Validation and clinical application of a high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of 10 anti-retrovirals in human peripheral blood mononuclear cells

This paper reports the validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method that allows the quantification of 10 antiretroviral (ARV) drugs in peripheral blood mononuclear cells (PBMCs) using 6 different isotopic internal standards (IS) and its clinical application. PBMCs are isolated from blood by density gradient centrifugation and drugs are extracted with a 60% methanol (MeOH) solution containing the 6 IS. The cell extract is then injected in the HPLC system and analytes are separated on a Symmetry Shield RP18 2.1 mm × 50 mm column. The different molecules are then detected by MS/MS in electrospray positive or negative ionisation modes and data are recorded using the multiple reaction monitoring (MRM) mode. Calibration curves are constructed in the range of 0.25–125 ng/ml of cell extract by a 1/x2 weighted quadratic regression. The regression coefficients obtained are always greater than 0.99 and back calculated values always comprised in the range of ±15% from their nominal concentration. Mean extraction recoveries are greater than 80% for all analytes and the method is accurate and precise with CV and bias lower than 9.4%. The lower limits of quantification (LLOQ) of the different drugs range from 0.0125 to 0.2 ng/ml of cell extract. This method was successfully applied to a cohort of 98 HIV-infected patients treated with Kaletra® (400/100 mg of lopinavir/ritonavir (LPV/RTV) twice a day, n = 48) or with Stocrin® (600 mg once a day, n = 50) and has been tested for cellular quantification of tipranavir (TPV) in 2 patients treated with Aptivus® (500 mg twice a day). The patients treated by Kaletra® showed mean cell-associated concentrations (CC) of 1819.0 and 917.2 ng/ml, for LPV and RTV, respectively. Patients treated with Stocrin® showed mean CC of 2388.11 ng/ml while both patients under Aptivus® showed TPV CC of 4322.7 and 1078.0 ng/ml, respectively. This method can be used to analyze ARV drug concentrations within the target tissue.