Development and validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous analysis of androgens, estrogens, glucocorticoids and progestagens in human serum.

We present a liquid chromatography tandem mass spectrometry method for the simultaneous analysis of 16 endogenous steroids (androgens, estrogens, glucocorticoids and progestogens) in human serum. Samples (250 µl of matrix) were extracted with t-butylmethyl ether prior to LC-MS/MS analysis. The chromatographic separation was achieved on a reversed-phase column using a methanol-water gradient. The HPLC was coupled to a triple quadrupole mass spectrometer equipped with an electrospray ionization source with acquisition in multiple reaction monitoring mode. The method was validated using surrogate matrices and human serum samples. The specificity of the method was confirmed for all of the considered steroids; linearity was also assessed (R2 > 0.99, lack-of-fit test) in the ranges of concentrations investigated. The lower limits of quantification were in the range 10-400 pg/ml depending on the target steroid. Accuracy was in the range 85-115% for all target steroids except for the lower limit of quantitation levels where it was 80-120%. The extraction recovery was always >65%. No significant matrix effects were observed. To test the reliability of the method, the analysis of serum samples collected from 10 healthy subjects (5 M/5F) was performed. The present method can be used to identify the trajectories of deviation from the concentration normality ranges applied to disorders of the gonadal and adrenal axes.

Influence of synthetic isoflavones on selected urinary steroid biomarkers: Relevance to doping control.

In this work we have investigated the influence of the intake of two synthetic isoflavones, methoxyisoflavone and ipriflavone, on the urinary concentration of endogenous steroids, and on their relative ratios, of doping relevance. Specifically, the concentrations of testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio), 5α-androstan-3α,17α-diol (5αAdiol), 5ß-androstan-3α,17α-diol (5ßAdiol), and the ratios T/E, A/T, A/Etio, 5αAdiol/5ßAdiol, 5αAdiol/E, were considered, in the framework of the Steroidal Module of the Athlete Biological Passport (ABP). The above set of parameters were complemented by the urinary levels of luteinizing hormone (total LH) and the ratio between T and LH (T/total LH), to assess the possible effects on the biosynthesis of the mentioned steroids. Five healthy Caucasian male volunteers were selected for the study. Urine samples were collected before and during the administration of (i) methoxyisoflavone (Methoxyisoflavone, MyProtein) and (ii) ipriflavone (Osteofix ®, Chiesi Farmaceutici). For the analysis of the urinary steroid profile, after enzymatic hydrolysis with ß-glucuronidase from Escherichia Coli (E. Coli) and liquid-liquid extraction with tert-buthylmethyl ether, all samples were analyzed by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS), while for the determination of total LH all urine samples were directly analyzed by a chemiluminescent immunometric assay technique (Siemens Immulite 2000 LH). Our results show that the administration of either methoxyisoflavone or ipriflavone causes an alteration of the urinary concentrations and concentration ratios of the investigated steroids, in the range 55-80% from the baseline values. Furthermore, an oversecretion of LH after the daily intake of methoxyisoflavone or ipriflavone was also recorded in all volunteers, corresponding to an increase in the biosynthesis and excretion of T and some of its metabolites. These changes trigger a disregulation in the pattern of urinary excretion of the steroids included in the Steroidal Module of the ABP, which makes more difficult the interpretation of the longitudinal steroid profile based on the definition of individual normality ranges for each athlete. Our data are also consistent with previous evidence regarding the in vitro effects of natural and synthetic isoflavones, suggesting that their monitoring in doping control routine analysis would be very beneficial for the result management activities.

Controlled administration of dehydrochloromethyltestosterone in humans: Urinary excretion and long-term detection of metabolites for anti-doping purpose.

