A specific method for the measurement of cyclosporin A in human whole blood by liquid chromatography-tandem mass spectrometry.

Therapeutic monitoring of the immunosuppressant cyclosporin A (CsA) is routinely performed by immunoassays to make individual dosage adjustments for patients after organ transplantation. High-performance liquid chromatography with ultraviolet detection (HPLC-UV) has been used as the reference method. However, HPLC-UV methods frequently suffer from chromatographic interferences that affect accuracy and reproducibility. A sensitive, specific HPLC-tandem mass spectrometry (HPLC/MS/MS) method for the quantitation of CsA has been developed. One hundred microliters CsA whole blood sample containing cyclosporin C (CsC) as the internal standard was extracted with ethyl ether. High-performance liquid chromatography separation was accomplished on an RP-C18 narrow-bore column at 50 degrees C with a linear gradient elution followed by on-line ion-spray ionization MS/MS analysis. The standard curve was established in the range of 10 to 1000 microg/l (r = 0.9989, n = 8). Limits of detection and quantitation were 1 microg/l and 5 microg/l, respectively. Imprecision was <4% across three control levels. Cyclosporine A recovery averaged 88%. Six metabolites: AM1, AM9, AM4N, Am1c, AM1a, and AM19 were identified with this method. AM1, AM9, and AM1c were further differentiated with a modification to the MS/MS conditions. This method was used in a comparison study with an HPLC-UV method: HPLC = 1.055 LC/MS/MS + 7.05 (microg/l), (Sy/x = 25.7), r2 = 0.982. With its high degree of sensitivity and specificity, this LC/MS/MS method offers a valuable reference method for immunoassay evaluation and a tool for metabolite investigation.