Thomas A. Baillie

High-throughput, accurate mass liquid chromatography/tandem mass spectrometry on a quadrupole time-of-flight system as a ‘first-line’ approach for metabolite identification studies

Throughput for drug metabolite identification studies has been increased significantly by the combined use of accurate mass liquid chromatography/tandem mass spectrometry (LC/MS/MS) data on a quadrupole time-of-flight (QTOF) system and targeted data analysis procedures. Employed in concert, these tools have led to the implementation of a semi-automated high-throughput metabolite identification strategy that has been incorporated successfully into lead optimization efforts in drug discovery. The availability of elemental composition data on precursor and all fragment ions in each spectrum has greatly enhanced confidence in ion structure assignments, while computer-based algorithms for defining sites of biotransformation based upon mass shifts of diagnostic fragment ions have facilitated identification of positions of metabolic transformation in drug candidates. Adoption of this technology as the first-line approach for in vitro metabolite profiling has resulted in the analysis of as many as 21 new chemical entities on one day from diverse structural classes and therapeutic programs.

Bridging cheminformatic metabolite prediction and tandem mass spectrometry.

Despite recent technological advances, the analysis of biological samples for metabolite identification purposes often requires prior knowledge of the metabolite masses to successfully acquire high quality mass spectral data in the presence of intense background and interfering matrix signals. This, in turn, necessitates prior knowledge of the metabolite structure, which in most cases can be predicted on the basis of the potential routes of metabolism of those functional groups present in the molecule. The following discussion highlights the significance of knowledge of the metabolite mass in facilitating the detection and structural elucidation of drug metabolites.

Fractional mass filtering as a means to assess circulating metabolites in early human clinical studies

Recent changes in the regulatory environment have led to a need for new methods to assess circulating human drug metabolites in early clinical studies with respect to their potential toxicological impact. The specific goals of such studies are to determine if the metabolites present in human plasma following administration of a drug candidate also are observed in plasma from the animal studies employed for preclinical toxicological evaluation, and to estimate corresponding exposure margins (animal:human) for the major metabolites. Until recently, the accepted best practice for the characterization of circulating drug metabolites utilized liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based methodologies, in conjunction with authentic chemical standards, for the detection and quantitative analyses of metabolites predicted from both animal studies and experiments with human liver preparations in vitro. While this approach is satisfactory for anticipated biotransformation products, metabolites that were not expected to circulate in human plasma frequently escape detection. Current accurate mass instruments enable the use of the technique of fractional mass filtering to detect both expected and unexpected metabolites in a rapid, less resource-intensive and more robust manner. Application of this technology to several clinical development programs at Merck Research Laboratories has demonstrated the value of fractional mass filtering in the assessment of circulating drug metabolites in early clinical trials.