In vitro interaction cocktail assay for nine major cytochrome P450 enzymes with 13 probe reactions and a single LC/MSMS run: analytical validation and testing with monoclonal anti‐CYP antibodies

Abstract

A sensitive and rugged LC/MSMS method was developed for a comprehensive in vitro metabolic interaction screening assay with N-in-1 approach reported earlier. A cocktail consisting of ten cytochrome P450 (CYP)-selective probe substrates with known kinetic, metabolic and interaction properties in vivo was incubated in a pool of human liver microsomes, and metabolites of melatonin (CYP1A2), coumarin (CYP2A6), bupropion (CYP2B6), amodiaquine (CYP2C8) tolbutamide (CYP2C9), omeprazole (CYP2C19 and CYP3A4), dextromethorphan (CYP2D6), chlorzoxazone (CYP2E1), midazolam (CYP3A4) and testosterone (CYP3A4) were simultaneously analysed with a single LC/MSMS run. Altogether, 13 metabolites and internal standard phenacetin were analysed in multiple reaction mode. Polarity switching mode was utilized to acquire negative ion mode electrospray data for hydroxychlorzoxazone and positive ionization data for the rest of the analytes. Fast gradient elution was applied, giving total injection cycle of 8 min. The method was modified for two different LC/MSMS systems, and was validated for linear range, detection limit, accuracy and precision for each metabolite. In addition, cocktail inhibition system was further tested using monoclonal anti-CYP antibodies as inhibitors for each probe reaction.

<i>In vitro</i> inhibitory activities of the extract of <i>Hibiscus sabdariffa</i> L. (family malvaceae) on selected cytochrome p450 isoforms

“…The enzyme activities in the presence of inhibitors (ethanolic extract of Hibiscus sabdariffa) were compared with the vehicle incubations. This assay method was validated using positive controls (Turpeinen et al, 2005;Tolonen et al, 2007) To evaluate the possible matrix effects to LC/MS/MS analysis of probe metabolites by the complex plant extract, extra control incubations where the extract was spiked after termination of metabolic reactions were performed and no matrix effects were observed at concentration at or below 100 µg/ml for any of the probe metabolites. Peak area of 7-hydroxycoumarin was 74% and peak area of hydroxychlorzoxazone was about 52%, when 1000 µg/ml of extract was added to the control incubation, suggesting some matrix suppression for these two probe metabolites.…”

“…The analysis of probe metabolites from CYP-specific marker reactions were conducted with a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method modified from an earlier paper where the inter and intraday variations of method and precision for all metabolites and the limit of quantification (LOQ) had been validated (Tolonen et al, 2007). Briefly, a Waters Alliance 2695 HPLC system (Waters Corp., Milford, MA) was used together with a Phenomenex Kinetex C18 column (2.1 × 50 mm column with 2.6 μm particle size) and an on-line filter at 40°C.…”

Metabolism of Pesticides by Human Cytochrome P450 Enzymes In Vitro – A Survey

“…A decrease in probe metabolite formation produced by inhibition is usually analyzed by LC-UV, LC-MS or fluorometry. In the cocktail assay, several CYP-selective probes are incubated with human liver microsomes and analyzed by LC-MS-MS (Tolonen et al 2007;Turpeinen et al 2006;Turpeinen et al 2005;Tolonen et al 2005). …”

Current methods of cytochrome P450 analysis

“…Определение активности различных изоформ цитохромов Р450 осуществляется на основании количества образующегося продукта реакции, при этом для детекции используется в основном масс-спектрометр с тройным квадруполем (QQQ) [74][75][76][77][78][79][80][81][82][83][84][85][86][87]. QQQ состоит из двух квадруполей и находящейся между ними ячейки соударений.…”

“…Наличие двух фильтров позволяет получить селективный сигнал исследуемого вещества и повысить чувствительность определения. Анализ литературных данных о наиболее часто используемых субстратах цитохромов Р450 и масс-спектрометрических параметрах, используемых для детектирования суммирован в таблице 3 [74][75][76][77][78][79][80][81][82][83][84][85][86][87].…”