title: Mass spectrometric characterization of glucuronides formed by a new concept, combining Cunninghamella elegans with TEMPO.
abstract: A new concept for the production of drug glucuronides is presented and the products formed were characterized using ultra high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). Glucuronic acid conjugates are important phase II metabolites of a wide range of drugs. There is a lack of commercially available glucuronides and classic synthetic methods are tedious and expensive. Thus, new methods of glucuronide synthesis are needed. Selective androgen receptor modulators (SARMs) of the aryl propionamide class were used as model compounds and were incubated with the fungus Cunninghamella elegans which was previously known to conjugate drugs with glucose. The resulting glucoside metabolites were then oxidized with tetramethylpiperidinyl-1-oxy (TEMPO). UPLC-HRMS analysis showed that the peaks corresponding to the glucosides had disappeared after the reaction and were replaced by peaks with m/z consistent with the corresponding glucuronic acid conjugates. The MS/MS spectra of the reaction products were investigated and the observed fragment ion pattern corroborated the suggested structural change. A comparison in terms of retention times and product ion spectra between the glucuronides formed by the new method and those produced by liver microsomes indicated that the conjugates from the two different sources were identical, thus demonstrating the human relevance of the presented technique. Furthermore, the glucuronides formed by the presented method were readily hydrolyzed by β-glucuronidase which further gave evidence as to the fact that they were of β configuration. The investigated method was easy to perform, required a low input of work and had a low cost.
authors.affiliation: Division of Analytical Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala University, Box 574, SE-75123 Uppsala, Sweden. Axel.Rydevik@farmkemi.uu.se
authors.forename: Axel
authors.initials: A
authors.lastname: Rydevik
authors.forename: Ulf
authors.initials: U
authors.lastname: Bondesson
authors.forename: Mario
authors.initials: M
authors.lastname: Thevis
authors.forename: Mikael
authors.initials: M
authors.lastname: Hedeland
mesh_terms: D000577:Amides / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D020093:Cunninghamella / Q000378:metabolism*; D003497:Cyclic N-Oxides / Q000737:chemistry* / Q000378:metabolism*; D005960:Glucosides / Q000737:chemistry / Q000378:metabolism; D020723:Glucuronic Acid / Q000737:chemistry / Q000378:metabolism; D020719:Glucuronides / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007477:Ions / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D050217:Metabolic Detoxication, Phase II / Q000502:physiology; D008862:Microsomes, Liver / Q000378:metabolism
chemical_list: D000577:Amides; D003497:Cyclic N-Oxides; D005960:Glucosides; D020719:Glucuronides; D007477:Ions; D020723:Glucuronic Acid; C034666:propionamide; C003959:TEMPO
keywords: Glucuronides; HRMS; High resolution mass spectrometry; NADPH; SARM; TEMPO; UDPGA; UPLC; high resolution mass spectrometry; nicotinamide adenine dinucleotide phosphate; selective androgen receptor modulator; tetramethylpiperidinyl-1-oxy; uridine 5′-diphosphoglucuronic acid