Identification of Drug Metabolites with Infrared Ion Spectroscopy – Application to Midazolam in vitro Metabolism** Rianne E. van Outersterp, Jonathan Martens, Giel Berden, Arnaud Lubin, Filip Cuyckens, et al. Chemistry Methods, 2023 The identification of biotransformation products of drug compounds is a crucial step in drug development. Over the last decades, liquid chromatography‐mass spectrometry (LC‐MS) has become the method of choice for metabolite profiling because of its high sensitivity and selectivity. However, determining the full molecular structure of the detected metabolites, including the exact biotransformation site, remains challenging on the basis of MS alone. Here we explore infrared ion spectroscopy (IRIS) as a novel MS‐based method for the elucidation of metabolic pathways in drug metabolism research. Using the drug midazolam as an example, we identify several biotransformation products directly from an in vitro drug incubation sample. We show that IR spectra of the aglycone MS/MS fragment ions of glucuronide metabolites establish a direct link between detected phase I and phase II metabolites. Moreover, using quantum‐chemically computed IR spectra of candidate structures, we are able to assign the exact sites of biotransformation in absence of reference standards. Additionally, we demonstrate the utility of IRIS for structural elucidation by identifying several ring‐opened midazolam derivatives formed in an acidic environment.
Strategies and analytical workflows to extend the dynamic range in quantitative LC-MS/MS analysis Emmanuel Njumbe Ediage, Tania Aerts, Arnaud Lubin, Filip Cuyckens, Lieve Dillen, et al. Bioanalysis, 2019 Aim: To evaluate alternative analytical strategies to extend the dynamic range in quantitative LC-MS/MS. Methodology & results: Two approaches based on prior or no prior knowledge of expected exposure levels were evaluated. These approaches make use of two analytical strategies, which include the use of more than one injection volume or dilution of sample extract with solvents or solvent mixtures. A total of 16 compounds with varying logP values were classified into polar and nonpolar groups and used in this evaluation. From the two analytical strategies, three workflows were derived. Conclusion: All three workflows were successfully evaluated and resulted in good accuracy (80-120%) for all the compound groups.
