The basis of a potential method for confirming intake of four natural androgens (testosterone, epitestosterone, dihydrotestosterone, and dehydroepiandrosterone is presented. The method relies on isolating from urine a steroid fraction containing androstenediol and androstanediol metabolites of these natural steroids and analyzing their 13C content by gas chromatography, combustion, isotope ratio mass spectrometry. The steroids were recovered from urine by conjugate hydrolysis with a Helix pomatia preparation (sulfatase and beta-glucuronidase), Girard T reagent separation to obtain a nonketonic fraction, and Sephadex LH-20 chromatography for purification. Metabolites appropriate for all of the natural steroids could be separated (as diacetates) by gas chromatography on a DB-17 capillary column viz.: 5 alpha (and beta)-androstane-3 alpha,17 alpha-diol (epitestosterone as precursor); 5 alpha (and beta)-androstane-3 alpha,17 beta-diol (testosterone as precursor); 5-androstene-3 beta,17 beta-diol (dehydroepiandrosterone precursor); and 5 alpha-androstane-3 alpha,17 beta- (and 17 alpha-) diol (dihydrotestosterone precursor). Measurement of the 13C content of the specific analytes after ingestion of the androgen precursors demonstrated a lowering of delta 13C/1000 value compared to normal values. Typically, in the male individual studied, delta 13C/1000 values for all components were -26 to -27 before drug administration and -29 to -30 at 6 h after, the latter values reflecting those obtaining for commercial synthetic steroid compared to in vivo synthesized steroid. While generally the metabolism of the steroids was as expected, this was not the case for 5 alpha-dihydrotestosterone. A major metabolite was 5 alpha-androstane-3 alpha,17 alpha-diol, which had presumably been formed by 17 beta/17 alpha isomerization, a process previously known for unnatural anabolics but not for natural hormones. The isolation, purification, and isotope ratio mass spectrometry techniques described may form the basis of a general method for confirming natural steroid misuse by sports participants.

Doses equivalent to 18, 72 and 216 mg testosterone (T)/week were administered for 6 months to eugonadal men. Urinary excretions of androgen glucuronides (G) were quantitatively analyzed by gas chromatography-mass spectrometry with stable isotope dilution during periods of control (without hormone treatment), T administration and recovery. The lowest dosage T administration did not affect the androgen profile, while higher dosages generally increased urinary excretions of T metabolites (TG, T sulfate, glucuronides of androsterone, etiocholanolone, 5 alpha- and 5 beta-androstane-3 alpha,17 beta-diol) and decreased excretions of conjugates of epitestosterone (ET) and its precursor androgen 5-androstene-3 beta,17 alpha-diol. A dose-dependent decrease of urinary LH in response to T was also observed. The ratio (T/ET)G, which is the sole official criterium for assessment of T self-administration by athletes, increased above the threshold value of 6 in most of the subjects, but not all, after the two highest dosage T regimens, and returned to normal during the recovery period. False positive or negative testing emphasizes the need for improvement of testing procedures. In this regard, valuable complementary information may be gained from ratios such as TG/ET(Total), TG/LH, (T/5-androstene-3 beta,17 alpha-diol)G, (5 alpha/5 beta)androstane-3 alpha,17 alpha-diol and (5 alpha/5 beta)androstane-3 alpha,17 beta-diol.

The interpretation of the steroidal module of the Athlete Biological Passport (ABP) in female athletes is complex due to the large variation of the endogenous urinary steroids. The menstrual cycle seems to be one of the largest confounders of the steroid profile. The duration of the different phases in the menstrual cycle differs between women and are difficult to predict only by counting days after menstruation. Here we have determined the follicle, ovulation and luteal phases, by assessing the menstrual hormones in serum samples collected from 17 healthy women with regular menses. Urine samples were collected three times per week during two consecutive cycles to measure the urinary steroid concentrations used in the ABP. The metabolite that was mostly affected by the menstrual phases was epitestosterone (E), where the median concentration was 133 % higher in the ovulation phase compared to the follicle phase (p < 0.0001). The women with a large coefficient of variation (CV) in their first cycle also had a large CV in their second cycle and vice versa. The inter-individual difference was extensive with a range of 11-230 % difference between the lowest and the highest T/E ratio during a cycle. In conclusion, E and ratios with E as denominator are problematic biomarkers for doping in female athletes. The timing of the sample collection in the menstrual cycle will have a large influence on the steroid profile. The results of this study highlight the need to find additional biomarkers for T doping in females.

