Methyltestosterone Research Articles

Cryopreservation of sperm from natural and sex-reversed orange-spotted grouper (Epinephelus coioides)

The shortage of males and/or sperm has been an impediment to the aquaculture of orange-spotted grouper (Epinephelus coioides). This study reversed orange-spotted grouper females into males using hormone implants. A cryopreservation protocol for sperm was developed using normal males, and then using similar procedures the cryopreservation of sperm from sex-reversed males was compared. Immature, young and mature female fish were injected with 4 mg kg−1 BW 17α methyltestosterone as implants and the gonad development stage was monitored over a 120-day period. All treated females converted into functional males within 120 days of the experimental period. Younger females (2Y) were all males within 30 days, although not all were capable of fertilizing fresh ova until day 60. The time after injection to sex reversal in immature fish was 50% shorter than in older females. Postthaw fertilization (81%, 82%) and hatching (45%, 47%) of cryopreserved sperm from natural males were the highest in trehalose (15–20%) with 150 mmol NaCl treatment; however, it was less than the control (89% fertilization and 69% hatch). There was no difference in the postthaw fertilization and the hatch percentages between sex-reversed male sperm (64% and 46% respectively) compared with natural male sperm (59% and 49%). The findings of this study suggest the potential use of sex-reversed males and cryopreserved sperm for commercial production of orange-spotted grouper seed for aquaculture.

Optimization of LC method for the determination of testosterone and epitestosterone in urine samples in view of biomedical studies and anti-doping research studies

A sensitive and rapid liquid chromatographic (LC) method for the simultaneous determination of testosterone (T) and epitestosterone (E) in human urine samples has been developed and elaborated. The ratio of the both steroids (T/E) in human urine is a widely used as doping control indicator. A sample pretreatment by solid-phase extraction (SPE) after hydrolysis using 36% hydrochloric acid for determination of total level of T has been applied. Unconjugated (free) form of the both androgens were determined without hydrolysis steps, what makes novelty of the method, because simplifies the proposed procedure. In turn, the measurements of urinary free T and E provided the diagnostic information for excess adrenal production of steroids. The proposed LC assay was evaluated by analyzing a series of urine samples containing T, E and methyltestosterone (MT) as internal standard at the range of concentration 2–300 ng −1 mL of both analyzed hormones. The proposed method was fully validated for specificity, linearity, limits of detection and quantitation, precision and trueness according to the current requirements concerning analytical methods. Interestingly, the developed LC method allows to obtain a sensitive enhancement with respect to UV detection with the quantitation limit for T and E equaled 2 ng mL −1. The method was selective and reliable for identity and enable to detect changes of endogenous levels of T and E in urine independently of fluctuations characteristic for both analyzed endogenous hormone level in plasma.

Long-term detection of methyltestosterone (ab-) use by a yeast transactivation system

The routinely used analytical method for detecting the abuse of anabolic steroids only allows the detection of molecules with known analytical properties. In our supplementary approach to structure-independent detection, substances are identified by their biological activity. In the present study, urines excreted after oral methyltestosterone (MT) administration were analyzed by a yeast androgen screen (YAS). The aim was to trace the excretion of MT or its metabolites in human urine samples and to compare the results with those from the established analytical method. MT and its two major metabolites were tested as pure compounds in the YAS. In a second step, the ability of the YAS to detect MT and its metabolites in urine samples was analyzed. For this purpose, a human volunteer ingested of a single dose of 5 mg methyltestosterone. Urine samples were collected after different time intervals (0-307 h) and were analyzed in the YAS and in parallel by GC/MS. Whereas the YAS was able to trace MT in urine samples at least for 14 days, the detection limits of the GC/MS method allowed follow-up until day six. In conclusion, our results demonstrate that the yeast reporter gene system could detect the activity of anabolic steroids like methyltestosterone with high sensitivity even in urine. Furthermore, the YAS was able to detect MT abuse for a longer period of time than classical GC/MS. Obviously, the system responds to long-lasting metabolites yet unidentified. Therefore, the YAS can be a powerful (pre-) screening tool with the potential that to be used to identify persistent or late screening metabolites of anabolic steroids, which could be used for an enhancement of the sensitivity of GC/MS detection techniques.

