Rapid and accurate liquid chromatography and tandem mass spectrometry method for the simultaneous quantification of ten metabolic reactions catalyzed by hepatic cytochrome P450 enzymes

Abstract

The hepatic cytochrome P450 enzymes play a central role in the biotransformation of endogenous and exogenous substances. A sensitive high-throughput liquid chromatography with tandem mass spectrometry assay was developed and validated for the simultaneous quantification of the products of ten metabolic reactions catalyzed by hepatic cytochrome P450 enzymes. After the substrates were incubated separately, the samples were pooled and analyzed by liquid chromatography with tandem mass spectrometry using an electrospray ionization source in the positive and negative ion modes. The method exhibited linearity over a broad concentration range, insensitivity to matrix effects, and high accuracy, precision, and stability. The novel method was successfully applied to study the kinetics of phenacetin-O deethylation, coumarin-7 hydroxylation, bupropion hydroxylation, taxol-6 hydroxylation, omeprazole-5 hydroxylation, dextromethorphan-O demethylation, tolbutamide-4 hydroxylation, chlorzoxazone-6 hydroxylation, testosterone-6β hydroxylation, and midazolam-1 hydroxylation in rat liver microsomes.

Substrate Hunting for the Myxobacterial CYP260A1 Revealed New 1α‐Hydroxylated Products from C‐19 Steroids

“…Hydroxylation of testosterone by human CYPsr eviewed by Agematu et al,[43] and Niwa et al,[49] and by the more recently reported human,[50][51][52] and bacterial[44,[53][54][55][56] CYPs is shown. Hydroxylation of testosterone by human CYPsr eviewed by Agematu et al,[43] and Niwa et al,[49] and by the more recently reported human,[50][51][52] and bacterial[44,[53][54][55][56] CYPs is shown.…”

“…Scheme2.Possiblep ositions of testosterone hydroxylation by different cytochromes P450. Hydroxylation of testosterone by human CYPsr eviewed by Agematu et al,[43] and Niwa et al,[49] and by the more recently reported human,[50][51][52] and bacterial[44,[53][54][55][56] CYPs is shown. The new 1a-hydroxylase activity of CYP260A1 from S. cellulosum So ce56i sa lso included.ChemBioChem 2016, 17,90-101 www.chembiochem.org 2 016 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim…”

Cyp3a11‐mediated testosterone‐6β‐hydroxylation decreased, while UGT1a9‐mediated propofol O‐glucuronidation increased, in mice with diabetes mellitus

“…Simultaneous determination of acetaminophen, 6‐hydroxychlorzoxazone, 7‐hydroxycoumarin, dextrorphan, 5‐hydroxyomeprazole, 1‐hydroxymidazolam, 6β‐hydroxytestosterone, hydroxylbupropion and 4‐hydroxytolbutamide by liquid chromatography with tandem mass spectrometry (LC–MS/MS) was performed as described previously . Briefly, LC separations were achieved using a Hypersil Gold C 18 (100 mm × 2.1 mm, 3 μm) with a guard column (10 mm × 2.1 mm, 3 μm).…”

“…The reaction was initiated by adding NADPH (1 m m ). After incubation at 37 °C for 30 min, the reaction was terminated and analysed as described previously .…”

Ultrasensitive quantification of the CYP2E1 probe chlorzoxazone and its main metabolite 6-hydroxychlorzoxazone in human plasma using ultra performance liquid chromatography coupled to tandem mass spectrometry after chlorzoxazone microdosing

“…Several methods utilizing the benefits of the LC-MS/MS technique have been developed to evaluate enzymatic activities when cocktail mixtures of different CYP substrates were administered [21,22], typically achieving quantitation levels between 10 and 1500 ng mL −1 [21,23]. However, multi-analyte assays do not reach sensitive optimum lower limits of quantification (LLOQ) and for chlorzoxazone the lowest reported quantification value is 0.1 ng mL −1 [24], hampering a full pharmacokinetics monitoring of chlorzoxazone microdoses.…”