Keywords: ABP; T/E; doping in sports; menstrual cycle; steroid profile; testosterone.

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. Discovery of novel biomarkers for early HCC from other liver diseases such as cirrhosis is of great clinical benefit. In this study, a novel steroid hormone metabolomic method based on liquid chromatography-mass spectrometry combined with logistic regression analysis was applied to study the steroid hormone disorders and to screen potential urinary steroid hormone biomarkers of early HCC. Thirty-six urinary steroid hormones were detected and quantified in healthy controls, cirrhotic patients, and early HCC patients. Heat map analysis and multivariate statistical analysis suggested severe disorders of steroid hormone network and holistically decreased urinary steroid hormone pattern in cirrhotic and early HCC patients. Logistic regression analysis reveals that a panel of two urinary steroid hormones (epitestosterone and allotetrahydrocortisol) displayed excellent diagnostic capability for distinguishing early HCC from cirrhosis with area under the curve (AUC) = 0.938 of receiver operating characteristic (ROC) analysis. These results help to overcome the disadvantage of lower sensitivity and specificity of alpha-fetoprotein for distinguishing early HCC from cirrhosis. Our work shows that steroid hormone metabolomics is a promising biomarker tool for biomarker study of early HCC.

The six forms of the 17alpha-hydroxy steroid dehydrogenase purified from rabbit liver cytosol have very similar physical properties. The molecular weights of all the enzymes were within 3% of the average mol.wt of 39 600. Only one of the six enzymes showed a significant difference in amino acid composition. All but one form of the 17alpha-hydroxy steroid dehydrogenases exhibited greater activities towards the androgen, epitestosterone, than towards oestrogen substrates. With oestrogen substrates one enzyme displayed a high specificity towards the substrate oestradiol-17alpha 3-glucuronide. This high activity was lost if the glucuronic acid moiety was removed or replaced by glucose or galacturonic acid. The other enzyme forms had approximately equal activity toward oestradiol-17alpha and its glucuronide or glucoside derivative. However, substitution of galacturonic acid at C-3 of oestradiol-17alpha substantially decreased the activity of all but one enzyme form.

The optimum conditions for hydrolysing conjugated metabolites of steroid hormones in bovine urine were performed with Helix pomatia juice, beta-glucuronidase from bovine liver and preparations of limpets and abalone entrails using response surface methodology. The experimental design and empirical modelling used allowed us to assess the main effects of factors (time, temperature, pH and enzyme quantity) and to predict the optimum conditions for each enzyme preparation. Confirmatory experiments were applied to check the predicted values and to validate the model. The comparison of the enzyme preparation efficiency for various conjugate steroids and the study of possible by-product synthesis led us to select abalone entrails to hydrolyse natural dehydroepiandrosterone, etiocholanolone, epitestosterone; 17 alpha-estradiol and estrone in bovine urine. The optimum conditions were found to be 20 h at 42 degrees C with the pH adjusted to 5.2 and using 12,000 units of enzyme preparation.

Dihydrotestosterone (DHT), a biologically active metabolite of testosterone, may be misused in sports to benefit from its anabolic and psychotropic effects. After DHT application, a significant increase of the glucuronides of DHT and its metabolites can be expected for a certain time period depending upon dose, formulation, route of administration, and in case of percutaneous administration the chainlength of the ester. DHT and its metabolites can be monitored by gas-chromatography/mass spectrometry (GC/MS) after enzymatic hydrolysis and trimethylsilylation. To investigate the extent of the alteration of the urinary steroid profile after DHT application, timely controlled experiments have been performed with: a) oral application of [16,16,17-2H3]-DHT, and b) sublingual application of a 25 mg dose of DHT. In the experiment with [16,16,17-2H3]-DHT within 24 hours about 44% of the applied dose was recovered after hydrolysis with beta-glucuronidase from E. coli as di- or tri-deuterated 5 alpha-androstane glucuronides: androsterone (33.2%), 5 alpha-androsta-ne-3 alpha,17 beta-diol (2.5%), 5 alpha-androstane-3 beta, 17 beta-diol (0.9%), DHT (7.2%). Hydrolysis with beta-glucuronidase/arylsulfatase from Helix Pomatia resulted in a about 10% higher yield except for DHT. In the study with sublingual application of 25 mg of DHT the extent of the recovery of DHT and its metabolites was in the same range as for the deuterated DHT. The urinary glucuronide concentrations of DHT, androsterone (AND), 5 alpha-androstane-3 alpha, 17 beta-diol (5 alpha A3 alpha D) and 5 alpha-androstane-3 beta,17 beta-diol (5 alpha A3 beta D) and their ratios to etiocholanolone (ETIO), 5 beta-androstane-3 alpha, 17 beta-diol (5 beta A3 alpha D) and epitestosterone (EPIT) were increased for up to 48 hours after application. For doping control purposes concentrations of DHT, 5 alpha A3 alpha D, 5 alpha A3 beta D and ratios of 5 alpha-metabolites to non 5 alpha-metabolites such as DHT/ETIO, DHT/EPIT, 5 alpha A3 alpha D/5 beta A3 alpha D, 5 alpha A3 beta D/5 beta A3 alpha D, and AND/ETIO outside the reference ranges are a proof for DHT application. Reference ranges for Asian and Caucasian male and female athletes are calculated from data bases of the Asian Games 1994, the previous Asian Games 1990 and the routine doping control samples of Caucasian athletes measured in Cologne 1994. At the occasion of the 1994 Asian Games in Hiroshima alterations in the concentrations and ratios of the DHT depending parameters for outside there reference ranges have been found and have been sanctioned on this basis by the Medical Commission of the Organisation of Olympic Council of Asia (OCA).

