title: 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 5 μL 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 2 s. The APCI parameters were set to negative needle voltage (-2 μA), carrier gas temperature (30°C) and air flow rate (3 L min(-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 -40 V. The analysis was achieved within 9 s 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: 23.5 ng mL(-1) (accuracy=112.3%; RSD=9.6%; n=6) and a blank+6 point linear regression up to 965 ng mL(-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.
authors.affiliation: CHU, Hôpital A. Michalon, Laboratoire de Pharmacologie-Toxicologie, BP217, Grenoble 38043, France. Electronic address: JFJourdil@chu-grenoble.fr.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Jourdil
authors.forename: Pierre
authors.initials: P
authors.lastname: Picard
authors.forename: Cécile
authors.initials: C
authors.lastname: Meunier
authors.forename: Serge
authors.initials: S
authors.lastname: Auger
authors.forename: Françoise
authors.initials: F
authors.lastname: Stanke-Labesque
mesh_terms: D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000592:standards; D016572:Cyclosporine / Q000097:blood* / Q000592:standards; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood* / Q000592:standards; D011786:Quality Control; D012015:Reference Standards; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000592:standards; D053719:Tandem Mass Spectrometry / Q000592:standards
chemical_list: D007166:Immunosuppressive Agents; D016572:Cyclosporine
keywords: Cyclosporin A; Laser diode thermal desorption; Liquid chromatography; Tandem mass spectrometry