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

Abstract

ABSTRACT:Recent regulatory guidance suggests that metabolites identified in human plasma should be present at equal or greater levels in one of the animal species used in safety assessments. In this report, a high-performance liquid chromatography-tandem mass spectrometry method is described whereby quantitative comparisons of exposures to metabolites between species can be obtained in the absence of authentic standards of the metabolites, calibration curves, and other attributes of standard bioanalytical methods. This novel method was tested using six drug-metabolite combinations. Plasma samples from animals are mixed with control plasma from humans and vice versa to remove possible differential effects of matrices. Through multiple ion monitoring-triggered enhanced product ion (EPI) scans, all metabolites were qualitatively confirmed, and daughter ions were selected for the most sensitive mass transitions to trigger EPI scans.Direct comparisons of metabolites in animal versus human plasma were achieved by calculating the peak area ratios of the metabolites versus an internal standard. Linearity of instrument responses was established by serial dilution. A statistical analysis demonstrated that experimentally measured ratios of the parent and metabolites in rat versus human correlated well with the nominal ratios of concentrations using linear regression with an average slope of 0.99 ؎ 0.08 (r ‫؍‬ 0.994 ؎ 0.005). This analysis showed that if the experimentally determined ratio of mass spectrometer responses is >2.0, then the actual exposure ratio is unity or greater (p < 0.01). This method offers timeand resource-sparing advantages to ascertaining metabolite exposure comparisons between humans and laboratory animal species. A strategy for application of this approach within standard drug development processes is described.

Meeting Report: Metabolites in Safety Testing (MIST) Symposium—Safety Assessment of Human Metabolites: What’s REALLY Necessary to Ascertain Exposure Coverage in Safety Tests?

“…A simple HPLC-MS/MS method was presented whereby quantitative comparisons of exposures to metabolites between animal and human can be obtained in the absence of authentic standards of the metabolites, calibration curves, and other attributes of standard bioanalytical methods (10,13). A statistical analysis showed that if the experimentally determined, animal-to-human MS response ratio is ≥2.0 then the actual exposure ratio is unity or greater (p<0.01).…”

“…The currently available techniques may be integrated to reliably find and structurally identify the metabolites from the plasma and urine samples that are already collected in typical phase I clinical trials. And semiquantitation of metabolites using liquid chromatography (LC)-UV, LC/MS/MS peak area ratio comparison (10,11,13), radiolabeled calibrant (5-7), and quantitative NMR standards (8,9,14) can be employed to compare the exposures to the metabolites in animals to humans. This allows a sponsor to comply with regulatory expectations for metabolite safety assessment without the need to wait for the conventional 14 C-ADME studies that are usually conducted in phases II or III.…”

“…These include the application of radiometrically calibrated metabolite standards (5-7) or creating metabolite standards from biological sources that are quantitated using nuclear magnetic resonance (NMR) spectroscopy (8,9). Finally, demonstration of relative exposures to metabolites across species can be accomplished using HPLCmass spectrometer (MS) peak area comparisons from animal and human plasma extracts (10,11).…”

A decade of drug metabolite safety testing: industry and regulatory shared learning

“…Some of these approaches include mixed matrix LC-MS/MS peak area comparisons across species [13], NMR-based techniques [14,15], and bioanalytical methods with radiocalibrants [16]. LC-MS peak area comparisons between animals and humans are routinely used by some sponsors to demonstrate that metabolite concentrations in animals exceed plasma concentrations in humans.…”

Late-occurring and Long-circulating Metabolites of GABAAα2,3 Receptor Modulator AZD7325 Involving Metabolic Cyclization and Aromatization: Relevance to MIST Analysis and Application for Patient Compliance

“…One approach adjusts the LC-MS signal of a metabolite with its MS signal response factor relative to the parent drug, as determined by correlating the MS and radiochromatographic signal responses in radiolabeled metabolite studies (Yu et al, 2007;Yi and Luffer-Atlas, 2010). Another method more efficient in time and resources is the direct comparison of the LC-MS signal of metabolite samples between different species after adding blank plasma of different species to each other to eliminate potential differences in sample matrix effect (Gao et al, 2010;Ma et al, 2010, Haglund et al, 2014.…”