BACKGROUND

Epi-testosterone (epiT) is the 17alpha-epimer of testosterone. It has been found at similar level as testosterone in human biological fluids. This steroid has thus been used as a natural internal standard for assessing testosterone abuse in sports. EpiT has been also shown to accumulate in mammary cyst fluid and in human prostate. It was found to possess antiandrogenic activity as well as neuroprotective effects. So far, the exact pathway leading to the formation of epiT has not been elucidated.

RESULTS

In this report, we describe the isolation and characterization of the enzyme 17alpha-hydroxysteroid dehydrogenase. The name is given according to its most potent activity. Using cells stably expressing the enzyme, we show that 17alpha-HSD catalyzes efficienty the transformation of 4-androstenedione (4-dione), dehydroepiandrosterone (DHEA), 5alpha-androstane-3,17-dione (5alpha-dione) and androsterone (ADT) into their corresponding 17alpha-hydroxy-steroids : epiT, 5-androstene-3beta,17alpha-diol (epi5diol), 5alpha-androstane-17alpha-ol-3-one (epiDHT) and 5alpha-androstane-3alpha,17alpha-diol (epi3alpha-diol), respectively. Similar to other members of the aldo-keto reductase family that possess the ability to reduce the keto-group into hydroxyl-group at different position on the steroid nucleus, 17alpha-HSD could also catalyze the transformation of DHT, 5alpha-dione, and 5alpha-pregnane-3,20-dione (DHP) into 3alpha-diol, ADT and 5alpha-pregnane-3alpha-ol-20-one (allopregnanolone) through its less potent 3alpha-HSD activity. We also have over-expressed the 17alpha-HSD in Escherichia coli and have purified it by affinity chromatography. The purified enzyme exhibits the same catalytic properties that have been observed with cultured HEK-293 stably transfected cells. Using quantitative Realtime-PCR to study tissue distribution of this enzyme in the mouse, we observed that it is expressed at very high levels in the kidney.

CONCLUSIONS

The present study permits to clarify the biosynthesis pathway of epiT. It also offers the opportunity to study gene regulation and function of this enzyme. Further study in human will allow a better comprehension about the use of epiT in drug abuse testing; it will also help to clarify the importance of its accumulation in breast cyst fluid and prostate, as well as its potential role as natural antiandrogen.

BACKGROUND

The administration of gonadotrophins is prohibited in sport but the effect in men of recently available recombinant hCG and LH on serum and urine concentrations of gonadotrophins and androgens has not been systematically evaluated in the antidoping context.

OBJECTIVE

To determine the time-course of recombinant LH (rhLH) and hCG (rhCG) on blood and urine hormone profiles in men to develop effective tests to detect rhLH and rhCG doping.

METHODS

Two randomized controlled studies with a 2 x 2 factorial design.

METHODS

Academic research centre.

METHODS

Healthy male volunteers aged 18-45 years.

