 10 to 3000 ng/mL respectively with correlation coefficients greater than 0.9963 using a 1/x2  weighted least square regression analysis of standard plots associated with eight-point calibration standards. The precision and mean accuracy were within the acceptable limits.