Steroid isotopic standards for gas chromatography-combustion isotope ratio mass spectrometry (GCC-IRMS)

Carbon isotope ratio (CIR) analysis of urinary steroids using gas chromatography-combustion isotope ratio mass spectrometry (GCC-IRMS) is a recognized test to detect illicit doping with synthetic testosterone. There are currently no universally used steroid isotopic standards (SIS). We adapted a protocol to prepare isotopically uniform steroids for use as a calibrant in GCC-IRMS that can be analyzed under the same conditions as used for steroids extracted from urine. Two separate SIS containing a mixture of steroids were created and coded CU/USADA 33-1 and CU/USADA 34-1, containing acetates and native steroids, respectively. CU/USADA 33-1 contains 5α-androstan-3β-ol acetate (5α-A-AC), 5α-androstan-3α-ol-17-one acetate (androsterone acetate, A-AC), 5β-androstan-3α-ol-11, 17-dione acetate (11-ketoetiocholanolone acetate, 11k-AC) and 5α-cholestane (Cne). CU/USADA 34-1 contains 5β-androstan-3α-ol-17-one (etiocholanolone, E), 5α-androstan-3α-ol-17-one (androsterone, A), and 5β-pregnane-3α, 20α-diol (5βP). Each mixture was prepared and dispensed into a set of about 100 ampoules using a protocol carefully designed to minimize isotopic fractionation and contamination. A natural gas reference material, NIST RM 8559, traceable to the international standard Vienna PeeDee Belemnite (VPDB) was used to calibrate the SIS. Absolute δ¹³C_VPDB and Δδ¹³C_VPDB values from randomly selected ampoules from both SIS indicate uniformity of steroid isotopic composition within measurement reproducibility, SD(δ¹³C) < 0.2‰. This procedure for creation of isotopic steroid mixtures results in consistent standards with isotope ratios traceable to the relevant international reference material.     

Steroid metabolism by mouse submaxillary glands. I. in vitro metabolism of testosterone and 4-androstene-3,17-dione

Tritiated 4-androstene-3,17-dione and testosterone were incubated with submaxillary gland homogenates of 6 month old male mice. In 15 and 180 minute incubations fortified with NADPH, submaxillary tissue converted 4-androstene-3,17-dione predominantly to androsterone and, to a lesser extent, testosterone, 17β-hydroxy-5α-androstan-3-one and 5α-androstane-3α, 17β-diol. Testosterone was converted primarily to 5α-androstane-3α, 17β-diol when exogenous NADPH was available; trace amounts of 4-androstene-3,17-dione, 17β-hydroxy-5α-androstan-3-one and androsterone were also formed. When a NADPH-generating system was omitted from the incubation medium both 4-androstene-3,17-dione and testosterone were poorly metabolized by submaxillary tissue; the amounts of reduced metabolites accumulating were markedly reduced.     

Profiling of urinary steroids by gas chromatography-mass spectrometry detection and confirmation of androstenedione administration using isotope ratio mass spectrometry

Androstenedione (4-androstene-3,17-dione) is banned by the World Anti-Doping Agency (WADA) as an endogenous steroid. The official method to confirm androstenedione abuse is isotope ratio mass spectrometry (IRMS). According to the guidance published by WADA, atypical steroid profiles are required to trigger IRMS analysis. However, in some situations, steroid profile parameters are not effective enough to suspect the misuse of endogenous steroids. The aim of this study was to investigate the atypical steroid profile induced by androstenedione administration and the detection of androstenedione doping using IRMS. Ingestion of androstenedione resulted in changes in urinary steroid profile, including increased concentrations of androsterone (An), etiocholanolone (Etio), 5α-androstane-3α,17β-diol (5α-diol), and 5β-androstane-3α,17β-diol (5β-diol) in all of the subjects. Nevertheless, the testosterone/epitestosterone (T/E) ratio was elevated only in some of the subjects. The rapid increases in the concentrations of An and Etio, as well as in T/E ratio for some subjects could provide indicators for initiating IRMS analysis only for a short time period, 2-22 h post-administration. However, IRMS could provide positive determinations for up to 55 h post-administration. This study demonstrated that, 5β-diol concentration or Etio/An ratio could be utilized as useful indicators for initiating IRMS analysis during 2-36 h post-administration. Lastly, Etio, with slower clearance, could be more effectively used than An for the confirmation of androstenedione doping using IRMS.     