METHODS

In the rhLH study, men were randomized into (i) either of two single doses of rhLH (75 IU or 225 IU), and (ii) suppression of endogenous LH and testosterone by nandrolone or no suppression. In the rhCG study, men were randomized into (i) either of two single doses of rhCG (250 or 750 microg), and (ii) suppression of endogenous LH and testosterone by nandrolone decanoate (ND) or no suppression. ND suppression comprised a single dose of 200 mg ND 3 days prior to, and in the rhCG study an additional dose 1 day after gonadotrophin injection.

METHODS

Serum and urine hCG, LH, T, T : LH ratio, urine epitestosterone (E) and urine T : E ratio.

RESULTS

Neither rhLH dose produced a significant increase in serum or urine LH or T or in the T : E or T : LH ratios regardless of ND-induced suppression of endogenous LH and T. Nor did an even higher dose (750 IU) in three healthy men with unsuppressed gonadal axis. These findings were confirmed with two different commercial LH immunoassays together with adjustment for any influence of urine sediment and dilution. Both rhCG doses produced a steep, dose-proportional increase in serum and urine hCG with increases in serum and urine T and suppression of serum and urine LH, regardless of hCG dose. Serum but not urine T was lowered by ND suppression. The T : LH ratio showed a progressive increase unrelated to rhCG dose or ND suppression, whereas both rhCG and ND suppression minimally increased T : E ratio.

CONCLUSIONS

Both rhCG doses produce a striking increase in serum hCG and T with suppression of serum LH but, at single doses up to 750 IU, rhLH has no influence on serum or urine LH or T. Effective rhLH doping, which relies on a sustained increases in endogenous T, would require much higher and more frequent daily rhLH doses. Use of LH immunoassays optimized for serum to detect rhLH doping by urine LH measurement requires more standardization and validation and, at present, is unreliable. The T : LH ratio is, however, a useful screening test for hCG doping although its utility requires further evaluation.

Mass spectrometric and tandem mass spectrometric behavior of eight anabolic steroid glucuronides were examined using electrospray (ESI) and atmospheric pressure chemical ionization (APCI) in negative and positive ion mode. The objective was to elucidate the most suitable ionization method to produce intense structure specific product ions and to examine the possibilities of distinguishing between isomeric steroid glucuronides. The analytes were glucuronide conjugates of testosterone (TG), epitestosterone (ETG), nandrolone (NG), androsterone (AG), 5alpha-estran-3alpha-ol-17-one (5alpha-NG), 5beta-estran-3alpha-ol-17-one (5beta-NG), 17alpha-methyl-5alpha-androstane-3alpha,17beta-diol (5alpha-MTG), and 17alpha-methyl-5beta-androstane-3alpha,17beta-diol (5beta-MTG), the last four being new compounds synthesized with enzyme-assisted method in our laboratory. High proton affinity of the 4-ene-3-one system in the steroid structure favored the formation of protonated molecule [M + H]+ in positive ion mode mass spectrometry (MS), whereas the steroid glucuronides with lower proton affinities were detected mainly as ammonium adducts [M + NH4]+. The only ion produced in negative ion mode mass spectrometry was a very intense and stable deprotonated molecule [M - H]- . Positive ion ESI and APCI MS/MS spectra showed abundant and structure specific product ions [M + H - Glu]+, [M + H - Glu - H2O]+, and [M + H - Glu - 2H2O]+ of protonated molecules and corresponding ions of the ammonium adduct ions. The ratio of the relative abundances of these ions and the stability of the precursor ion provided distinction of 5alpha-NG and 5beta-NG isomers and TG and ETG isomers. Corresponding diagnostic ions were only minor peaks in negative ion MS/MS spectra. It was shown that positive ion ESI MS/MS is the most promising method for further development of LC-MS methods for anabolic steroid glucuronides.

High intensity strength training causes changes in steroid hormone concentrations. This could be altered by the muscular contraction type: eccentric or concentric. The aim of this study was to compare the effect of the completion of a short concentric (CON) and concentric/eccentric (CON/ECC) trial on the urinary steroid profile, both with the same total work. 18 males performed the trials on an isokinetic dynamometer (BIODEX III) exercising quadriceps muscles, right and left, on different days. Trial 1 (CON): 4 x 10 Concentric knee extension + relax knee flexion, speed 600/second; rest 90 seconds between each series and 4 minutes between each leg exercise. Trial 2 (CON/ECC): 4 x 5 concentric knee extension + Eccentric knee flexion under similar conditions. Urine samples were taken before the exercise and one hour after finishing it. Androsterone, Etiocholanolone, DHEA, Androstenedione, Testosterone, Epitestosterone, Dehydrotestosterone, Estrone, B-Estradiol, Tetrahydrocortisone, Tetrahydrocortisol, Cortisone and Cortisol (free, glucoconjugated and sulfoconjugated) urinary values were determined using gas chromatography/mass spectrometry techniques. No significant differences were noted in Total Work and Average Peak Torque, although Maximum Peak Torque in the CON/ECC trial was higher than in the CON trial. These results demonstrate no changes in the steroid profile before and after trials, or when comparing CON to CON/ECC trials. The data suggest that eccentric contractions do not cause hormonal changes different to the ones produced by concentric contractions, when they are performed in strength short trials with the same total workload.

