Quantitative analysis of androst-4-ene-3,6,17-trione and metabolites in human urine after the administration of a food supplement by liquid chromatography/ion trap-mass spectrometry

6-OXO®, a new nutritional supplement commercially available on the internet, is sold as an aromatase-inhibitor and contains androst-4-ene-3,6,17-trione as active ingredient. This anabolic steroid is a prohibited substance in sports. Androst-4-ene-3,6,17-trione is metabolised to androst-4-ene-6α-ol-3,17-dione and androst-4-ene-6α,17β-diol-3-one. A fast, sensitive and accurate LC/MS method was developed and validated for the quantification of androst-4-ene-3,6,17-trione and its metabolites in urine. The method is capable of determining the stereochemical position of the hydroxy-group at C-6 of the metabolites and consists of a liquid-liquid extraction step with diethylether after enzymatic hydrolysis, followed by separation on a reversed phase column. Ionisation of the analytes is carried out using atmospheric pressure chemical ionisation. The limit of quantification of the method was 5 ng/mL for all compounds. The accuracy ranged from 14.8 to 1.3% for androst-4-ene-3,6,17-trione, 9.4 to 1.6% for androst-4-ene-6α-ol-3,17-dione and 4.1 to 3.2% for androst-4-ene-6α,17β-diol-3-one in the range of 5-1000 ng/mL. Using this method androst-4-ene-6α-ol-3,17-dione was identified as a major urinary metabolite, whereas androst-4-ene-6α,17β-diol-3-one as a minor metabolite. While the parent compound is predominantly excreted in conjugated form, both metabolites are solely excreted as conjugates.