HPLC-MS method for the quantification of nine anti-HIV drugs from dry plasma spot on glass filter and their long term stability in different conditions

A bioanalytical method for the determination of most commonly prescribed protease inhibitors (saquinavit. atazanavir, amprenavir, darunavii, lopmavir and ritonavir) and non-nucleoside reverse transcriptase inhibitors (etravirine, efavirenz and nevirapine) was developed, modifying our pi emus HPLC-MS chromatographic run, validated and a complete short and long term stability evaluation was carried out. One hundred microlities of plasma were distributed on a collection glass paper fillet (Glass-Microfibre from Sartorius), then the filter underwent thermal treatment. both for drying and for HIV inactivation, and stored at loom temperature. 4 degrees C and -20 degrees C. The analytes were extracted from the filter disc using tert-butylmethylether with basic pH, after the addition of the Internal standards quinoxaline The extract was dried, reconstructed and the chromatographic separation was performed on a reversed-phase C-18 column (150 mm x 2 0 mm) and the analytes were quantified using a single quadrupole mass spectrometer. The method was validated considering the concentration ranges encountered in clinical trials and the routine clinical practice The assay was linear over the concentration ranges tested Accuracies ranged from 92.1% to 111.9% and intra-day and inter-day relative standard deviation for all quality control levels ranged from 02 to 129 and 3 1 to 14 4, respectively Analytes in dried plasma spots were stable tot longer time when dried/inactivation step was carnal out before storage compared to samples not dried/inactivated berm e the analysis The dried/inactivation step allows shipment of samples at room temperature without any risks, therefore the developed and validated method enables an easy and cheap sample shipment tot therapeutic drug monitoring and pharmacokinetic studies (C) 2010 Elsevier B V All rights reserved