The use of stable carbon isotope analysis to detect the abuse of testosterone in cattle

The use of stable carbon isotope analysis to detect the administration of a nabolic steroids to cattle was investigated, Samples were extracted by soli d-phase extraction on C-18 cartridges. Stable isotope ratios (C-13:C-12) we re measured by gas chromatography-isotope ratio mass spectrometry (GC-IRMS) of the underivatised extracts. A programmed temperature vaporiser (PTV) in jector was installed in the GC-IRMS system, which conferred a number of adv antages. First, it allowed large volumes of sample to be injected whilst th e injector liner was cool. The solvent was subsequently vented to the atmos phere prior to transfer of the sample to the GC column. Thus a significantl y greater amount of sample could be presented for analysis, thereby increas ing the sensitivity. Second, by this means virtually all the solvent could be removed prior to analysis. This eliminates solvent peak tailing, which c an be a major problem in GC-IRMS. Finally, the PTV allowed the use of highe r initial GC oven temperatures, which in turn facilitated the analysis of u nderivatised steroids by reducing the GC run time and improving the chromat ographic peak shape. The carbon isotope composition of 5 beta-androstane-3 alpha,17 alpha-diol, the major metabolite of testosterone found in bovine b ile, was measured in bile samples from untreated cattle and from cattle inj ected intramuscularly with testosterone or a mixture of testosterone esters . There was considerable inter-animal variation in the values obtained and there was no significant difference between samples from treated and untrea ted animals. However, when the isotopic composition of the metabolite was n ormalised with respect to that of an endogenous reference compound (cholest erol) in the same sample, the difference between treated and untreated anim als became statistically significant.