体内DHEA及其相关类固醇激素的检测与评价
摘要
脱氢表雄酮(dehydroepiandrosterone,DHEA)是人体分泌的一种重要生理活性物质,属内源性类固醇激素。类固醇激素(Anabolic steroids),又称甾体同化激素,是近年来使用最频繁与广泛的运动兴奋剂,因此,DHEA等类固醇激素的检测一直是运动兴奋剂检测的热点。尿液与血液是当今兴奋剂检测领域最为普遍采用的检材,但这些检材存在检测时限短等难以克服的缺陷,因此探寻新的检材成为兴奋剂检测研究的热点。毛发具有检测时限长、易于保存、能够反映用药历史等优势,已在滥用药物的检测领域得到了较为广泛的应用。因此,进行毛发中类固醇激素检测的研究,为兴奋剂检测提供技术储备,具有重要的意义。
类固醇激素按照来源不同分为两大类:外源性类固醇激素以及内源性类固醇激素。兴奋剂检测在对待这两类激素的策略上有着本质的区别。针对外源性类固醇激素相对比较简单,仅需在检材中检出该类物质即可判为阳性。而内源性激素因人体内存在此类物质,给研究和结果判定带来了复杂性。因此,作为分析方法应用和结果评价的重要环节,必须考察中国人群体内内源性类固醇激素的存在状况,考察中国人群尿液及毛发中内源性类固醇激素的分布情况;探讨通过尿液、毛发分析判断内源性类固醇滥用的可行性,从而为内源性类固醇滥用的分析和评价提供方法和依据。
本文对中国人群体内(尿液、毛发)中DHEA及其相关物质的检测、分布及结果评价进行了系统的研究,建立了较为完整的体内DHEA及其相关物质的分析、确认、评价体系。主要包括以下几个方面:
一、尿液中DHEA及其相关物质的分析与研究
1.建立了液相色谱-串联质谱法(LC-MS/MS)测定尿液中的DEHA及其相关物质的方法。尿样经液-液提取后,采用Cosmosil C18色谱柱,并以甲醇-0.1%甲酸缓冲液(含0.02 mol/L乙酸铵)(体积比为68∶32)为流动相,以三重四极杆串联质谱多反应监测扫描方式对脱氢表雄酮(DHEA)、睾酮、表睾酮、雄酮和苯胆烷醇酮等5种激素成分进行检测。方法的最低检出限为0.01～10ng/mL,平均回收率为96.7%～106.5%,日内和日间相对标准偏差(RSD)分别小于7%和11%。应用所建立的方法测定了健康志愿者口服DHEA后尿液中内源性类固醇激素的变化情况,结果表明该方法样品处理简便,色谱分离完全,结果准确可靠,可应用于体液中类固醇激素的常规分析。
2.考察了中国健康成人尿液中DHEA及其相关内源性类固醇激素的水平,揭示服用DHEA后尿液中DHEA的代谢规律以及对其他相关内源性类固醇激素水平的影响。结果表明志愿者尿液中的睾酮浓度明显低于我国和国外运动员。单剂量服用DHEA后,尿液中DHEA、雄酮和苯胆烷醇酮的浓度有明显的升高,达峰时间分别为:3.86±0.64h、3.43±0.90h和3.57±0.90h。因此可以推断尿液中DHEA等内源性类固醇激素水平因种属、年龄、性别、身体素质等因素存在较大差异。监测滥用外源性的DHEA时,其尿液中的DHEA浓度是最有效的技术指标。
二、毛发中DHEA及其相关物质筛选方法的建立
1.建立了气相色谱-串联质谱法(GC-MS/MS)测定毛发中的DHEA及其相关类固醇激素的筛选方法。头发经碱水解后液-液提取,利用MSTFA衍生化试剂衍生化后以串联四极杆质谱多反应监测扫描方式对脱氢表雄酮、睾酮、表睾酮、雄酮、苯胆烷醇酮、诺龙、甲基异睾酮、甲基睾酮、去氢甲基睾酮、乙诺酮等10种激素成分进行检测。方法的最低检出限为0.2-5pg/mg,平均回收率为71.0%～110.5%,日内和日间相对标准偏差(RSD)分别小于17%和18%。结果表明该方法样品处理简便,色谱分离完全,结果准确可靠,可应用于毛发中类固醇激素的常规分析。
2.应用建立方法测定了健康人头发中DHEA及其相关内源性类固醇激素。此外,按照NIST所列方法自行制备了类固醇激素的阳性毛发,应用所建立的方法对阳性毛发中去氢甲基睾酮与乙诺酮进行了测定。
三、中国健康人头发中DHEA及其相关类固醇激素的检测与评价
1.应用所建立的筛选方法测定了88名不同性别和年龄的中国健康人头发中DHEA及其相关内源性类固醇激素含量。
2.考察了不同性别年龄健康中国人头发中DHEA及其相关内源性类固醇激素分布情况,结果表明头发中雄酮、表睾酮、苯胆烷醇酮的含量明显低于DHEA与睾酮的含量;男性头发中睾酮的含量在20-40年龄段达到峰值随后随着年龄的增长下降;男性女性头发中睾酮含量没有明显区别。
四、DHEA在豚鼠毛发中的分布研究
1.测定了豚鼠毛发中内源性DHEA的含量。取单色、双色或三色毛发豚鼠共22只,剃去背部取样区的毛发,测定空白毛发中DHEA的含量,结果表明豚鼠毛发中内源性DHEA含量与毛发颜色没有明显关系,与人头发中DHEA含量相比较没有明显区别。
2.考察了单次注射DHEA后豚鼠毛发中DHEA含量变化过程。选取背部单色、双色或三色毛发豚鼠各6只,给药前剃光背部取样区毛发,以20mg/kg剂量单次腹腔注射DHEA,隔天剃毛,分别包装保存,连续7次,测定DHEA的含量。结果表明毛发中DHEA的含量高峰出现在给药后的第二天。
3.考察了多次给药后豚鼠毛发中DHEA含量的变化过程。选取背部黑色毛发豚鼠共18只,分成高、中、低3组。给药前剃光背部取样区毛发,以20mg/kg剂量注射DHEA,连续注射3天,第一次给药后第二天剃毛并隔天取毛,分别包装保存,连续7次,测定毛发中DHEA的含量。结果表明三个剂量组中DHEA含量均在末次给药后第二天达到峰值,毛发中DHEA的含量与给药剂量相关,低剂量组毛发中DHEA含量变化不明显。
4.毛发颜色对注射DHEA后豚鼠毛发中DHEA含量的影响,取背部长有不同毛发色块(黑、白、棕)的豚鼠6只,给药前剃光背部取样区毛发,以20mg/kg剂量腹腔注射DHEA,给药后第二天剃毛,测定所得各色毛发中DHEA的含量。结果表明同体豚鼠毛发中DHEA的含量与颜色有关,含量从大到小依次为黑色、棕色、白色。
综上所述,本文的创新点及研究的意义如下:
1.本文所建立的尿液中DHEA及其相关内源性类固醇激素的定性定量分析方法灵敏、专属性强、为国内首创,该方法的检测限与定量限明显低于其他研究报导结果,总体指标达到国际先进水平。
2.首次测定了中国人群毛发中DHEA及其相关内源性类固醇激素的含量,为阳性结果的判定提供了依据。讨论了中国人群不同年龄、性别毛发中内源性类固醇激素含量的分布。为毛发中内源性类固醇激素结果的判定提供了依据。
3.首次对DHEA在豚鼠毛发中的分布及时间过程进行了研究,结果表明给于外源性DHEA后黑色毛发中的DHEA结合率较高。并且中国人黑色头发适合毛发中内源性类固醇激素的检测。
Dehydroepiandrsterone (DHEA), a very important biological active prehorone, is a kind of endogenous steroid. In recent years, DHEA and other related steroids have always been widely and frequently used so that the detection of these steroids has become very hot. In this field, although urine and blood are 2 commonly adopted specimens they have many shortcomings that very difficult to be overcame. Recently, hair, a variety of specimen other than blood and urine, has been purposed to drug exposure because hair has a long detection time limit, can be easily conserved and indicate the history of drug exposre. So the study of the determination of steroids in hair which is to provide evidence to help hair become the specimen of anti-doping is of great importance.
Anabolic steroids can be devided into 2 types: exdogenous steroids and endogenous steroids. The strategies on the detection of these 2 types of steroids are totally different. To determine exogenous steroids, if the steroids can be detected the results is positive. To determine endogenouse steroids, because the steroids exist in the body the judgement of the result is very diffictult. Before the dtermination of the endogenouse steroids, we must study the conditions of endogenous steroid in human body and discuss the feasibility of deciding endogenous steroids abuse and then provide evidence and methods information for the analysis and evaluation of endogenous steroids abuse.
