Identification of drug metabolites with infrared ion spectroscopy – application to midazolam in vitro metabolism

Abstract

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.

Targeted small molecule identification using heartcutting liquid chromatography–infrared ion spectroscopy

“…We recently showed the application of IRIS in drug metabolite profiling by identifying metabolites of the drug midazolam (MDZ). 45 Midazolam undergoes a hydroxylation reaction, resulting in 1'-hydroxymidazolam (1'-OH-MDZ, m/z 342), which undergoes a second metabolic reaction to form a glucuronide, 1'-hydroxymidazolam-O-glucuronide (1'-OH-MDZ-O-gluc, m/z 518, see Figure 4a). Identification of the metabolites of MDZ involved recording IR spectra of the two ions, but also of the glucuronide-loss CID MS/MS fragment (the aglycone) of 1'-OH-MDZ-O-gluc.…”