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Modified pentamer formation assay for measurement of tacrolimus and its active metabolites: comparison with liquid chromatography–tandem mass spectrometry and microparticle enzyme-linked immunoassay (MEIA-II)

¹ Abteilung Klinische Chemie, Georg-August-Universitaet Goettingen, D-37075 Goettingen, Germany.

² Abbott Diagnostics Division, Abbott Laboratories, Abbott Park, IL 60064.
^a Address correspondence to this author at: Abteilung Klinische Chemie, Zentrum Innere Medizin, Georg-August-Universitaet Goettingen, D-37075 Goettingen, Germany. Fax 49551-398551; e-mail varmstro{at}med.uni-goettingen.de.

A modified pentamer formation assay (PFA) for quantification of tacrolimus and active metabolites after extraction from whole blood is described. The lower limit of detection was 2 µg/L. Intraassay precision (CV) was 5.7–13.7%, and the interassay CV was 6.1–14.9%. Tacrolimus trough concentrations in 104 whole blood specimens from liver and kidney transplant recipients were compared with results from HPLC–tandem mass spectrometry (LC/MS/MS) and microparticle enzyme immunoassay (MEIA-II). Data were analyzed by difference plots and are presented as median (95% confidence intervals) of the method differences. MEIA-II results were on average 2.00 µg/L (range, -0.08 to 5.17 µg/L) higher than LC/MS/MS, whereas PFA results were only 1.07 µg/L (range, -2.62 to 5.33 µg/L) higher. Of 104 specimens tested, 25 displayed differences 3 µg/L between MEIA-II and PFA: median difference, 4.65 µg/L (range, 3.01–8.79 µg/L). The corresponding median difference between PFA and LC/MS/MS was -0.91 µg/L (range, -4.11 to 0.85 µg/L), and the difference between MEIA-II and LC/MS/MS was 3.67 µg/L (range, 1.88–6.34 µg/L), suggesting the presence of inactive metabolites that caused a positive bias in the immunoassay. In contrast, similar median differences were observed for the remaining 79 specimens: MEIA-II minus LC/MS/MS, 1.78 µg/L (range, -0.45 to 4.11 µg/L); PFA minus LC/MS/MS, 1.90 µg/L (range, -1.70 to 5.50 µg/L). Active tacrolimus metabolites may have contributed to the higher apparent tacrolimus concentrations in these specimens.

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

				N. W. Brown, C. E. Gonde, J. E. Adams, and J. M. Tredger Low Hematocrit and Serum Albumin Concentrations Underlie the Overestimation of Tacrolimus Concentrations by Microparticle Enzyme Immunoassay versus Liquid Chromatography-Tandem Mass Spectrometry Clin. Chem., March 1, 2005; 51(3): 586 - 592. [Abstract] [Full Text] [PDF]

				F. Streit, V. W. Armstrong, and M. Oellerich Rapid Liquid Chromatography-Tandem Mass Spectrometry Routine Method for Simultaneous Determination of Sirolimus, Everolimus, Tacrolimus, and Cyclosporin A in Whole Blood Clin. Chem., June 1, 2002; 48(6): 955 - 958. [Full Text] [PDF]