This paper systematically studied the detection, distribution and evaluation of DHEA and related steroids in vivo among Chinese people and established a complete analasys, conformation and eavalation system for DHEA and related steroids. The comments are as below:
1. The analysis and study of DHEA and related steroids in urine
(1) A method was developed for the determination of endogenous steroids in urine with methyltestosterone as internal standard. After liquid-liquid extraction, the urine sample was chromatographed on a Cosmosil C_(18) column with a mixture of methanol and ammonium acetate-formic acid (68:32, v/v) as mobile phase, then detected with liquid chromatography-tandem mass Spectrometry (LC-MS/MS) system with electrospray ionization (ESI) in multi-reaction monitoring (MRM) mode. The detection limits ranged from 0.01 ng/mL (for testosterone and epitestosterone) to 10 ng/mL (for androsterone and etiocholanolone). The recoveries ranged from 96.7% to 106.5% and intra- and inter-day precisions (measured as relative standard deviations) were less than 7% and 11%, respectively. With simple and fast sample preparation, the method was sensitive and specific for simultaneous determination of these 5 kinds of endogenous steroids in urine. The method has been successfully applied in pharmacokinetic study and was thus a potential alternative for gas chromatography-mass Spectrometry (GC-MS) based procedures in routine analysis of endogenous steroids such as DHEA in human urine.
(2) Studied the level of DHEA in Chinese humans'urine and revealed the influence of oral DHEA to the metabolism of DHEA in urine and the level of other endogenous steroids in rine. The results showed that the level of testosterone in Chinese humans'urine is lower than Chinese and foreign athletes. After single oral administration of DHEA, the levels of DHEA, androsterone, etiocholanolone raised and the peak of the level were 3.86±0.64h、3.43±0.90h and 3.57±0.90h separately. Through these results we concluded that the levels of DHEA and other related endogenous steroids varied because of ethnic group, age, gender and physical quality. The concentration of DHEA in urine could be the most effective criteria for the determination of DHEA administration.
2. The establish of the screening method of DHEA and related steroids in hair
(1) A method was developed for the screening of DHEA and related steroids in hair with d3-testosterone as internal standard. After liquid-liquid extraction, the hair sample was derivatived by MSTFA, then detected with gas chromatography-tandem mass Spectrometry (GC-MS/MS) system in MRM mode. The detection limits ranged from 0.2 pg/mg to 5 pg/mg . The recoveries ranged from 71.0% to 110.5% and intra- and inter-day precisions (measured as relative standard deviations) were less than 17% and 18%, respectively. With simple and fast sample preparation, the method was sensitive and specific for simultaneous determination of these 10 steroids in hair. The method could be a potential method in routine analysis of steroids such as DHEA in human hair.
(2) Determined DHEA and other related endogenous steroids in healthy Chinese people's hair. After making positive hair following the instruction published by NIST, we determined methandienone and norethandrolone in the positive hair.
3. The determination and evaluation of DHEA and related steroids in Chinese healthy people's hair.
(1) Determined the concentration of DHEA and other related steroids in 88 Chinese healthy people's hair divided by different age group and gender.
(2) Studied distribution condition of DHEA and related endogenous steroids in 88 healthy Chinese people'hair divided by different age group and gender. The results showed that the concentration of androsterone, epitestosterone, etiocholanolone was lower than that of DHEA and testosterone, the concentration of testosterone in men's hair reached peak in the age of 20 to 40 and there was no obvious difference between men and women's hair.
4. The study of distribution of DHEA in ginea pig's hair
(1) Determined the concentration of endogenous DHEA in ginea pig's hair. Divided 22 ginea pigs into 3 groups: single color, double colors and triple colors. Determined the concentration of endogenous DHEA after taking the hair on genia pigs' back. The result showed that there was no significant difference between different color and the concentration of DHEA was as high as that of humans' hair.
(2) Studied the changing process of DHEA concentration in genia pigs' hair after single administration. Divided 6 ginea pigs into 3 groups: single color, double colors and triple colors. After shaving out the hair on genia pigs' back give a dose of 20mg/kg DHEA through intraperitoneal injection and then collected the hair every 2 days which lasted 7 times. Determined the concentration of DHEA after taking the hair on genia pigs' back. The result showed that the concentration reached the peak on the second day after the administration.
(3) Determined the changing process of DHEA concentration in genia pigs'hair after multiple administration. Divided 18 ginea pigs into 3 groups through the dose. After shaving out the hair on genia pigs' back give doses of 20mg/kg, 4mg/kg and 0.8 mg/kg DHEA through intraperitoneal injection 1 time/day(3 days). Took the hair on the second day after the administration and then took every 2 days which lasted 7 times. Determined the concentration of DHEA after taking the hair on genia pigs' back. The result showed that the changing process of the 3 groups were nearly the same and the changing of DHEA was not significant in the low dose group.
(4) Sudied the influce of hair color to the DHEA concentration after DHEA administration. Chose 6 genia pigs with 3 different hair color on the back. After shaving out the hair on the back gave a dose of 20mg/kg through intraperitoneal injection. Took the hair every 2 days which lasted 7 times and determined the DHEA concention in the hair. The result showed that the DHEA concentration in the hair changed because of the hair color and the concentrations were black, brown and white from high to low.
Innovation points and significance are as below:
1. The quatitation and determination method for DHEA and related steroids in urine were sensitive and specific. The LOD and LOQ were significantly low than the other study and overall indicator reached advanced international level.
2. First determined DHEA and related steroids in Chinese people's hair and provided evidence for the judgement of positive case. Discussed the distribution of endogenous steroids in Chinses people's hair among different age and gendor. Provided evidence for the detection of endogenous steroids in the field of anti-doping.
3. First studied the distribution and changing process of DHEA in genia pigs's hair. The results showed that the exogenous DHEA binded with black hair more easily than the other hair color and Chines people's hair were suitable for the detection of endogenous steroids.
类固醇激素按照来源不同分为两大类:外源性类固醇激素以及内源性类固醇激素。兴奋剂检测在对待这两类激素的策略上有着本质的区别。针对外源性类固醇激素相对比较简单,仅需在检材中检出该类物质即可判为阳性。而内源性激素因人体内存在此类物质,给研究和结果判定带来了复杂性。因此,作为分析方法应用和结果评价的重要环节,必须考察中国人群体内内源性类固醇激素的存在状况,考察中国人群尿液及毛发中内源性类固醇激素的分布情况;探讨通过尿液、毛发分析判断内源性类固醇滥用的可行性,从而为内源性类固醇滥用的分析和评价提供方法和依据。
本文对中国人群体内(尿液、毛发)中DHEA及其相关物质的检测、分布及结果评价进行了系统的研究,建立了较为完整的体内DHEA及其相关物质的分析、确认、评价体系。主要包括以下几个方面:
一、尿液中DHEA及其相关物质的分析与研究
1.建立了液相色谱-串联质谱法(LC-MS/MS)测定尿液中的DEHA及其相关物质的方法。尿样经液-液提取后,采用Cosmosil C18色谱柱,并以甲醇-0.1%甲酸缓冲液(含0.02 mol/L乙酸铵)(体积比为68∶32)为流动相,以三重四极杆串联质谱多反应监测扫描方式对脱氢表雄酮(DHEA)、睾酮、表睾酮、雄酮和苯胆烷醇酮等5种激素成分进行检测。方法的最低检出限为0.01～10ng/mL,平均回收率为96.7%～106.5%,日内和日间相对标准偏差(RSD)分别小于7%和11%。应用所建立的方法测定了健康志愿者口服DHEA后尿液中内源性类固醇激素的变化情况,结果表明该方法样品处理简便,色谱分离完全,结果准确可靠,可应用于体液中类固醇激素的常规分析。
2.考察了中国健康成人尿液中DHEA及其相关内源性类固醇激素的水平,揭示服用DHEA后尿液中DHEA的代谢规律以及对其他相关内源性类固醇激素水平的影响。结果表明志愿者尿液中的睾酮浓度明显低于我国和国外运动员。单剂量服用DHEA后,尿液中DHEA、雄酮和苯胆烷醇酮的浓度有明显的升高,达峰时间分别为:3.86±0.64h、3.43±0.90h和3.57±0.90h。因此可以推断尿液中DHEA等内源性类固醇激素水平因种属、年龄、性别、身体素质等因素存在较大差异。监测滥用外源性的DHEA时,其尿液中的DHEA浓度是最有效的技术指标。
二、毛发中DHEA及其相关物质筛选方法的建立
1.建立了气相色谱-串联质谱法(GC-MS/MS)测定毛发中的DHEA及其相关类固醇激素的筛选方法。头发经碱水解后液-液提取,利用MSTFA衍生化试剂衍生化后以串联四极杆质谱多反应监测扫描方式对脱氢表雄酮、睾酮、表睾酮、雄酮、苯胆烷醇酮、诺龙、甲基异睾酮、甲基睾酮、去氢甲基睾酮、乙诺酮等10种激素成分进行检测。方法的最低检出限为0.2-5pg/mg,平均回收率为71.0%～110.5%,日内和日间相对标准偏差(RSD)分别小于17%和18%。结果表明该方法样品处理简便,色谱分离完全,结果准确可靠,可应用于毛发中类固醇激素的常规分析。
2.应用建立方法测定了健康人头发中DHEA及其相关内源性类固醇激素。此外,按照NIST所列方法自行制备了类固醇激素的阳性毛发,应用所建立的方法对阳性毛发中去氢甲基睾酮与乙诺酮进行了测定。
三、中国健康人头发中DHEA及其相关类固醇激素的检测与评价
1.应用所建立的筛选方法测定了88名不同性别和年龄的中国健康人头发中DHEA及其相关内源性类固醇激素含量。
2.考察了不同性别年龄健康中国人头发中DHEA及其相关内源性类固醇激素分布情况,结果表明头发中雄酮、表睾酮、苯胆烷醇酮的含量明显低于DHEA与睾酮的含量;男性头发中睾酮的含量在20-40年龄段达到峰值随后随着年龄的增长下降;男性女性头发中睾酮含量没有明显区别。
