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

Bodnar-Broniarczyk, Magdalena, Pawiński, Tomasz, Kunicki, Paweł K.
Journal of chromatography 2019 v.1104 pp. 220-227
acetonitrile, ammonium, blood sampling, high performance liquid chromatography, isotope labeling, methyl ethers, monitoring, spectrometers, tacrolimus, tandem mass spectrometry, zinc sulfate
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 TAC13C,D2 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 + NH4]+ 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 TAC13C,D2. 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 (<3.09% and <3.63% for TAC13C,D2 and ASC, respectively) and accuracy (99.55–100.63% and 97.35–101.71% for TAC13C,D2 and ASC, respectively). Similar ARs were found for all three compounds yielding: 74.89–76.36% for TAC, 78.37% for TAC13C,D2 and 75.66% for ASC. Significant ME were observed yielding on the average: −16.04% and −29.07% for TAC, −16.64% for TAC13C,D2 and −28.41% for ASC. Consequently, PE was 64.11% and 53.12% for TAC, 65.35% for TAC13C,D2 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 TAC13C,D2 and ASC, respectively.An IS ascomycin presented a performance equivalent to TAC13C,D2 in LC–MS/MS method developed for TAC monitoring.