Fractionation of free and conjugated steroids for the detection of boldenone metabolites in calf urine with ultra-performance liquid chromatography/tandem mass spectrometry

For over a decade there has been an intensive debate on the possible natural origin of boldenone (androst-1,4-diene-17beta-ol-3-one, 17beta-boldenone) in calf urine and several alternative markers to discriminate between endogenously formed boldenone and exogenously administered boldenone have been suggested. The currently approved method for proving illegal administration of beta-boldenone(ester) is the detection of beta-boldenone conjugates. In the presented method the sulphate, glucuronide and free fractions are separated from each other during cleanup on a SAX column to be able to determine the conjugated status of the boldenone metabolites. The sulphate and glucuronide fractions are submitted to hydrolysis and all three fractions are further cleaned up on a combination of C18/NH2 solid-phase extraction (SPE) columns. Chromatographic separation of the boldenone metabolites was achieved with a Waters Acquity UPLC instrument using a Sapphire C18 (1.7 microm; 2x50 mm) column within 5 min. Detection of the analytes was achieved by electrospray ionisation tandem mass spectrometry. The decision limits of this method, validated according to Commission Decision 2002/657/EC, were 0.08 ng mL(-1) for androsta-1,4-diene-3,17-dione, 0.13 ng mL(-1) for androst-4-ene-3,17-dione, 0.11 ng mL(-1) for 17alpha-boldenone, 0.07 ng mL(-1) for 17beta-boldenone, 0.24 ng mL(-1) for 5beta-androst-1-en-17beta-ol-3-one and 0.58 ng mL(-1) for 6beta-hydroxy-17beta-boldenone. Because of the fractionation approach used in this method there is no need for conjugated reference standards which often are not available. The disadvantage of needing three analytical runs to determine the conjugated status of each of the metabolites was overcome by using fast chromatography.     

Biotransformation of (+)-androst-4-ene-3,17-dione

Fermentation of (+)-androst-4-ene-3,17-dione (1) with Curvularia lunata for 10 days yielded five oxidative and reductive metabolites, androsta-1,4-diene-3,17-dione (2), 17beta-hydroxyandrosta-1,4-dien-3-one (3), 11alpha-hydroxyandrost-4-ene-3,17-dione (4), 11alpha,17beta-dihydroxyandrost-4-en-3-one (5) and 15alpha-hydroxyandrosta-1,4-dien-17-one (6). The structures of these metabolites were elucidated on the basis of spectroscopic techniques. These microbially transformed products were assayed against the clinically important enzymes, tyrosinase and prolyl endopeptidase.     

The ultimate veal calf reference experiment: hormone residue analysis data obtained by gas and liquid chromatography tandem mass spectrometry

A lifetime controlled reference experiment has been performed using 42 veal calves, 21 males and 21 females which were fed and housed according to European regulations and common veterinary practice. During the experiment feed, water, urine and hair were sampled and feed intake and growth were monitored. Thus for the first time residue analysis data were obtained from guaranteed lifetime-untreated animals. The analysis was focused on the natural hormones estradiol and testosterone and their metabolites, on 17beta- and 17alpha-nortestosterone, on 17beta- and 17alpha-boldenone and androsta-1,4-diene-3,17-dione (ADD), and carried out by gas chromatography tandem mass spectrometry (GC/MS/MS), an estrogen bioassay and liquid chromatography (LC) MS/MS. Feed, water and hair samples were negative for the residues tested. Female calf urines showed occasionally low levels of 17alpha-estradiol and 17alpha-testosterone. On one particular sampling day male veal calf urines showed very high levels of 17alpha-testosterone (up to 1000 ng mL(-1)), accompanied by lower levels of estrone and 17beta-testosterone. Despite these extreme levels of natural testosterone, 17beta-boldenone was never detected in the same urine samples; even 17alpha-boldenone and ADD were only occasionally beyond CCalpha (maximum levels 2.7 ng mL(-1)). The data from this unique experiment provide a set of reference values for steroid hormones in calf urine and demonstrate that 17beta-boldenone is not a naturally occurring compound in urine samples.     