四、DHEA在豚鼠毛发中的分布研究
1.测定了豚鼠毛发中内源性DHEA的含量。取单色、双色或三色毛发豚鼠共22只,剃去背部取样区的毛发,测定空白毛发中DHEA的含量,结果表明豚鼠毛发中内源性DHEA含量与毛发颜色没有明显关系,与人头发中DHEA含量相比较没有明显区别。
2.考察了单次注射DHEA后豚鼠毛发中DHEA含量变化过程。选取背部单色、双色或三色毛发豚鼠各6只,给药前剃光背部取样区毛发,以20mg/kg剂量单次腹腔注射DHEA,隔天剃毛,分别包装保存,连续7次,测定DHEA的含量。结果表明毛发中DHEA的含量高峰出现在给药后的第二天。
3.考察了多次给药后豚鼠毛发中DHEA含量的变化过程。选取背部黑色毛发豚鼠共18只,分成高、中、低3组。给药前剃光背部取样区毛发,以20mg/kg剂量注射DHEA,连续注射3天,第一次给药后第二天剃毛并隔天取毛,分别包装保存,连续7次,测定毛发中DHEA的含量。结果表明三个剂量组中DHEA含量均在末次给药后第二天达到峰值,毛发中DHEA的含量与给药剂量相关,低剂量组毛发中DHEA含量变化不明显。
4.毛发颜色对注射DHEA后豚鼠毛发中DHEA含量的影响,取背部长有不同毛发色块(黑、白、棕)的豚鼠6只,给药前剃光背部取样区毛发,以20mg/kg剂量腹腔注射DHEA,给药后第二天剃毛,测定所得各色毛发中DHEA的含量。结果表明同体豚鼠毛发中DHEA的含量与颜色有关,含量从大到小依次为黑色、棕色、白色。
综上所述,本文的创新点及研究的意义如下:
1.本文所建立的尿液中DHEA及其相关内源性类固醇激素的定性定量分析方法灵敏、专属性强、为国内首创,该方法的检测限与定量限明显低于其他研究报导结果,总体指标达到国际先进水平。
2.首次测定了中国人群毛发中DHEA及其相关内源性类固醇激素的含量,为阳性结果的判定提供了依据。讨论了中国人群不同年龄、性别毛发中内源性类固醇激素含量的分布。为毛发中内源性类固醇激素结果的判定提供了依据。
3.首次对DHEA在豚鼠毛发中的分布及时间过程进行了研究,结果表明给于外源性DHEA后黑色毛发中的DHEA结合率较高。并且中国人黑色头发适合毛发中内源性类固醇激素的检测。
Dehydroepiandrsterone (DHEA), a very important biological active prehorone, is a kind of endogenous steroid. In recent years, DHEA and other related steroids have always been widely and frequently used so that the detection of these steroids has become very hot. In this field, although urine and blood are 2 commonly adopted specimens they have many shortcomings that very difficult to be overcame. Recently, hair, a variety of specimen other than blood and urine, has been purposed to drug exposure because hair has a long detection time limit, can be easily conserved and indicate the history of drug exposre. So the study of the determination of steroids in hair which is to provide evidence to help hair become the specimen of anti-doping is of great importance.
Anabolic steroids can be devided into 2 types: exdogenous steroids and endogenous steroids. The strategies on the detection of these 2 types of steroids are totally different. To determine exogenous steroids, if the steroids can be detected the results is positive. To determine endogenouse steroids, because the steroids exist in the body the judgement of the result is very diffictult. Before the dtermination of the endogenouse steroids, we must study the conditions of endogenous steroid in human body and discuss the feasibility of deciding endogenous steroids abuse and then provide evidence and methods information for the analysis and evaluation of endogenous steroids abuse.
This paper systematically studied the detection, distribution and evaluation of DHEA and related steroids in vivo among Chinese people and established a complete analasys, conformation and eavalation system for DHEA and related steroids. The comments are as below:
1. The analysis and study of DHEA and related steroids in urine
(1) A method was developed for the determination of endogenous steroids in urine with methyltestosterone as internal standard. After liquid-liquid extraction, the urine sample was chromatographed on a Cosmosil C_(18) column with a mixture of methanol and ammonium acetate-formic acid (68:32, v/v) as mobile phase, then detected with liquid chromatography-tandem mass Spectrometry (LC-MS/MS) system with electrospray ionization (ESI) in multi-reaction monitoring (MRM) mode. The detection limits ranged from 0.01 ng/mL (for testosterone and epitestosterone) to 10 ng/mL (for androsterone and etiocholanolone). The recoveries ranged from 96.7% to 106.5% and intra- and inter-day precisions (measured as relative standard deviations) were less than 7% and 11%, respectively. With simple and fast sample preparation, the method was sensitive and specific for simultaneous determination of these 5 kinds of endogenous steroids in urine. The method has been successfully applied in pharmacokinetic study and was thus a potential alternative for gas chromatography-mass Spectrometry (GC-MS) based procedures in routine analysis of endogenous steroids such as DHEA in human urine.
(2) Studied the level of DHEA in Chinese humans'urine and revealed the influence of oral DHEA to the metabolism of DHEA in urine and the level of other endogenous steroids in rine. The results showed that the level of testosterone in Chinese humans'urine is lower than Chinese and foreign athletes. After single oral administration of DHEA, the levels of DHEA, androsterone, etiocholanolone raised and the peak of the level were 3.86±0.64h、3.43±0.90h and 3.57±0.90h separately. Through these results we concluded that the levels of DHEA and other related endogenous steroids varied because of ethnic group, age, gender and physical quality. The concentration of DHEA in urine could be the most effective criteria for the determination of DHEA administration.
2. The establish of the screening method of DHEA and related steroids in hair
(1) A method was developed for the screening of DHEA and related steroids in hair with d3-testosterone as internal standard. After liquid-liquid extraction, the hair sample was derivatived by MSTFA, then detected with gas chromatography-tandem mass Spectrometry (GC-MS/MS) system in MRM mode. The detection limits ranged from 0.2 pg/mg to 5 pg/mg . The recoveries ranged from 71.0% to 110.5% and intra- and inter-day precisions (measured as relative standard deviations) were less than 17% and 18%, respectively. With simple and fast sample preparation, the method was sensitive and specific for simultaneous determination of these 10 steroids in hair. The method could be a potential method in routine analysis of steroids such as DHEA in human hair.
(2) Determined DHEA and other related endogenous steroids in healthy Chinese people's hair. After making positive hair following the instruction published by NIST, we determined methandienone and norethandrolone in the positive hair.
3. The determination and evaluation of DHEA and related steroids in Chinese healthy people's hair.
(1) Determined the concentration of DHEA and other related steroids in 88 Chinese healthy people's hair divided by different age group and gender.
(2) Studied distribution condition of DHEA and related endogenous steroids in 88 healthy Chinese people'hair divided by different age group and gender. The results showed that the concentration of androsterone, epitestosterone, etiocholanolone was lower than that of DHEA and testosterone, the concentration of testosterone in men's hair reached peak in the age of 20 to 40 and there was no obvious difference between men and women's hair.
4. The study of distribution of DHEA in ginea pig's hair
(1) Determined the concentration of endogenous DHEA in ginea pig's hair. Divided 22 ginea pigs into 3 groups: single color, double colors and triple colors. Determined the concentration of endogenous DHEA after taking the hair on genia pigs' back. The result showed that there was no significant difference between different color and the concentration of DHEA was as high as that of humans' hair.
(2) Studied the changing process of DHEA concentration in genia pigs' hair after single administration. Divided 6 ginea pigs into 3 groups: single color, double colors and triple colors. After shaving out the hair on genia pigs' back give a dose of 20mg/kg DHEA through intraperitoneal injection and then collected the hair every 2 days which lasted 7 times. Determined the concentration of DHEA after taking the hair on genia pigs' back. The result showed that the concentration reached the peak on the second day after the administration.
(3) Determined the changing process of DHEA concentration in genia pigs'hair after multiple administration. Divided 18 ginea pigs into 3 groups through the dose. After shaving out the hair on genia pigs' back give doses of 20mg/kg, 4mg/kg and 0.8 mg/kg DHEA through intraperitoneal injection 1 time/day(3 days). Took the hair on the second day after the administration and then took every 2 days which lasted 7 times. Determined the concentration of DHEA after taking the hair on genia pigs' back. The result showed that the changing process of the 3 groups were nearly the same and the changing of DHEA was not significant in the low dose group.
(4) Sudied the influce of hair color to the DHEA concentration after DHEA administration. Chose 6 genia pigs with 3 different hair color on the back. After shaving out the hair on the back gave a dose of 20mg/kg through intraperitoneal injection. Took the hair every 2 days which lasted 7 times and determined the DHEA concention in the hair. The result showed that the DHEA concentration in the hair changed because of the hair color and the concentrations were black, brown and white from high to low.
