气相色谱质谱联用法对运动员尿液中内源性类固醇水平特征的研究

Statistical analysis of endogenous steroids profile of athletes for the study of the characteristics and rules of steroid metabolism indices in urine by GC-MS

目的：建立气相色谱-质谱联用（GC-MS）同时测定尿液中6种内源性类固醇激素水平的方法,对广东省运动员体内的类固醇激素代谢特征进行统计归纳和比较分析,为广东省运动员尿液中类固醇激素的生物护照的建立提供数据支持。方法：选取1 474名广东省运动员（包括814名男队员,660名女队员）在赛前训练期间的尿样,并对其中主要的6种内源性类固醇进行酶解、提取及衍生化后采用气质联用（GC-MS）方法定量分析。分析采用HP-1MS色谱柱（17 m×0.2 mm,0.11μm）,恒流模式,使用氦气为载气,流量为1 m L·min-1,进样量：1μL;分流比：10∶1;进样口温度和EI离子源温度分别为280℃和200℃;质谱SCAN模式定性确证,扫描范围m/z 40~460;选择离子模式（SIM）下定量分析。结果：广东省女运动员尿液中6种类固醇激素浓度及睾酮/表睾酮比值平均值均显著低于国外女运动员（P〈0.05）,其中睾酮浓度平均值仅为国外女运动员的12.2%;广东省男运动员尿液中雄酮、本胆烷醇酮、睾酮、5β-雄烷-3α,17β-二醇4种类固醇激素浓度及睾酮/表睾酮比值平均值均显著低于国外男运动员（P〈0.05）,而表睾酮浓度平均值明显高于国外男运动员（P〈0.05）,5α-雄烷-3α,17β-二醇浓度平均值与国外男运动员基本持平（P〉0.05）。广东省男、女运动员尿液中类固醇5α-雄烷-3α,17β-二醇/5β-雄烷-3α,17β-二醇浓度比值则均显著高于国外男、女运动员的水平（P〈0.05）,雄酮/本胆烷醇酮浓度比值与国外运动员基本持平。结论：本研究选取6种内源性类固醇激素进行气质联用分析,并通过与国内外运动员相关代谢指标的对比分析,对广东运动员训练期间的尿液中类固醇代谢特征进行归纳,为广东省运动员尿液内源性类固醇指标生物护照的建立提供数据支持。

Objective：To establish a method for statistical analyzing of 6 kinds of urinary endogenous steroids of athletes from Guangdong province using gas chromatography-mass spectrometry.The characteristics of in vivo hormone metabolism were summarized and analyzed,so as to support the establishment of biological passport of urine steroids for athlete（ASP,Athlete Steroidal Passport）from Guangdong province.Methods：Urine samples were collected from 1 474 athletes（including 814 male and 660 female athletes）from Guangdong province in the stage of competition training period.Six kinds of endogenous androgens were enzymatic hydrolyzed and extracted,then detected by GC-MS for semi-quantitative analysis.The analysis was performed on an HP-1 μs column（17 m×0.2 mm,0.11 μm）with helium as the carrier gas.The flow rate was set at 1 m L·min-1,the injection volume was 1 μL,and the split ratio was 10 ∶ 1.The inlet temperature and ion source temperature were controlled at 280 ℃ and 200 ℃,respectively.A full scan mode（SCAN）was performed for qualitative analysis with scanning range of m/z 40-460 and selected ion monitoring（SIM）for quantitative analysis.Results：The total concentrations of the steroids as well as the ratio of testosterone/epitestosterone in urine of Guangdong female athletes were significantly lower than the relevant data of female foreign athletes（P〈0.05）.Notably,the concentration of testosterone was only 12.2% of that of female foreign athletes.The total concentrations of androsterone,etiocholanolone,testosterone,5β-androstane-3α,17β-diol and testosterone/epitestosterone in urine of Guangdong male athletes were significantly lower than the relevant data of male foreign athletes（P〈0.05）,while the concentration of epitestosterone in male athletes was significantly higher than male foreign athletes（P〈0.05）and the concentration of 5α-androstane-3α,17β-diol in male athletes were roughly equal to that of male foreign athletes.The concentration ratio of 5α-androstane-3α,17β-diol/5β-androstane-3α,17β-diol of both Guangdong female and male athletes was significantly higher than foreign athletes（P〈0.05）.The concentration ratio of androsterone/etiocholanolone was roughly equal to that of foreign athletes.Conclusion：The developed method of this study is accurate and reliable for the qualitative and quantitative profile analysis by GC-MS.Characteristics of steroid metabolism in urines of Guangdong athletes during the training period are summarized through the comparison of related metabolism indexes of athletes both at home and abroad,providing data support for the establishment of athlete steroidal passport for Guangdong athletes.