Seized designer supplement named "1-Androsterone": identification as 3β-hydroxy-5α-androst-1-en-17-one and its urinary elimination

New analogues of androgens that had never been available as approved drugs are marketed as “dietary supplement” recently. They are mainly advertised to promote muscle mass and are considered by the governmental authorities in various countries, as well as by the World Anti-doping Agency for sport, as being pharmacologically and/or chemically related to anabolic steroids.In the present study, we report the detection of a steroid in a product seized by the State Bureau of Criminal Investigation Schleswig-Holstein, Germany. The product “1-Androsterone” of the brand name “Advanced Muscle Science” was labeled to contain 100 mg of “1-Androstene-3b-ol,17-one” per capsule. The product was analyzed underivatized and as bis-TMS derivative by GC-MS. The steroid was identified by comparison with chemically synthesized 3β-hydroxy-5α-androst-1-en-17-one, prepared by reduction of 5α-androst-1-ene-3,17-dione with LS-Selectride (Lithium tris-isoamylborohydride), and by nuclear magnetic resonance. Semi-quantitation revealed an amount of 3β-hydroxy-5α-androst-1-en-17-one in the capsules as labeled.Following oral administration to a male volunteer, the main urinary metabolites were monitored. 1-Testosterone (17β-hydroxy-5α-androst-1-en-3-one), 1-androstenedione (5α-androst-1-ene-3,17-dione), 3α-hydroxy-5α-androst-1-en-17-one, 5α-androst-1-ene-3α,17β-diol, and 5α-androst-1-ene-3β,17β-diol were detected besides the parent compound and two more metabolites (up to now not finally identified but most likely C-18 and C-19 hydroxylated 5α-androst-1-ene-3,17-diones). Additionally, common steroids of the urinary steroid profile were altered after the administration of “1-Androsterone”. Especially the ratios of androsterone/etiocholanolone and 5α-/5β-androstane-3α,17β-diol and the concentration of 5α-dihydrotestosterone were influenced. 3α-Hydroxy-5α-androst-1-en-17-one appears to be suitable for the long-term detection of the steroid (ab-)use, as this characteristic metabolite was detectable in screening up to nine days after a single administration of one capsule.     

Effect of oxidizing adulterants on human urinary steroid profiles

Steroid profiling is the most versatile and informative technique adapted by doping control laboratories for detection of steroid abuse. The absolute concentrations and ratios of endogenous steroids including testosterone, epitestosterone, androsterone, etiocholanolone, 5α-androstane-3α,17β-diol and 5β-androstane-3α,17β-diol constitute the significant characteristics of a steroid profile. In the present study we report the influence of various oxidizing adulterants on the steroid profile of human urine. Gas chromatography–mass spectrometry analysis was carried out to develop the steroid profile of human male and female urine. Oxidants potassium nitrite, sodium hypochlorite, potassium permanganate, cerium ammonium nitrate, sodium metaperiodate, pyridinium chlorochromate, potassium dichromate and potassium perchlorate were reacted with urine at various concentrations and conditions and the effect of these oxidants on the steroid profile were analyzed. Most of the oxidizing chemicals led to significant changes in endogenous steroid profile parameters which were considered stable under normal conditions. These oxidizing chemicals can cause serious problems regarding the interpretation of steroid profiles and have the potential to act as masking agents that can complicate or prevent the detection of the steroid abuse.     

Sertoli cells from immature rats : in vitro stimulation of steroid metabolism by FSH.

Sertoli cells from 10 day old rats convert androstenedione to testosterone and 5α-androstane-3α,17β-diol, testosterone to 17β-hydroxy-5α-androstan-3-one and 5α-androstane-3α,17β-diol, and 17β-hydroxy-5α-androstan-3-one to 5α-andro-stane-3α,17β-diol after 72 hours $\text{in vitro}$. Conversions of androstenedione to testosterone and 5α-androstane-3α,17β-diol, and testosterone to 5α-androstane-3α,17β-diol were 2 to 3 times greater in FSH treated cultures. Steroid conversion was not stimulated significantly by LH or TSH. The results are interpreted as evidence that in young rats Sertoli steroid metabolism is stimulated by FSH, that Sertoli cells are an androgen target and that FSH may induce or facilitate Sertoli androgen responsiveness.     