Innovation points and significance are as below:
1. The quatitation and determination method for DHEA and related steroids in urine were sensitive and specific. The LOD and LOQ were significantly low than the other study and overall indicator reached advanced international level.
2. First determined DHEA and related steroids in Chinese people's hair and provided evidence for the judgement of positive case. Discussed the distribution of endogenous steroids in Chinses people's hair among different age and gendor. Provided evidence for the detection of endogenous steroids in the field of anti-doping.
3. First studied the distribution and changing process of DHEA in genia pigs's hair. The results showed that the exogenous DHEA binded with black hair more easily than the other hair color and Chines people's hair were suitable for the detection of endogenous steroids.
引文
[1]卢昌亚.运动兴奋剂概论[M].上海:上海科学技术出版社,1999:101
[2]Corrigan B.DHEA and sports[J]Clin J Sport Med,2002,12(4):236-241
[3]Labrie F,Luu-The V,Belange Ar.Is dehydroepiandrosterone a hormone?[J].J Endocrino,2005,187(2):169-196
[4]Dehennin L,Ferry M,Lafarge P.Oral administration of dehydroepian drosterone to healthy men:alteration of the urinary androgen profile and conseq uences for the detection of abuse in sport by gas chromatography-mass spectrum etry[J].Steroids 1998,63(5):80-87
[5]WADA Project Team.Reporting and evaluation guidance for testosterone,epitest osterone,T/E ratio and other endogenous steroids[EB/OL],http:// www.wada-ama.org/rtecontent/document/end_s teroids_aug _ 04.pdf
[6]Choi M H,Kim K R,Chung B C.Simultaneous determination of urinary androgen glucuronides by high temperature gas chromatography-mass spectrometry with selected ion monitoring[J].Steroids,2000,65(1):54-59
[7]Guan F,Uboh C E,Soma L R.Detection,quantification and confirmation of anabolic steroids in equine plasma by liquid chromatography and tandem mass spectrometry[J].J Chromatogr B,2005,829(1):56-68
[8]WADA Project Team.Minimum required performence limits for detection of prohibited substance.[EB/OL].http://www.wada-ama.org/rtecontent/document/perflimits2.pdf.2004-01-15
[9]王静竹 吴侔天 张亦农.运动员尿中类固醇激素水平的研究[J].中国运动医学杂志.2004,23:200-203
[10]L.Starka.Epitestosterone[J].J Ster Bioch Mol Bio 2003,87:27-34
[11]Jenny Jakobsson..Large Differences in Testosterone Excretion in Korean and Swedish Men Are Strongly Associated with a UDP-Glucuronosyl Transfe rase 2B17Polymorphism J ClinEndocrin & Metab 2005.91(2):687-693
[12]Paul J.Perryl,John H.Maclndoe,._Detection of anabolic steroid administration:ratio of urinary testosterone to epitestosterone vs the ratio of urinary testosterone to luteinizing hormone Clin Chem.1997,43:731-735
[13]Donike M,Ueki M,Kuroda Y Detection of dihydrotestosterone(DHT) doping:alterations in the steroid profile and reference ranges for DHT and its 5α-metabolites[J].J Sports Med Phys Fitness 1995,35:235-250
[14]Geyer H.,U.Mareck-Engelke,W.Schanzer,M.Donike Proceedings of the 14th Cologne Workshop on dope analysis,12th to 17th March 1996,M.Donike, H. Geyer, A.Gotzmann, U. Mareck-Engelke[R], Sport und Buch Strausse Edition Sport, Koln 1997: p. 139
[15]Bosy TZ, Moore KA, Poklis A. The effect of oral dehydroepiandroster one on the urine testosterone/epitestosterone ratio (T/E) in human male volunteers[J]. J Anal Toxicol 1998, 22: 455-459
[16] R. Kronstrand, S. Fo¨rsberg, B. Kagedal, J. Ahlner, G. Larson, Relationship between melanin and codeine concentrations in hair after oral administration[R] Proceedings of the annual meeting of the American Academy of Forensic Scien es, 1999
[17] Lauriane R, Fabrice M, Yoann D. Development and validation of a multi-residue method for the detection of a wide range of hormonal anabolic compounds in hair using gas chromatography-tandem mass Spectrometry [J].Anal Chim Acta. 2007,586 93-104
[18] Kintz P, Cirimele V, Jeanneau T, Ludes B. Identification of testosterone and testosterone esters in human hair[J]. J Anal Toxicol 1999,23:352-356
[19] Scherer CR, Reinhardt G Nachweis sechs endogener Steroide in menschli chen Haaren mit GC/MS und Isotopenverdu nnungsanalyse Abstract[R]. Althoff H, editor. 74. Jahrestagung der Deutschen Gesellschaft fu¨r Rechtsmediz in, Aachen, 1995
[20] Jean G, Marjorie C, Gilbert P. Determination of Endo genous Levels of GHB in Human Hair. Are there Possibilities for the Identificat ion of GHB Administration through Hair Analysis in Cases of Drug-Facilitated Sexual Assault[J]? J Anal Toxicol, 2003[8], 27:574-590
[21] Henderson G, Harkey M, Zhou C, III, Incorporat ion of isotopically labeled cocaine and metabolites into human hair: 1. Dose-re sponse relationships[J]. J. Anal. Toxicol. 1996,20: 1-12.
[22] Rollins D, Wilkins D, Krueger G, Codeine disposition in human hair after single and multiple doses.[J] Eur. J. Clin. Pharmacol. 1996,50(1): 391-397
[23] Nakahara Y, Takahashi K, Kikura R, Hair analysis for drugs of abuse. X. Effects of physiochemical properties of drugs on the incorporation rates into hair, Biol. Pharm. Bull[J]. 1995,18(9): 1223-1227
[24] Ho¨ld K, Wilkins D, Crouch D Detection of stanozolol in hair by negative ion chemical ionization mass spectrometry[J] J. Anal. Toxicol. 20 (1996) 345-349.
[25] M.J. Wheeler, Y.-B. Zhong, A.T. Kicman, S.B. Courts, The measurement of testosterone in hair, J.Endocrinol. 1998,159(1) 5-8
[26]Labrie F,Belanger A,Cusan L,Physiological changes in dehydroepiandroste rone are not reflected by serum levels of active androgens and estrogens but of their metabolites:Intracrinology[J].J Clin Endo Metab.1997,82(2):2403-2409
[27]M.P.Manns Age-related changes in serum concentrations of SHBG,testost erone,estrogens and IGF-I in men:results from cross-sectional investigation on healthy subjects and calculations from previously reported studies[R],vorgelegt von Vitali Gorenoi aus St.Petersburg chem.Leningrad,2002.
[28]Andre,Tchernof,Fernand L Dehydroepiandrosterone,obesity and cardio vascular disease risk:a review of human studies.Euro J Endocrin 2004,151(1):1-14.
[29]C.Jurado,P.Kintz,M.Menendez,M.Repetto,Influence of cosmetic treatment of hair on drug testing[J].Int.J.Leg.Med.1997(2):159-163.
[30]L.Po "tsch,G.Skopp,Stability of opiates in hair fibers after exposure to cosmetic treatment[J]Forensic Sci.Int.1996,81(2) 95-102.
[31]J.Seguraa,S.Pichinib,S.H.Penga,X.Hair analysis and detectab ility of single dose administration of androgenic steroid esters[J].Foren Sci Int 2000,1070:347-359
[32]Tatsuya H,Yujin S,Kazutake S.Determination of salivary dehydroepiand rosterone using liquid chromatography-tandem mass spectrometry combined with charged derivatization[J].J Chrom B,2007,846(1):195-201
[33]Jean Pierre G,Marjorie C,Gilbert P.Determination of Endogenous Levels of GHB in Human Hair.Are there Possibilities for the Identification of GHB Administration through Hair Analysis in Cases of Drug- Facilitated Sexual Assau It[J]? Journal of Analytical Toxico 2003,27(8):574-580
[34]Shen Min,Xiang Ping,Shen baohua.Detection ofmeperidine and its metab Olites in the hair of meperidine addits[J].J Foren Sci Int.1999,1030:159-171
[35]沈敏,向平等.头发中抗抑郁药以及抗精神病药的检测及评价.法医学杂质[J].2000,16(3):214-217
[36]姜宴,沈敏,赵子琴等 甲基苯丙胺在豚鼠毛发中分布及转化的初步研究[J].法医学杂志2001,4(4):214-217
[37]Segura J,Possibilities of ELISA methodologies for hair analysis[EB/OL],R.A.de Zeeuw,I.Al Hosani,S.Al Munthiri,A.Maqbool(Eds.),Proceedings of the 1995International Conference and Workshop.Abu Dhabi,Hair Analysis in Forensic Toxicology,1995,:351
[38]Gaillard Y,Balland A,Doucet F,Pe'pin G.Detection of illegal clenbuterol use in calves using hair analysis.Application in meat quality control[J].J.Chr omatogr.B 1997,703():85-95.
[39]Gleixner A,Sauerwein H,Meyer H,Detection of the anabolic b adr enoceptor agonist clenbuterol 2in human scalp hair by HPLC/EIA[J].Clin.Chem.1996,42(11)1869-1871.