In this cross-sectional study on 140 subjects, several testosterone and epitestosterone metabolites have been analyzed by gas chromatography-mass spectrometry associated with stable isotope dilution, a technique requested for doping analysis in general, and detection of exogenous testosterone supply in particular. Urinary excretions of luteinizing hormone, testosterone and epitestosterone glucuronides and sulfates, as well as glucuronides of 5-androstene-3 beta, 17 alpha-diol, 5 alpha- and 5 beta-androstane-3 alpha, 17 alpha-diol and the corresponding 17 beta-isomers, present similar patterns of increase throughout pubertal development, from stage 1 up to stage 5. Excretion levels are significantly different in general between stages 1, 2, 3 and 4, the highest percentage increase being observed between stages 3 and 4. None of the ratios of testosterone to epitestosterone glucuronides are beyond the threshold value of 6, where testosterone abuse by athletes is suspected. No particular pubertal stage exceeded this critical value with a probability higher than p = 0.006, a value that was determined on the whole population. This is consistent with the non-significant differences between correlation slopes of regression curves, relating either testosterone or epitestosterone glucuronide to chronological age. The ratio of testosterone glucuronide to luteinizing hormone increases significantly throughout puberty and this might be a limitation to the widespread use of this ratio for the detection of testosterone misuse.(ABSTRACT TRUNCATED AT 250 WORDS)

Six laboratories in six countries collaborated to investigate the analytical method for estimating the testosterone to epitestosterone ratio (T/E) in urine by gas chromatography/mass spectrometry in the context of detecting the application of T as a doping agent in sport. The protocol specified many but not all details of reagents and instrument conditions. The design included the distribution and analysis of four urines with different T/E values, three replicates per value, and one standard. The ranges of mean T/E values for the four urines estimated by peak area (PA) were 0.32-0.42, 0.72-0.94, 0.91-1.14 and 3.19-5.48. The analyses of variance for these data and for the peak height (PH) data were significant for the laboratory factor (p < 0.0001). In addition there was a significant interaction between the urine factor and the laboratory factor which indicates the complexity of the analysis. T/E calculated using PA was not significantly different from that using PH. For within-laboratory precision all values for PH and PA were < 8.3%, and for between-laboratory precision all values were < 11.7% except for one (20.1%). The data represent a baseline for future experiments designed to elucidate the sources of within-and between-laboratory variance, and to harmonize estimates of T/E.

According to the annual report of the World Anti-Doping Agency, steroids are the most frequently detected class of doping agents. Detecting the misuse of endogenously occurring steroids, i.e. steroids such as testosterone that are produced naturally by humans, is one of the most challenging issues in doping control analysis. The established thresholds for urinary concentrations or concentration ratios such as the testosterone/epitestosterone quotient are sometimes inconclusive owing to the large biological variation in these parameters.For more than 15 years, doping control laboratories focused on the carbon isotope ratios of endogenous steroids to distinguish between naturally elevated steroid profile parameters and illicit administration of steroids. A variety of different methods has been developed throughout the last decade and the number of different steroids under investigation by isotope ratio mass spectrometry has recently grown considerably. Besides norandrosterone, boldenone was found to occur endogenously in rare cases and the misuse of corticosteroids or epitestosterone can now be detected with the aid of carbon isotope ratios as well. In addition, steroids excreted as sulfoconjugates were investigated, and the first results regarding hydrogen isotope ratios recently became available.All of these will be presented in detail within this review together with some considerations on validation issues and on identification of parameters influencing steroidal isotope ratios in urine.

OBJECTIVE

To study the effect and time profile of different doses of testosterone enanthate on the blood lipid profile and gonadotropins.

