TuP-10
THE QUANTITATION OF CYCLOSPORIN A IN WHOLE BLOOD BY HPLC - ELECTROSPRAY - TANDEM MASS SPECTROMETRY
PJ Taylor1, PT Martin1, SV Lynch2, CE Jones1, and SM Pond1,3
Departments of 1Clinical Pharmacology, 2Surgery, and 3Medicine, Princess Alexandra Hospital, Qld 4102
Cyclosporin A (CSA) is an immunosuppressant drug that has revolutionised the field of solid organ transplantation. However, clinical usage is hindered by the narrow therapeutic window. Thus, therapeutic drug monitoring (TDM) is used routinely for patients receiving CSA. Several techniques have been developed to measure CSA, the main two being immunoassays and HPLC with UV detection. We have developed a HPLC-electrospray-tandem mass spectrometry method for the quantitation of CSA in whole blood. Whole blood (100 µl) was treated with 200 µl acetonitrile:water (70:30 v/v) containing 18.2 ng of cyclosporin C, the internal standard (IS). Samples were mixed and centrifuged. The supernatant was applied to C18 solid phase extraction cartridges (Waters) which had been preconditioned with methanol (5 ml) and water (5 ml). The loaded cartridges were washed sequentially with water (5 ml), 50% methanol/water (2 ml) and heptane (2 ml). The analytes were eluted with 50% isopropyl alcohol/heptane (1 ml) and the eluant dried under air at 60 °C. Chromatographic separation was achieved with a 2.1 mm i.d. x 100 mm C8 Alltima column (Alltech), at 70°C, using a mobile phase of 72% methanol/28% 0.05 M ammonium acetate buffer (pH 5.1). A flow rate of 300 µl/min was split post column (1:8 into the mass spectrometer) to achieve a chromatographic run time of 13 minutes. Detection was by multiple reactant monitoring (CSA m/z 1203.0-->425.4, IS m/z 1221.0-->425.4) in positive ionisation mode. To produce predominantly protonated species of the analytes, the orifice potential was set to 100 V. Quantitation was by peak area ratios against the IS. The assay was linear from 5 µg/l to 1000 µg/l with accuracy between 98.5% to 102.3% over the range studied. Imprecision in terms of the total coefficients of variation was 11.1% at 10 µg/l (mean=10.23 µg/l, n=12) and 2.8% at 800 µg/l (mean=781.7 µg/l, n=12). Absolute recovery of CSA and IS was 70.0% (n=12) and 85.4% (n=18), respectively. The limit of quantitation of 140 pg (on column), is 50-100 times lower than reported for current HPLC methods. The small sample requirements and high sensitivity of this method will facilitate many potential applications in TDM in paediatric patients, human and animal pharmacokinetic studies and measurement of concentrations in small biopsy samples.