A non-paradoxical dose response to 17α-methyltestosterone by Nile tilapia Oreochromis niloticus (L.): effects on the sex ratio, growth and gonadal development

Commercial tilapia production is dependent on monosex culture, commonly obtained through the inclusion of an androgen in the diet for a brief period soon after hatch. To determine a minimum effective dose and identify the problems associated with over-dosing, Nile tilapia Oreochromis niloticus fry were fed diets containing methyltestosterone (MT) at rates up to 1200 mg kg−1 of diet for 28 days. The minimum effective dose for ≥95% males was 14 mg MT kg−1 diet. Percent phenotypic males increased as the rate increased from 3.75 (80%) to 30 mg kg−1 MT (99%). Methyltestosterone given at rates of 120 mg kg−1 or more reduced efficacy but did not result in a reduced frequency of males relative to that of non-treated fish. The term ‘paradoxical feminization’ does not adequately describe the observed sex ratios, where no fish were feminized but the efficacy of MT at high doses to masculinize females was reduced. At 1200 mg MT kg−1, the frequency of females (48 ± 1%) was not different from that in the non-treated population. The mechanism for the reduced efficacy is not clear and is not adequately explained as an aromatization of androgen to oestrogen response.

Safety and efficacy of low-dose esterified estrogens and methyltestosterone, alone or combined, for the treatment of hot flashes in menopausal women: a randomized, double-blind, placebo-controlled study

This study evaluated safety and efficacy of esterified estrogens and methyltestosterone administered alone or in combination for the treatment of hot flashes in menopausal women. The 0.30-mg esterified estrogens and 0.30-mg methyltestosterone combination was the lowest effective dose, and our results are consistent with the known safety profile of estrogen and androgen combination products.

Influence of reproductive status, sex hormones and temperature on plasma IGF-I concentrations in sunshine bass ( Morone chrysops × Morone saxatilis)

Insulin-like growth factor-I (IGF-I) concentrations in male and female sunshine bass ( Morone chrysops × Morone saxatilis) were determined in March, early April, and late April in outdoor ponds at a commercial farm. Female fish were always larger than male fish; however, plasma IGF-I concentrations tended to be higher in male fish and increased as pond temperature and feeding increased in both sexes. Gonadal development was greatest in both sexes in March and declined to a regressed state by the end of April and the same pattern of change occurred with plasma estrogen and testosterone. Growth and IGF-I concentrations in sunshine bass fed estrogen, methyl testosterone, or a control diet were also determined. Growth was reduced in fish fed both sex hormones. Fish fed the control diet had the highest IGF-I levels, androgen-fed fish had intermediate levels, and estrogen-fed fish had the lowest IGF-I concentrations after 4 weeks on the diet. Plasma IGF-I concentrations appeared to respond to increasing temperature in the ponds, and were inversely related to gonadal development and sex hormones. Exogenous sex hormones resulted in a decrease in plasma IGF-I, feeding activity and growth.

Histopathology of the Testis as a Sensitive Tool to Detect Potentially Adverse effects of Environmental Chemicals on Male Fertility in Japanese Quail

Histopathology proved a sensitive tool to detect adverse effects of environmental chemicals on testis morphology and function in Japanese quail even if there was no evidence of reduced fertility. This became apparent in one-generation studies with the fungicides vinclozolin and epoxiconazole in which microscopic findings, such as atrophy or epithelial degeneration, paralleled a reduction in spermatid count whereas testis weight was not altered and reproductive success not compromised at the same dose levels. In a study with methyl testosterone in which fertility was in fact reduced, lower spermatid count was accompanied by a strong decrease in testis weight. Histological examination of the testis and counting of spermatids might be suitable and sensitive methods to elucidate the mechanism if a male-mediated decline in reproductive success is suspected.

In vitro study of Organization for Economic Co-operation and Development (OECD) endocrine disruptor screening and testing methods- establishment of a recombinant rat androgen receptor (rrAR) binding assay

The androgen receptor (AR) binding assay can be used to determine the ability of probable endocrine disruptors (EDs) to compete with synthetic androgen methyltrienolone (R1881) for binding to recombinant rat AR (rrAR). In this study, we assessed AR binding of various chemicals using Lexius Freyberger's method. The rank of relative binding affinity (RBA, IC(50)) on the tested chemicals was trenbolone 1.3 x 10(-8) M (RBA 138) > dihydrotesterone (DHT) 1.8 x 10(-8) M (RBA 100) > methyl testosterone 5.7 x 10(-8) M (RBA 31.6) > nonylphenol (NP) 1.3 x 10(-5) M (RBA 0.14) > bisphenol A (BPA) 1.1 x 10(-4) M (RBA 0.016) > isobutyl paraben 3.1 x 10(-4) M (RBA 0.0058) > butyl paraben 6.2 x 10(-4) M (RBA 0.0029) > propyl paraben 9.7 x 10(-4) M (RBA 0.0019). However, di(n-butyl) phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP), known anti-androgenic chemicals, did not show any significant AR binding activity. Our data suggests that in vitro AR binding assay may be useful as a screening tool for potential EDs.

