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 x 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/x(2) 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.
Elens
L
L
Louvain Center for Toxicology and Applied Pharmacology (LTAP), Université Catholique de Louvain, Avenue E. Mounier 53.02, 1200 Brussels, Belgium. laure.elens@uclouvain.be
Veriter
S
S
Yombi
J C
JC
Di Fazio
V
V
Vanbinst
R
R
Lison
D
D
Wallemacq
P
P
Vandercam
B
B
Haufroid
V
V
D000328:Adult; D019380:Anti-HIV Agents / Q000032:analysis* / Q000627:therapeutic use; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D016903:Drug Monitoring / Q000379:methods*; D005260:Female; D006678:HIV / Q000187:drug effects; D015658:HIV Infections / Q000188:drug therapy* / Q000821:virology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D008297:Male; D008875:Middle Aged; D053719:Tandem Mass Spectrometry / Q000379:methods*
D019380:Anti-HIV Agents