Dehydrochloromethyltestosterone (DHCMT) is an anabolic-androgenic steroid that was developed by Jenapharm in the 1960s and was marketed as Oral Turinabol®. It is prohibited in sports at all times; nevertheless, there are several findings by anti-doping laboratories every year. New long-term metabolites have been proposed in 2011/12, which resulted in adverse analytical findings in retests of the Olympic games of 2008 and 2012. However, no controlled administration trial monitoring these long-term metabolites was reported until now. In this study, DHCMT (5 mg, p.o.) was administered to five healthy male volunteers and their urine samples were collected for a total of 60 days. The unconjugated and the glucuronidated fraction were analyzed separately by gas chromatography coupled to tandem mass spectrometry. The formation of the described long-term metabolites was verified, and their excretion monitored in detail. Due to interindividual differences there were several varieties in the excretion profiles among the volunteers. The metabolite M3, which has a fully reduced A-ring and modified D-ring structure, was identified by comparison with reference material as 4α-chloro-17ß-hydroxymethyl-17α-methyl-18-nor-5α-androstan-13-en-3α-ol. It was found to be suitable as long-term marker for the intake of DHCMT in four of the volunteers. In one of the volunteers, it was detectable for 45 days after single oral dose administration. However, in two of the volunteers M5 (already published as long-term metabolite in the 1990s) showed longer detection windows. In one volunteer M3 was undetectable but another metabolite, M2, was found as the longest detectable metabolite. The last sample clearly identified as positive was collected between 9.9 and 44.9 days. Furthermore, the metabolite epiM4 (partially reduced A-ring and a modified D-ring structure which is epimerized in position 17 compared to M3) was identified in the urine of all volunteers with the help of chemically synthesized reference as 4-chloro-17α-hydroxymethyl-17ß-methyl-18-nor-androsta-4,13-dien-3ß-ol. It may serve as additional confirmatory metabolite. It is highly recommended to screen for all known metabolites in both fractions, glucuronidated and unconjugated, to improve identification of cheating athletes. This study also offers some deeper insights into the metabolism of DHCMT and of 17α-methyl steroids in general.

New Insights into the Metabolism of Methyltestosterone and Metandienone: Detection of Novel A-Ring Reduced Metabolites.

Metandienone and methyltestosterone are orally active anabolic-androgenic steroids with a 17α-methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis. Following the previously reported detection of long-term metabolites with a 17ξ-hydroxymethyl-17ξ-methyl-18-nor-5ξ-androst-13-en-3ξ-ol structure in the chlorinated metandienone analog dehydrochloromethyltestosterone ("oral turinabol"), in this study we investigated the formation of similar metabolites of metandienone and 17α-methyltestosterone with a rearranged D-ring and a fully reduced A-ring. Using a semi-targeted approach including the synthesis of reference compounds, two diastereomeric substances, viz. 17α-hydroxymethyl-17ß-methyl-18-nor-5ß-androst-13-en-3α-ol and its 5α-analog, were identified following an administration of methyltestosterone. In post-administration urines of metandienone, only the 5ß-metabolite was detected. Additionally, 3α,5ß-tetrahydro-epi-methyltestosterone was identified in the urines of both administrations besides the classical metabolites included in the screening procedures. Besides their applicability for anti-doping analysis, the results provide new insights into the metabolism of 17α-methyl steroids with respect to the order of reductions in the A-ring, the participation of different enzymes, and alterations to the D-ring.

Improving the detection of anabolic steroid esters in human serum by LC-MS.

The detection of the abuse of pseudo-endogenous steroids in sport is articulated in two different levels: an initial testing procedure, based on the longitudinal evaluation of the urinary androgenic steroid profile by gas-chromatography mass spectrometry (GC-MSn), and a confirmation analysis, based on the differentiation between the endogenous and exogenous origin of the pseudo-endogenous steroids by gas-chromatography coupled to isotopic ratio mass spectrometry (GC/C/IRMS). The abuse of pharmaceutical preparations displaying a carbon isotopic composition values within a range similar to those reported for endogenous urinary steroids makes more difficult the application of GC/C/IRMS technique. To overcome this limitation, the direct detection of an intact synthetic anabolic steroid ester in blood matrices (plasma and/or serum) could supply the unequivocal proof of exogenous administration of pseudo-endogenous steroids. Here we are presenting a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the analysis of 14 testosterone (T) esters and 2 nandrolone (Nand) esters in human serum. Sample pre-treatment consisted of protein precipitation, liquid-liquid extraction and derivatization. The formation of three different derivatives (oxime derivatives, Girard P and Girard T hydrazones) is considered, in order to guarantee an improvement in the detection capability of the assay with respect to underivatized compounds. Once the most suitable derivative was selected, the method was validated, according to the World Anti-Doping Agency (WADA) criteria, in terms of specificity, linearity, limit of detection (LOD), extraction recovery, matrix effect (ion suppression/enhancement), carry over and autosampler stability. The formation of Girard P hydrazones of T and Nand esters provides the best results compared to the underivatized compounds, oxime and Girard T derivatives, respectively. The presented analytical method is specific for all considered compounds and linear in the range of concentrations investigated (0.25-10 ng/mL). The LODs are between 0.03 and 0.30 ng/mL, the extraction recovery higher than 70 % for all esters and no remarkable matrix effect, expressed in terms of ion enhancement and ion suppression, was observed. Finally, the developed and validate method was applied in the analysis of serum samples collected after the administration of a single dose (40 mg, 1 capsule) of testosterone undecanoate (Andriol ®) demonstrating its applicability.

