Simultaneously quantifying parent drugs and screening for metabolites in plasma pharmacokinetic samples using selected reaction monitoring information‐dependent acquisition on a QTrap instrument

Abstract

Bioanalytical support of plasma pharmacokinetic (PK) studies for drug discovery programs primarily involves the quantitative analysis of dosed compounds using liquid chromatography/atmospheric pressure ionization tandem mass spectrometry (LC/MS/MS) operated in selected reaction monitoring (SRM) mode. However, there is a growing need for information on the metabolism of new chemical entities (NCEs), in addition to the time-concentration profiles from these studies. In this paper, we present a novel approach to not only quantify parent drugs with SRM, but also simultaneously screen for metabolites using a hybrid triple quadrupole/linear ion trap (QqQ(LIT)) instrument. This was achieved by incorporating both the conventional SRM-only acquisition of parent compounds and the SRM-triggered information-dependent acquisition (IDA) of potential metabolites within the same scan cycle during the same LC/MS/MS run. Two test compounds were used to demonstrate the applicability of this approach. Plasma samples from PK studies were processed by simple protein precipitation and the supernatant was diluted with water before injection. The fast scanning capability of the linear ion trap allowed for the information-dependent acquisition of metabolite MS/MS spectra (<1 s/scan), in addition to the collection of adequate data points for SRM-only channels. The MS/MS spectra obtained from potential metabolites in post-dose samples correlated well with the spectra of the parent compounds studied, therefore providing additional confirmatory structure information without the need for repetitive analyses. Relative quantitative time-concentration profiles of identified metabolites were also obtained. Furthermore, this articulated SRM+SRM-IDA approach generated equivalent quantitative results for parent compounds to those obtained by conventional SRM-only analysis. This approach has been successfully used to support discovery PK screening programs.

Characterization of Flavonoid Metabolites in Rat Plasma, Urine, and Feces after Oral Administration of Semen Ziziphi Spinosae Extract by HPLC-Diode-Array Detection (DAD) and Ion-Trap Mass Spectrometry (MSn)

“…The results show that the total peaks and corresponding peak areas in metabolic chromatograms were different when collected at different periods, and the most abundant metabolites were found at 4, 12, and 12 h in plasma, urine, and feces samples, respectively, corresponding to the cases of components during physiological disposition. [9][10][11][12][13] To obtain further metabolic information, the biological samples with predominant metabolic compounds were further analyzed and elucidated by HPLC-ESI-MS n . The base peak chromatograms of plasma (A), urine (C), and feces (E) and UV chromatograms of plasma (B), urine (D), and feces (F) with the most abundant metabolites are presented in Fig.…”

“…The blood was centrifuged at 3500 rpm for 15 min to separate plasma, then the plasma of each group was mixed together as the sampling schedule and stored at Ϫ70°C before analysis. [9][10][11][12][13][14] Bio-Samples Preparation To release the protein-binding medicinal composition, 50 ml of 20% acetic acid was added into 2 ml plasma sample and mixed by vortex, followed by 8 ml methanol added and vortexed again. Then, the solution was centrifuged at 3500 rpm for 15 min.…”

“…Five microliters of the filtrate was injected into the HPLC system for HPLC-DAD-MS n analysis. [9][10][11][12][13][14] The blank samples of feces, urine, and plasma were processed with the same methods as described above. All samples were stored at Ϫ70°C until analysis.…”

A Simple Liquid Chromatography-Tandem Mass Spectrometry Method to Determine Relative Plasma Exposures of Drug Metabolites across Species for Metabolite Safety Assessments

“…The hybrid quadrupole linear ion trap mass spectrometer (QTRAP) has been used to quantitate the parent drug and simultaneously screen metabolites in biological matrix (Xia et al, 2003;Li et al, 2005). Multiple ion monitoring (MIM) can be used to trigger an enhanced product ion (EPI) scan to identify a large number of metabolites in a single chromatographic run (Yao et al, 2008).…”

Study of Isobaric Interference in Quantification of Citrulline by Parallel Fragmentation Monitoring

“…5,6) SRM is mainly carried out with liquid chromatography electrospray ionization triple-quadrupole (LC-ESI-QqQ) MS platform. Unambiguous identi cation of small molecules is one of the current challenges for the analytical laboratories.…”