Validation of a UHPLC-MS/MS Method to Quantify Twelve Antiretroviral Drugs within Peripheral Blood Mononuclear Cells from People Living with HIV

Abstract

Recently, anti-HIV treatment has achieved high efficacy and tolerability. Nevertheless, few data are available about the intracellular penetration of antiretrovirals, partly due to the technical challenges related to intracellular quantification. This work aimed to validate an ultra-high performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method for the simultaneous quantification of maraviroc, nevirapine, rilpivirine, dolutegravir, raltegravir, cobicistat, darunavir, ritonavir, atazanavir, efavirenz, elvitegravir, and etravirine within peripheral blood mononuclear cells (PBMCs) and apply it to samples from patients. PBMCs were isolated by density gradient on cell preparation tubes (CPT). Samples were prepared by addition of internal standards (IS), sonication, centrifugation, and drying. Reconstituted extracts underwent chromatographic separation by reversed phase UHPLC and detection was performed by electrospray ionization and multiple reaction monitoring. Method validation followed FDA and EMA guidelines, showing acceptable accuracy, precision, recovery and IS-normalized matrix effect. The application to 56 samples from patients undergoing antiretroviral treatment provided description of intracellular penetration, showing method eligibility for future studies.