Microbial transformation of (+)-androsta-1 ,4-diene-3,17-dione by Cephalosporium aphidicola

Fermentation of (+)-androsta-1,4-diene-3,17-dione ([structure: see text]) with Cephalosporium aphidicola for 8 days yielded oxidative and reductive metabolites, androst-4-ene-3,17-dione ([structure: see text]), 17beta-hydroxyandrosta-1,4-diene-3-one ([structure: see text]), 11alpha-hydroxyandrosta-1,4-diene-3,17-dione ([structure: see text]), 11alpha-hydroxyandrost-4-ene-3,17-dione ([structure: see text]), 11alpha,17beta-dihydroxyandrost-4-ene-3-one ([structure: see text]) and 11alpha,17beta-dihydroxyandrosta-1,4-diene-3-one ([structure: see text]). The fermentation of [structure: see text] with Fusarium lini also yielded metabolites [structure: see text]. The structures of these metabolites were elucidated on the basis of spectroscopic techniques.     

New synthesis of estradiol from androsta-1,4-diene-3,17-dione

A new method for the aromatization of ring A in androsta-1,4-diene-3,17-dione, available from sterols by means of the microbiological degradation of the side chain, was developed. The method consists of the reduction of androsta-1,4-diene-3,17-dione to the corresponding dienediol followed by double C,O-deprotonation of ring A, accompanied by expulsion of the 19-methyl group and formation of estradiol in a high yield. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 599–601, March, 1999.     

Detection of androst-4-ene-3,6,17-trione (6-OXO) and its metabolites in urine by gas chromatography-mass spectrometry in relation to doping analysis

The metabolism and excretion of androst-4-ene-3,6,17-trione after administration of the 'nutritional' supplement 6-OXO was investigated by gas chromatography-mass spectrometry (GC-MS) in full-scan mode. The parent drug androst-4-ene-3,6,17-trione and androst-4-ene-6alpha,17beta-diol-3-one and androst-4-ene-6alpha-ol-3,17-dione were detected in the post-administration urine samples. Because androst-4-ene-3,6,17-trione is an anabolic steroid and an aromatase inhibitor, this substance is regarded as a doping agent. Hence, a selective and sensitive GC-MS method in selected ion monitoring mode for the detection of the TMS-enol-TMS-ether derivatives of these substances was developed and validated for doping control purposes. The limit of detection (LOD) of the investigated compounds ranged from 5 to 10 ng/mL. Using this method, the detection time for androst-4-ene-3,6,17-trione and androst-4-ene-6alpha,17beta-diol-3-one was 24 h, while androst-4-ene-6alpha-ol-3,17-dione could be detected up to 37 h after administration of the dose recommended by the manufacturer.     

Totalsynthese optisch aktiver Steroide. XIII. Mitteilung. Synthese von 4,9(11)-Androstadien-3,17-dion

Synthesis of 4,9(11)-androstadiene-3,17-dione. The total synthesis of 4,9(11)-androstadiene-3,17-dione ( 23 ) is described starting from (1 S, 7 aS)-1-t-butoxy-7a-methyl-5,6,7,7a-tetrahydroindan-5-one ( 8 ) as a CD-building block. The key step involves alkylation of 8 with 1-chloro-7,7-o-phenylenedioxy-octane-3-one ( 14 ) a novel AB-synthon to give the diketone 15 . 15 is converted to the secosteroid 20 via the diene enol ether 17 using a previously described procedure. Low temperature methylation of 20 affords predominantly the 10β-methyl compound 21 , which is readily transformed into the title dione 23 .     

