Quantitative determination of alkylated quaternary amines and their n-hydroxylated metabolites in an enzyme incubation matrix by liquid chromatography electrospray ionization mass spectrometry

• Published in: Biomedical chromatography Vol. 19; no. 8; pp. 579 - 585
• Main Authors: Holmes, Victoria E., Shaw, P. Nicholas, Ortori, Catharine A., Barrett, David A.
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01.10.2005
• Subjects: aliphatic quaternary amines; Alkylation; Amines - analysis; CYP4A1; Cytochrome P-450 Enzyme System - metabolism; Hydrolysis; hydroxylated metabolites; in vitro metabolism; liquid chromatography electrospray mass spectrometry; Reference Standards; Spectrometry, Mass, Electrospray Ionization - methods
• ISSN: 0269-3879; 1099-0801
• DOI: 10.1002/bmc.483

A simple, rapid and sensitive reversed‐phase liquid chromatography method coupled to electrospray ionization mass spectrometry has been developed for studying the in vitro metabolism of the long‐chain quaternary ammonium compounds dodecyltrimethylamine, tetradecyltrimethylamine and hexadecyltrimethylamine. Samples were prepared from the biological matrix by a simple protein precipitation stage. The separation was performed using a BDS Hypersil C8 3 µm particle size (100 × 3 mm i.d.) column with a fast gradient separation (60% B to 100% B) using a mobile phase of 10 mm aqueous ammonium acetate (pH 4.0, with 0.06% triethylamine; (A)–acetonitrile (B) at 0.7 mL min−1. To minimize contamination of the MS source a switching value was used to divert the solvent front to waste. Decylammonium bromide was used as the internal standard and analytes were identified and quantified by positive ion electrospray selected ion monitoring of their intact molecular cations. The assay had a limit of quantitation of 0.25 µm (6.25 pmol on column) and was linear over the range 0.25–100 µm assay concentration for this series of long‐chain quaternary amines. The precision of intra‐ and inter‐day assays was better than 19% and the accuracy was between 93 and 109%. The method was used to assess the in vitro metabolism of the quaternary amines by wild‐type cytochrome P450 enzyme CYP4A1 and mutants in an artifical membrane system. Copyright © 2005 John Wiley & Sons, Ltd.