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)

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 mu g/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 we re compared with results from HPLC-tandem mass spectrometry (LC/MS/MS) and microparticle enzyme immunoassay (MEIA-II). Data were analyzed by differenc e plots and are presented as median (95% confidence intervals) of the metho d differences. MEIA-II results were on average 2.00 mu g/L (range, -0.08 to 5.17 mu g/L) higher than LC/MS/MS, whereas PFA results were only 1.07 mu g /L (range, -2.62 to 5.33 mu g/L) higher. Of 104 specimens tested, 25 displa yed differences greater than or equal to 3 mu g/L between MEIA-II and PFA: median difference, 4.65 mu g/L, (range, 3.01-8.79 mu g/L). The correspondin g median difference between PFA and LC/MS/MS was -0.91 mu g/L, (range, -4.1 1 to 0.85 mu g/L), and the difference between MEIA-II and LC/MS/MS was 3.67 mu g/L (range, 1.88-6.34 mu g/L), suggesting the presence of inactive meta bolites that caused a positive bias in the immunoassay. In contrast, simila r median differences were observed for the remaining 79 specimens: MEIA-II minus LC/MS/MS, 1.78 mu g/L (range, -0.45 to 4.11 mu g/L); PFA minus LC/MS/ MS, 1.90 mu g/L (range, -1.70 to 5.50 mu g/L). Active tacrolimus metabolite s may have contributed to the higher apparent tacrolimus concentrations in these specimens.