Testosterone metabolism revisited: discovery of new metabolites

The metabolism of testosterone is revisited. Four previously unreported metabolites were detected in urine after hydrolysis with KOH using a liquid chromatography–tandem mass spectrometry method and precursor ion scan mode. The metabolites were characterized by a product ion scan obtained with accurate mass measurements. Androsta-4,6-dien-3,17-dione, androsta-1,4-dien-3,17-dione, 17-hydroxy-androsta-4,6-dien-3-one and 15-androsten-3,17-dione were proposed as feasible structures for these metabolites on the basis of the mass spectrometry data. The proposed structures were confirmed by analysis of synthetic reference compounds. Only 15-androsten-3,17-dione could not be confirmed, owing to the lack of a commercially available standard. That all four compounds are testosterone metabolites was confirmed by the qualitative analysis of several urine samples collected before and after administration of testosterone undecanoate. The metabolite androsta-1,4-dien-3,17-dione has a structure analogous to that of the exogenous anabolic steroid boldenone. Specific transitions for boldenone and its metabolite 17β-hydroxy-5β-androst-1-en-3-one were also monitored. Both compounds were also detected after KOH treatment, suggesting that this metabolic pathway is involved in the endogenous detection of boldenone previously reported by several authors.     

Characterization of boldione and its metabolites in human urine by liquid chromatography/electrospray ionization mass spectrometry and gas chromatography/mass spectrometry

Boldione (1,4-androstadiene-3,17-dione) is a direct precursor (prohormone) to the anabolic steroid boldenone (1,4-androstadiene-17beta-ol-3-one). It is advertised as a highly anabolic/androgenic compound promoting muscularity, enhancing strength and overall physical performance, and is available on the Internet and in health stores. This work was undertaken to determine and characterize boldione and its metabolites in human urine, using both liquid chromatography with electrospray ionization mass spectrometry and gas chromatography with mass spectrometry and derivatization. Boldione and its three metabolites were detected in dosed human urine after dosing a healthy volunteer with 100 mg boldione. The excretion studies showed that boldione and its metabolites were detectable in urine for 48 h after oral administration, with maximum excretion rates after 1.8 and 3.6 h (boldenone case). The amounts of boldione and boldenone excreted in urine from this 100 mg dose were 34.45 and 15.95 mg, respectively.     

Trace analysis of 28 steroids in surface water, wastewater and sludge samples by rapid resolution liquid chromatography-electrospray ionization tandem mass spectrometry

A sensitive rapid resolution liquid chromatography-tandem mass spectrometry (RRLC-MS/MS) method, combined with solid-phase extraction, ultrasonic extraction and silica gel cartridge cleanup, was developed for 28 steroids including 4 estrogens (estrone (E1), 17β-estradiol (E2), 17α-ethynyl estradiol (EE2), diethylstilbestrol (DES)), 14 androgens (androsta-1,4-diene-3,17-dione (ADD), 17α-trenbolone, 17β-trenbolone, 4-androstene-3,17-dione, 19-nortestoserone, 17β-boldenone, 17α-boldenone, testosterone (T), epi-androsterone (EADR), methyltestosterone (MT), 4-hydroxy-androst-4-ene-17-dione (4-OHA), 5α-dihydrotestosterone (5α-DHT), androsterone (ADR), stanozolol (S)), 5 progestagens (progesterone (P), ethynyl testosterone (ET), 19-norethindrone, norgestrel, medroxyprogesterone (MP)), and 5 glucocorticoids (cortisol, cortisone, prednisone, prednisolone, dexamethasone) in surface water, wastewater and sludge samples. The recoveries of surface water, influents, effluents and sludge samples were 90.6-119.0% (except 5α-DHT was 143%), 44.0-200%, 60.7-123% and 62.6-138%, respectively. The method detection limits for the 28 analytes in surface water, influents, effluents and freeze-dried sludge samples were 0.01-0.24 ng/L, 0.02-1.44 ng/L, 0.01-0.49 ng/L and 0.08-2.06 ng/g, respectively. This method was applied in the determination of the residual steroidal hormones in two surface water of Danshui River, 12 wastewater and 8 sludge samples from two wastewater treatment plants (Meihu and Huiyang WWTPs) in Guangdong (China). Ten analytes were detected in surface water samples with concentrations ranging between 0.4 ng/L (17β-boldenone) and 55.3 ng/L (5α-DHT); twenty analytes in the wastewater samples with concentrations ranging between 0.3 ng/L (P) and 621 ng/L (5α-DHT); and 12 analytes in the sludge samples with concentrations ranging between 1.6 ng/g (E1) and 372 ng/g (EADR).     

