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Study of Determination and Evaluation of DHEA and Other Related Steroids in Vivo

Author: WangMengZuo
Tutor: ShenMin;XiangPing;ShenBaoHua
School: Fudan University
Course: Forensic
Keywords: anabolic steroids urine hair LC-MS/MS GC-MS/MS
CLC: D919
Type: Master's thesis
Year: 2008
Downloads: 166
Quote: 0
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Abstract


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 C18 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.

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