[40]Kelly RC,Mieczkowski T,Sweeney SA,Bourland JA.Hair analysis for drugs of abuse.Hair Color and race differentials or systematic differences in drug preferences[J]? Foren Sci Int.2000,107(1):63-86
[41]Mieczkowski T,Newel R.Statistical examination of hair color as a poten tial biasing factor in hair analysis[J].Foren Sci Int.2000,107(1):13-38
[42]闫谨,王杉,密捷波 兴奋剂与兴奋剂检测研究进展[J]。大学化学2003,18(2):10-20
[43]Kintz P,Cirimele V,Sachs H.Testing for anabolic steroids in hair from two bodybuilders[J].Foren Sci Intl,1999,101(3):209-213
[1] Labrie F, Luu-The V, Belange Ar. Is dehydroepiandrosterone a hormone[J]? J Endocrin. 2005,87(2):169-196
[2] Dehennin L, Ferry M, Lafarge P, Peres G, Lafarge JP. Oral administration of dehydroepiandrosterone to healthy men: alteration of the urinary androgen profile and consequences for the detection of abuse in sport by gas chromatography-mass spectrometry[J]. Steroids 1998,63(5): 80-87.
[3] Belange A,Candas B, Dupont A. Changes in serum concentrations of conjugated and unconjugated steroids in 40-to80-year-old men[J]. J Clin Cndocrinol Metab 1994,79: 1086-1090
[4] Nestler JE, Barlascini CO, Clore JN et al. Dehydroepiandrosterone reduces serum low density lipoprotein levels and body fat but does not alter insulin sensitivity in normal men[J]. J Clin Endocrin Metab 1988,66: 57-61.
[5] Welle S, Jozefowicz R, Statt M. Failure of dehydroepiandrosterone to influence energy and protein metabolism in humans[J]. J Clin Endocrin Met. 1990, 71(5): 1259-1264
[6) Corrigan Brian, BM, SB. DHEA and Sport[J]. Clin J Sport Med. 2002,12(4): 236-241
[7] Etienne-Emile B, Paul R. Dehydroepiandrosterone (DHEA) and dehydroepi androsterone sulfate (DHEAS) as neuroactive neurosteroids[J] Proc. Natl. Acad. Sci. USA. 1998, 95(8): 4089-4091
[8] Peter H. Jellinck. Dehydroepiandrosterone (DHEA) metabolism in the brain: Identification by liquid chromatography/mass Spectrometry of the delta-4-isomerof DHEA and related steroids formed from androstenedione by mouse BV2 microglia[J]. J Ster Biochem& Molecu Bio 2006, 98(1): 41-47
[9] Yorio M, Ryo K, Shushi H. A simple, accurate, and sensitive assay method of dehydroepiandrosterone sulfate: application for quantitative determination in human breast cyst and duct fluids[J]. Steroids. 2002, 67(5): 333-338
[10] Le Bail, Lotfi H, Charles L. Conversion of dehydroepiandrosterone sulfate at physiological plasma concentration into estrogens in MCF-7 cells[J]. Steroids 2002, 67(13): 1057-1064
[11] Yasushi M, Shigemasa Y, Akira H. Simultaneous determination of dehydr oepiandrosterone and its 7-oxygenated metabolites in human serum by high-resolution gas chromatography-mass spectrometry[J]. Steroids 2004,69(13): 817-824
[12] Brian D, Frank Z,. Stanczyk P. Pharmacokinetics of dehydroepiandroste rone and its metabolites after long-term daily oral administration to healthy young men[J]. Ferti Steri 2004,81(3):595-604
[13] WADA Project Team. Reporting and evaluation guidance for testosterone, epitest osterone, T/E ratio and other endogenous steroids[EB/OL]. http:// www.wada-ama.org/ rtecontent/document/end_s teroids_aug _ 04.pdf
[14] Rodrigo A, Thomas E. Chapman. A rapid screening assay for measuring urinary androsterone and etiocholanolone 5 13C (%) values by gas chromatogr aphy/combustion/isotope ratio mass spectrometry[J]. Rapid Commun Mass Spectrom. 2000(23): 2294-2299
[15] Kintz P, Nele S. Use of Alternative Specimens: Drugs of Abuse in Saliva and Doping Agents in Hair Therapeutic Drug Monitoring[J]. 2002,24(2): 239-246
[16] Hill M, Lapc'ik O, Havl'ikov'a H. 7-Hydroxydehydroepiandrosterone epimers in human serum and saliva. Comparison of gas chromatography-mass Spectrometry and radioimmunoassay[J]. J Chromatogr 2001,935(): 297-307.
[17] Laurent R. Is there a place for hair analysis in doping controls[J]? Foren Science Inter. 2000,107(1): 309-323
[18] Kintz P, Cirimele V, Ludes B. Physiological concentrations of DHEA in human hair[J]. J Anal Toxicol 1999, 23(1): 424-8.
[19] Kintz P, Vincent C, Marc D. Dehydroepiandrosterone (DHEA) and Testosterone Concentrations in Human Hair after Chronic DHEA Supplementation Clinical Chemistry[J]. 2000,46(3): 414-415
[20] Shimada K, Tanaka M, Nambara T. Determination of 17-ketosteroid sulphates in serum by high-performance liquid chromatography with electrochemical detection using pre-column derivatization. J. Chromatogr[J]. 1984, 307(1): 23-28
[21] Wu M, Takagi K, Okuyama S. Determination of 17-ketosteroid sulphates in serum by high-performance liquid chromatography with electrochemical detection using pre-column derivatization[J]. J. Chromatogr. 1986, 377: 121.
[22] Tagawa N, Tamanaka J, Fujinami A. Serum dehydroepiandrosterone, dehydroepiandrosterone sulfate, and pregnenolone sulfate concentrations in patients with hyperthyroidism and hypothyroidism[J] Clin.Chem. 2000, 46: 523-528
[23] Asaba H, Hosoya K, Takanaga H. Blood-brain barrier is involved in the efflux transport of a neuroactive steroid, dehydroepiandrosterone sulfate, via organic anion transporting polypeptide[J]. J. Neurochem. 2000, 75(5): 1907-1916
[24] Lavallee B, Provost P, Belanger A. Formation of pregnenolone and dehydroepiandrosterone-fatty acid esters by lecithin-cholesterol acyltransferase in human plasma high density lipoproteins[J]. Biophys Acta 996, 1299(3): 306-312
[25] Kelly C , Thomas H, Lee H. Nanoelectrospray Mass Spectrometry and Precursor Ion Monitoring for Quantitative Steroid Analysis and Attomole Sensitivity[J]. Anal Chem. 1999, 71(13): 2358-2363
[26] Payne DW, Holtzclaw WD, Adashi EY. A convenient, unified scheme for the differential extraction of conjugated and unconjugated serum C19 steroids on Sep-Pak C18-cartridges[J]. J. Steroid Biochem. 1989, 33(2): 289
[27] Boschi S, Iasio R, Mesini P. Measurement of steroid hormones in plasma by isocratic high performance liquid chromatography coupled to radioimmunoassay[J]. Clin Chim Acta 1994, 231(1): 107-113
[28] Raeside J, Renaud R, Christie H. Postnatal decline in gonadal secretion of dehydroepiandrosterone and 3β-hydroxyandrosta-5,7-dien-17-one in the newborn foal[J]. Endocrinol. 1997, 155: 277-282
[29] Causon R, Collins S, Fry D. Determination of urinary dehydroepian drosterone sulphate by combined high-performance liquid chromatography and radioimmunoassay[J]. J. Chromatogr. 1982, 227: 485-492
[30] Liere P, Akwa Y, Weil-Engerer S. Validation of an analytical procedure to measure trace amounts of neurosteroids in brain tissue by gas chromatography-mass spectrometry[J]. J. Chromatogr. B 2000.739(2): 301-312
[31] Qi Jia, Mei-Feng H, Zhao-Xing P, Quantification of urine 17-ketosteroid sulfates and glucuronides by high-performance liquid chromatography-ion trap mass spectroscopy[J]. J Chromatogr B, 2001, 750(): 81-91
[32] Nahoul K, Kottler M. Relationships between androgen and estrogen sulfate in breast cyst fluid[J]. Clin Chem Acta. 1992, 209:179-187
[33] Chasalow FI, Blethen SL, Bradlow L. Dehydroepiandrosterone sulfate (DHEA-S) and DHEA-S-like compound in fibrocystic disease of the breast[J]. Steroids. 1988, 52(3): 205-215
[34] Oldrl ich, LapcI oA K, Richard H. Immunoassay of 7-hydroxysteroids: 2. Radioimmunoassay of 7a-hydroxy-dehydroepiandrost erone[J]. J Ster Biochem & Mol Bio. 1999,71:231-237
[35] Richard H, Martin H, Ivan S. Immunomodulatory 7-hydroxylated metabolites of dehydroepiandrosterone are present in human semen[J].J Ster Biochem & Mol Bio.2000,75(4):273-276
[36]Wolthersa B,Kraan G.Clinical applications of gas chromatography and gas chromatography-mass spectrometry of steroids[J].J Chromatog.A.1999,843():247-274
[37]Man C.Simultaneous determination of urinary androgen glucuronides by high temperature gas chromatography-mass spectrometry with selected ion monitoring[J].Steroids.2000,65(1):54-59
[38]Dehennin L,Lafarge P,Dailly D.Combined profile of androgen glucuro- and sulfoconjugates in postcompetition urine of sportsmen:a simple screening procedure using gas chromatography-mass spectrometry[J].J Chromatog B.1996,687(1):85-91
