High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy. I. Development of direct injection/on-line guard cartridge extraction/tandem mass spectrometry for the simultaneous detection of CYP probe substrates and their metabolites

Abstract

A highly efficient direct injection/on‐line guard cartridge extraction/tandem mass spectrometry (DI‐GCE/MS/MS) method utilizing electrospray polarity switching was developed for the simultaneous detection of probe substrates and marker metabolites of seven human hepatic cytochrome P450 (CYP) isozymes: CYP1A2, 2A6, 3A4, 2C9, 2C19, 2D6 and 2E1. Microsomal incubations were terminated with formic acid, centrifuged, and the resulting supernatants were injected for analysis by DI‐GCE/MS/MS. This method employed an extremely short C18 cartridge (4 mm in length) which allowed rapid cleanup of sample matrices while retaining the analytes an appropriate time (2.0–2.2 min). From 1.5 to 2.7 min the effluent was directed to the mass spectrometer for detection otherwise diverted to waste. As a result of the efficient on‐line extraction, matrix (e.g., salts and proteins) suppression was minimized. In addition, no visible source contamination was observed and system performance (chromatographic and mass spectrometric) did not significantly deteriorate after 500 consecutive injections. Electrospray polarity switching was strategically executed on a Micromass Quattro II mass spectrometer by establishing dummy ion transitions to protect the analytes from the interference of the overwhelming noise which was unavoidable for the first transition scanned following each polarity switch. This unique strategy led to the simultaneous detection of seven CYP probe substrates and seven corresponding marker metabolites (12 by positive mode and 2 by negative mode). Copyright © 2000 John Wiley & Sons, Ltd.

Validated method for rapid inhibition screening of six cytochrome P450 enzymes by liquid chromatography–tandem mass spectrometry

“…Currently, the most widely employed incubation sample preparation is protein precipitation (PPT) [11][12][13][14][15][16][17][18][19]. Our initial approach for sample preparation was based on PPT, while it was found that liquid-liquid extraction provided a 10-fold higher sensitivity for the majority of the CYP probe substrate metabolites than PPT.…”

“…At present, numerous high-throughput approaches using fluorogenic substrates to measure CYP activities have been described [8,9], as well as substrate "cocktail" experiments that simultaneously measure more than one CYP activity [10][11][12][13][14][15][16][17][18]. Recently Kim et al [19] described a high-throughput method that allowed the simultaneous evaluation of the metabolic activities of nine major human CYP isoforms (i.e., CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) on their well-known specific probe substrates using human hepatic microsomes.…”

Analytical approaches to determine cytochrome P450 inhibitory potential of new chemical entities in drug discovery

“…Upon the development of a substrate cocktail biological incubation assay there was a need for a cocktail analytical assay with appropriate throughput and sensitivity to determine a test compound's CYP450 inhibitory potential. Several LC-MS/MS-based cocktail analytical assays have been reported in the literature [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28]. However, many of these methods suffer from limitations such as use of recombinant CYPs, S-mephenytoin 4 -Hydroxymephenytoin CYP1A2 Tacrine 1 -Hydroxytacrine clinically irrelevant probe substrates, intensive sample preparation, use of sample preparation HPLC columns, longer run times and higher protein (HLM) content in biological incubations that provides higher substrate turnover; however, could result in nonspecific binding.…”

Synchronized dual-polarity electrospray ionization mass spectrometry

“…Polarity switching [2] is an approach to monitor positive and negative ions alternatively by a mass spectrometer. It is utilized with liquid-chromatographic systems to identify protein modifications [3,4], metabolites [5], and environmental hazards [6], but the instrument performance declines due to duty cycle problems, and the few seconds required for signal recovery after every such switch.The best strategy to obtain positive and negative mass spectra is to employ contemporaneous dual-polarity (DP) ion detection [7,8]. The only DP ionization approach involving liquid samples is the field-induced droplet ionization (FIDI) [9], in which positive and negative liquid jets are produced from falling droplets within a strong d.c. electric field.…”

“…Polarity switching [2] is an approach to monitor positive and negative ions alternatively by a mass spectrometer. It is utilized with liquid-chromatographic systems to identify protein modifications [3,4], metabolites [5], and environmental hazards [6], but the instrument performance declines due to duty cycle problems, and the few seconds required for signal recovery after every such switch.…”