Influence of Indomethacin on Steroid Metabolism: Endocrine Disruption and Confounding Effects in Urinary Steroid Profiling of Anti-Doping Analyses.

Anabolic androgenic steroids (AAS) are prohibited as doping substances in sports by the World Anti-Doping Agency. Concentrations and concentration ratios of endogenous AAS (steroid profile markers) in urine samples collected from athletes are used to detect their administration. Certain (non-prohibited) drugs have been shown to influence the steroid profile and thereby sophisticate anti-doping analysis. It was shown in vitro that the non-steroidal anti-inflammatory drug (NSAID) indomethacin inhibits selected steroid-biotransformations catalyzed by the aldo-keto reductase (AKR) 1C3, which plays a key role in the endogenous steroid metabolism. Kinetic parameters for the indomethacin-mediated inhibition of the AKR1C3 catalyzed reduction in etiocholanolone were determined in vitro using two comparing methods. As NSAIDs are very frequently used (not only) by athletes, the inhibitory impact of indomethacin intake on the steroid metabolism was evaluated, and steroid profile alterations were detected in vivo (one male and one female volunteer). Significant differences between samples collected before, during or after the intake of indomethacin for selected steroid profile markers were observed. The presented results are of relevance for the interpretation of results from doping control analysis. Additionally, the administration of NSAIDs should be carefully reconsidered due to their potential as endocrine disruptors.

Validation of steroid sulfates deconjugation for metabolic studies. Application to human urine samples.

BACKGROUND: Urinary sulfate fraction of the anabolic androgenic steroids is not analyzed routinely in anti-doping analyses but has demonstrated in the last years an increasing interest among the anti-doping community. Sulfate conjugates are linked to plasma proteins increasing the residence time in the body compared to glucuro-conjugated metabolites, and then their analyses may allow improving the detection time window of specific metabolites. Hydrolysis of sulfates can be made enzymatically or chemically and can be challenging, depending on the strategy selected. METHODS: Hydrolysis by solvolysis was validated for metabolic studies, focusing on setting a quality control able to assess the hydrolytic step. To the internal standards mixture, androsterone-D4 and etiocholanolone-D5 sulfate were added. The proposed protocol was applied over samples collected after dehydroepiandrosterone (DHEA) administrations. RESULTS: The stability of the structures showed good results, and no evident formation of degradation products was observed. Internal standard to monitor the efficiency of hydrolysis, recovery, and retention time was successfully introduced. Additional analytes (4ß-hydroxy-DHEA, 5-androstene-3ß,17ß-diol and 5α-androstane-3ß,17ß-diol) were found to be affected besides of DHEA and epiandrosterone (epiA) as previously described. CONCLUSIONS: Results in terms of linearity, precision, and accuracy, showed that the method is suitable to quantify seven analytes in urine in the sulfated fraction. The validated method was successfully applied to urine samples after administration of DHEA to detect this compound in the sulfate fraction and preliminarily to negative samples from athletes of both sexes, to determine Q1 and Q3 inter-quartiles. A quality control assessment for the hydrolysis efficiency was established for every individual sample.

Detection of clostebol in sports: Accidental doping?

