RT Journal Article SR Electronic T1 Determination of Acyl-, O-, and N-Glucuronide using Chemical Derivatization Coupled with Liquid Chromatography - High Resolution Mass Spectrometry JF Drug Metabolism and Disposition JO Drug Metab Dispos FD American Society for Pharmacology and Experimental Therapeutics SP DMD-AR-2022-000832 DO 10.1124/dmd.122.000832 A1 Yukuang Guo A1 Abhi Shah A1 Eugene Oh A1 Swapan Chowdhury A1 Xiaochun Zhu YR 2022 UL http://dmd.aspetjournals.org/content/early/2022/03/03/dmd.122.000832.abstract AB Glucuronidation is the most common phase II metabolic pathway to eliminate small molecule drugs from the body. However, determination of glucuronide structure is quite challenging by mass spectrometry due to its inability to generate structure informative fragments about the site of glucuronidation. Herein we described a simple method to differentiate acyl-, O-, and N-glucuronides using chemical derivatization. The idea is that derivatization of acyl-, O- or N-glucuronide of a molecule results in predictable and different numbers of derivatized functional groups, which can be determined by the mass shift using mass spectrometry. The following two reactions were applied to specifically derivatize carboxyl and hydroxyl groups that are present on the aglycone and its glucuronide metabolite. Carboxyl groups were activated by thionyl chloride followed by esterification with ethanol. Hydroxyl groups were derivatized via silylation by 1-(trimethylsilyl)imidazole. The mass shift per derivatized carboxyl and hydroxyl group was +28.031 Da and +72.040 Da, respectively. This approach was successfully validated using commercial glucuronide standards including benazepril acyl-glucuronide, raloxifene O-glucuronides, and silodosin O-glucuronide. In addition, this approach was applied to determine the type of glucuronide metabolites that were isolated from liver microsomal incubation, where alvimopan and diclofenac acyl-glucuronides, darunavir, haloperidol, and propranolol O-glucuronides, and darunavir N-glucuronide were identified. Lastly, this approach was successfully utilized to elucidate the definitive structure of a clinically observed metabolite, soticlestat O-glucuronide. In conclusion, a novel efficient and cost-effective approach was developed to determine acyl-, O-, and N-glucuronide using chemical derivatization coupled with liquid chromatography-high resolution mass spectrometry. Significance Statement The method described in this study can differentiate acyl-, O-, and N-glucuronides and allow for elucidation of glucuronide structures when multiple possibilities of glucuronidation exist. The type of glucuronidation information is particularly useful for a drug candidate containing carboxyl group(s), which can form reactive acyl-glucuronides. Additionally, the method can potentially be used for the definitive structure elucidation for a compound containing a single carboxyl, hydroxyl, or amino group even when multiple types of functional groups are present for glucuronidation.