In vitro conversion of testosterone-4-14C to androgens of the 5alpha-androstane series by a normal human ovary

In a previous communication (1) the identification of Δ⁴ -3-oxo-steroids and estrogens as metabolites of testosterone-4-¹⁴C incubated with normal post-ovulatory human ovaries was reported. Thin-layer chromatography of the extracts of those ovaries which contained no corpus luteum yielded zones of radioactivity which were not associated with any of these products. Detailed investigation of these zones from the extract of one of these glands resulted in identification of the following radiometabolites of the 5α-androstane series: 5α-androstane-3,17-dione, androsterone, 3β-hydroxy-5α-androstan-17-one, 17β-hydroxy-5α-androstan-3-one, 5α-androstane-3ga, 17β-diol and 5α-androstane-3β, 17β-diol. The capacity of a normal human ovary to produce these 5α-reduced androgens, especially the potent 17β-hydroxy-steroids, suggests a regulatory role of these compounds in ovarian function.     

A molecularly imprinted receptor for separation of testosterone and epitestosterone, based on a steroidal cross-linker

A series of molecularly imprinted polymers have been prepared and investigated as stationary phases in high performance liquid chromatography for the separation of testosterone and epitestosterone using non-polar mobile phases. The polymers were imprinted using 5α-dihydrotestosterone as template, and all retain testosterone more strongly than its 17α-OH epimer. The best polymer was prepared using trifluoromethylacrylic acid as functional monomer (interacting with the template via hydrogen bonds), divinylbenzene as ‘inert’ cross-linker, and chloroform as porogen. It also included a steroid-based cross-linker, which may interact with the template via van der Waals interactions to lend additional ‘shape selectivity’. A 250 × 4.6 mm column packed with this polymer gave baseline resolution of testosterone and epitestosterone (15 μg each) in under 20 min. Preparation of the steroid based cross-linker included the selective reduction of 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) to the 3α,17β-diol using K-selectride.     

Preparation of some C 19 steroid-protein conjugates

17-Carboxymethoximino derivatives of DHA (1), androsterone and etiocholanolone, as well as the 7-carboxymethoximino derivatives of 3β-hydroxy-androst-5-ene-7,17-dione and 7-keto-androst-5-ene-3β, 17β-diol have been prepared and conjugated to BSA for use in producing antisera to the corresponding C₁₉ steroids.     

Urinary marker of oral pregnenolone administration

Pregnenolone (PREG) can potentially be abused by athletes to maintain an equilibration of the steroidal environment after sex steroids administrations. Five men volunteers orally ingested 50 mg PREG to determine optimal urinary markers for detection of this steroid. Our findings show that ingestion of PREG has no significant effects on the testosterone/epitestosterone (T/E) and testosterone/luteinizing hormone (T/LH) ratios, whereas variable changes on the carbon isotopic values of three T metabolites: androsterone, etiocholanolone, 5beta-androstane-3alpha,17beta-diol (5beta-androstanediol) together with 16(5alpha)-androsten-3alpha-ol (androstenol) and 5beta-pregnane-3alpha,20alpha-diol (pregnanediol) have been observed. The difference between the carbon isotopic values (delta13C-values) of androstenol and pregnanediol is potentially the most reliable marker of exogenous PREG administration in males. For all subjects, the differences differ by 3.0 per thousand or more over a period of about 10 h and for both of them the detection window for positivity is extended over 40 h.     