베스테르 철갑상어 치어 성전환을 위한 17α-methyltestosterone과 estradiol-17β 경구투여 효과

The purpose of this study was to examine the effects of oral-administered sex hormone for hybrid sturgeon, bester juvenile. The bester juveniles (2 months after hatching) were received a diet containing various doses of <TEX>$17\alpha$</TEX>-methyltestosterone (MT) or estradiol-<TEX>$17\beta$</TEX> (<TEX>$E_2$</TEX>) for 6 months. Somatic growth of bester sturgeon juvenile did not show significant differences between experimental and control groups (27.9-30.5 cm; 125.1-161.7 g), although survival percentages showed a decreasing tendency in MT-treated animals. By histological examination, germ cells were recorded as smooth type in MT-treated fish and uneven type of germinal epithelium in <TEX>$E_2$</TEX>-treated animals. Their sex ratios were 5:4:1 (male: female: undifferentiation) in control and low dose of MT-treated fish (1 mg/kg), and 9:1:0 in fish treated with high dose of MT (10 mg/kg), whereas the ratios were reversed by both low and high doses of <TEX>$E_2$</TEX> treatment, recorded as 2:8:0. Gonadal areas were not significantly differed in all trials (424,600.4 - 1,039,656.3 <TEX>${\mu}m^2$</TEX>). Total number of germ cells, number of germ cells per gonadal areas and number of germ cells per area were significantly higher to 144.7-148.7 cells/section, 374.0-408.5 <TEX>$cells/mm^2$</TEX> and 1,599.5-1,670.9 <TEX>$cells/mm^2$</TEX> in <TEX>$E_2$</TEX> treatment than those of others (30.4-63.9 cells/section, 148.4-226.9 <TEX>$cells/mm^2$</TEX> and 850.0-1,050.6 <TEX>$cells/mm^2$</TEX>), respectively. And somatic growth according to their gender was not significantly differed between male and female.

PERFORMANCES OF POPULATIONS OF SEX REVERSED TILAPIA AND GENETICALLY MALE TILAPIA IN FINGERLING PONDS

One of the most popular techniques in mass production of all or nearly all male tilapia population is through sex reversal. However, cross mating of YY super male with regular brood-stock is gaining popularity and has been practiced by aqua-culturists. This study was conducted to evaluate the performance of 2 populations of sex reversed tilapia (sex reversed with dietary application of 17-α methyl testosterone, SRT-1, and sex reversed with dietary administration of imidazole, SRT-2) and 1 population of genetically male tilapia (GMT) derived from YY super male until the end of fingerling rearing period. Mixed sex tilapia (MST) derived from normal mating was used as the control population. The results showed that the daily growth rates of SRT-1, SRT-2, GMT, and MST were 0.12, 0.11, 0.20, and 0.07 g/day, with survival rates were 57.83%, 64.67%, 39.67%, and 68.78%, respectively. The GMT has higher harvest yield than the other populations; SRT-1 (+15.73%), SRT-2 (+6.37%), and MST (+20.82%).

Chemical fate and biological effects of several endocrine disrupters compounds in two echinoderm species

Two echinoderm species, the sea urchin Paracentrotus lividus and the feather star Antedon mediterranea, were exposed for 28 days to several EDCs: three putative androgenic compounds, triphenyltin (TPT), fenarimol (FEN), methyltestosterone (MET), and two putative antiandrogenic compounds, p,p'-DDE (DDE) and cyproterone acetate (CPA). The exposure nominal concentrations were from 10 to 3000 ng L(-1), depending on the compound. This paper is an attempt to join three different aspects coming from our ecotoxicological tests: (1) the chemical behaviour inside the experimental system; (2) the measured toxicological endpoints; (3) the biochemical responses, to which the measured endpoints may depend. The chemical fate of the different compounds was enquired by a modelling approach throughout the application of the 'Aquarium model'. An estimation of the day-to-day concentration levels in water and biota were obtained together with the amount assumed each day by each animal (uptake in microg animal(-1) d(-1) or ng g-wet weight(-1) d(-1)). The toxicological endpoints investigated deal with the reproductive potential (gonad maturation stage, gonad index and oocyte diameter) and with the regenerative potential (growth and histology). Almost all the compounds exerted some kind of effect at the tested concentrations, however TPT was the most effective in altering both reproductive and regenerative parameters (also at the concentration of few ng L(-1)). The biochemical analyses of testosterone (T) and 17beta-estradiol (E(2)) also showed the ability of the selected compounds to significantly alter endogenous steroid concentrations.

