| Objective:Formestane, Boldenone,19-Norandrosterone are endogenous steroids, 13C- Testosterone is a new kind of endogenous steroids doping. The isotope mass spectrometry (IRMS) is the routine method for the detection of endogenous steroids. Doping testing laboratory in our country has not yet established carbon isotope ratio detection method for Formestane, Boldenone,19-Norandrosterone and 13C-Testosterone. The purpose of this study was to set up detection methods for Formestane, Boldenone,19-Norandrcsterone and 13C labeled Testosterone with IRMS. Formestane, Boldenone, Nandrolon and Testosterone doped with 13C-Testosterone were chosen as the test doping in order to identify characteristic parameters for initiating confirmation analysis of doping.Method:Exploring and establishing the high performance liquid chromatography separation and purification conditions and IRMS operating conditions in order to determine the isotope ratio mass spectrometry detection methods for Formestane, Boldenone,19-Nornandrosterone and 13C-Testosterone. These detection methods were evaluated. Recruited 31 subjects, divided into F, B, N, T, a total of four groups. In the group F, group B, group N were 8 subjects while 7 in the group T. Group F received a single oral foemestane preparation of 20 mg, Group B received a single oral boldenone preparation of 40 mg, Group N received a single oral nandrolone preparation of 20 mg, and collect all the subjects for 3 days in 12 urine samples. Urine endogenous steroids’ concentration and their isotope ratios were analyzed for the detection study. Group T received a single oral testosterone formulation of 80 mg doped with 13C labeled testosterone,15 urine samples were collected for 4 days. Purification and concentration in urine androserone (An), Removing 13C labeled carbon atom in the An molecules with the chemical reaction methods to get the new compounds ANAD, measuring the difference between An and ANAD isotopic ratios in the urine, the statistical analysis was performed in order to study the detection rules.Results:(1) The detection methods are simple and high operating efficiency. The RSDs for precisions, ruggedness and linearity were less than 2%, learnt through the method validation. (2) After administrating the Formestane, Boldenone, Nandrolone, the value of the concentration and the isotope ratio in urine An, Etio, PD, T, ET fluctuated in the normal range (3)The hours in which IRMS detection of urine sample showed positive results of Formestane is 22, while Boldenone is 37,19-Norandrosterone and 19-Noretiocholanolone is 27. (4) The cut-off value of the difference of An and ANAD isotope ratio is 1.2 ‰ to determination whether the use of 13C- Testosterone.Conclusion:(1)Set up the carbon isotope ratio detection method for Formestane, Boldenone,19-Norandrosterone and 13C-Testosterone. (2)The difference of isotope ratio value between the Formestane and the endogenous reference compound PD can determine whether the intake of exogenous formestane.(3) The difference of isotope ratio value between the Boldenone or Boldenone Metabolite and the endogenous reference compound PD can determine whether the intake of exogenous boldenone. (4) The difference of isotope ratio value between the 19-Norandrosterone or 19-Noretiocholanolone and the endogenous reference compound PD can determine whether the intake of exogenous 19-Norandrosterone or 19-Noretiocholanolone.(5) The difference between AN and ANAD isotope ratio determine whether the intake of 13C-Testosterone. |
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