Validation of an UPLC-MS/MS Method for Quantitative Analysis of Raltegravir in Human Plasma Samples

Background: An ultra-performance liquid chromatography-tandem mass spectrometry method was developed for the quantification of raltegravir (RTG) plasma concentrations in samples from HIV patients treated with the drug. Methods: Plasma samples were extracted by liquid-liquid extraction followed by evaporation to dryness and reconstitution in mobile phase. The chromatographic separation was carried out on an AQUITY UPLC C-18 column with an isocratic mobile phase consisting of water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid (50:50 vol/vol). The detection was performed on a triple quadrupole tandem mass spectrometer using multi-reaction monitoring via electrospray ionization source with positive ionization mode. Results: Under these conditions, a single chromatographic run could be completed within 1 minute. The method was validated by estimating the precision and the accuracy for inter- and intra-day analysis in the concentration range of 5-2560 ng/mL. The method was linear over the investigated range with all the correlation coefficients, r, greater than 0.995 on 5 replicates. The intra- and inter-day precision (percentage of coefficient of variation) ranged from 2.4% to 11.2%, and the inaccuracy (percent of relative standard deviation) ranged from 2.5% to 12.9%. No significant matrix effect was observed. The mean recovery value of RTG was 80%. Conclusions: This rapid and sensitive method was validated and could be applied to pharmacokinetic studies for the determination of RTG concentrations in human plasma samples.