Investigation of the feeding effect on the 13C/12C isotope ratio of the hormones in bovine urine using gas chromatography/combustion isotope ratio mass spectrometry

The effect of the feeding on the 13C/12C isotope ratio of four endogenous steroid hormones testosterone (T), epi-testosterone (epi-T), dehydroepiandrosterone (DHEA) and etiocholanolone (ETIO) in bovine urine was investigated. An analytical method to determine the accurate isotope ratio was developed including an extensive clean up followed by enrichment of the analytes in two steps of HPLC fractionation. Feeding experiments with four young animals were performed using C3 and C4 plants (grass, maize silage, hay, etc.) over a time period of about 280 days. One cattle was used as a control animal with no change of its diet over the full period. The detection of the 13C/12C isotope ratio of the acetylated extracts was performed by gas chromatography/combustion isotope ratio mass spectrometry. After the first change of the feeding from C4 to C3 plants significant changes of the δ 13C ‰ values were observed from the −19 to −23‰ level to the −24 to −32‰ level for etiocholanolone and epi-testosterone in urine of three animals, whereas the DHEA values remained under the level of the two metabolites. Testosterone could not be detected with GC–C–IRMS due to its low concentration in young animals. After the second change of the diet from C3 to C4 plants (after 222 days), the measured δ 13C ‰ values have been stabilised at the original level. The results show that in case of the feeding with only C3 plants the endogenous δ values of −32‰ can be reached. In this case the contribution of exogenous material with a δ value of −32‰ could not be detected independently of the concentration. If the diet contains C4 plants the difference or the ratio of the δ 13C ‰ values becomes the determinant in the discriminatory power. For validation of the method a human and a cattle were treated with testosterone and the δ 13C ‰ values were measured in incurred human and cattle urine.