Studies related to the origin of C18 neutral steroids isolated from extracts of urine from the male horse: the identification of urinary 19-oic acids and their decarboxylation to produce estr-4-en-17beta-ol-3-one (19-nortestosterone) and estr-4-ene-3,17-dione (19-norandrost-4-ene-3,17-dione) during sample processing

For almost two decades we have known that enzymatic hydrolysis of "normal" urine samples from the entire male horse using Escherichia coli (E. coli) followed by solvolysis (ethyl acetate:methanol:sulphuric acid) results in the detection of significant amounts of estr-4-ene-3,17-dione (19-norandrost-4-ene-3,17-dione) along with estr-4-en-17beta-ol-3-one (19-nortestosterone, nandrolone) in extracts of the hydrolysed urine and that both steroids are isolated from the solvolysis fraction. This solvolysis process is targeted at the steroid sulphates. Also we have shown that 19-norandrost-4-ene-3,17-dione and 19-nortestosterone are isolated from testicular tissue extracts. Subsequently, evidence was obtained that 19-nortestosterone detected in extracts of "normal" urine from male horses may not be derived from the 17beta-sulphate conjugate. However, following administration of 19-nortestosterone based proprietary anabolic steroids to all horses (males, females and castrates), the urinary 19-nortestosterone arising from the administration is excreted primarily as the 17beta-sulphate conjugate. Thus, if the 19-nortestosterone-17beta-sulphate conjugate arises only following administration this has interesting implications for drug surveillance programmes to control administration of 19-nortestosterone based anabolic preparations to male horses. These results have led us to consider that the precursors to 19-nortestosterone and 19-norandrost-4-ene-3,17-dione, present in the urine prior to the hydrolysis steps, have the same basic structure except for the functionality at the 17-position. We have used preparative high pressure liquid chromatography (LC) and LC fractionation to separate these precursors from the high amounts of oestrogenic sulphates present in "normal" urine from the entire male horse. Purified fractions have then been studied by liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) to identify the precursors.     

Metabolism of androsta‐1,4,6‐triene‐3,17‐dione and detection by gas chromatography/mass spectrometry in doping control

The urinary metabolism of the irreversible aromatase inhibitor androsta‐1,4,6‐triene‐3,17‐dione was investigated. It is mainly excreted unchanged and as its 17β‐hydroxy analogue. For confirmation, 17β‐hydroxyandrosta‐1,4,6‐trien‐3‐one was synthesized and characterized by nuclear magnetic resonance (NMR) in addition to the parent compound. In addition, several reduced metabolites were detected in the post‐administration urines, namely 17β‐hydroxyandrosta‐1,4‐dien‐3‐one (boldenone), 17β‐hydroxy‐5β‐androst‐1‐en‐3‐one (boldenone metabolite), 17β‐hydroxyandrosta‐4,6‐dien‐3‐one, and androsta‐4,6‐diene‐3,17‐dione. The identification was performed by comparison of the metabolites with reference material utilizing gas chromatography/mass spectrometry (GC/MS) of the underivatized compounds and GC/MS and GC/tandem mass spectrometry (MS/MS) of their trimethylsilyl (TMS) derivatives. Alterations in the steroid profile were also observed, most obviously in the androsterone/testosterone ratio. Even if not explicitly listed, androsta‐1,4,6‐triene‐3,17‐dione is classified as a prohibited substance in sports by the World Anti‐Doping Agency (WADA) due to its aromatase‐inhibiting properties. In 2006 three samples from human routine sports doping control tested positive for metabolites of androsta‐1,4,6‐triene‐3,17‐dione. The samples were initially found suspicious for the boldenone metabolite 17β‐hydroxy‐5β‐androst‐1‐en‐3‐one. Since metabolites of androst‐4‐ene‐3,6,17‐trione were also present in the urine samples, it is presumed that these findings were due to the administration of a product like ‘Novedex Xtreme’, which could be easily obtained from the sport supplement market. Copyright © 2008 John Wiley & Sons, Ltd.     

