Identification and analysis of nine metabolites of cyclosporine in whole blood by liquid chromatography. 1: Purification of analytical standards and optimization of the assay.

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Abstract
We describe an extraction and an isocratic "high-performance" liquid-chromatographic (HPLC) separation of cyclosporine (CsA) and nine metabolites (M1, M8, M17, M18, M21, M25, M26, M203-218, and MUNDF1) from whole blood. Metabolites (for standards) were purified from human bile with liquid-liquid and solid-phase extractions, chromatographed on a cyanopropyl (CN) semipreparative HPLC column, and further purified on octyl, CN, and silica columns. The identity of each metabolite was verified with authentic standards on three chemically different HPLC columns and on the basis of cross-reactivity data from radioimmunoassay. For the routine analytical method, 1 mL of whole blood is diluted, hemolyzed, and applied to a Bond Elut CN (500 mg) cartridge to extract CsA, metabolites, and cyclosporin C, the internal standard. Interferences are removed by using four wash solutions and an additional cartridge of octyldecyl sorbent introduced prior to elution. Analytes are separated on a Zorbax CN analytical column maintained at 58 degrees C, with detection at 214 nm. Analytical recovery, as tested with three lots of CN sorbent, ranged from 47% to 95% for the 10 cyclosporines. Between-run CVs are less than 10% at 200 micrograms/L (concentration of each compound) and the standard curves are linear to 1500 micrograms/L. We also report a study of the separation mechanisms.