Quantification of cyclosporine A in peripheral blood mononuclear cells by liquid chromatography-electrospray mass spectrometry using a column-switching approach

Abstract

As a potential alternative to cyclosporine A (CsA) monitoring in whole blood, a sensitive and selective method was developed for quantifying this immunosuppressive drug in human peripheral blood mononuclear cells (PBMCs) by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). PBMCs were isolated from whole blood by density gradient centrifugation. After purification, cell counts were performed to express CsA amounts per single cell. The pelleted cells were then lysed and CsA was extracted with methanol (MeOH) containing 27-demethoxy-sirolimus as internal standard. After evaporation of the supernatant under nitrogen, the residue was reconstituted in MeOH, further diluted with water and injected onto a column-switching unit. On-line solid-phase extraction was performed using a C8 column with an acidic aqueous mobile phase containing 5% MeOH. The analytes were transferred in the back-flush mode on a C18 column with 65% MeOH and the chromatographic separation performed with a MeOH gradient (65–90%). The detection was carried out with a single quadrupole analyzer and the sodium adducts [M + Na] + were monitored for quantification. This sensitive method was fully validated in the range of 5–400 ng/mL. This allowed the measurement of very small CsA amounts present in cells up to 0.5 fg/PBMC in clinical samples. Trueness (95.0–113.2%), repeatability (5.1–9.9%) and intermediate precision (7.0–14.7%) were found to be satisfactory. This method represents a new potential tool for therapeutic drug monitoring of CsA and could be used in clinical conditions if the utility of intracellular measurements is confirmed in prospective clinical trials.