Development and validation of an UPLC-MS/MS bioanalytical method for simultaneous quantification of the antiretroviral drugs dolutegravir, elvitegravir, raltegravir, nevirapine and etravirine in human plasma.

Dolutegravir, elvitegravir, raltegravir, nevirapine and etravirine are antiretroviral drugs used as part of combined antiretroviral treatment for HIV-infection. For quantification of these drugs in human K2EDTA plasma samples an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) bioanalytical method was developed and validated. Stable isotope labeled internal standards were used for each analyte. Simple protein precipitation with methanol was implemented to prepare plasma samples of at least 50 μL. The method was validated for dolutegravir, elvitegravir, raltegravir, nevirapine and etravirine over the ranges 9.7-9700, 52-10,470, 9.7-9730, 73-14,680 and 15-3010 ng/mL, respectively. Within-run and between-run accuracy and precision were within ±15% of the nominal concentration for quality controls at high, medium and low concentrations, and within ±20% at the lower limit of quantification for all analytes. Recovery was ≥76% and reproducible. Long-term stability of patient plasma samples was demonstrated for at least 12 months at -40 °C (4 months for etravirine). Currently, this robust method with a run time of 10 min is used in clinical research and for therapeutic drug monitoring of these frequently used antiretroviral drugs.