Zhang Ying, Tobias Herbert J, Brenna J Thomas
Cornell University, Division of Nutritional Sciences, Savage Hall, Ithaca, NY 14853, United States.
Steroids. 2009 Mar;74(3):369-78. doi: 10.1016/j.steroids.2008.10.001. Epub 2008 Oct 17.
Carbon isotope ratio (CIR) analysis of urinary steroids using gas chromatography-combustion isotope ratio mass spectrometry (GCC-IRMS) is a recognized test to detect illicit doping with synthetic testosterone. There are currently no universally used steroid isotopic standards (SIS). We adapted a protocol to prepare isotopically uniform steroids for use as a calibrant in GCC-IRMS that can be analyzed under the same conditions as used for steroids extracted from urine. Two separate SIS containing a mixture of steroids were created and coded CU/USADA 33-1 and CU/USADA 34-1, containing acetates and native steroids, respectively. CU/USADA 33-1 contains 5alpha-androstan-3beta-ol acetate (5alpha-A-AC), 5alpha-androstan-3alpha-ol-17-one acetate (androsterone acetate, A-AC), 5beta-androstan-3alpha-ol-11, 17-dione acetate (11-ketoetiocholanolone acetate, 11k-AC) and 5alpha-cholestane (Cne). CU/USADA 34-1 contains 5beta-androstan-3alpha-ol-17-one (etiocholanolone, E), 5alpha-androstan-3alpha-ol-17-one (androsterone, A), and 5beta-pregnane-3alpha, 20alpha-diol (5betaP). Each mixture was prepared and dispensed into a set of about 100 ampoules using a protocol carefully designed to minimize isotopic fractionation and contamination. A natural gas reference material, NIST RM 8559, traceable to the international standard Vienna PeeDee Belemnite (VPDB) was used to calibrate the SIS. Absolute delta(13)C(VPDB) and Deltadelta(13)C(VPDB) values from randomly selected ampoules from both SIS indicate uniformity of steroid isotopic composition within measurement reproducibility, SD(delta(13)C)<0.2 per thousand. This procedure for creation of isotopic steroid mixtures results in consistent standards with isotope ratios traceable to the relevant international reference material.
采用气相色谱 - 燃烧 - 同位素比率质谱法（GCC - IRMS）对尿甾体进行碳同位素比率（CIR）分析是一种公认的检测合成睾酮非法使用的方法。目前尚无普遍使用的甾体同位素标准品（SIS）。我们采用了一种方案来制备同位素均匀的甾体，用作GCC - IRMS中的校准物，其可在与从尿液中提取的甾体相同的条件下进行分析。制备了两种单独的含有甾体混合物的SIS，并分别编码为CU/USADA 33 - 1和CU/USADA 34 - 1，分别含有醋酸酯和天然甾体。CU/USADA 33 - 1包含5α - 雄甾烷 - 3β - 醇醋酸酯（5α - A - AC）、5α - 雄甾烷 - 3α - 醇 - 17 - 酮醋酸酯（雄甾酮醋酸酯，A - AC）、5β - 雄甾烷 - 3α - 醇 - 11,17 - 二酮醋酸酯（11 - 酮 - 本胆烷醇酮醋酸酯，11k - AC）和5α - 胆甾烷（Cne）。CU/USADA 34 - 1包含5β - 雄甾烷 - 3α - 醇 - 17 - 酮（本胆烷醇酮，E）、5α - 雄甾烷 - 3α - 醇 - 17 - 酮（雄甾酮，A）和5β - 孕烷 - 3α,20α - 二醇（5βP）。使用精心设计的方案制备每种混合物并将其分装到约100个安瓿中，以尽量减少同位素分馏和污染。使用可溯源至国际标准维也纳 - 皮迪 - 贝列尼特（VPDB）的天然气参考物质NIST RM 8559来校准SIS。从两种SIS中随机选择的安瓿中得到的绝对δ(13)C(VPDB)和Δδ(13)C(VPDB)值表明，在测量再现性范围内甾体同位素组成均匀，SD(δ(13)C)＜0.2‰。这种制备同位素甾体混合物的方法产生了与同位素比率一致的标准品，其可溯源至相关的国际参考物质。
