Isotope–labeled versus analog internal standard in LC–MS/MS method for tacrolimus determination in human whole blood samples – A compensation of matrix effects

Abstract The aim of this work was to develop and to validate LC–MS/MS method for tacrolimus (TAC) determination in whole blood samples using two different types of internal standards (IS): an isotope-labeled TAC 13 C,D 2 and a structural analog ascomycin (ASC). Matrix effects (ME) were evaluated to determine their influence on validation parameters. The LC–MS/MS analyses were performed using a 4000 QTRAP® mass spectrometer (AB Sciex) coupled to a HPLC 1260 Infinity system (Agilent Technologies). The [M + NH 4 ] + adducts were monitored with mass transitions of: 821.5 → 768.4  m / z for TAC, 809.5 → 756.4  m / z for ASC, and 824.6 → 771.5  m / z for TAC 13 C,D 2 . Blood samples were treated with 0.1 mol/L zinc sulfate - acetonitrile (50:50, v/v) then extracted with tert -butyl methyl ether. ME evaluations were performed by preextraction addition (n = 6), postextraction addition (n = 8), and repeated measures (n = 8) of reference solutions, separately for both ISs. ME, absolute recovery (AR) and process efficiency (PE) were calculated. Low (1.5 ng/mL) and high (16 ng/mL) TAC concentrations were tested. The method was successfully calibrated in a range of: 0.5–20 ng/mL. Both ISs provided satisfactory imprecision ( 13 C,D 2 and ASC, respectively) and accuracy (99.55–100.63% and 97.35–101.71% for TAC 13 C,D 2 and ASC, respectively). Similar ARs were found for all three compounds yielding: 74.89–76.36% for TAC, 78.37% for TAC 13 C,D 2 and 75.66% for ASC. Significant ME were observed yielding on the average: −16.04% and −29.07% for TAC, −16.64% for TAC 13 C,D 2 and −28.41% for ASC. Consequently, PE was 64.11% and 53.12% for TAC, 65.35% for TAC 13 C,D 2 and 54.18% for ASC. ME for TAC were perfectly compensated (TAC/IS ratio) in each sample resulting in mean percent value of: 0.89% and −0.97% for TAC 13 C,D 2 and ASC, respectively. An IS ascomycin presented a performance equivalent to TAC 13 C,D 2 in LC–MS/MS method developed for TAC monitoring.