Androgen Replacement Therapy in Turner Syndrome: A Pilot Study

Women with Turner syndrome (TS) have reduced levels of androgens due to ovarian failure. Morbidity associated with TS, such as bone fragility, metabolic changes, obesity, neurocognitive profile, and sexual problems may partly relate to androgen insufficiency and improve on androgen replacement therapy (ART). The objective of the study was to determine the effect of androgens on morbidity in TS. Fourteen TS women (aged 17-27 yr) participated in a randomized, double-blind, placebo-controlled crossover pilot. The study was conducted in a hospital outpatient clinic between December 2001 and July 2004. TS patients were on estrogen/progestin replacement therapy. Subjects received oral 1.5 mg methyl testosterone (ART) or placebo for 1 yr and the alternative for another year. The study compared body composition as a primary outcome, and physiology, biochemistry, visceral fat, cognition, and quality of life (QOL) as secondary outcomes. ART as compared with placebo reduced total cholesterol, triglycerides, and high-density lipoprotein cholesterol. It improved bone mineral density, increased lean body mass, and decreased fat mass. ART improved attention, reaction time, and verbal memory and had no effect on executive functions and spatial cognition. Patients reported improved QOL, including general health, coping with stress, and sexual desire. Androgen insufficiency plays a role in TS-impaired body composition, neurocognition, and QOL, and these aspects improve with ART, which was safe and effective when given for 1 yr.

Influence of ethynylestradiol and methyltestosterone on the hypothalamo–hypophyseal–gonadal axis of adult air-breathing catfish, Clarias gariepinus

Adult male and female air-breathing catfish Clarias gariepinus were treated with ethynylestradiol (EE 2) and methyltestosterone (MT) at concentrations of 1 μg/L, respectively for 21 days. EE 2 treatment caused disappearance of spermatids/sperm from several testicular lumen/lobules in males while MT treatment to females led to precocious ovarian development. EE 2 caused significant fluid retention in all tissues including peritoneal cavity and seminal vesicles. Immunocytochemical localization of catfish GnRH (cfGnRH) and luteinizing hormone (LH) in preoptic area-hypothalamus (POA-H) and pituitary, respectively, revealed decreased immunoreactivity (ir-) following EE 2 treatment in males. MT treatment however caused no observable change in cfGnRH ir- and a significant increase in LH ir- in females. Semi-quantitative RT-PCR analysis indicated that cfGnRH transcripts in POA-H decreased significantly following EE 2 and MT treatment in males and females, respectively. Levels of POA-H dopamine (inhibitory monoamine for gonadotropin [GTH] synthesis and release) increased following EE 2 and MT treatment in males and females while levels of serotonin and norepinephrine (GTH-stimulatory monoamines) decreased significantly. The results demonstrate a direct in vivo effect of sex steroid analogs on cfGnRH–LH axis and monoaminergic system vis-à-vis on gonads in addition to probable direct action on gonads.

Methyltestosterone

Methyltestosterone

Growth and survival of tilapia Oreochromis niloticus (Linnaeus, 1758) submitted to different temperatures during the process of sex reversal

The objective of this research was to evaluate the sex reversal technique using 17α--methyltestosterone (MT) hormone, submitted to temperature modification of fry Nilo tilapia storage, aiming to get the data of sex reversal combined with growth performance and fry survival. The experiment was performed at UFLA Fish Culture Station, using tilapia fry (0,008 ± 0.002 g e 0,9 ± 0.1 cm) obeying a totally randomized experimental delineation in a factorial scheme 4x4, in 4 temperatures (26º, 28º, 30º, 32ºC) and 4 hormonal doses (0, 20, 40, 60mg of MT/kg of ration) during 28 days, with 5 repetitions. As temperature raised, weight gain rate, size and survival increased (p&lt;0.01); however, this temperature raise was not effective in modifying males ratio (p&gt;0.01), which occurred only due to the used hormone treatment. The dose of 40 mg of MT/kg of ration provided similar results to those of 60mg of MT/kg of ration. Hence, the temperature band from 26º to 32ºC does not affect sex reversal rate, but temperatures around 30ºC improves the performance of tilapias related to the growth and survival, and the dose of 40 mg of MT/kg of ration is enough to achieve monosex populations.

