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1-Aminobenzotriazole (ABT) is a non-isoform specific, time-dependent inhibitor of cytochrome P450 (CYP) enzymes used extensively in preclinical studies to determine the relative contribution of oxidative metabolism. Although ABT has been widely used, the extent and duration of its inhibitory effect is not well understood. The purpose of this study is to characterize ABT inhibition of CYP in rats at both the hepatic and intestinal levels. In vivo studies using midazolam (p.o. and i.v.), as a probe for CYP activity, demonstrated that CYP inhibition was not complete even at the highest dose (300 mg/kg). Additional in vivo studies demonstrated that even at 26 h following ABT administration, there was significant CYP inhibition remaining. In vitro studies, conducted in both rat liver microsomes and rat hepatocytes, confirm that ABT is a time-dependent inhibitor of rat CYP orthologs. However, in rat liver microsomes, there was more than 15% CYP activity remaining following a 60 min preincubation at 2 mm ABT and 5-10% of CYP activity was remaining in rat hepatocytes suspended in rat plasma following a 60 min preincubation at 2 mm ABT. 1-Aminobenzotriazole is a useful tool in elucidating the oxidative component of metabolism in preclinical species; however, conclusions made from the preclinical use of ABT should not operate under the assumption that CYP enzymatic activity is completely inhibited. Copyright © 2016 John Wiley & Sons, Ltd.

RATIONALE Drug discovery samples are routinely analyzed using liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods on triple quadrupole mass spectrometers employing multiple reaction monitoring (MRM). In order to improve analysis throughput, quantitation of small molecules on a quadrupole time-of-flight (QqTOF) instrument using TOF scan and high-resolution MRM (MRM-HR) modes was evaluated in this study. METHODS Cassette dosed plasma and brain samples from nine compounds were extracted using a protein precipitation method. Separation was achieved by reversed-phase liquid chromatography. Mass spectrometric analysis was performed using TOF scan and high-resolution MRM approaches on a QqTOF mass spectrometer with turbo-ionspray ionization. Results were compared to those obtained on a triple quadrupole mass spectrometer. RESULTS The dynamic range varied depending on compounds and instruments and was similar between the MRM on QqQ and full TOF scan mode on QqTOF. Linear or quadratic regression and 1/x(2) weighting were used. Resolution on the QqTOF instrument was around 32000 and mass accuracy was within 4.4 ppm. The MRM-HR method showed better sensitivity compared to the TOF scan method, and was comparable to the MRM on a QqQ mass spectrometer. Assay accuracy was within ±25%. CONCLUSIONS A TOF scan method allowed the use of the generic method without compound-specific optimization and was an alternative choice for routine high-throughput quantitation of small molecules. The MRM-HR method on the QqTOF showed good sensitivity which was comparable to that obtained by the MRM method on the triple quadrupole mass spectrometer.