The detection of clostebol misuse in sports has been growing recently, especially in Italy, due to the ample availability of pharmaceutical formulations containing clostebol acetate (Trofodermin®) and the use of more sensitive instrumentation by the antidoping laboratories. Most of these cases have been claimed to be related to a nonconscious use of the drug or through contact with relatives or teammates using it. We have investigated, through the application of the well-known and currently used gas chromatographic mass spectrometric procedures, the likelihood of these allegations and have demonstrated that after a single transdermal administration of 5 mg of clostebol acetate and a transient contact with the application area, it is possible to generate adverse analytical findings in antidoping controls. We have reviewed the Phase I and Phase II clostebol metabolism in order to generate evidences that may help the sport authorities reviewing these cases. The main clostebol metabolite (4-chloro-androst-4-en-3α-ol-17-one, M1) generally used at the screening level as well as other three metabolites (M2-M4) are mainly excreted as glucuronides, whereas M5 (4ζ-chloro-5ζ-androstan-3ß-ol-17-one) is predominantly excreted as sulfate. Neither the 5α-reductases activity (impaired by the presence of the chlorine in C4) nor specific sulfotransferases present in the skin allowed a clear distinction of the administration route. Studies with a larger number of volunteers and probably investigating another physiological fluid allowed in antidoping such as blood are needed for a deeper investigation. It is not unreasonable to establish a reporting level for M1, maybe creating some false negatives but excluding nonintentional doping scenarios.

Development and application of analytical procedures for the GC-MS/MS analysis of the sulfates metabolites of anabolic androgenic steroids: The pivotal role of chemical hydrolysis.

In this work, we present a gas-chromatography tandem mass spectrometry (GC-MS/MS) method for the identification of the sulfo-conjugate metabolites of pseudo-endogenous steroids (endogenous steroids when administered exogenously). We have preliminarily evaluated the performances of different preparations of sulfatases from Pseudomonas aeruginosa and Helix pomatia, characterized by various origins and catalytic activities, and compared the efficacy of the enzymatic hydrolysis with chemical hydrolysis, performed with a mixture of ethyl acetate, methanol, and sulphuric acid. A procedure for the selective isolation of steroid conjugates from the urine matrix has been designed and optimized, based on the "sequential" extraction of the glucuro-conjugated and of the sulfo-conjugated fractions, performed by two different direct methods, i.e. by ion paired extraction or solid-phase extraction. More specifically, the former method is based on the use of N,N-dimethylephedrinium bromide as the ion paired extraction reagent, while the latter on the use of WAX® (weak anion exchange) cartridges. The performance of the newly developed procedure has been assessed by the analysis of real urine excretion samples collected after the oral intake of a single dose of dehydroepiandrosterone (DHEA) or androstenedione (AED), measuring the concentration of epiandrosterone (EpiA) sulfate. Our results have shown the following: (i) although the yields of chemical hydrolysis and enzymatic hydrolysis are in some cases quite similar, the former is generally preferable since it results in the quantitative cleavage of sulfate moiety; (ii) ion paired extraction has been selected as the most reliable method for direct isolation of sulfate steroids from urine matrices; (iii) EpiA sulfate allows to prolong the detectability of DHEA and AED when compared to routinely used steroidal target compounds.

A further insight into methyltestosterone metabolism: New evidences from in vitro and in vivo experiments.

RATIONALE: Although the metabolism of methyltestosterone (MT) has been extensively studied since the 1950s using different techniques, the aim of this study was to investigate the hydroxylation in positions C2, C4 and C6 after in vitro experiments and in vivo excretion studies using gas chromatography time-of-flight (GC/TOF) and gas chromatography/tandem mass spectrometry (GC/MS/MS). The results could be influenced by the mass spectrometric analyser used. METHODS: Incubations were carried out with human liver microsomes and six enzymes belonging to the cytochrome P450 family using MT as a substrate. The trimethylsilyl derivatives of the samples were analysed using GC/TOF and GC/MS/MS once the correct MS/MS transitions had been selected, mainly for 6-hydroxymethyltestosterone (6-OH-MT) to avoid artefact interferences. A urinary excretion study was then performed after the administration of a 10 mg single oral dose of MT to a volunteer. RESULTS: The formation of hydroxylated metabolites of MT in the C6, C4 and C2 positions after both in vitro and in vivo experiments was observed. Sample evaluation using GC/TOF showed an interference for 6-OH-MT that could only be resolved in GC/MS/MS by monitoring specific transitions. The transitory detection of these hydroxylated metabolites in urine agrees with previous investigations that had described this metabolic route as being of little significance. CONCLUSIONS: In doping analysis, the formation of 4-hydroxymethyltestosterone (oxymesterone) from MT cannot be underestimated. Although it is only detected as a minor and short-term excretion metabolite, it cannot be overlooked as it was found in both in vitro and in vivo experiments. The use of a combination of different mass spectrometric instruments allowed reliable conclusions to be reached, and it was shown that special attention must be given to artefact formation.

