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

Abstract

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 μg/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 μl of sample in zinc sulfate–methanol–acetonitrile mixture containing IS, followed by centrifugation. HPLC was performed on ChromSpher π column, 30 mm × 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 μg/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 °C and for at least 20 days at −20 °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.

Introduction

Therapeutic drug monitoring (TDM) of immunosuppressive drugs is a must, dictated by narrow therapeutic range of these compounds, low dose-concentration correlation, and necessity of continuous use after organ transplantation. To most common immunosuppressive drugs belong: cyclosporine A (CsA), tacrolimus (TCR), sirolimus (SIR), and mycophenolic acid (MPA). These drugs were monitored in our hospital using immunoassays or HPLC. However, the use of immunoassays is costly and the comparison of results with those obtained by more specific HPLC or liquid chromatography–tandem mass spectrometry (LC–MS–MS) procedures show generally overestimation, caused by limited selectivity of all assays. This was reported for TCR [1], [2], MPA [3], SIR [4], [5], [6], [7], [8], and CsA [9].

For these reasons, the use of LC–MS–MS became very common in the last years, at first for single immunosuppressants [1], [9], [10], [11], later for multiple drugs [12], [13], [14], [15], [16], [17]. The latter approach allowed simultaneous monitoring of several drugs, administered for the patient. Simultaneous determination of immunosuppressants was limited to whole blood samples. On the other hand, MPA is determined in plasma, and the assay procedures published for MPA differ widely from those used for other immunosuppressants. Recently, Annesley et al. [18] described LC–MS–MS method for determination of MPA and its glucuronide (MPAG), using the same SPE cartridges and mobile phase components as for determination of CsA, SIR, and TCR in the whole blood.

The purpose of the present study was to present a method for simultaneous determination of CsA, TCR, and SIR in whole blood as well as MPA in plasma, using the same pretreatment procedure and same analytical conditions. This procedure contributes to further simplification of the TDM of immunosuppressive drugs since it allows determination all drugs involved in one analytical procedure.