Protein Precipitation Method for Determination of Zileuton in Human Plasma by LC-MS/MS

• Published in: Journal of pharmaceutical innovation 2020-12, Vol.15 (4), p.581-590
• Main Authors: Armoudjian, Yeghig, Mikayelyan, Astghik, Zakaryan, Hasmik, Aleksanyan, Armine, Alaverdyan, Harutyun
• Format: Article
• Language: English
• Subjects: Biochemical Engineering; Biomedical and Life Sciences; Biomedicine; Biotechnology; Industrial and Production Engineering; Original Article; Pharmacology/Toxicology
• ISSN: 1872-5120; 1939-8042
• DOI: 10.1007/s12247-019-09403-6

Purpose The main aim of this study was to establish a rapid and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of Zileuton in human plasma. Methods Zileuton was extracted from human plasma samples by protein precipitation with acidified acetonitrile. Following protein precipitation (PPT), analyte separation was done using Phenomenex Kinetex™ XB-C18 (50 × 2.1 mm, 1.7 μm) column using isocratic elution with a mobile phase of 0.1% formic acid (50%) and 0.1% formic acid:acetonitrile 2:3 V:V (50%) at a flow rate of 0.4 mL/min and an injection volume of 5 μL. The detection was performed on a triple quadrupole mass spectrometer by multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 237.0 → 161.0, 241.0 → 165.0 for Zileuton and Zileuton-D4 in positive electrospray ionization mode, correspondingly. The method was validated over a concentration range of 20.0–8000.0 ng/mL on SCIEX Triple Quad 4500 MS System. Total run time was 2.1 min. Results The method was fully validated. Precision and accuracy results were as follows: the overall %Accuracy for intra batch LLOQ QC was within 93.7–109.5%, for the rest of QCs 98.8–111.6%, and the highest %CV for LLOQ QC was 10.1%, and for the rest of QCs 5.3%. The variety of tests for matrix effects, extraction efficiency, selectivity, linearity, sensitivity, recovery, and stability were also performed. Conclusions A rapid, simple, and specific LC-ESI-MS method for determination of Zileuton in human plasma has been described. The method has several advantages compared to the previously reported methods, including shorter analysis time, better sensitivity, simple sample preparation, wider range of concentrations, and use of isotopic internal standard. The developed and validated method can be successfully applied for the bioequivalence/pharmacokinetic studies of Zileuton.