A novel approach to perform metabolite screening during the quantitative LC–MS/MS analyses of in vitro metabolic stability samples using a hybrid triple‐quadrupole linear ion trap mass spectrometer

Abstract

In vitro metabolic stability experiments using microsomes or other liver preparations are important components in the discovery and lead-optimization stages of compound selection in the pharmaceutical industry. Currently, liquid chromatography-tandem mass spectrometric (LC-MS/MS) support of in vitro metabolic stability studies primarily involves the monitoring of disappearance of parent compounds, using selected reaction monitoring (SRM) on triple-quadrupole instruments. If moderate to high turnover is observed, separate metabolite identification experiments are then conducted to characterize the biotransformation products. In this paper, we present a novel method to simultaneously perform metabolite screening in addition to the quantitative stability measurements, both within the same chromatographic run. This is accomplished by combining SRM and SRM-triggered, information-dependent acquisition (IDA) of MS/MS spectra on a hybrid triple-quadrupole linear ion trap (QqQLIT) mass spectrometer. Microsomal stability experiments using model compounds, bufuralol, propranolol, imipramine, midazolam, verapamil and diclofenac, were used to demonstrate the applicability of our approach. This SRM + SRM-IDA approach generated metabolic stability results similar to those obtained by conventional SRM-only approach. In addition, MS/MS spectra from potential metabolites were obtained with the enhanced product ion (EPI) scan function of LIT during the same injection. These spectra were correlated to the spectra of parent compounds to confirm the postulated structures. The time-concentration profiles of identified metabolites were also estimated from the acquired data. This approach has been successfully used to support discovery programs.

Evaluation of Metabolically Stabilized Angiotensin IV Analogs as Procognitive/Antidementia Agents

“…To provide a context for dihexa's clearance rate, the stability of piroxicam (60.2 ml/min/mg), verapamil (112.5 ml/min/mg), and 7-ethoxycoumarin (136.7 ml/min/mg) was also monitored as high-, moderate-, and lowmetabolized standards, respectively. The clearance rates previously indicated were within the published ranges for these often-employed standards (Di et al, 2004;Shou et al, 2005;Lu et al, 2006;Behera et al, 2008). The clearance time courses for dihexa and the standards fit curves defining single-phase exponential decay processes with R 2 values between of 0.96 and 0.99 (Supplemental Fig.…”

‘N‐in‐one’ strategy for metabolite identification using a liquid chromatography/hybrid triple quadrupole linear ion trap instrument using multiple dependent product ion scans triggered with full mass scan

“…18,19 Even though the fast trap mode scan enables multiple scan functions simultaneously in IDA setting such as one EMS survey scan and multiple dependant EPI scans, as reviewed by Hopfgartner et al, 17 there has not been report of such an application. Although similar approaches have been used in three-dimensional ion traps in data-dependent acquisition (DDA) fashion for years, our strategy is still unique in that the Q-Trap generates true triple quadrupole type product ion spectra without low-mass cut-off which provide more structural information.…”

Comparison of sub‐2‐μm particle columns for fast metabolite ID

“…The developed methods were also suitable for determination of major metabolites of compounds with moderate metabolic turnover like propranolol [4].…”

“…Recent advances have demonstrated the interest in utilizing different modes of chromatography and conditions in order to reduce analytical run times. Kantharaj et al [3] and Shou et al [4] applied fast gradient chromatography using 3.1 lm particle col-umns coupled to IT MS with analytical run times of 5 -6 min.…”