13C/12C Ratios of endogenous urinary steroids investigated for doping control purposes

In order to detect the misuse of endogenous anabolic steroids such as testosterone by athletes a total of n = 1734 suspicious urine samples were investigated by gas chromatography/combustion/isotope ratio mass spectrometry throughout the years 2005, 2006 and 2007. The ¹³C/¹²C ratio of a target substance (androsterone, a testosterone metabolite) was compared to the ¹³C/¹²C ratio of an endogenous reference compound (11β‐hydroxyandrosterone). N = 1340 samples were investigated due to elevated testosterone/epitestosterone ratios, with n = 87 (6.5%) exceptional findings regarding their isotopic ratios. An additional n = 164 samples were investigated because of elevated dehydroepiandrosterone concentrations, with n = 2 (1.2%) exceptional findings. The remainder were subjected to isotope ratio analysis because of elevated androsterone levels or because this was requested by sports federations. Significant differences between female and male samples were found for the ¹³C/¹²C ratios of androsterone and 11β‐hydroxyandrosterone but not for samples taken in or out of competition. A further n = 645 samples originating from other World Anti‐Doping Agency accredited laboratories, mainly throughout Europe as well as South America, South Africa and Southeast Asia, were investigated. The ¹³C/¹²C ratios of the urinary steroids differ significantly for each geographical region, reflecting the dietary status of the individuals. The system stability over time has been tested by repeated injections of a standard solution and repeated processing of frozen stored blank urine. Despite a drift over time in absolute ¹³C/¹²C ratios, no significant change in the difference of ¹³C/¹²C (11β‐hydroxyandrosterone) minus ¹³C/¹²C (androsterone) could be observed. Copyright © 2009 John Wiley & Sons, Ltd. Copyright © 2009 John Wiley & Sons, Ltd.     

Carbon isotope ratio mass spectrometry for detection of endogenous steroid use: A testing strategy

Isotope ratio mass spectrometry (IRMS) testing is performed to determine if an atypical steroid profile is due to administration of an endogenous steroid. Androsterone (Andro) and etiocholanolone (Etio), and/or the androstanediols (5α‐ and 5β‐androstane‐3α,17β‐diol) are typically analyzed by IRMS to determine the ¹³C/¹²C ratio. The ratios of these target compounds are compared to the ¹³C/¹²C ratio of an endogenous reference compound (ERC) such as 5β‐pregnane‐3α,20α‐diol (Pdiol). Concentrations of Andro and Etio are high so ¹³C/¹²C ratios can easily be measured in most urine samples. Despite the potentially improved sensitivity of the androstanediols for detecting the use of some testosterone formulations, additional processing steps are often required that increase labour costs and turnaround times. Since this can be problematic when performing large numbers of IRMS measurements, we established thresholds for Andro and Etio that can be used to determine the need for additional androstanediol testing. Using these criteria, 105 out of 2639 urine samples exceeded the Andro and/or Etio thresholds, with 52 of these samples being positive based on Andro and Etio IRMS testing alone. The remaining 53 urine samples had androstanediol IRMS testing performed and 3 samples were positive based on the androstanediol results. A similar strategy was used to establish a threshold for Pdiol to identify athletes with relatively ¹³C‐depleted values so that an alternative ERC can be used to confirm or establish a true endogenous reference value. Adoption of a similar strategy by other laboratories can significantly reduce IRMS sample processing and analysis times, thereby increasing testing capacity. Copyright © 2013 John Wiley & Sons, Ltd.     

The effect of athletes` hyperhydration on the urinary ‘steroid profile’ markers in doping control analysis

The urinary ‘steroid profile’ in doping control analysis is a powerful tool aimed at detecting intra‐individual deviations related to the abuse of endogenous steroids. Factors altering the steroid profile include, among others, the excessive fluid intake leading to low endogenous steroids concentrations compared to an individual's normal values. Cases report the use of hyperhydration by athletes as a masking method during anti‐doping urine sample collection. Seven healthy physically active non‐smoking Caucasian males were examined for a 72‐hour period using water and a commercial sports drink as hyperhydration agents (20 mL/kg body weight). Urine samples were collected and analyzed according to World Anti‐Doping Agency (WADA) technical documents. Although, significant differences were observed on the endogenous steroid concentrations under the studied hyperhydration conditions, specific gravity adjustment based on a reference value of 1.020 can eliminate the dilution induced effect. Adjustment methods based on creatinine and urinary flow rate were also examined; however, specific gravity was the optimum method in terms of effectiveness to adjust concentrations close to the baseline steroid profile and practicability. No significant effect on the urinary steroid ratios was observed with variability values within 30% of the mean for the majority of data. Furthermore, no masking on the detection ability of endogenous steroids was observed due to hyperhydration. It can be concluded that any deviation on the endogenous steroid concentrations due to excessive fluid intake can be compensated by the specific gravity adjustment and therefore, hyperhydration is not effective as a masking method on the detection of the abuse of endogenous steroids.     

Mass spectrometric characterization of urinary hydrafinil metabolites for routine doping control purposes

Little information on the human metabolism and urinary elimination of hydrafinil (9-fluorenol) exists. In order to support preventive anti-doping activities concerning compounds such as hydrafinil, a pilot elimination study was conducted with three healthy male volunteers receiving a single oral dose of 50 mg of hydrafinil. Urine samples were collected prior to and up to 72-h post-administration and were subjected to both gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry, which allowed for the identification of the intact drug as well as Phase I and Phase II metabolites, primarily hydroxylated and/or glucuronidated or sulfo-conjugated hydrafinil. The identity of these metabolites was corroborated by high-resolution/high-accuracy tandem mass spectrometry, and the applicability of routine doping control workflows for the detection of hydrafinil and its main metabolites was assessed. Therefore, two findings of hydrafinil and its metabolites were recorded, which concerned out-of-competition doping control samples and, hence, were not pursued with confirmatory analyses. Yet, the initial testing procedure results indicate that hydrafinil might require consideration in sports drug testing programs to ensure its detection, if classified as prohibited by the World Anti-Doping Agency (WADA).     