METHODS

Twenty-five healthy male volunteers aged 27-43 years were given 500 mg, 250 mg, and 125 mg of testosterone enanthate as single intramuscular doses of Testoviron(®) Depot. Luteinizing hormone (LH), follicle-stimulating hormone (FSH), blood lipid profile (total cholesterol, plasma [p-] low-density lipoprotein, p-high-density lipoprotein [HDL], p-apolipoprotein A1 [ApoA1], p-apolipoprotein B, p-triglycerides, p-lipoprotein(a), serum [s-] testosterone, and 25-hydroxyvitamin D3) were analyzed prior to, and 4 and 14 days after dosing. Testosterone and epitestosterone in urine (testosterone/epitestosterone ratio) were analyzed prior to each dose after a washout period of 6-8 weeks.

CONCLUSIONS

All doses investigated suppressed the LH and FSH concentrations in serum. LH remained suppressed 6 weeks after the 500 mg dose. These results indicate that testosterone has a more profound endocrine effect on the hypothalamic-pituitary-gonadal axis than was previously thought. There was no alteration in 25-hydroxyvitamin D3 levels after testosterone administration compared to baseline levels. The 250 and 500 mg doses induced decreased concentrations of ApoA1 and HDL, whereas the lowest dose (125 mg) did not have any effect on the lipid profile.

CONCLUSIONS

The single doses of testosterone produced a dose-dependent increase in serum testosterone concentrations together with suppression of s-LH and s-FSH. Alterations in ApoA1 and HDL were observed after the two highest single doses. It is possible that long-time abuse of anabolic androgenic steroids will lead to alteration in vitamin D status. Knowledge and understanding of the side effects of anabolic androgenic steroids are important to the treatment and care of abusers of testosterone.

The present study was performed to investigate the influence of the intake of selective oestrogen receptor modulators on the urinary endogenous steroids profile. For this purpose the circadian variability of luteinizing hormone, follicle-stimulating hormone, testosterone, 5α-androstan-3α,17β-diol, 5β-androstan-3α,17β-diol, epitestosterone, 4-androstenedione, androsterone and etiocholanolone were measured on eight subjects (four males and four females) by gas chromatography-mass spectrometry and chemiluminescent immunometric assay techniques before and after oral administration of multiple doses of either tamoxifen (80 mg for 2 days) or toremifene (120 mg for 2 days) or clomiphene (100 mg for 2 days). The individual baseline variability of the steroids studied was set up by collecting the urine samples every 3 h, for 3 days prior to the treatment; whereas the evaluation of the effects of the oral administration of multiple doses of selective oestrogen receptor modulators on the steroid urinary profile was assessed by collecting urine samples every three hours for at least five days from the first administration. The results of our measurements showed that, only in male subjects, the relative urinary concentrations of testosterone, epitestosterone and 4-androstenedione were significantly altered generally after the second day of drug administration. While no significant effects were recorded in both sexes on the luteinizing hormone, follicle-stimulating hormone, androsterone, etiocholanolone, 5α-androstan-3α,17β-diol and 5β-androstan-3α,17β-diol urinary levels and on testosterone/epitestosterone, 5α-androstan-3α,17β-diol/5β-androstan-3α,17β-diol and androsterone/etiocholanolone ratios.

Direct detection of several steroid glucuronide and sulfate conjugates was achieved with electrospray reversed-phase HPLC-mass spectrometry. Separation of steroid 17-OH or 5-H epimers conjugated with glucuronide or sulfate could be achieved using gradient elution. Testosterone glucuronide, testosterone sulfate, epitestosterone sulfate and epitestosterone glucuronide were chromatographically resolved, although significant variation in solvent strength was observed between methanol and acetonitrile. Positive ionization mode MS and MS-MS spectra were employed to obtain both quantitative and structural information. Some differences were noted with respect to steroid structure and adduct formation, including significant differences in the stability of epimers in the declustering region of the interface. Negative ionization mode, although having lower limits of detection, did not provide useful structural information in either the MS or MS-MS mode. Using a packed capillary column (300 microns I.D.), a detection limit of 25 pg was achieved for epitestosterone glucuronide.

