Maurer and Meyer (2016) examined high-resolution mass spectrometry (HRMS) and its application in clinical toxicology and forensic toxicology. They reviewed papers published since 2013 covering HRMS applications in human toxicology. In particular, they covered clinical and forensic toxicology, mainly for pharmaceuticals, drugs of abuse (DOAs) and toxins in the field of drug metabolism (including enzyme kinetics, drug screening and quantification in human body samples).
Drug metabolism studies are an important part of drug discovery and development, and of toxicological risk assessment. Liquid chromatography–HRMS (LC-HRMS) provides benefits such as high selectivity and specificity, providing better sensitivity. It also allows measurement of the elemental composition of molecules or fragments formed during ionization. Maurer and Meyer discuss HRMS applications in drug metabolism in DOA such as cannabinoids, stimulants, hallucinogens and opioids. They report that in vitro studies have used a variety of human liver preparations (including microsomes and cytosol, S9 fractions, primary hepatocytes and cell cultures), or heterologously expressed single phase I and II enzymes (for example, in yeast or insect cells for enzyme kinetics). They found that the best preparation for testing new psychoactive substances (NPSes) in vitro is primary hepatocytes. Recently, researchers used HRMS to identify phase I and II metabolites in urine of humans and/or rats. There are two major strategies used:
- Analytical procedures generally consisted of an Orbitrap mass spectrometer (Thermo Fisher) with ion traps or quadrupoles in front.
- LC–quadrupole time of flight (LC-QTOF) was used to analyze samples in combination with HRMS, mostly with specific tools such as mass defect filtering.
The authors found that HRMS is useful for quantifying substrates and products in enzyme kinetic studies if very low concentrations need to be determined. They note two particular studies, one in which the authors used software-aided cytochrome P450 (CYP) reaction phenotyping and kinetic analysis using an LC-triple-TOF assay. This allowed them to detect the CYP enzymes responsible for substrate depletion and metabolite formation, identify metabolite structures based on high-resolution tandem MS (HRMS/MS) fragment analysis and metabolic steps, and determine the initial relative formation rates of major metabolites. Another study used LC-HRMS-QTOF to define a novel pathway implicated in forming reactive cocaine protein adducts.
Maurer and Meyer then critically discuss pros and cons of low-resolution MS and HRMS for NPS screening approaches in urine, blood and hair samples, as well as in urine-containing soil collected during rave parties. Based on their review of the literature, they concluded that non-targeted screening procedures could easily be performed by HRMS. However, most authors still developed targeted screening procedures. They also discuss the advantage of sequential window acquisition of all theoretical fragment ion spectra for very fast scanning (QTOF) instruments. They suggest it is a promising option for non-targeted HRMS screening in clinical and forensic toxicology. In particular, they note LC-HRMS as the best suited for multi-component target methods and for general unknown screening. Furthermore, LC-HRMS/MS allows differentiating isobaric compounds. The downsides of HRMS include expense, insufficiency of data evaluation software for some applications (e.g., broad screening) and the requirement for skilled operators.
The authors conclude overall that HRMS devices are a very good all-around tool for applications in human toxicology, especially in clinical and forensic toxicology. They provide very high identification power and easy development of qualitative and quantitative methods.
1. Maurer, H.H., and Meyer, M.R. (2016) “High‑resolution mass spectrometry in toxicology: Current status and future perspectives,” Archives of Toxicology, 90(9) (pp. 2161–2172), doi: 10.1007/s00204-016-1764-1.