In vitro cytochrome P450 activity: Development and validation of a sensitive high performance liquid chromatography–tandem mass spectrometry method for the quantification of six probe metabolites after derivatization with pyridine-3-sulfonyl chloride in an aqueous environment

A high‐throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography–tandem mass spectrometry

“…This saved sample preparation as well as time compared with other methods (Testino and Patonay, ; Bhoopathy et al ., ; Li et al ., ; De Bock et al ., ). Compared with other systems, this system used 200 μL of 0.5 mg/mL microsome protein for each run (Testino and Patonay, ; Atkinson et al ., ; De Bock et al ., ). Comparing with reported cocktail methods where validation was not fully rounded (Testino and Patonay, ; Kim et al ., ; Otten et al ., ), our method was validated in various aspects, including matrix effects, linearity, LLOQ, accuracy, precision and stability, which sufficiently supported its efficacy.…”

A sensitive and high‐throughput LC‐MS/MS method for inhibition assay of seven major cytochrome P450s in human liver microsomes using an in vitro cocktail of probe substrates

“…All metabolites were eluted within 7 min and separated in their individual MRM channels. This saved time compared with previously published methods (De Bock et al ., ; Dixit et al ., ; Yin et al ., ).…”

“…A large volume of organic solvent is typically used, which dilutes the sample (Otten et al ., ), affects the peak shape of acetaminophen and decreases the sensitivity of metabolites. By using a small volume of stop reagent (25 μL in 250 μL incubation volume; De Bock et al ., ), sample handling was minimized and dilution of the resulting extract was avoided, which increased the sensitivity of the detection.…”

“…Incubation mixtures (250 μL) contained 0.1 m potassium phosphate buffer (PBS, pH 7.4), 3 m m MgCl 2 , 0.25 mg protein/mL of hepatic microsomes, inhibitor and substrate or substrate cocktail (final concentrations in Table ); an NADPH regeneration system (1 m m NADP+, 5 m m glucose 6‐phosphate and 1 U/mL glucose 6‐phosphate dehydrogenase) was added after pre‐incubation of 5 min at 37 °C. Following incubation at 37 °C for 5, 10, 15 and 30 min, the reaction was terminated by adding 25 μL of stop reagent (H 2 O–CH 3 CN–HCOOH; 42:55:3; v/v/v; De Bock et al ., ), containing180 n m internal standard (IS: verapamil), and cooling the mixtures on ice. Subsequently, the samples were centrifuged for 15 min at 20,000 g (4 °C) and 200 μL of the supernatant was analyzed by LC‐MS/MS.…”

“…This study presents an efficient cocktail approach that uses low protein concentration and minimizes compound‐to‐compound interactions among substrates as well. This approach utilizes a sensitive and selective LC‐MS/MS method with a shorter run time compared with the methods that have been previously published (De Bock et al ., ; Dixit et al ., ; Yin et al ., ). To validate this approach, seven CYP‐specific inhibitors that are recommended by US Food and Drug Administration () were tested and IC 50 values were calculated and compared with cocktail assay and single assay.…”