Determination of the ratio of testosterone to epitestosterone (T/E) in urine is used to detect testosterone administration in athletes, with a ratio>> 6 considered as evidence of an offense. We show that administration of ketoconazole, which inhibits testosterone biosynthesis, may be useful for differentiating between an athlete who is using testosterone and one who naturally gives a ratio>> 6. In a control subject pretreated with testosterone, ketoconazole caused the ratio to increase; conversely, it caused a decrease in the ratio in an athlete under investigation. Repeated administration of ketoconazole to two normal men caused a decrease in the ratio due to a large decrease in the urinary excretion rate of testosterone relative to epitestosterone. Stimulation with human chorionic gonadotropin exacerbated the differences in excretion rates. A single administration of ketoconazole to six normal men caused the T/E ratios to decrease significantly within 8 h, a suitable time scale for use in a dynamic test.

Testosterone (T) is the primary male sex hormone. In addition to the development of secondary sex characteristics, testosterone has anabolic effects including increases in muscle size and strength and increases in lean body mass, making it an attractive candidate to enhance athletic performance. In the case of exogenous administration of testosterone, the ratio of testosterone to its isomer, epitestosterone (E), is elevated. WADA has set a standard for T/E ratios of 4.0 as indicative of possible exogenous testosterone administration. Typically, a sample that screens for a T/E ratio above that threshold is then subjected to quantitative confirmation by GC/MS. This methodology, however, can limited due to sensitivity issues as well as a limited number of qualifying ions that can be used for unambiguous identification. We have developed a confirmation method which uses liquid/liquid extraction, followed by room temperature Girard P derivatization, and analysis using LC/MS-ToF. We observe a number of advantages over conventional GC/MS analysis. Analysis time is decreased. Sensitivity is increased, resulting in limits of detection of 2 and 0.5 ng/ml for testosterone and epitestosterone, respectively. The number of diagnostic qualifier ions is also increased allowing more confident identification of the analytes. Finally, while this method has been developed on a QToF instrument, it should be easily transferable to any tandem LC/MS/MS system.

Two chromatographic methods, reversed-phase liquid chromatography (LC) and immunoaffinity chromatography (IAC), were compared in the preparation of purified testosterone extracts suitable for gas chromatography-combustion/isotope ratio mass spectrometry (GC-C-IRMS) analysis. We have shown previously that GC-C-IRMS is a promising means of detection of testosterone misuse in sport. The two clean-up procedures afford sufficient recovery and adequate purity of testosterone. LC presents several advantages over IAC: access to other urinary steroids, longer column life, no need for special equipment and no antibody preparation. For IAC, the antibodies to testosterone must be selected with care for high affinity and low cross-reactivity. Nevertheless, IAC is of some interest in our experiments, the recovery is slightly better for low concentrations of urinary testosterone and IAC does not induce isotopic discrimination even in overloading experiments. This is the first report on sample preparation by IAC prior to GC-C-IRMS and carbon isotope ratio values for urinary epitestosterone. The carbon isotope ratio test can identify users' urines missed by the testosterone to epitestosterone ratio (T/E>> 6) test.

The most frequently used method to demonstrate testosterone abuse is the determination of the testosterone and epitestosterone concentration ratio (T/E ratio) in urine. Nevertheless, it is known that factors other than testosterone administration may increase the T/E ratio. In the last years, the determination of the carbon isotope ratio has proven to be the most promising method to help discriminate between naturally elevated T/E ratios and those reflecting T use. In this paper, an excretion study following oral administration of 40 mg testosterone undecanoate initially and 13 h later is presented. Four testosterone metabolites (androsterone, etiocholanolone, 5 alpha-androstanediol, and 5 beta-androstanediol) together with an endogenous reference (5 beta-pregnanediol) were extracted from the urines and the delta(13)C/(12)C ratio of each compound was analyzed by gas chromatography-combustion-isotope ratio mass spectrometry. The results show similar maximum delta(13)C-value variations (parts per thousand difference of delta(13)C/(12)C ratio from the isotope ratio standard) for the T metabolites and concomitant changes of the T/E ratios after administration of the first and the second dose of T. Whereas the T/E ratios as well as the androsterone, etiocholanolone and 5 alpha-androstanediol delta(13)C-values returned to the baseline 15 h after the second T administration, a decrease of the 5 beta-androstanediol delta-values could be detected for over 40 h. This suggests that measurements of 5 beta-androstanediol delta-values allow the detection of a testosterone ingestion over a longer post-administration period than other T metabolites delta(13)C-values or than the usual T/E ratio approach.

OBJECTIVE

Exogenous testosterone administration is classically detected by measuring the ratio of testosterone to epitestosterone in urine. Athletes are considered to be positive for drug abuse if the urinary testosterone to epitestosterone ratio is greater than 6. We aimed at investigating the urinary excretion of testosterone and epitestosterone during pubertal development.

