The use of gas chromatography–mass spectrometry/combustion/isotope ratio mass spectrometry to demonstrate progesterone treatment in bovines

•The first published method to demonstrate progesterone treatment in bovines using GC–MS/C/IMRS is described.•Abundance of pregnanediol-isomers in urine from pregnant and treated animals revealed target metabolites of progesterone.•Acetylation of the individual metabolites and reference compounds was compared to and preferred over an oxidation step•5-Androstene-3β,17α-diol was used as endogenous reference compound, 5β-pregnane-3α, 20α-diol was the selected metabolite.•Their difference in δ13CVPDB values allowed to differentiate between treated and untreated cows. Currently, no analytical method is available to demonstrate progesterone administration in biological samples collected in rearing animals, and therefore, tracking the abuse of this popular growth promoter is arduous. In this study, a method is presented to reveal progesterone (PG) treatment on the basis of carbon isotope measurement of 5β-pregnane-3α, 20α-diol (BAA-PD), a major PG metabolite excreted in bovine urine, by gas chromatography–mass spectrometry/combustion/isotope ratio mass spectrometry (GC–MS/C/IRMS). 5-Androstene-3β,17α-diol (AEdiol) is used as endogenous reference compound. Intermediate precisions (n=11) of 0.56‰ and 0.68‰ have been determined for AEdiol and BAA-PD, respectively. The analytical method was used for the very first time to successfully differentiate urine samples collected in treated and untreated animals.