Development of a GC/C/IRMS method - Confirmation of a novel steroid profiling approach in doping control

In doping control, an athlete can only be convicted with the misuse with endogenous steroids like testosterone (T), if abnormal values of steroid metabolites and steroid ratios are observed and if the subsequent analysis with isotope ratios mass spectrometry (IRMS) confirms the presence of exogenously administered androgens. In this work, we compare the results of a novel steroid profiling approach with the performance an in-house developed IRMS method. The developed IRMS has the advantage over other methods to be relatively short in time and with target compounds androsterone, etiocholanolone, 5β-androstane 3α,17β-diol and 5α-androstane 3α,17β-diol. Pregnanediol was used as an endogenous reference compound (ERC). Reference limits for the IRMS values were established and applied as decision limits for the evaluation of excretion urine from administration with oral T, T-gel, dihydrotestosterone (DHT) - gel and dehydroepiandrosterone (DHEA). Results indicated the importance of both androstanediols as important IRMS markers where relative values compared to an ERC (Δδ(13)C) yielded better detection accuracy than absolute δ(13)C-values. The detection times of all administered endogenous steroids were evaluated using the proposed thresholds. The results of traditional steroid profiling and a new approach based upon minor steroid metabolites monitoring introduced in a longitudinal framework were evaluated with IRMS. With traditional steroid profiling methods, 95% of the atypical samples could be confirmed whereas an additional 74% of IRMS confirmed was provided by a new biomarkers strategy. These results prove that the other steroid profiling strategies can improve the efficiency in detection of misuse with endogenous steroids.     

Clinical analysis on steroids. XII. Occurrence of D-homoannulation during the hot acid hydrolysis of 5β-pregnane-3α,20α-diol disulfate

Two D-homosteroids were isolated from the hydrolyzate of 5β-pregnane -3α,20α-diol disulfate (II) when it was refluxed in 3N hydrochloric acid. The structures of these steroids have been elucidated as 17α-methyl-D-homo-5β-androstane-3α, 17aβ-diol (VI) and 17α-methyl-17aγb-chloro-D-homo-5β-androstan-3α-ol (VIII) by instrumental analyses. The former was identical with a synthetic specimen derived from 5β-pregnane-3α,20β-diol di-sulfate (IV) by uranediol rearrangement. The main hydrolyzates obtained were 17α-ethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol (V) and 5β-pregnane-3α, 20α-diol (III).     

Longitudinal profiling of urinary steroids by gas chromatography/combustion/isotope ratio mass spectrometry: diet change may result in carbon isotopic variations

Longitudinal profiling of urinary steroids was investigated by using a gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) method. The carbon isotope ratio of three urinary testosterone (T) metabolites: androsterone, etiocholanolone, 5beta-androstane-3alpha,17beta-diol (5beta-androstanediol) together with 16(5alpha)-androsten-3alpha-ol (androstenol) and 5beta-pregnane-3alpha,20alpha-diol (5beta-pregnanediol) were measured in urine samples collected from three top-level athletes over 2 years. Throughout the study, the subjects were living in Switzerland and were residing every year for a month or two in an African country. (13)C-enrichment larger than 2.5 per thousand was observed for one subject after a 2-month stay in Africa. Our findings reveal that (13)C-enrichment caused by a diet change might be reduced if the stay in Africa was shorter or if the urine sample was not collected within the days after return to Switzerland. The steroids of interest in each sample did not show significant isotopic fractionation that could lead to false positive results in anti-doping testing. In contrast to the results obtained with the carbon isotopic ratio, profiling of urinary testosterone/epitestosterone (T/E) ratios was found to be unaffected by a diet change.     

Steroid metabolism in human breast cancer cell lines

The metabolism of 1,2-³H-androstenedione was studied in 2 cell lines, MCF-7 (estrogen responsive) and BT-20 (estrogen nonresponsive) over 48 hrs. Water soluble and unconjugated metabolites were separated by solvent partition and the former was submitted to chromatography on Sephadex LH-20 and enzyme hydrolysis. The resulting unconjugated steroids were separated by paper chromatography and identities were established by reverse isotope dilution. The unconjugated steroids initially obtained were separated by chromatography and identified by reverse isotope dilution. About 70% of the androstenedione was metabolized by both cell lines. However, the respective conversions to conjugates by MCF-7 and BT-20 were 31% and 0.32%. In the former, glucosiduronates predominated (94%) and consisted of androsterone (55%), etiocholanolone (9.4%) and androstanediol (5α-androstane-3α,17β-diol) (9.3%). Androsterone comprised most of the unconjugated metabolites in both cell lines. Androstanediol was found in both cell lines, 2% in MCF-7 and 12% in BT-20. Testosterone, 5α-androstane-3,17-dione and 3β-hydroxy-5α-androstan-17-one were isolated only from MCF-7. The metabolism of ³H-estriol was studied in a similar way. Both cell lines produced about equal amounts of estriol-3-sulfate (9%) and a compound with properties of estriol-3-glucosiduronate (0.15 – 0.5%). The results worthy of emphasis are: 1. The far greater conjugation of androgens exhibited by the MCF-7 cell lines as compared to the BT-20 cell lines; 2. In MCF-7, the high conversion of androstenedione to etiocholanolone (glucosiduronate form), a metabolite reported to form only in liver and sebaceous cysts; 3. The possible formation in both cell lines of estriol-3-glucosiduronate, normally a metabolite of the intestine.     