Development of a rapid normal-phase LC-positive ion APCI-MS method for simultaneous detection and quantitation of cholesterol, androst-4-ene-3,1 7-dione, and androsta-1,4-diene-3,17-dione

This paper describes the development of a normal-phase liquid chromatograph-UV-diode array detection-positive ion atmospheric pressure chemical ionization-mass spectrometry method for the simultaneous identification and quantitation of cholesterol, androst-4-ene-3,17-dione (AD), and androsta-1,4-diene-3,17-dione (ADD) in fermentation broths. The compounds detected under positive ion atmospheric pressure chemical ionization on a mass spectrometer by selected ion monitoring are separated by normal-phase high-performance liquid chromatography. [M+H]+ ions are taken into consideration for quantitation of AD and ADD, and [M-H2O+H]+ ions are considered for quantitation of cholesterol. The compounds are analyzed on a Si60 silica (5 microm, 125 x 4-mm i.d.) Merck column using a 2:3 isocratic mixture of isopropyl alcohol and hexane. The calibration curves resulting from the reference compounds in the concentration range of 100-5000 pg on column exhibit a good linear correlation (r2 > or = 0.996). The method is validated by analyzing six replicates of broth samples fortified with three compounds, namely, cholesterol, AD, and ADD, at 0.050 and 0.5 microg/g levels. The mean recoveries for the fortifications range from 90% to 98% with relative standard deviations in the range of 3.36% to 9.78%. The method is developed to study the qualitative as well as quantitative conversion of cholesterol to AD and ADD by a microorganism identified as Nocardia sp. These studies helped the investigation of the reaction kinetics, which showed that the molar biotransformation of cholesterol into AD and ADD was 21%, even when the reaction was prolonged for 96 h.     

Development and validation of a liquid chromatography-tandem mass spectrometry method for the separation of conjugated and unconjugated 17alpha- and 17beta-boldenone in urine sample

Natural occurrence or illegal treatment of boldenone (BOLD) presence in cattle urine is under debate within the European Union. Separation of conjugated and unconjugated forms of 17alpha-boldenone (alpha-BOLD) and 17beta-boldenone (beta-BOLD) and presence of related molecules as androsta-1,4-diene-3,17-dione (ADD) appear critical points for the decision of an illegal use. The aim of this study is a new analytical approach of BOLD and ADD confirmation in cattle urine. The separation between conjugated and unconjugated forms of BOLD was obtained by a preliminary urine liquid-liquid extraction step with ethyl acetate. In this step the organic phase extracts only unconjugated BOLD and ADD, while BOLD in conjugated form remain in urine phase. Afterwards the urine phase, contains conjugated BOLD, was subjected to an enzymatic deconjugation. Solid-phase extraction (OASIS-HLB Waters) was used for the purification and concentration of analytes in organic and urine phases and liquid chromatography ion electrospray tandem mass spectrometry (LC-MS-MS) was applied for the confirmation of BOLD and ADD, using deuterium-labelled 17beta-boldenone (BOLD-d3) as internal standard. The method was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/CE. The results obtained demonstrate that the developed method show very high specificity, precision, trueness and ruggedness. Decision limits (CCalpha) smaller than 0.5 ng mL(-1) were obtained for each analyte.     