Direct determination of anabolic steroids in pig urine by a new SPME–GC–MS method

A new solid phase microextraction (SPME) method coupled with gas chromatography–mass spectrometry (GC–MS) was developed for rapid determination of four anabolic steroids such as 3α-hydroxy-5α-androstane-17-one (HA), dihydrotestosterone (DHT), androstenedione (AD) and methyltestosterone (MT) in pig urine. SPME was used to extract the four anabolic compounds directly without derivatization. The optimum SPME sampling conditions were based on the home-made carbowax-divinylbenzene (CW-DVB) fiber coating during extraction at 40 °C for 50 min with 0.18 g/mL NaCl solution and 750 rpm stirring speed. The linear ranges of the proposed method were in the range of 8–640 pg/mL for HA and DHT and 16–510 pg/mL for AD and MT, respectively. The detection limits (S/N = 3) were from 2 to 8 pg/mL for the four anabolic steroids. This SPME method provided very high enrichment factors for the four anabolic steroids, which were 1063-fold and 965-fold for HA and DHT at the concentration of 8 pg/mL and 207-fold and 451-fold for AD and MT at the concentration of 16 pg/mL, respectively. The recoveries ranged from 71.3 to 121%, and the RSDs were lower than 12.9%. The method was sensitive and reliable for determination of trace anabolic steroids in biological samples.

Identification of multiple dmrt1s in catfish: localization, dimorphic expression pattern, changes during testicular cycle and after methyltestosterone treatment

The double sex and mab-3 related (DM) transcription factor 1 (dmrt1) plays an important role in testicular differentiation. Here, we report cloning of multiple dmrt1s, a full-length and two alternative spliced forms from adult catfish (Clarias gariepinus) testis, which encode predicted proteins of 287 (dmrt1a), 253 (dmrt1b) and 233 (dmrt1c) amino acid residues respectively. Interestingly, dmrt1c lacks the majority of the DM domain. Multiple dmrt1s (dmrt1a and dmrt1c) were obtained from Clarias batrachus as well. Tissue distribution (transcript and protein) of catfish dmrt1 revealed exclusive expression in testis. Semi-quantitative RT-PCR revealed the presence of multiple dmrt1s with high levels of dmrt1a in adult testis but not in ovary. Real-time RT-PCR analysis during testicular cycle showed higher levels of dmrt1 transcripts in preparatory and pre-spawning when compared with spawning and post-spawning phases. Immunocytochemical and immunofluorescence localization revealed the presence of catfish Dmrt1 protein in spermatogonia and spermatocytes, which indicates plausible role in spermatogenesis. Histological analysis indicated initiation of gonadal sex differentiation in catfish around 40-50 days after hatching. The potential role for dmrt1 in testicular differentiation is evident from its stage-dependent elevated expression in developing testis. Furthermore, dimorphic expressions of dmrt1s were evident at different stages of gonadal development or recrudescence in catfish. Treatment of methyl testosterone (MT) during early stages of gonadal sex differentiation resulted in adult males. Interestingly, we also obtained MT-treated fishes having ova-testis gonads. Analysis of dmrt1, sox9a, foxl2 and cyp19a1 expression patterns in MT-treated gonads revealed tissue-specific pattern. These results together suggest that multiple dmrt1s are testis-specific markers in catfish.

