Simple Extraction Protocol for Analysis of Immunosuppressant Drugs in Whole Blood

Abstract

The immunosuppressant drugs cyclosporine (cyclosporin A), tacrolimus, sirolimus, and everolimus are used to prevent organ rejection. Because commercial immunoassays are available only for cyclosporine and tacrolimus, HPLC (1)(2)(3), often in conjunction with mass spectrometry (MS or MS/MS) (4)(5)(6)(7), has been used to quantify these drugs. For MS/MS, sample preparation has often involved simple protein precipitation and solvent dissolution. Protocols have used zinc sulfate followed by acetonitrile (8), methanol (9), or acetone (10). Sometimes these reagents are premixed (11)(12)(13)(14). During in-house assay development we encountered several problems when zinc sulfate and acetonitrile were used. For example, many specimens clumped when blood was added to the zinc sulfate. Even after addition of acetonitrile and vortex-mixing, the clumps remained, necessitating manual dislodgement of the pellet. This occurred with lysed human blood specimens, lyophilized whole-blood controls (e.g., Bio-Rad Lyphochek), and College of American Pathologists proficiency testing specimens. We substituted methanol for acetonitrile with minor improvement. Another problem was that the recovery of sirolimus from whole blood using zinc sulfate followed by acetonitrile or methanol was <100% (9). In addition, the observed MS/MS responses for commercial calibrators and controls were often ∼30% higher than for human blood specimens. Here we present a new extraction protocol that improves absolute recoveries, provides excellent precision, and shows less ion suppression. Although the performance of this protocol for several immunosuppressants is described, we present example data only for sirolimus. We also describe a solid-phase extraction (SPE) that may be added to further enhance the cleanliness of extracts. For recovery and extraction experiments, we used cyclosporine from Novartis, tacrolimus from Fujisawa, and sirolimus from LC …