Fast and simultaneous determination of darunavir and eleven other antiretroviral drugs for therapeutic drug monitoring: Method development and validation for the determination of all currently approved HIV protease inhibitors and non-nucleoside reverse transcriptase inhibitors in human plasma by liquid chromatography coupled with electrospray ionization tandem mass spectrometry

For the quantification of all currently approved non-nucleoside reverse transcriptase inhibitors and protease inhibitors, including the new protease inhibitor darunavir and the active nelfinavir metabolite M8, an assay was developed, using liquid chromatography coupled with tandem mass spectrometry. The sample pretreatment consisted of a protein precipitation with a mixture of methanol and acetonitrile using only 100 μL plasma. Chromatographic separation was performed on a reversed-phase C18 column (150 X 2.0 mm, particle size 5 μm) with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.25 mL/min. The analytical run time was only 10 min. The triple quadrupole mass spectrometer was operated in the positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over a range of 0.1 to 20 μg/mL for amprenavir, atazanavir, efavirenz, indinavir, lopinavir, nelfinavir, the active nelfinavir metabolite M8, nevirapine and ritonavir, a range of 0.05 to 10 μg/mL for saquinavir and darunavir and a range of 0.5 to 100 μg/mL for tipranavir, based on observed concentration ranges in patients treated with these drugs. D5-squinavir, D6-indinavir, 13C6-efavirenz and dibenzepine were used as internal standards. The method was proven to be specific, accurate, precise and robust. Accuracies ranged from 88.5% to 102.2% and all precisions were less than 9.5%. Furthermore, the assay demonstrates a high sensitivity for all analytes and the stepwise gradient allows addition of new analytes into the same method. The method is now successfully applied for routine therapeutic drug monitoring and pharmacokinetic studies in HIV-infected patients.