F. André

Analytical strategies for the direct mass spectrometric analysis of steroid and corticosteroid phase II metabolites.

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.

Detection and identification of anabolic steroids in bovine urine by gas chromatography—mass spectrometry

Abstract Within the scope of the national plan for hormone control in France, a study was made to develop a system for the control of the illegal use of anabolic steroids for fattening purposes in animal production. A screening procedure for these residues in bovine urine was developed, based on C18 solid-phase extraction, silica gel purification and gas chromatographic—mass spectrometric analysis with selected ion monitoring. All the method parameters are discussed, especially the enzymatic and chemical hydrolysis steps and the choice of derivatives.