Abstract
Mass spectral library annotation of liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) data is a reliable approach for fast identification of organic contaminants and toxicants in complex environmental and biological matrices. While determining the exposure of humans or mammals, it is indispensable to include phase I and phase II metabolites (conjugates) along with the parent compounds, but often, tandem mass spectra for these are unavailable. In this study, we present and evaluate a strategy for annotating glucuronide conjugates in LC-HRMS/MS scans by applying a neutral loss search for detection, then truncating the spectra which we refer to as in silico deconjugation, and finally searching these against mass spectral libraries of the aglycones. The workflow was tested on a dataset of in vitro–generated glucuronides of reference standard mixtures and a dataset of 51 authentic urine samples collected from patients with known medication status, acquired on different instrumentations. A total number of 75 different glucuronidated molecular structures were identified by in silico deconjugation and spectral library annotation. We also identified specific molecular structures (sulfonamides, ether bonds, di-glucuronides), which resulted in slightly different fragmentation patterns between the glucuronide and the unconjugated compound. This led to a decreased spectral matching score and in some cases to a false-negative identification. Still, by applying this method, we revealed a reliable annotation of most common glucuronides, leading to a new strategy reducing the need for deconjugation steps or for recording many reference glucuronide spectra for screening approaches.Graphical
Highlights
Screening and non-targeted methods have increased in popularity and application for qualitative analysis of hundreds of compounds simultaneously [1]
Evaluating the impact of the collision energy with QqTOF reference spectra For successful application of the in silico deconjugation approach, the presence of the fragment generated by the Gluc-neutral loss (NL) in the glucuronide-specific MS/MS spectrum is necessary
We evaluated the impact of collision energy settings on the fragmentation patterns of glucuronides and the corresponding aglycones with a training set taken from an established MS/MS spectral library (WRTMSD) [21]
Summary
Screening and non-targeted methods have increased in popularity and application for qualitative analysis of hundreds of compounds simultaneously [1]. Mammalian metabolites can be grouped in phase I metabolites, occurring mainly from oxidation reactions and phase II metabolites, formed by conjugation reactions of parent compounds or phase I metabolites. Glucuronidation is one major conjugation reaction in phase II metabolism [3]. The conjugation of a xenobiotic compound increases its water solubility and enhances urinary excretion [5]. Based on the clearance mechanisms presented on RxList (drug index for prescription drug information, interactions and side effects), glucuronidation is listed as a relevant excretion pathway for approximately one in ten of the top 200 drugs prescribed [7]
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