Antifertility Effects of Orally Administration of Low Dose Gossypol Acetic Acid Combined with Methyltestosterone Plus Ethinyl Estradiol on Male Rat

Objective To investigate the feasibility and optimal regimen of orally administration of low dose gossypol acetic acid (GA) combined with methyltestosterone (MT) plus ethinyl estradiol (EE) for contraception in males. Methods Wistar male rats were randomly assigned into four groups, 20 in each group. Animals in group A or B were administered daily with 1% methyl cellulose or GA (12 mg/kg) suspended in 1% methyl cellulose, respectively. Rats in group C or D took firstly GA 12 mg/kg+MT 20 mg/kg+EE 0.1 mg/kg or MT 20 mg/kg+EE 0.1 mg/kg, in a suspension with 1% methyl cellulose, via gastric intubation. After the infertilities were initiated(6 weeks for group C, 8 weeks for group D), GA was served alone while MT+EE were withdrawn in rats of groups C and D. The treatment was ceased after 18 weeks and some males from group C were permitted to recover. Fertility testing, 10 males per group, was served for determining infertility or restoration of fertility in treated rats. Examinations of histology and biochemistry in treated rats were used to examine the morphologic influences on sperm, testis, epididymides and viscera, and biochemical changes in blood. The growth and development of F1 generation of the rats would also be tested in a series of behavioral tests. Results Ten rats from group C were infertile at week 6 after treatment, and the fulfilled infertility was maintained with low-dose GA (12 mg/kg) only daily. Six weeks after cessation of treatment, all of treated males recovered their fertility. However, 8 of 10 rats from group D were in sterility at 6th week of treatment and all at 8th week of treatment, but the infertility could not be kept with the similar dose GA alone later on. Moreover, no adverse effects were found in our present experiments. Conclusion Administration of oral low dose GA combined with MT and EE as loading dose could successfully induce infertility in short term, whereafter the efficacy could completely be maintained by similar low dose of GA alone for long term. The combined regimen should be safe, effective, reversible and long term, and then optimal and promised contraception for male.

Simultaneous determination of androgenic and estrogenic endpoints in the threespine stickleback ( Gasterosteus aculeatus) using quantitative RT-PCR

A method to evaluate the expression of three hormone responsive genes, vitellogenin (estrogens), spiggin (androgens), and an androgen receptor (ARβ) using real-time PCR in threespine stickleback is presented. Primers were designed from previously characterised spiggin and ARβ sequences, while a homology cloning strategy was used to isolate a partial gene sequence for stickleback vitellogenin (Vtg). Spiggin mRNA was significantly higher in kidneys of field-caught males compared to females by greater than five orders of magnitude while ARβ levels were only 1.4-fold higher in males. Female fish had four order of magnitude higher liver Vtg expression than wild-captured males. To determine the sensitivity of these genes to induction by hormones, male and female sticklebacks were exposed to 1, 10 and 100 ng/L of methyltestosterone (MT) or estradiol (E2) in a flow-through exposure system for 7 days. Spiggin induction in females, and Vtg induction in males were both detectable at 10 ng/L of MT and E2, respectively. MT exposure did not induce ARβ expression in the kidneys of female stickleback. In vitro gonadal steroid hormones production was measured in testes and ovaries of exposed stickleback to compare gene expression endpoints to an endpoint of hormonal reproductive alteration. Reduction in testosterone production in ovaries at all three MT exposure concentrations, and ovarian estradiol synthesis at the 100 ng/L exposure were the only effects observed in the in vitro steroidogenesis for either hormone exposure. Application of these methods to assess both androgenic, estrogenic, and anti-steroidogenic properties of environmental contaminants in a single fish species will be a valuable tool for identifying compounds causing reproductive dysfunction in fishes.

A new mixed micellar electrokinetic chromatography method for analysis of natural and synthetic anabolic steroids

A simple, rapid and low-costing new mixed surfactant MEKC method has been developed for the analysis of five neutral anabolic steroids in this paper. It was found that the bile salt coupling with Triton X-100 was a suitable bi-micellar surfactant for the separation of these anabolic steroids with similar structure. The separation conditions were optimized in detail. The five natural and synthetic anabolic steroids, such as androstenedione (AD), 19-norandrostenedione (NAD), 1,4-androstadiene-3,17-dione (ADD), methandrostenolone (MA) and methyltestosterone (MT) were separated and detected in an alkaline buffer system (pH 9.0) containing 15 mM Britton-Robinson (BR) buffer, 50 mM sodium cholate (SC) and 0.1% (v/v) Triton X-100 with detection wavelength at 241 nm and 18 kV of separation voltage. Under the optimal conditions, five coexistence neutral steroids were completely separated within 12 min with the detection limits ranged from 0.20 to 0.51 μg/mL. This method was successfully used for detection and confirmation of the anabolic steroid methandrostenolone in methandrostenolone tablets and in the real human urine, GC–MS method was applied to confirm the free methandrostenolone existence in the urine sample in order to validate the reliability of MEKC method.