LC-HRMS Biomarker Suspect Screening in Low Dose Studies

Abstract

Exposure biomarker development is a time consuming procedure, starting with identification of presumably appropriate metabolites of the xenobiotic in question. Investing into unsuitable biomarker candidates (i.e. metabolites which are formed in low proportion or are not formed at all) can be costly. Accordingly, efficient strategies for the identification of promising biomarker candidates are needed. For this purpose, liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) is often applied for metabolite screening in in vitro and animal in vivo screening approaches, typically involving high doses/concentrations of the xenobiotic.Given the limitations of in vitro models and interspecies differences in metabolism, we sought to improve the certainty of metabolite screening by analyzing urine samples from human studies with low oral dosing (i.e. toxicologically uncritical). Metabolite screening was performed by LC-Q-Orbitrap-MS combined with a commercial data mining software. Identification criteria were plausible excretion kinetics, product ion spectra, 13C isotopomer ratios, and retention times.We present results of a pilot study, which identified nine metabolites of the plasticizer di (2-ethylhexyl) terephthalate (DEHTP). Three of them (5OH-MEHTP, 5Oxo-MEHTP, and 5cx-MEPTP) are already established biomarkers of DEHTP exposure. Five additional, specific metabolites have not been described previously. In another example we successfully identified several tentative metabolites of the UV filter avobenzone. However, we also report examples, in which metabolite identification did not succeed by this approach (e.g. the plasticizer DEHA) and we highlight influencing factors (such as mobile phase pH, in source adduct formation and fragmentation) possibly leading to wrong conclusions.The presented approach is a valuable but not universal tool for biomarker development. In some cases, a more sensitive triple quadrupole screening can be superior.