Determination of anabolic steroids in creatine nutritional supplements after supercritical fluid extraction

The effects of various parameters, i.e. extraction pressure, temperature, time, and modifier on the efficiency of extraction were investigated using an analytical-scale supercritical fluid extraction system. An optimal set of conditions for the extraction and determination by gas chromatography-mass spectrometry of trimethylsilyl derivatives of 4-androstene-3,17-dione, 1,4-androstadiene-3,17-dione, nandrolone, and testosterone in nutritional supplements was developed. The optimum amount of creatine supplement was 1 g, while the optimum pressure and temperature were determined to be 35 MPa and 80 °C, respectively. The optimum dynamic extraction time was 45 min. The limit of detection (LOD) of the investigated compounds ranged from 5 to 25 ng · g⁻¹ of supplement, while recoveries ranged from 76.1 to 86.6%. Correspondence: Petra Mikulcikova, Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, nám. Cs. Legií 565, CZ 532 10 Pardubice, Czech Republic     

Evaluation of boldenone formation and related steroids transformations in veal faeces by liquid chromatography/tandem mass spectrometry

It is established that bovine urine can result positive for boldenone and androstadienedione in consequence of faecal contamination. The simple transfer of steroids to urine is one minor aspect of faecal contamination. A high de novo production of steroids in faeces after deposition and in faeces-contaminated urine is almost certainly due to microbial activity, although the precursor compounds and transformations leading to the presence of these illegal steroids are unclear. We developed a simple in vitro method - incubation of faecal matter suspended in 0.9% saline - to induce steroid transformations in faeces, and analyzed the products by liquid chromatography/tandem mass spectrometry, without the need for prior extraction. Norethandrolone was the internal standard. The linearity (R(2): 0.987-0.999), sensitivity (LODs: 0.3 to 1.0 ng/mL; LOQs: 1.0 to 3.0 ng/mL), precision (intra-day CVs: 2.6-8.2; inter-day CVs: 4.5-11.5) and accuracy (percentage recovery: 89-120%) were calculated for the studied steroids. Androstenedione, androstadienedione, alpha- and beta-boldenone, testosterone and epitestosterone transformations were investigated. Mutual interconversion of steroids was observed, although 17beta-hydroxy steroids had low stability compared with 17alpha-hydroxy and 17-keto steroids. The results suggest that this simple in vitro system may be an effective way of studying hormone transformations in faeces and, after analogue studies, in faeces-contaminated urine.     

6alpha-Methylandrostenedione: gas chromatographic mass spectrometric detection in doping control

In recent years products containing 6alpha-methylandrost-4-ene-3,17-dione have appeared on the sport supplement market. Scientific studies have proven aromatase inhibition and anabolic and mild androgenic properties; however, no preparation has been approved for medical use up to now. In sports 6alpha-methylandrost-4-ene-3,17-dione has to be classified as a prohibited substance according to the regulations of the World Anti-Doping Agency (WADA). For the detection of its misuse the metabolism was studied following the administration of two preparations obtained from the Internet (Formadrol and Methyl-1-Pro). Several metabolites as well as the parent compounds were synthesized and the structures of 3alpha-hydroxy-6alpha-methyl-5beta-androstan-17-one, 6alpha-methylandrost-4-ene-3,17-dione, and 5beta-dihydromedroxyprogesterone were confirmed by nuclear magnetic resonance (NMR) spectroscopy. The main metabolite, 3alpha-hydroxy-6alpha-methyl-5beta-androstan-17-one, was found to be excreted as glucuronide and was still detectable in microg/mL amounts until urine collection was terminated (after 25 h). Additionally, samples from routine human sports doping control had already tested positive for the presence of metabolites of 6alpha-methylandrost-4-ene-3,17-dione. Screening analysis can be easily performed by the existing screening procedure for anabolic steroids using 3alpha-hydroxy-6alpha-methyl-5beta-androstan-17-one as target substance (limit of detection <10 ng/mL). Its discrimination from the closely eluting drostanolone metabolite, 3alpha-hydroxy-2alpha-methyl-5alpha-androstan-17-one, is possible as the mono-TMS derivative.     

Identification of 4-hydroxyandrost-4-ene-3,17-dione metabolites in prostatic cancer patients by liquid chromatography-mass spectrometry.

