Bestimmung der Herkunft von Steroidhormonen

Abstract

In the context of the EU-project “ISOSTER Determination of the Origin of Hormones in Cattle” (funded by the European Commission, DG RTD “Growth” Programme, Project No. GRD1-2001-40085) a method was developed and validated to extract steroids from cattle faeces for GC-C-IRMS analysis. The aim of this GC-C-IRMS analysis is the detection of an administration of natural steroid hormones by isotope ratios. This method is principally based on the assumption of an alteration of isotope ratios through an administation. Whereas isotope ratios of the precursors reflect the animals’ basic value, isotope ratios of hormones and metabolites are influenced by the substance that was administered. Since the GC-C-IRMS measurements demand very clean extracts, the developed clean up procedure consits of different extensive purification steps to meet these requirements. The method consists of an extraction step with methanol by soxhlet or accelerated solvent extraction followed by precipitation and filtration. Filtrates are purified on polymer based solid phase extraction columns. Sulphate conjugates of steroids are hydrolysed with sulphuric acid in ethyl acetate and the extracts are washed with sodium hydroxide solution. The organic layer is further purified on an HPLC gradient system by an enrichment of the analytes on a restricted access material precolumn prior to their separation on a reversed phase material. The steroids are collected and the fractions are acetylated. The acetates are further separated and purified on a second reversed phase HPLC system. The steroids in the collected fractions of the second HPLC step are extracted with n-hexane. They are first analysed by GC-MS to confirm their identity subsequently followed by GC-C-IRMS analysis. The high requirements of GC-C-IRMS measurements on sample clean up, including high purity of extracts, prevention of isotopic discrimination and an sufficient amount of target analytes in the extracts of above 10 ng, are met by the method developed for at least one precursor and several hormones or their metabolites. In validation experiments it could be shown that the requirements on purity lead to a loss in recovery of analytes. No discrimination of isotopes during the clean up for dehydroepiandrosterone, 17α-estradiol, progesterone, epiandrosterone and etiocholanolone occured. Dependent on the concentration of the steroid, the extraction of up to 100 g of cattle faeces can be necessary to reach sufficient amounts of target steroids in the extracts. Due to the complex sample preparation the method is not suitable for routine analysis of large sample numbers, but it enables measurements of isotope ratios of at least one precursor and several hormones and metabolites.