METHODS

We performed a cross-sectional study of 141 normal male subjects between ages 8 and 26 years.

METHODS

We studied 141 subjects: 32 at stage 1 of Tanner, 27 at stage 2, 30 at stage 3, 25 at stage 4 and 27 at stage 5.

METHODS

Subjects performed a 24-hour urine collection. Urinary testosterone and epitestosterone were measured by gas chromatography-mass spectrometry with selected ion monitoring.

RESULTS

Urinary testosterone was 20.5 +/- 1.7 nmol/24 h (mean +/- SEM) at stage 1, 49 +/- 2.9 at stage 2, 98.8 +/- 3.4 at stage 3, 371.8 +/- 21.8 at stage 4 and 403.4 +/- 16.1 nmol/24h at stage 5. Urinary epitestosterone was 13.1 +/- 1.5 nmol/24h at stage 1, 29.1 +/- 3.3 at stage 2, 48.3 +/- 3.7 at stage 3, 156.3 +/- 14.8 at stage 4 and 221.1 +/- 18.6 nmol/24h at stage 5. The urinary excretions of both steroids increased significantly during puberty and were highly correlated with chronological age (P < 0.001). Comparison of the correlation slopes (P < 0.001) showed that the urinary profiles of testosterone and epitestosterone are not parallel during pubertal development. Two subjects presented a testosterone to epitestosterone ratio above 6, corresponding to a low urinary concentration of epitestosterone, without pathological explanation.

CONCLUSIONS

Testosterone and epitestosterone do not present the same urinary profiles throughout puberty. Marked increases of the testosterone to epitestosterone ratio can be observed at this period and may interfere with doping tests.

This study was performed on 16 professional racing cyclists to examine changes in urine concentrations of androgen hormones (testosterone, epitestosterone, androsterone, etiocholanolone, 11-hydroxy-androsterone and 11-hydroxy-etiocholanolone) and plasma sex hormone binding globulin (SHBG) after training and after competition. The urinary concentrations of androgen hormones decreased during the period of training and increased during competition, this being the reverse of what happened to SHBG plasma concentrations. These changes would suggest that physical activity may have an influence on the elimination of androgen hormones and on the synthesis of its transporting protein SHBG.

The screening of testosterone (T) misuse for doping control is based on the urinary steroid profile, including T, its precursors and metabolites. Modifications of individual levels and ratio between those metabolites are indicators of T misuse. In the context of screening analysis, the most discriminant criterion known to date is based on the T glucuronide (TG) to epitestosterone glucuronide (EG) ratio (TG/EG). Following the World Anti-Doping Agency (WADA) recommendations, there is suspicion of T misuse when the ratio reaches 4 or beyond. While this marker remains very sensitive and specific, it suffers from large inter-individual variability, with important influence of enzyme polymorphisms. Moreover, use of low dose or topical administration forms makes the screening of endogenous steroids difficult while the detection window no longer suits the doping habit. As reference limits are estimated on the basis of population studies, which encompass inter-individual and inter-ethnic variability, new strategies including individual threshold monitoring and alternative biomarkers were proposed to detect T misuse. The purpose of this study was to evaluate the potential of ultra-high pressure liquid chromatography (UHPLC) coupled with a new generation high resolution quadrupole time-of-flight mass spectrometer (QTOF-MS) to investigate the steroid metabolism after transdermal and oral T administration. An approach was developed to quantify 12 targeted urinary steroids as direct glucuro- and sulfo-conjugated metabolites, allowing the conservation of the phase II metabolism information, reflecting genetic and environmental influences. The UHPLC-QTOF-MS(E) platform was applied to clinical study samples from 19 healthy male volunteers, having different genotypes for the UGT2B17 enzyme responsible for the glucuroconjugation of T. Based on reference population ranges, none of the traditional markers of T misuse could detect doping after topical administration of T, while the detection window was short after oral TU ingestion. The detection ability of the 12 targeted steroids was thus evaluated by using individual thresholds following both transdermal and oral administration. Other relevant biomarkers and minor metabolites were studied for complementary information to the steroid profile, including sulfoconjugated analytes and hydroxy forms of glucuroconjugated metabolites. While sulfoconjugated steroids may provide helpful screening information for individuals with homozygotous UGT2B17 deletion, hydroxy-glucuroconjugated analytes could enhance the detection window of oral T undecanoate (TU) doping.