Liquid chromatography with thermospray mass spectrometry has proved to be an invaluable technique for the study of metabolic degradation of xenobiotics in complex biological fluids. This paper describes the detection of 4-hydroxyandrost-4-ene-3,17-dione and its metabolites in urinary extracts from prostatic cancer patients. Several metabolites were detected including 4 beta,5 alpha-dihydroxyandrostan-3,17-dione, 3,17-dihydroxyandrostan-4-ones and 3 alpha-hydroxy-5 beta-androstan-4,17-dione.     

Metabolic studies of methenolone acetate in horses

Methenolone acetate (17β-acetoxy-1-methyl-5α-androst-1-en-3-one), a synthetic anabolic steroid, is frequently abused in human sports. It is preferred for its therapeutic efficiency and lower hepatic toxicity compared with its 17α-alkylated analogs. As with other anabolic steroids, methenolone acetate may be used to enhance performance in racehorses. Metabolic studies on methenolone acetate have been reported for humans, whereas little is known about its metabolic fate in horses. This paper describes the investigation of in vitro and in vivo metabolism of methenolone acetate in racehorses.Studies on the in vitro biotransformation of methenolone acetate with horse liver microsomes were carried out. Methenolone (M1, 1-methyl-5α-androst-1-en-17β-ol-3-one) and seven other metabolites (M2-M8) were detected in vitro. They were 1-methyl-5α-androst-1-ene-3,17-dione (M2), 1-methyl-5α-androst-1-en-6-ol-3,17-dione (M3) and two stereoisomers of 1-methylen-5α-androstan-2-ol-3,17-dione (M4 and M5), 1-methyl-5α-androst-1-en-16-ol-3,17-dione (M6) and monohydroxylated 1-methyl-5α-androst-1-en-17-ol-3-one (M7 and M8). After oral administration of Primobolan^® (80 tablets × 5 mg of methenolone acetate each) to two thoroughbred geldings, the parent steroid ester was not detected in the post-administration urine samples. However, seven metabolites, namely M1, M6-M8, two stereoisomers of M7 (M9 and M10) and 1-methyl-5α-androst-1-en-17α-ol-3-one (M11), could be detected. The metabolic pathway for methenolone acetate is postulated. This study has shown that metabolite M1 could be targeted for controlling the abuse of methenolone acetate in horses.     

Identification of the aromatase inhibitors anastrozole and exemestane in human urine using liquid chromatography/tandem mass spectrometry

Anastrozole (2,2'-[5-(1H-1,2,4-triazol-1-ylmethyl)-1.3-phenylene]bis(2-methylpropionitrile)) and exemestane (6-methylenandrostan-1,4-diene-3,17-dione) are therapeutically used to treat hormone-sensitive breast cancer in postmenopausal women. For doping purposes they may be used to counteract adverse effects of an extensive abuse of anabolic androgenic steroids (gynaecomastia) and to increase plasma testosterone concentrations. Excretion study urine samples and spot urine samples from women suffering from metastatic breast cancer, being treated with anastrozole or exemestane, were collected and analyzed to develop/optimize a detection system for anastrozole and exemestane to allow the identification of athletes who do not comply with the internationally prohibited use of these cancer drugs. The assay was based on liquid-liquid extraction after enzymatic hydrolysis following liquid chromatography/tandem mass spectrometry (LC/MS/MS). Anastrozole, exemestane and its main metabolite (17-dihydroexemestane) were identified in urine by comparison of mass spectra and retention times with respective reference substances. An assay validation for the analysis of anastrozole and exemestane was performed regarding lower limits of detection (anastrozole: 0.02 ng/mL; exemestane: 3.1 ng/mL; dihydroexemestane: 0.5 ng/mL), interday precisions (6.6-11.1%, 4.9-9.1% and 5.6-8.3% for low [10 ng/mL], medium [50 ng/mL] and high [100 ng/mL] concentration) and recoveries (ranged from 85-97%).