[39]Pujos R,Flament-Waton M,Goetinck P.Optimizing the extraction and analysis of DHEA sulfate,corticosteroidsand androgens in urine:application to a study of the influence of corticosteroid intake on urinary steroid profiles[J].Anal Bioanal Chem.2004,3800:524-536
[40]Baulieu E,Neurosteroids:a novel function of the brain[J].Psychoneuroen docrin[J].1998,23(4):963-987
[41]Valle'e M,Rivera J,Koob G.Quantification of neurosteroids in rat plasma and brain following swim stress and allopregnanolone administration using negative chemical ionization gas chromatography/mass spectrometry[J].Anal.Biochem.2000,287(3):153-166
[42]王静竹,吴侔天,张亦农.气相色谱/燃烧炉/同位素比值质谱方法对尿中去氢表雄酮及其代谢物的检测.药学学报.2005,40(2):159-163
[43]David J,Bortsy Larry D,Bowers.Direct measurement of urinary testos terone and epitestosterone conjugates using high-performance liquidchroma tography/tandem mass spectrometry[J].J.Mass Spectrom.2000,35(1):50-61
[44]Katayama M,Nakane R,Matsuda Y.Determination of progesterone and 17-hydroxyprogesterone by high performance liquid chromatography after pre -column derivatization with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionohydra zide[J].Analyst.1998,123:2339-2342
[45]Ueno K,Umeda T.Electrochemical and chromatographic properties of selected hydrazine and hydrazide derivatives of carbonyl compounds[J].J.Chromatogr.1991,585(2):225-231
[46] Kuniko M , Yoko N, Kazutake S. Simultaneous determination of androste nediol 3-sulfate and dehydroepiandrosterone sulfate in human serum using isoto pe diluted liquid chromatography-electrospray ionizati on-mass spectrometry[J]. J Chromatogr B. 2003, 796: 121-130
[47] Masaharu N, Susumu Y, Kenji S. Determination of Dehydroepiandrost erone Sulphate in Biological Samples by Liquid Chromatography/Atmospheric Pressure Chemical Ionization-Mass Spectrometry using[7,7,16,16-2H4]-Deh ydroepiandrosterone Sulphateas an Internal Standard[J]. Biom Chromotog, 1998,12(4): 211-216
[48] Shackleton C, Kletke C, Wudy S. Dehydroepiandrosterone sulfate quantific ation in serum using high-performance liquid chromatography/mass spectrum etry and a deuterated internal standard: a technique suitable for routine use or as a reference[J]. Steroids. 1990, 55: 472-478
[49] Fuyu G, Uboh Cornelius E, Soma. Lawrence R. Detection, quantification and confirmation of anabolic steroids in equine plasma by liquid chromatography and tandem mass Spectrometry[J]. J Chromatogr B. 2005, 829(1): 56-68
[50] Bowers LD, Sanaullah. Direct measurement of steroid sulfate and glucuronide conjugates with high-performance liquid chromatography-mass spectrometry[J]. J Chromatogr B. 1996;687(1): 61-68
[51] Tatsuya H, Yujin S, Kazutake S. Determination of salivary dehydroepiandr osterone using liquid chromatography-tandem mass Spectrometry combined with charged derivatization[J]. J Chromatog B. 2007,846(1): 195-201
[52] Griffiths W, Liu S, Alvelius G, Sjo¨vall J. Derivatisation for the characterization of neutral oxosteroids by electrospray and matrix-assisted laser desorption/ionisation tandem mass Spectrometry: the Girard P derivative[J]. Rapid Commun. Mass Spectrom. 2003,17(): 924-935.
[53] Kelly C, Thomas H, Lee H. Mass Spectrometry and Precursor Ion Monitoring for Quantitative Steroid Analysis and Attomole Sensitivity[J]. Anal. Chem. 1999,71:2358-2363
[54] A.T. Cawleya, E.R. Hineb, GJ. Searching for new markers of endogenous steroid administration in athletes: "looking outside the metabolic box" [J]. Foren Sci Inter. 2004,143: 103-114
[55] Peter Van Eenoo, Frans T. Delbeke. Metabolism and excretion of anabolic steroids in doping control—New steroids and new insights[J]. J Ster Biochem & Mol. 2006,101 (4): 161-178
[56]NorikoT, Junko T,Aya F. Serum Dehydroepiandrosterone, Dehydroepia ndrosterone Sulfate, and Pregnenolone Sulfate Concentrations in Patients with Hyperthyroidism and Hypothyroidism[J]. Clin Chem.,2000,46(11): 523-528
[57] Schmidt M, Kreutz M, Loffler G Conversion of dehydroepiandrosterone to downstream steroid hormones in macrophages[J]. J Endocrin. 2000,164(2): 161-169
[2]Corrigan B.DHEA and sports[J]Clin J Sport Med,2002,12(4):236-241
[3]Labrie F,Luu-The V,Belange Ar.Is dehydroepiandrosterone a hormone?[J].J Endocrino,2005,187(2):169-196
[4]Dehennin L,Ferry M,Lafarge P.Oral administration of dehydroepian drosterone to healthy men:alteration of the urinary androgen profile and conseq uences for the detection of abuse in sport by gas chromatography-mass spectrum etry[J].Steroids 1998,63(5):80-87
[5]WADA Project Team.Reporting and evaluation guidance for testosterone,epitest osterone,T/E ratio and other endogenous steroids[EB/OL],http:// www.wada-ama.org/rtecontent/document/end_s teroids_aug _ 04.pdf
[6]Choi M H,Kim K R,Chung B C.Simultaneous determination of urinary androgen glucuronides by high temperature gas chromatography-mass spectrometry with selected ion monitoring[J].Steroids,2000,65(1):54-59
[7]Guan F,Uboh C E,Soma L R.Detection,quantification and confirmation of anabolic steroids in equine plasma by liquid chromatography and tandem mass spectrometry[J].J Chromatogr B,2005,829(1):56-68
[8]WADA Project Team.Minimum required performence limits for detection of prohibited substance.[EB/OL].http://www.wada-ama.org/rtecontent/document/perflimits2.pdf.2004-01-15
[9]王静竹 吴侔天 张亦农.运动员尿中类固醇激素水平的研究[J].中国运动医学杂志.2004,23:200-203
[10]L.Starka.Epitestosterone[J].J Ster Bioch Mol Bio 2003,87:27-34
[11]Jenny Jakobsson..Large Differences in Testosterone Excretion in Korean and Swedish Men Are Strongly Associated with a UDP-Glucuronosyl Transfe rase 2B17Polymorphism J ClinEndocrin & Metab 2005.91(2):687-693
[12]Paul J.Perryl,John H.Maclndoe,._Detection of anabolic steroid administration:ratio of urinary testosterone to epitestosterone vs the ratio of urinary testosterone to luteinizing hormone Clin Chem.1997,43:731-735
[13]Donike M,Ueki M,Kuroda Y Detection of dihydrotestosterone(DHT) doping:alterations in the steroid profile and reference ranges for DHT and its 5α-metabolites[J].J Sports Med Phys Fitness 1995,35:235-250
[14]Geyer H.,U.Mareck-Engelke,W.Schanzer,M.Donike Proceedings of the 14th Cologne Workshop on dope analysis,12th to 17th March 1996,M.Donike, H. Geyer, A.Gotzmann, U. Mareck-Engelke[R], Sport und Buch Strausse Edition Sport, Koln 1997: p. 139
[15]Bosy TZ, Moore KA, Poklis A. The effect of oral dehydroepiandroster one on the urine testosterone/epitestosterone ratio (T/E) in human male volunteers[J]. J Anal Toxicol 1998, 22: 455-459
[16] R. Kronstrand, S. Fo¨rsberg, B. Kagedal, J. Ahlner, G. Larson, Relationship between melanin and codeine concentrations in hair after oral administration[R] Proceedings of the annual meeting of the American Academy of Forensic Scien es, 1999
[17] Lauriane R, Fabrice M, Yoann D. Development and validation of a multi-residue method for the detection of a wide range of hormonal anabolic compounds in hair using gas chromatography-tandem mass Spectrometry [J].Anal Chim Acta. 2007,586 93-104
[18] Kintz P, Cirimele V, Jeanneau T, Ludes B. Identification of testosterone and testosterone esters in human hair[J]. J Anal Toxicol 1999,23:352-356
[19] Scherer CR, Reinhardt G Nachweis sechs endogener Steroide in menschli chen Haaren mit GC/MS und Isotopenverdu nnungsanalyse Abstract[R]. Althoff H, editor. 74. Jahrestagung der Deutschen Gesellschaft fu¨r Rechtsmediz in, Aachen, 1995
[20] Jean G, Marjorie C, Gilbert P. Determination of Endo genous Levels of GHB in Human Hair. Are there Possibilities for the Identificat ion of GHB Administration through Hair Analysis in Cases of Drug-Facilitated Sexual Assault[J]? J Anal Toxicol, 2003[8], 27:574-590
[21] Henderson G, Harkey M, Zhou C, III, Incorporat ion of isotopically labeled cocaine and metabolites into human hair: 1. Dose-re sponse relationships[J]. J. Anal. Toxicol. 1996,20: 1-12.
