Ultra-fast cyclosporin A quantitation in whole blood by Laser Diode Thermal Desorption – Tandem Mass Spectrometry; comparison with High Performance Liquid Chromatography–Tandem Mass Spectrometry

Abstract

In the last decade the quantitation of immunosuppressive drugs has seen vast improvements in analytical methods, optimizing time, accuracy of analysis and cost. Laser Diode Thermal Desorption (LDTD) coupled to Atmospheric Pressure Chemical ionization–tandem mass spectrometry (APCI-MS/MS) represents a technological breakthrough that removes the chromatographic separation step and thereby significantly increases the analytical throughput for the quantitation of cyclosporin A (CsA) in whole blood for therapeutic drug monitoring (TDM).A simple protein precipitation step was used prior to depositing 5μL of the extract on a 96-well LazWell™ plate and CsA was quantified by LDTD-APCI-MS/MS.The laser pattern was set to ramp from 0 to 45% laser power within 2s. The APCI parameters were set to negative needle voltage (−2μA), carrier gas temperature (30°C) and air flow rate (3Lmin−1). The negative ion single reaction monitoring transitions for CsA and its internal standard cyclosporin D (CsD) were respectively m/z 1201.1/1088.9 and m/z 1214.8/1102.8; obtained with a collision energy of −40V.The analysis was achieved within 9s from sample to sample.The extraction procedure yielded high recovery (92%; RSD=9.4%, n=6). The lower limit of quantitation was fixed at the first level of calibration: 23.5ngmL−1 (accuracy=112.3%; RSD=9.6%; n=6) and a blank+6 point linear regression up to 965ngmL−1 was used. Using 4 levels of quality control (QC), intra-day assays (n=6) ranged from 93.5 to 95.7% (bias) and from 3.4 to 13.1% (RSD) while inter-day assays (n=6) ranged from 92.9 to 105.3% (bias) and from 4.9 to 7.5% (RSD). An inter-sample contamination of CsA of 2.3% was calculated that was considered negligible with respect to the range of CsA concentrations. Whole blood samples (120) from patients under CsA treatment were analyzed by LDTD-APCI-MS/MS and HPLC–ESI-MS/MS, the gold standard reference method for CsA quantification. Both methods agreed (P≥0.99), with a coefficient of correlation of 0.99 (95% confidence interval 0.982–0.991). The Passing–Bablok regression revealed no significant deviation from linearity (Cusum test, P=0.11). This method seems suitable for use in TDM of CsA.