In silico deconjugation of glucuronide conjugates enhances tandem mass spectra library annotation of human samples

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 Abstract

How Wastewater Reflects Human Metabolism─Suspect Screening of Pharmaceutical Metabolites in Wastewater Influent

“…The fact that glucuronidated metabolites tend to break apart at the site of conjugation during fragmentation was used to specifically identify glucuronide and sulfate metabolites by neutral loss search in the positive and negative ionization mode. 44 Besides neutral losses, specific MS2 fragment search for glucuronide and glutathione conjugates was conducted in the negative ionization mode (see SI-C2.13 ). Together, these approaches were able to further support the identification of paracetamol-sulfate (ESI+), tapentadol- O -sulfate (ESI+), diphenhydramine- N -glucuronide (ESI+) and lamotrigine- N2 -glucuronide (ESI+), which were detected in all analyzed influent samples with concentrations ranging from 10 to 1000 ng/L.…”

Immobilised-enzyme microreactors for the identification and synthesis of conjugated drug metabolites

“…Its respective mass spectrum shows fragment m/z = 212, representing a loss of m/z = 176, which is characteristic for glucuronide conjugates. 39 Effluent collected from the UGT1a1 ow reactor incubated with both UDP-GA and resorun (2 h, 0.1 mL min −1 ) contained a metabolite identied as resorun-glucuronide, based on m/z = 388 and a retention time of 8.0 min (Fig. 10c).…”

Wastewater-based epidemiology for the assessment of population exposure to chemicals: The need for integration with human biomonitoring for global One Health actions

“…These are primarily: selection of specific human metabolic biomarkers, characteristics of urban water cycle including sewer systems (population size and mobility in a WWTP catchment, flow rate), and understanding the fate of biomarkers (stability, biotransformation, sorption). Novel screening approaches for human biomonitoring of a multitude of contaminant metabolites in urine based on high-throughput in vitro incubation, suspect and non-target Liquid Chromatography and High Resolution Mass Spectrometry (LC-HRMS) screening involving in silico deconjugation and automated data evaluation [23] , [24] create new opportunities to discover new relevant biomarkers providing insights to so far unknown exposures in both HBM as well as WBE.…”