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)

Abstract

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.