Influence of Pain Killers on the Urinary Anabolic Steroid Profile.

Anabolic androgenic steroids (AAS) are prohibited as performance-enhancing drugs in sports. Among them, testosterone and its precursors are often referred to as "pseudoendogenous" AAS, that is, endogenous steroids that are prohibited when administered exogenously. To detect their misuse, among other methods, the World Anti-Doping Agency-accredited laboratories monitor the steroid profile (concentrations and concentration ratios of endogenous steroids, precursors and metabolites) in urine samples collected from athletes in and out of competition. Alterations in steroid profile markers are used as indicators for misuse of anabolic steroids in sports. Therefore, especially their metabolic pathways with possible interactions are crucial to elucidate. As steroid metabolism is very complex, and many enzymes are involved, certain non-prohibited drugs may influence steroid metabolite excretion. One important group of steroid-metabolizing enzymes is aldo-keto reductases (AKRs). An inhibition of them by non-steroidal anti-inflammatory drugs (NSAIDs), which are neither prohibited nor monitored, but frequently used drugs in sports, was demonstrated in vitro. Thus, this work aims to investigate the influence of NSAID intake on the urinary steroid profile. Kinetic and inhibitory studies were performed using 5α-dihydrotestosterone as substrate. The results obtained from in vitro experiments show that ibuprofen inhibits AKR1C2 and thus influences steroid biotransformation. For in vivo investigations, urine samples prior, during and postadministration of ibuprofen were analyzed using routine methods to monitor the steroid profile. Changes in markers of the steroid profile of volunteers were observed. The combination of in vitro and in vivo results suggests that monitoring of ibuprofen may be useful in doping control analysis. The presented work illustrates the importance to consider co-administration of (non-prohibited) drugs during antidoping analysis. Intake of multiple substances is likely leading to interfering effects. Divergent results in antidoping analysis may therefore be observed and misinterpretation of analytical data may occur. Similar considerations may be appropriate for other fields of forensic applications.

Validation of an ultra-sensitive detection method for steroid esters in plasma for doping analysis using positive chemical ionization GC-MS/MS.

The standard approach to detect misuse with testosterone in sport is based on the determination and evaluation of the urinary steroid profile followed by the confirmation of atypical profiles using isotope ratio mass spectrometry. The detection capacity of these methods can be attenuated by confounding factors or testosterone preparations with endogenous isotopic fingerprints. An alternative detection method for misuse of an endogenous steroid in sports is the direct detection of the administered steroid ester present in most preparations. Thus unambiguous proof for doping misuse can be delivered. In this work, the sensitivity of gas chromatography coupled to a triple quadrupole with chemical ionization (GC-CI-MS/MS) is applied to detect trace levels of 10 testosterone and 2 nandrolone esters in plasma for in human doping analysis. The detection method was developed employing a liquid-liquid extraction and HPLC cleanup step before analysis on the GC-CI-MS/MS. The quantitative method was validated in a linear range of 100-2000 pg/ml and proved to be selective, reproducible and very sensitive with limits of detection as low as to 10 pg/ml. A clinical study with the administration of testosterone undecanoate in 3 volunteers was carried out and the compound was detectable up to 86 days after administration.

Effects of transdermal administration of testosterone gel on the urinary steroid profile in hypogonadal men: Implications in antidoping analysis.

