Quantification of Tacrolimus and Three Demethylated Metabolites in Human Whole Blood Using LC-ESI-MS/MS

Background: A bioalanytical method for the quantification of tacrolimus (TAC) and 3 metabolites, 13-O, 15-O, and 31-O-demethylated TAC (M-I, M-III, and M-II) in human whole blood using liquid chromatography, electrospray ionization, tandem mass spectrometry (LC-ESI-MS/MS) was developed and validated. Method: The analytes were extracted from 85 mu L of blood by protein precipitation followed by solid-phase extraction and a concentration step. The analytes and the internal standard (IS, ascomycin) were separated on a C18 column using a slow gradient mobile phase elution, with an analysis time of 3.3 minutes. The ammonium-adduct ions with transitions of m/z 821.5 > 768.7 (TAC), 807.5 > 754.7 (M-I, M-III, M-II), and 809.4 > 756.7 (IS) were measured in selected reaction monitoring mode using electrospray ionization. Results: Measuring ranges were 0.1-50 ng/mL for M-II, M-III, and TAC and 0.15-39 ng/mL for M-I. Imprecision in quantification was <20% for all analytes, whereas accuracy was within +/- 20%. Recovery was calculated to be >50% for all analytes. The sample's stability was proven for 1 month at -20 degrees C and 72 hours at room temperature. Three freeze-thaw cycles had no significant effect on the stability. The prepared samples were stable at least 16 hours at 8 degrees C. Analysis of 53 patient samples resulted in average concentrations of 7.2 for TAC, 0.8 for M-I, 0.4 for M-III, and 0.2 ng/mL for M-II. The total metabolite concentration was 17% (4%-52%) of the TAC concentration. The TAC concentration measured by LC-MS/MS was 36.1% +/- 27.1% lower than by immunochemical (enzyme multiplied immunoassay technique) analysis. When adding the metabolite crossreactivity in the presence of TAC, the difference between the 2 methods was still 29.8% +/- 28.3%, indicating that the overestimation of TAC concentration of enzyme multiplied immunoassay technique compared with liquid chromatography-tandem mass spectrometry cannot only be ascribed to the demethylated metabolites. Conclusions: An LC-ESI-MS/MS method for the quantitative analysis of TAC and 3 metabolites, using a 2-step sample preparation was successfully developed, validated, and applied on 53 patient samples.