Biotransformation of testosterone and other androgens by suspension cultures of Nicotiana tabacum “Bright yellow”

It has been shown that the cultured cells of Nicotiana tabacum “Bright Yellow” are capable of transforming testosterone to Δ⁴-androstene-3, 17-dione, 5α-androstan-17β-ol-3-one, 5α-androstane-3β, 17β-diol, its dipalmitate and 3- and 17-monoglucosides, epiandrosterone, its palmitate and glucoside, testosterone glucoside. 5α-Androstane-3β, 17β-diol dipalmitate and 3- and 17-monoglucosides, epiandrosterone palmitate and glucoside, and testosterone glucoside have been found for the first time as metabolites of testosterone in plant systems. Δ⁴-Androstene-3,17-dione was converted to testosterone. 5α-Androstan-17β-ol-3-one, which has been recognized as an active form of testosterone in mammals, was also detected. It has also been demonstrated that [4-¹⁴C]testosterone is actively incorporated in these transformations.     

Determination of urine steroid profile in untrained men to evaluate recovery after a strength training session

Intense physical exercise is an important modifier of hormone metabolism. The aim of this study was to evaluate the variations in the urine profile of glucuroconjugated steroids (androgens, estrogens, and corticosteroids) as a consequence of a session of strength exercises. The subjects were a group (N = 20) of untrained male university students. They performed 3 sets of 10 repetitions, with a 3-minute recovery time between sets, at 70-75% of 1 repetition maximum (1RM). Four urine samples were collected per subject: before the session, immediately after, 3 hours after, and 48 hours after the session. They were assayed using a gas chromatograph coupled with a mass spectrometer. The concentrations of the different hormones were determined according to the urine creatinine level (ng steroid per mg creatinine). The substances assayed were testosterone, epitestosterone (Epit), androstenedione, dehydroepiandrosterone (DHEA), androsterone, etiocholanolone, beta-estradiol, estrone, tetrahydrocortisone (THE), and tetrahydrocortisol (THF). The results showed a significant decline after exercise with respect to the rested state in the urinary excretion of testosterone, Epit, DHEA, androsterone, and etiocholanolone. At 48 hours, there was a significant increase in the urinary excretion of Epit, androstenedione, androsterone, etiocholanolone, estrone, and THE. The androsterone + etiocholanolone/THE + THF ratio decreased after exercise, increased significantly (p < 0.05) at 3 hours, and returned to near resting levels at 48 hours. The data suggest that the performing a strength session at 70-75% of maximum strength provoked a state of fatigue in the subjects, from which they recovered 48 hours after the exercise.     

Alteration in the normal pattern of serum testosterone and 5α-androstane-3α,17β-diol in the immature male rat following chronic treatment with luteinizing hormone releasing hormone or luteinizing hormone

A major component of sexual maturation in the male rat is a progressive decline in serum concentrations of 5α-androstane-3α,17β-diol (3α-diol) and a concomitant increase in testicular testosterone biosynthesis and secretion. Chronic administration of synthetic luteinizing hormone releasing hormone (LHRH) or luteinizing hormone (LH)/human chorionic gonadotropin (hCG) to immature male rats has been shown to result in a delay in sexual maturation as evidenced by decreased sex accessory gland weights and altered testicular testosterone production. We have examined the postulate that such treatments may either reverse or retard the normal developmental pattern of serum testosterone and 3α-diol concentrations. Chronic $\text{in vivo}$ treatment of 28 day old immature male rats for 2 weeks with daily injections of either 0.5 μg of LHRH, 1.0 μg of LHRH, or 30 μg of LH was found to result in significant reductions in weights of the seminal vesicles and ventral prostate glands and diminutions in serum testosterone concentrations. Serum content of 3α-diol was either unchanged or slightly elevated in the LHRH treated animals and increased significantly in the LH treated animals. These data suggest that either a reversal of or retardation in the normal developmental pattern of serum testosterone and 3α-diol content has been achieved in the immature male rat by chronic LHRH or LH treatment.     