Testosterone is one of the most abused pseudo-endogenous anabolic steroids in sport doping. The current method adopted to detect the abuse of testosterone and other pseudo-endogenous steroids (endogenous steroids when administered exogenously) is first based on the longitudinal monitoring of several urinary biomarkers, which constitute the so called "steroidal module" of the Athlete Biological Passport (ABP): atypical samples undergo a confirmation analysis based on the measurement of the 13C/12C isotopic ratio of selected target compounds, to distinguish their endogenous or exogenous origin. At the same time, testosterone administration can be allowed in athletes diagnosed with hypogonadism, provided they are granted a therapeutic use exemption by the relevant medical authority. In this pilot study we have investigated whether the approach based on the preliminary determination of the urinary steroid profile, in the format considered in the steroidal module of the ABP, also integrated with the inclusion of the sulfo-conjugates and of additional target steroids, can retain its validity also in the case of hypogonadal athletes. We have studied the effects of a single low dose (40 mg) of testosterone gel (T-gel) on the urinary concentration of the markers of steroidal module of the ABP, as well as on some additional steroid markers. The study was based on the analysis of urinary samples from 19 non-hospitalized hypogonadal men, 10 of them with late-onset hypogonadism (LOH), collected before, after 4 h and after 24 h the transdermal self-administration of 40 mg of T-gel. None of the patient had any co-morbidities possibly affecting the urinary excretion of the steroidal markers. The steroidal markers were quantified by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) after the enzymatic hydrolysis of the respective glucuro-conjugates and the chemical hydrolysis of the respective sulfo-conjugates. Targeted GC-MS/MS analysis was carried out operating in electron impact (EI) ionization mode, with acquisition in multiple reaction monitoring (MRM) mode. Our preliminary results show that, as expected, the treatment with T-gel leads, in all hypogonadal men, to an increase of the urinary concentration of the glucuro-conjugate metabolites of testosterone and its main metabolites, with special relevance to those with 5α-reduction. Furthermore, samples collected from non-LOH hypogonadal men showed an increase also in the levels of epitestosterone glucuronide, testosterone sulfate and epitestosterone sulfate. Apart from their biochemical and pharmacological relevance, these outcomes could be leveraged to refine the analytical strategy currently followed in the antidoping field for the analysis of the urinary steroidal markers, with potential implications also in other forensic and/or clinical investigations.

An investigation on the metabolic pathways of synthetic isoflavones by gas chromatography coupled to high accuracy mass spectrometry.

RATIONALE: Isoflavones are a group of flavonoids that may be of interest in sport doping because they can be used by athletes in the recovery periods after the administration of anabolic steroids, with the aim of increasing the natural production of luteinizing hormone (LH) and, consequently, the biosynthesis of endogenous androgens. METHODS: The in vivo metabolism of methoxyisoflavone (5-methyl-7-methoxyisoflavone) and ipriflavone (7-isopropoxyisoflavone), respectively present in a dietary supplement and in a pharmaceutical preparation, was investigated. The study was carried out by the analysis of urinary samples collected from male Caucasian subjects before, during and after the oral administration of methoxyisoflavone or ipriflavone. After enzymatic hydrolysis and liquid-liquid extraction, all urinary samples were analyzed by gas chromatography/quadrupole time-of-flight (qTOF MS system/qTOF) electron ionization mass spectrometry (EI-MS). RESULTS: Eight metabolites of methoxyisoflavone and six metabolites of ipriflavone were isolated. The corresponding accurate mass spectra are specific for isoflavone structures and revealed also a retro-Diels-Alder fragmentation. CONCLUSIONS: When excreted in large amounts, the urinary metabolites of methoxyisoflavone and ipriflavone can be traced to potential confounding factors in doping analysis. As methoxyisoflavone and ipriflavone have been shown to inhibit the enzyme aromatase, thus interfering with the normal metabolic pathways of testosterone, the detection of their intake, by screening for the presence of their main metabolites in urine, might be helpful in routine doping control analysis.

Synthetic isoflavones and doping: A novel class of aromatase inhibitors?

Anectodical information suggests that flavonoids may be widely used among athletes for their multiple biochemical and pharmacological effects. We have evaluated in vitro the effects of two synthetic isoflavones, methoxyisoflavone and ipriflavone, on the catalytic activity of human aromatase (CYP19), the enzyme catalyzing the conversion of androgens (ie, testosterone or androstenedione) to estrogens (ie, estradiol and estrone). The potential inhibitory effect was evaluated by measuring the rate of aromatization of testosterone, monitored by gas chromatography-mass spectrometry (GC-MS), both in the presence and in the absence of methoxyisoflavone or ipriflavone, comparing their effects with those of synthetic aromatase inhibitors (formestane, anastrozole, and aminoglutethimide) presently included in the list of prohibited substances and methods, and of natural flavonoids (chrysin, quercetin, and daidzein), that are known inhibitors of CYP19. The preliminary results of our in vitro study show that methoxyisoflavone and ipriflavone act as competitive inhibitors of aromatase, the degree of inhibition measured in vitro being of the same order of magnitude of that of the aromatase inhibitors commonly used in anti-estrogenic therapies. Our preliminary in vitro results indicate that, in principle, a sufficiently large intake of isoflavones could alter the kinetics of the dynamic equilibria between androgens and estrogens, suggesting their monitoring in doping control routine analysis.