Automated, Fast, and Sensitive Quantification of Drugs in Human Plasma by LC/LC-MS

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Abstract
An analytic assay based on automated sample preparation and liquid chromatography (LC) coupled with electrospray mass spectrometry (ESI-MS) was developed for the quantification of 6 protease inhibitors (PIs) and 3 nonnucleoside reverse transcriptase inhibitors (NNRTIs). The 6 PIs, amprenavir, indinavir, ritonavir, lopinavir, nelfinavir, and saquinavir, as well as the three NNRTIs, nevirapine, efavirenz, and delavirdine, require a succinct analysis technique for therapeutic drug monitoring in HIV/AIDS patients. After protein precipitation, samples were loaded on a C8, 10 x 4-mm extraction column, washed, and, after activation of the column-switching valve, backflushed onto the 30 x 2.1 mm C8 analytic column. [M+H] ions were detected in the selected ion mode. A nonlinear fit (y(-1) = a + b/x, all r2 > 0.999) for amprenavir, indinavir, ritonavir, lopinavir, nelfinavir, and saquinavir and a linear fit (y = ax + b, all r2 > 0.999) for nevirapine, efavirenz, and delavirdine led to best regression. Absolute recoveries were as follows: PIs > 81%; NNRTIs > 76%. Interday and intraday precision were <12.5% for the PIs and <11.7% for the NNRTIs. Interday and intraday accuracy were <12.2% for the PIs and <14.9% for the NNRTIs. Limits of quantification were 20, 40, 50, 40, 40, 20, and 100 microg/L for amprenavir, indinavir, ritonavir, lopinavir, nelfinavir, saquinavir, and the NNRTIs, respectively. The assay allows fast analysis of patient samples for therapeutic drug monitoring (TDM) and has successfully been used for TDM and pharmacokinetic drug-drug interactions studies.