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

Magdalena Bodnar-Broniarczyk; Tomasz Pawiński; Paweł K Kunicki

doi:10.1016/j.jchromb.2018.11.026

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

J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Jan 1:1104:220-227. doi: 10.1016/j.jchromb.2018.11.026. Epub 2018 Nov 28.

Authors

Magdalena Bodnar-Broniarczyk¹, Tomasz Pawiński², Paweł K Kunicki³

Affiliations

¹ Department of Drug Chemistry, Faculty of Pharmacy, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland. Electronic address: magdalena.bodnar-broniarczyk@wum.edu.pl.
² Department of Drug Chemistry, Faculty of Pharmacy, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland. Electronic address: tomasz.pawinski@wum.edu.pl.
³ Department of Drug Chemistry, Faculty of Pharmacy, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland; Clinical Pharmacology Unit, Department of Medical Biology (previous name: Department of Clinical Biochemistry), Institute of Cardiology, Alpejska 42, 04-628 Warsaw, Poland. Electronic address: pawel.kunicki@wum.edu.pl.

PMID: 30530114
DOI: 10.1016/j.jchromb.2018.11.026

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 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.

Keywords: Internal standard; LC–MS/MS; Matrix effects; Tacrolimus; Validation.

MeSH terms

Chromatography, Liquid / methods*
Humans
Limit of Detection
Linear Models
Reference Standards
Reproducibility of Results
Tacrolimus / blood*
Tandem Mass Spectrometry / methods*

Substances

Tacrolimus