IRMS detection of testosterone manipulated with C labeled standards in human urine by removing the labeled C

Isotope ratio mass spectrometry (IRMS) is applied to confirm testosterone (T) abuse by determining the carbon isotope ratios (δ¹³C value). However, ¹³C labeled standards can be used to control the δ¹³C value and produce manipulated T which cannot be detected by the current method. A method was explored to remove the ¹³C labeled atom at C-3 from the molecule of androsterone (Andro), the metabolite of T in urine, to produce the resultant (A-nor-5α-androstane-2,17-dione, ANAD). The difference in δ¹³C values between Andro and ANAD (Δδ¹³C_Andro–ANAD, ‰) would change significantly in case manipulated T is abused. Twenty-one volunteers administered T manipulated with different ¹³C labeled standards. The collected urine samples were analyzed with the established method, and the maximum value of Δδ¹³C_Andro–ANAD post ingestion ranged from 3.0‰ to 8.8‰. Based on the population reference, the cut-off value of Δδ¹³C_Andro–ANAD for positive result was suggested as 1.2‰. The developed method could be used to detect T manipulated with 3-¹³C labeled standards.