Metabolism of 17β-hydroxy-2-hydroxymethylene-5α-androstan-3-one in the rabbit

An acidic metabolite, 2α-carboxy-5α-androstane-3α, 16α, 17αtriol and two neutral metabolites, 2α-hydroxymethyl-5α-androstane-3α, 17α-diol, and 2α-hydroxymethyl-5α-androstane-3α, 16α, 17α-triol have been identified in the urine of rabbits orally dosed with 17β-hydroxy-2-hydroxymethylene-5α-androstan-3-one. 2α-Hydroxymethyl-5α-androstane-3α, 16α, 17α-triol was previously obtained from the urine of rabbits dosed with 17β-hydroxy-2α-methyl-5α-androstan-3-one. The acidic metabolite was the major urinary excretion product.     

Androgen metabolism by rat epididymis. 2. Metabolic conversion of 3H-testosterone in vitro

The epididymis of adult rats metabolizes ³H-testosterone by experiments $\text{in}$$\text{vitro}$. After incubation of slices from epididymal tissue for 2 hrs at 37°C, 8% of the total radioactivity was found in the water-soluble fraction, whereas 92% in the ether soluble fraction (free steroids). The free steroids were examined further and the following metabolites identified: $\text{testosterone}$ (17β-hydroxy-4-androsten-3-one) 10,4%, $\text{androstendione}$ (4-androstene-3,17-dione) 6,2%, $\text{5α-A-dione}$ (5α-androstane-3,17-dione) 7,3%, $\text{DHT}$ (17β-hydroxy-5α-androstane-3-one) 39,3%, $\text{3α-diol}$ (5α-androstane-3α,17β-diol) 22,7%, $\text{3β-diol}$ (5α-androstane-3β,17β-diol) 4,6% and androsterone(3α-hydroxy-5α-androstan-17-one) 8,9%. The relative amount of each metabolite is given in per cent of the total radioactivity in the ether soluble fraction. When segments (caput, corpus, cauda) of epididymis were incubated in the same way, differences in steroid metabolism were demonstrated. Characteristic for caput epididymidis was high formation of DHT (58,4%) and 3α-diol (23,5%). Corpus epididymidis showed lower formation of DHT (50,6%) and 3α-diol (12,7%), but an approximately 3 times higher formation of 5α-A-dione (12,0%) than caput (3,4%) and cauda (3,5%). Cauda epididymis showed the lowest formation of DHT (38,3%), whereas 3α-diol (29,1%) and androsterone (11,4%) formation were relatively high. The ratio between 17β-hydroxy metabolites (DHT and androstanediols) and 17-keto metabolites were much higher in the caput (8,8) than in the corpus (3,2) and cauda (3,6), indicating a higher 5α-reductase activity in this segment.     

Synthesis of 5α-Androstane-3α,16α,17β-triol from 3β-hydroxy-5-androsten-17-one

5α-Androstane-3α, 16α 17β-triol was synthesized from 3β-hy-droxy-5-androsten-17-one. The procedure Involved catalytic hydrogenation of 3β-hydroxy-5-androsten-17-one to 3β-hydroxy-5α-androstan-17-one. This was followed by conversion of the 3β-hydroxy group to 3α-benzoyloxy group by the Mitsunobu reaction. Further treatment with isopropenyl acetate yielded 5α-androsten-16-ene-3α, 17-diol 3-benzoate 17-acetate. This was then converted to 3α, 17-dihydroxy-5α-androstan-16-one 3-benzoate 17-acetate via the unstable epoxide intermediate after treatment with $\text{m}$-cloroperoxybenzoic acid. LiAlH₄ reduction of this compound formed 5α-androstane-3α, 16α, 17β-trlol. ¹H and ¹³C NMR of various steroids are presented to confirm the structure of this compound.