[22] Rollins D, Wilkins D, Krueger G, Codeine disposition in human hair after single and multiple doses.[J] Eur. J. Clin. Pharmacol. 1996,50(1): 391-397
[23] Nakahara Y, Takahashi K, Kikura R, Hair analysis for drugs of abuse. X. Effects of physiochemical properties of drugs on the incorporation rates into hair, Biol. Pharm. Bull[J]. 1995,18(9): 1223-1227
[24] Ho¨ld K, Wilkins D, Crouch D Detection of stanozolol in hair by negative ion chemical ionization mass spectrometry[J] J. Anal. Toxicol. 20 (1996) 345-349.
[25] M.J. Wheeler, Y.-B. Zhong, A.T. Kicman, S.B. Courts, The measurement of testosterone in hair, J.Endocrinol. 1998,159(1) 5-8
[26]Labrie F,Belanger A,Cusan L,Physiological changes in dehydroepiandroste rone are not reflected by serum levels of active androgens and estrogens but of their metabolites:Intracrinology[J].J Clin Endo Metab.1997,82(2):2403-2409
[27]M.P.Manns Age-related changes in serum concentrations of SHBG,testost erone,estrogens and IGF-I in men:results from cross-sectional investigation on healthy subjects and calculations from previously reported studies[R],vorgelegt von Vitali Gorenoi aus St.Petersburg chem.Leningrad,2002.
[28]Andre,Tchernof,Fernand L Dehydroepiandrosterone,obesity and cardio vascular disease risk:a review of human studies.Euro J Endocrin 2004,151(1):1-14.
[29]C.Jurado,P.Kintz,M.Menendez,M.Repetto,Influence of cosmetic treatment of hair on drug testing[J].Int.J.Leg.Med.1997(2):159-163.
[30]L.Po "tsch,G.Skopp,Stability of opiates in hair fibers after exposure to cosmetic treatment[J]Forensic Sci.Int.1996,81(2) 95-102.
[31]J.Seguraa,S.Pichinib,S.H.Penga,X.Hair analysis and detectab ility of single dose administration of androgenic steroid esters[J].Foren Sci Int 2000,1070:347-359
[32]Tatsuya H,Yujin S,Kazutake S.Determination of salivary dehydroepiand rosterone using liquid chromatography-tandem mass spectrometry combined with charged derivatization[J].J Chrom B,2007,846(1):195-201
[33]Jean Pierre G,Marjorie C,Gilbert P.Determination of Endogenous Levels of GHB in Human Hair.Are there Possibilities for the Identification of GHB Administration through Hair Analysis in Cases of Drug- Facilitated Sexual Assau It[J]? Journal of Analytical Toxico 2003,27(8):574-580
[34]Shen Min,Xiang Ping,Shen baohua.Detection ofmeperidine and its metab Olites in the hair of meperidine addits[J].J Foren Sci Int.1999,1030:159-171
[35]沈敏,向平等.头发中抗抑郁药以及抗精神病药的检测及评价.法医学杂质[J].2000,16(3):214-217
[36]姜宴,沈敏,赵子琴等 甲基苯丙胺在豚鼠毛发中分布及转化的初步研究[J].法医学杂志2001,4(4):214-217
[37]Segura J,Possibilities of ELISA methodologies for hair analysis[EB/OL],R.A.de Zeeuw,I.Al Hosani,S.Al Munthiri,A.Maqbool(Eds.),Proceedings of the 1995International Conference and Workshop.Abu Dhabi,Hair Analysis in Forensic Toxicology,1995,:351
[38]Gaillard Y,Balland A,Doucet F,Pe'pin G.Detection of illegal clenbuterol use in calves using hair analysis.Application in meat quality control[J].J.Chr omatogr.B 1997,703():85-95.
[39]Gleixner A,Sauerwein H,Meyer H,Detection of the anabolic b adr enoceptor agonist clenbuterol 2in human scalp hair by HPLC/EIA[J].Clin.Chem.1996,42(11)1869-1871.
[40]Kelly RC,Mieczkowski T,Sweeney SA,Bourland JA.Hair analysis for drugs of abuse.Hair Color and race differentials or systematic differences in drug preferences[J]? Foren Sci Int.2000,107(1):63-86
[41]Mieczkowski T,Newel R.Statistical examination of hair color as a poten tial biasing factor in hair analysis[J].Foren Sci Int.2000,107(1):13-38
[42]闫谨,王杉,密捷波 兴奋剂与兴奋剂检测研究进展[J]。大学化学2003,18(2):10-20
[43]Kintz P,Cirimele V,Sachs H.Testing for anabolic steroids in hair from two bodybuilders[J].Foren Sci Intl,1999,101(3):209-213
[1] Labrie F, Luu-The V, Belange Ar. Is dehydroepiandrosterone a hormone[J]? J Endocrin. 2005,87(2):169-196
[2] Dehennin L, Ferry M, Lafarge P, Peres G, Lafarge JP. Oral administration of dehydroepiandrosterone to healthy men: alteration of the urinary androgen profile and consequences for the detection of abuse in sport by gas chromatography-mass spectrometry[J]. Steroids 1998,63(5): 80-87.
[3] Belange A,Candas B, Dupont A. Changes in serum concentrations of conjugated and unconjugated steroids in 40-to80-year-old men[J]. J Clin Cndocrinol Metab 1994,79: 1086-1090
[4] Nestler JE, Barlascini CO, Clore JN et al. Dehydroepiandrosterone reduces serum low density lipoprotein levels and body fat but does not alter insulin sensitivity in normal men[J]. J Clin Endocrin Metab 1988,66: 57-61.
[5] Welle S, Jozefowicz R, Statt M. Failure of dehydroepiandrosterone to influence energy and protein metabolism in humans[J]. J Clin Endocrin Met. 1990, 71(5): 1259-1264
[6) Corrigan Brian, BM, SB. DHEA and Sport[J]. Clin J Sport Med. 2002,12(4): 236-241
[7] Etienne-Emile B, Paul R. Dehydroepiandrosterone (DHEA) and dehydroepi androsterone sulfate (DHEAS) as neuroactive neurosteroids[J] Proc. Natl. Acad. Sci. USA. 1998, 95(8): 4089-4091
[8] Peter H. Jellinck. Dehydroepiandrosterone (DHEA) metabolism in the brain: Identification by liquid chromatography/mass Spectrometry of the delta-4-isomerof DHEA and related steroids formed from androstenedione by mouse BV2 microglia[J]. J Ster Biochem& Molecu Bio 2006, 98(1): 41-47
[9] Yorio M, Ryo K, Shushi H. A simple, accurate, and sensitive assay method of dehydroepiandrosterone sulfate: application for quantitative determination in human breast cyst and duct fluids[J]. Steroids. 2002, 67(5): 333-338
[10] Le Bail, Lotfi H, Charles L. Conversion of dehydroepiandrosterone sulfate at physiological plasma concentration into estrogens in MCF-7 cells[J]. Steroids 2002, 67(13): 1057-1064
[11] Yasushi M, Shigemasa Y, Akira H. Simultaneous determination of dehydr oepiandrosterone and its 7-oxygenated metabolites in human serum by high-resolution gas chromatography-mass spectrometry[J]. Steroids 2004,69(13): 817-824
[12] Brian D, Frank Z,. Stanczyk P. Pharmacokinetics of dehydroepiandroste rone and its metabolites after long-term daily oral administration to healthy young men[J]. Ferti Steri 2004,81(3):595-604
[13] WADA Project Team. Reporting and evaluation guidance for testosterone, epitest osterone, T/E ratio and other endogenous steroids[EB/OL]. http:// www.wada-ama.org/ rtecontent/document/end_s teroids_aug _ 04.pdf
[14] Rodrigo A, Thomas E. Chapman. A rapid screening assay for measuring urinary androsterone and etiocholanolone 5 13C (%) values by gas chromatogr aphy/combustion/isotope ratio mass spectrometry[J]. Rapid Commun Mass Spectrom. 2000(23): 2294-2299
[15] Kintz P, Nele S. Use of Alternative Specimens: Drugs of Abuse in Saliva and Doping Agents in Hair Therapeutic Drug Monitoring[J]. 2002,24(2): 239-246
[16] Hill M, Lapc'ik O, Havl'ikov'a H. 7-Hydroxydehydroepiandrosterone epimers in human serum and saliva. Comparison of gas chromatography-mass Spectrometry and radioimmunoassay[J]. J Chromatogr 2001,935(): 297-307.
[17] Laurent R. Is there a place for hair analysis in doping controls[J]? Foren Science Inter. 2000,107(1): 309-323
[18] Kintz P, Cirimele V, Ludes B. Physiological concentrations of DHEA in human hair[J]. J Anal Toxicol 1999, 23(1): 424-8.
