Simultaneous LC-MS-MS determination of cyclosporine A, tacrolimus, and sirolimus in whole blood as well as mycophenolic acid in plasma using common pretreatment procedure.

The purpose of the study was to develop rapid and simple procedure for simultaneous determination of cyclosporine A (CsA), tacrolimus (TCR), and sirolimus (SIR) in whole blood and mycophenolic acid (MPA) in plasma. Ascomycin (ASCO), cyclosporine D (CsD), and desmethoxysirolimus (DMSIR) were used as internal standards (IS) for TCR, CsA and MPA, and SIR, respectively. In the method development, six-level blood calibrators were used for CsA (range 47-1725 ng/ml), TCR (range 2.1-38.8 ng/ml), and SIR (range 2.4-39.6 ng/ml). Four-level calibrators were used for MPA (range 0.15-5.48 microg/ml). Four levels of quality control (QC) standards were used for blood samples, together with two levels of QC standards in plasma. All QC standards and calibrators were obtained from commercial sources. Sample preparation based on precipitation of 50 microl of sample in zinc sulfate-methanol-acetonitrile mixture containing IS, followed by centrifugation. HPLC was performed on ChromSpher pi column, 30 mm x 3 mm, in ballistic gradient of ammonium formate buffer-methanol at 0.8 ml flow rate. Following gradient elution profile was applied: 0-1.2 min at 30% methanol (divert valve to waste), 1.21-3.1 min 97% methanol (divert valve to detector), 3.11-3.7 min 30% methanol (divert valve to waste). ESI-MS-MS (MRM) was done on TSQ Quantum instrument with ESI source in positive ion mode. Ammoniated adducts of protonated molecules were used as precursor ions for all analytes but MPA. For this compound sodium adduct was used. Following transitions were monitored: for CsA m/z 1220-1203; for CsD 1234-1217; for SIR 931.6-864.5 and 882.6; for DMSIR 902-834.5; for TCR 821.5-768.5 and 785.5; for ASCO 809.5-756; for MPA 343-211.6; for MPA-glucuronide 514-306 and 211.6. The limits of quantitation were: 1 ng/ml for TCR and SIR, 20 ng/ml for CsA, and 0.1 microg/ml for MPA. Post-column infusion experiments showed that no positive or negative peaks appeared after injection of matrix in the elution range of target compounds. General signal suppression caused by matrix ranged from 20-40%, and was caused mainly by zinc sulfate present in deproteinizing solution. Extracted samples were stable for 2 days at 4 degrees C and for at least 20 days at -20 degrees C. MPA was fully separated from its glucuronide, which was eluted at around 0.7-0.8 min and directed to the waste. Some mutual cross-contribution of CsD and CsA was observed (below 1%), other IS did not contribute to target compounds and vice versa. Observations of chromatograms from patients taken single therapy demonstrated that possible metabolites of CsA, TCR, or SIR did not interfere with target compounds or IS.