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This Article Full Text Full Text (PDF) All Versions of this Article: clinchem.2004.037416v1 50/10/1845    most recent Submit an electronic Letter to the Editor about this paper Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (14) Citing Articles via Google Scholar Google Scholar Articles by Annesley, T. M. Articles by Clayton, L. Search for Related Content PubMed PubMed Citation Articles by Annesley, T. M. Articles by Clayton, L. Related Collections Drug Monitoring and Toxicology Automation and Analytical Techniques

Simple Extraction Protocol for Analysis of Immunosuppressant Drugs in Whole Blood

¹ University of Michigan Health Systems, 1500 East Medical Center Dr., Ann Arbor, MI 48109-0054;

^aauthor for correspondence: e-mail annesley@umich.edu

The first 20% of the full text of this article appears below.

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 . . . [Full Text of this Article]

The following articles in journals at HighWire Press have cited this article:

				T. M. Annesley Methanol-Associated Matrix Effects in Electrospray Ionization Tandem Mass Spectrometry Clin. Chem., October 1, 2007; 53(10): 1827 - 1834. [Abstract] [Full Text] [PDF]

				K. L. Napoli Organic solvents compromise performance of internal standard (ascomycin) in proficiency testing of mass spectrometry-based assays for tacrolimus. Clin. Chem., April 1, 2006; 52(4): 765 - 766. [Full Text] [PDF]

				P. J. Taylor, S. R. Brown, D. P. Cooper, P. Salm, M. R. Morris, P. I. Pillans, and S. V. Lynch Evaluation of 3 Internal Standards for the Measurement of Cyclosporin by HPLC-Mass Spectrometry Clin. Chem., October 1, 2005; 51(10): 1890 - 1893. [Full Text] [PDF]

				G. Khoschsorur Simultaneous Measurement of Sirolimus and Everolimus in Whole Blood by HPLC with Ultraviolet Detection Clin. Chem., September 1, 2005; 51(9): 1721 - 1724. [Full Text] [PDF]

				T. M. Annesley and L. T. Clayton Quantification of Mycophenolic Acid and Glucuronide Metabolite in Human Serum by HPLC-Tandem Mass Spectrometry Clin. Chem., May 1, 2005; 51(5): 872 - 877. [Abstract] [Full Text] [PDF]

				T. M. Annesley Application of Commercial Calibrators for the Analysis of Immunosuppressant Drugs in Whole Blood Clin. Chem., February 1, 2005; 51(2): 457 - 460. [Full Text] [PDF]