title: Analytical strategies for the direct mass spectrometric analysis of steroid and corticosteroid phase II metabolites.
abstract: The use of steroid hormones as growth promoters remains illegal in Europe. A classical approach used to control their utilization consists to measure the parent drug in target biological matrices. However, this strategy may fail when the parent drug is submitted to extensive metabolism reactions. For urine and tissue samples, chemical or enzymatic hydrolysis is usually applied in order to deconjugate glucuronide and sulfate phase II metabolites. But this treatment lead to the loss of information such as nature and relative proportions of the different conjugated forms, which can be useful, for example, to discriminate an endogenous production from an exogenous administration for natural hormones, or for other clinical or biochemical specific applications. For these purposes, direct measurement of conjugated metabolites using liquid chromatography-tandem mass spectrometry may represent a solution of choice. In this context, the mass spectrometric behavior of 14 steroid and corticosteroid phase II metabolites after electrospray ionization was investigated. Their fragmentation pathways in tandem mass spectrometry revealed some specificities within the different group of conjugates. A specific acquisition program (MRM mode) was developed for the unambiguous identification of the studied reference compounds. A more generic method (Parent Scan mode) was also developed for fishing approaches consisting to monitor several fragment ions typical of each conjugate class. A reverse phase HPLC procedure was also proposed for efficient retention and separation of the studied compounds. Finally, a protocol based on quaternary amine SPE was developed, permitting the separation of free, glucuronide, and sulfate fractions. Preliminary results on biological samples demonstrated the suitability of this analytical strategy for direct measurement of dexamethasone glucuronide and sulfate residues in bovine urine.
authors.affiliation: Laboratoire d'Etude des Résidus et Contaminants dans les Aliments (LABERCA), Ecole Nationale Vétérinaire de Nantes (ENVN), USC Institut National de la Recherche Agronomique (INRA), BP 50707, 44307 Nantes Cedex 3, France. laberca@vet-nantes.fr
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Antignac
authors.forename: Aline
authors.initials: A
authors.lastname: Brosseaud
authors.forename: Isabelle
authors.initials: I
authors.lastname: Gaudin-Hirret
authors.forename: François
authors.initials: F
authors.lastname: André
authors.forename: Bruno Le
authors.initials: BL
authors.lastname: Bizec
mesh_terms: D000305:Adrenal Cortex Hormones / Q000737:chemistry* / Q000378:metabolism / Q000652:urine*; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D003907:Dexamethasone / Q000031:analogs & derivatives / Q000652:urine; D008401:Gas Chromatography-Mass Spectrometry; D020719:Glucuronides / Q000737:chemistry; D006868:Hydrolysis; D007477:Ions; D013058:Mass Spectrometry / Q000379:methods*; D008956:Models, Chemical; D013256:Steroids / Q000737:chemistry* / Q000652:urine*; D015813:Substance Abuse Detection / Q000379:methods*; D013431:Sulfates / Q000652:urine
chemical_list: D000305:Adrenal Cortex Hormones; D020719:Glucuronides; D007477:Ions; D013256:Steroids; D013431:Sulfates; C095328:dexamethasone glucuronide; D003907:Dexamethasone