[19] Kintz P, Vincent C, Marc D. Dehydroepiandrosterone (DHEA) and Testosterone Concentrations in Human Hair after Chronic DHEA Supplementation Clinical Chemistry[J]. 2000,46(3): 414-415
[20] Shimada K, Tanaka M, Nambara T. Determination of 17-ketosteroid sulphates in serum by high-performance liquid chromatography with electrochemical detection using pre-column derivatization. J. Chromatogr[J]. 1984, 307(1): 23-28
[21] Wu M, Takagi K, Okuyama S. Determination of 17-ketosteroid sulphates in serum by high-performance liquid chromatography with electrochemical detection using pre-column derivatization[J]. J. Chromatogr. 1986, 377: 121.
[22] Tagawa N, Tamanaka J, Fujinami A. Serum dehydroepiandrosterone, dehydroepiandrosterone sulfate, and pregnenolone sulfate concentrations in patients with hyperthyroidism and hypothyroidism[J] Clin.Chem. 2000, 46: 523-528
[23] Asaba H, Hosoya K, Takanaga H. Blood-brain barrier is involved in the efflux transport of a neuroactive steroid, dehydroepiandrosterone sulfate, via organic anion transporting polypeptide[J]. J. Neurochem. 2000, 75(5): 1907-1916
[24] Lavallee B, Provost P, Belanger A. Formation of pregnenolone and dehydroepiandrosterone-fatty acid esters by lecithin-cholesterol acyltransferase in human plasma high density lipoproteins[J]. Biophys Acta 996, 1299(3): 306-312
[25] Kelly C , Thomas H, Lee H. Nanoelectrospray Mass Spectrometry and Precursor Ion Monitoring for Quantitative Steroid Analysis and Attomole Sensitivity[J]. Anal Chem. 1999, 71(13): 2358-2363
[26] Payne DW, Holtzclaw WD, Adashi EY. A convenient, unified scheme for the differential extraction of conjugated and unconjugated serum C19 steroids on Sep-Pak C18-cartridges[J]. J. Steroid Biochem. 1989, 33(2): 289
[27] Boschi S, Iasio R, Mesini P. Measurement of steroid hormones in plasma by isocratic high performance liquid chromatography coupled to radioimmunoassay[J]. Clin Chim Acta 1994, 231(1): 107-113
[28] Raeside J, Renaud R, Christie H. Postnatal decline in gonadal secretion of dehydroepiandrosterone and 3β-hydroxyandrosta-5,7-dien-17-one in the newborn foal[J]. Endocrinol. 1997, 155: 277-282
[29] Causon R, Collins S, Fry D. Determination of urinary dehydroepian drosterone sulphate by combined high-performance liquid chromatography and radioimmunoassay[J]. J. Chromatogr. 1982, 227: 485-492
[30] Liere P, Akwa Y, Weil-Engerer S. Validation of an analytical procedure to measure trace amounts of neurosteroids in brain tissue by gas chromatography-mass spectrometry[J]. J. Chromatogr. B 2000.739(2): 301-312
[31] Qi Jia, Mei-Feng H, Zhao-Xing P, Quantification of urine 17-ketosteroid sulfates and glucuronides by high-performance liquid chromatography-ion trap mass spectroscopy[J]. J Chromatogr B, 2001, 750(): 81-91
[32] Nahoul K, Kottler M. Relationships between androgen and estrogen sulfate in breast cyst fluid[J]. Clin Chem Acta. 1992, 209:179-187
[33] Chasalow FI, Blethen SL, Bradlow L. Dehydroepiandrosterone sulfate (DHEA-S) and DHEA-S-like compound in fibrocystic disease of the breast[J]. Steroids. 1988, 52(3): 205-215
[34] Oldrl ich, LapcI oA K, Richard H. Immunoassay of 7-hydroxysteroids: 2. Radioimmunoassay of 7a-hydroxy-dehydroepiandrost erone[J]. J Ster Biochem & Mol Bio. 1999,71:231-237
[35] Richard H, Martin H, Ivan S. Immunomodulatory 7-hydroxylated metabolites of dehydroepiandrosterone are present in human semen[J].J Ster Biochem & Mol Bio.2000,75(4):273-276
[36]Wolthersa B,Kraan G.Clinical applications of gas chromatography and gas chromatography-mass spectrometry of steroids[J].J Chromatog.A.1999,843():247-274
[37]Man C.Simultaneous determination of urinary androgen glucuronides by high temperature gas chromatography-mass spectrometry with selected ion monitoring[J].Steroids.2000,65(1):54-59
[38]Dehennin L,Lafarge P,Dailly D.Combined profile of androgen glucuro- and sulfoconjugates in postcompetition urine of sportsmen:a simple screening procedure using gas chromatography-mass spectrometry[J].J Chromatog B.1996,687(1):85-91
[39]Pujos R,Flament-Waton M,Goetinck P.Optimizing the extraction and analysis of DHEA sulfate,corticosteroidsand androgens in urine:application to a study of the influence of corticosteroid intake on urinary steroid profiles[J].Anal Bioanal Chem.2004,3800:524-536
[40]Baulieu E,Neurosteroids:a novel function of the brain[J].Psychoneuroen docrin[J].1998,23(4):963-987
[41]Valle'e M,Rivera J,Koob G.Quantification of neurosteroids in rat plasma and brain following swim stress and allopregnanolone administration using negative chemical ionization gas chromatography/mass spectrometry[J].Anal.Biochem.2000,287(3):153-166
[42]王静竹,吴侔天,张亦农.气相色谱/燃烧炉/同位素比值质谱方法对尿中去氢表雄酮及其代谢物的检测.药学学报.2005,40(2):159-163
[43]David J,Bortsy Larry D,Bowers.Direct measurement of urinary testos terone and epitestosterone conjugates using high-performance liquidchroma tography/tandem mass spectrometry[J].J.Mass Spectrom.2000,35(1):50-61
[44]Katayama M,Nakane R,Matsuda Y.Determination of progesterone and 17-hydroxyprogesterone by high performance liquid chromatography after pre -column derivatization with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionohydra zide[J].Analyst.1998,123:2339-2342
[45]Ueno K,Umeda T.Electrochemical and chromatographic properties of selected hydrazine and hydrazide derivatives of carbonyl compounds[J].J.Chromatogr.1991,585(2):225-231
[46] Kuniko M , Yoko N, Kazutake S. Simultaneous determination of androste nediol 3-sulfate and dehydroepiandrosterone sulfate in human serum using isoto pe diluted liquid chromatography-electrospray ionizati on-mass spectrometry[J]. J Chromatogr B. 2003, 796: 121-130
[47] Masaharu N, Susumu Y, Kenji S. Determination of Dehydroepiandrost erone Sulphate in Biological Samples by Liquid Chromatography/Atmospheric Pressure Chemical Ionization-Mass Spectrometry using[7,7,16,16-2H4]-Deh ydroepiandrosterone Sulphateas an Internal Standard[J]. Biom Chromotog, 1998,12(4): 211-216
[48] Shackleton C, Kletke C, Wudy S. Dehydroepiandrosterone sulfate quantific ation in serum using high-performance liquid chromatography/mass spectrum etry and a deuterated internal standard: a technique suitable for routine use or as a reference[J]. Steroids. 1990, 55: 472-478
[49] Fuyu G, Uboh Cornelius E, Soma. Lawrence R. Detection, quantification and confirmation of anabolic steroids in equine plasma by liquid chromatography and tandem mass Spectrometry[J]. J Chromatogr B. 2005, 829(1): 56-68
[50] Bowers LD, Sanaullah. Direct measurement of steroid sulfate and glucuronide conjugates with high-performance liquid chromatography-mass spectrometry[J]. J Chromatogr B. 1996;687(1): 61-68
[51] Tatsuya H, Yujin S, Kazutake S. Determination of salivary dehydroepiandr osterone using liquid chromatography-tandem mass Spectrometry combined with charged derivatization[J]. J Chromatog B. 2007,846(1): 195-201
[52] Griffiths W, Liu S, Alvelius G, Sjo¨vall J. Derivatisation for the characterization of neutral oxosteroids by electrospray and matrix-assisted laser desorption/ionisation tandem mass Spectrometry: the Girard P derivative[J]. Rapid Commun. Mass Spectrom. 2003,17(): 924-935.
[53] Kelly C, Thomas H, Lee H. Mass Spectrometry and Precursor Ion Monitoring for Quantitative Steroid Analysis and Attomole Sensitivity[J]. Anal. Chem. 1999,71:2358-2363
[54] A.T. Cawleya, E.R. Hineb, GJ. Searching for new markers of endogenous steroid administration in athletes: "looking outside the metabolic box" [J]. Foren Sci Inter. 2004,143: 103-114
[55] Peter Van Eenoo, Frans T. Delbeke. Metabolism and excretion of anabolic steroids in doping control—New steroids and new insights[J]. J Ster Biochem & Mol. 2006,101 (4): 161-178
[56]NorikoT, Junko T,Aya F. Serum Dehydroepiandrosterone, Dehydroepia ndrosterone Sulfate, and Pregnenolone Sulfate Concentrations in Patients with Hyperthyroidism and Hypothyroidism[J]. Clin Chem.,2000,46(11): 523-528
[57] Schmidt M, Kreutz M, Loffler G Conversion of dehydroepiandrosterone to downstream steroid hormones in macrophages[J]. J Endocrin. 2000,164(2): 161-169