LC-MS/MS-Based Approach for Obtaining Exposure Estimates of Metabolites in Early Clinical Trials Using Radioactive Metabolites as Reference Standards

Abstract

An LC-MS/MS-based approach that employs authentic radioactive metabolites as reference standards was developed to estimate metabolite exposures in early drug development studies. This method is useful to estimate metabolite levels in studies done with non-radiolabeled compounds where metabolite standards are not available to allow standard LC-MS/MS assay development. A metabolite mixture obtained from an in vivo source treated with a radiolabeled compound was partially purified, quantified, and spiked into human plasma to provide metabolite standard curves. Metabolites were analyzed by LC-MS/MS using the specific mass transitions and an internal standard. The metabolite concentrations determined by this approach were found to be comparable to those determined by valid LC-MS/MS assays. This approach does not requires synthesis of authentic metabolites or the knowledge of exact structures of metabolites, and therefore should provide a useful method to obtain early estimates of circulating metabolites in early clinical or toxicological studies.

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

“…There have been approaches described that offer an ability to measure drug metabolites without the synthesis of an authentic standard. These have included the generation of a radiolabeled calibration standard of metabolite from a biological source (Zhang et al, 2007;Leclercq et al, 2009) or establishing a stock solution concentration of metabolite that has been isolated with 1 H NMR and using that solution for the construction of standard curves (Vishwanathan et al, 2009). It has been proposed that LC-MS/MS peak areas can be used as a relative concentration comparison between animals and humans to demonstrate the coverage of metabolite exposure in animals, and some bioanalytical issues have been discussed and remain to be resolved (Leclercq et al, 2009;Walker et al, 2009).…”

The Science of Laboratory and Project Management in Regulated Bioanalysis

“…The US FDA has allowed the use of tiered assays to investigate early metabolism. This approach allows individuals to use nonvalidated assays, often with biologically sourced metabolites as reference calibrators, to estimate exposures across species [5,6]. Both the International Conference on Harmonisation and Metabolites in Safety Testing guidance require steady-state coverage in at least one primary toxicology species for circulating human metabolites [7,8].…”

Analytical Method Development and Validation in Accordance to the Regulatory Guidelines

“…NMR signal response is independent of structure, allowing isolated metabolites to be quantified by comparing their response to that of a known added amount of parent drug. In another approach, radiochemical detection has been used to calibrate stock solutions of metabolites [31]. This approach requires separation of metabolites, as, unlike NMR or MS, detection is nonspecific.…”

“…One approach previously mentioned uses metabolites from radiolabeled animal or human in vitro studies spiked into human plasma as standards for quantification [31]. To ensure accurate quantification, the radiolabeled profile must separate all metabolites from each other.…”