Usefulness of serum androgen isotope ratio mass spectrometry (IRMS) to detect testosterone supplementation in women

The detection of testosterone intake is facilitated by monitoring the urinary steroid profile in the athlete biological passport. This technique can be used with confidence to identify target samples for isotope ratio mass spectrometry. Regrettably, most research has been performed on male subjects resulting in a method that does not account for females' steroid concentration and/or variation. This study evaluates the usefulness of the carbon isotope ratio (CIR) in serum of female subjects. Two steroid sulphates are targeted in serum, androsterone and epiandrosterone. Both exhibit statistically significant depletion of their CIR after 10 weeks of daily (10 mg) transdermal testosterone administration. Of the 21 female subjects, samples from six individuals were identified as adverse analytical findings; additionally, four were found atypical considering the serum CIR. The urinary athlete biological passport was not sufficiently sensitive to identify target serum samples for isotope ratio mass spectroscopy. Of the six with a suspicious passport, only two could be confirmed using the serum CIR of androsterone and epiandrosterone. This study shows that CIR analysis in serum cannot be considered the sole confirmatory solution to detect testosterone doping in women due to low sensitivity. However, this analysis has the potential to be used as a complementary method in certain situations to confirm exogenous testosterone in women.     

Towards a robust test to detect gene doping for anabolic enhancement in human athletes

Success in gene therapy in treating human disease makes this technology attractive to enhance athletic performance, creating the need for gene doping detection. In 2021, World Anti-Doping Agency (WADA) approved the first gene doping test. Here, we describe a new method to detect doping with four additional genes, follistatin, growth hormone 1, growth hormone–releasing hormone and insulin-like growth factor 1, that may improve performance by increasing muscle size and strength. The method utilises four hydrolysis probe-based polymerase chain reaction (PCR) assays that target the transgenes based on the coding sequence of the four endogenous genes. The assays are specific, reproducible and capable to detect five copies of transgene in the presence of very similar endogenous gene in 25,000 times excess. To underpin reliable and comparable routine method performance by doping testing laboratories, a synthetic reference material for the method was designed and generated following the ISO Guide 35. The complete method was validated in blood samples using plasma as extraction matrix and QIAamp DNA blood midi DNA extraction kit. All blood samples from different donors (n = 8) simulated to be negative or positive (1500 transgene copies spiked per millilitre of blood) for the transgenes were reported correctly. The new method that targets four additional genes will extend the capabilities of laboratories involved in doping control to protect athletes' health, fairness and equality.     

Non‐targeted acquisition strategy for screening doping compounds based on GC‐EI‐hybrid quadrupole‐Orbitrap mass spectrometry: A focus on exogenous anabolic steroids

This is a first look at a non‐targeted screening method based on Orbitrap gas chromatography–mass spectrometry (GC–MS) technology for a large number of banned compounds in sports found in urine, including exogenous anabolic steroids, β‐agonists, narcotics, stimulants, hormone modulators, and diuretics. A simple sample preparation was processed in four steps: an enzymatic hydrolysis, liquid–liquid extraction, evaporation, and trimethylsilylation. All compounds were able to meet the World Anti‐Doping Agency's sensitivity criteria with mass accuracies less than 1 ppm and with sufficient points across the peak by running the Orbitrap GC–MS in full‐scan mode. In addition, we discuss our initial findings of using a full‐scan selected ion monitoring‐tandem mass spectrometry (SIM‐MS/MS) approach as a way to obtain lower detection limits and reach desirable selectivity for some exogenous anabolic steroids.     

Urinary concentrations of AICAR and mannitol in athlete population

Both AICAR and mannitol are prohibited for use in sports, but no decisive criteria that would guide anti‐doping laboratories on data interpretation have been established so far. In an attempt to help harmonize reporting and management of analytical findings, reference population data collected for US athletes are presented. Upon analysis of 12 377 samples, mean urinary AICAR concentration was found to be 647 ± 365 ng/mL with median value of 574 ng/mL, 99th percentile at 1786 ng/mL and 99.7th percentile at 2151 ng/mL. Based on these results, we suggest that any sample with AICAR concentration greater than 2000 or 2500 ng/mL be analyzed by carbon isotope ratio mass spectrometry to establish the origin. Urinary mannitol concentrations demonstrate larger variation with the mean value of 72 ± 140 μg/mL and median at 41 μg/mL (n = 6407). While the 99.7th percentile for mannitol was measured to be 1094 μg/mL, the population data alone is not sufficient to suggest a threshold value. It is also shown that the use of mannitol as a sweetener in amounts of up to 20 g per day results in a urinary concentration of about 14 mg/mL. As only intravenous mannitol is prohibited in sports, controlled excretion studies are needed to see whether intravenous administration could in fact be discriminated from dietary intake. An important observation is that mannitol present in mg/mL quantities significantly increases urine specific gravity, which makes a widely accepted normalization approach not applicable.     

Inter‐individual variation of the urinary steroid profiles in Swedish and Norwegian athletes

The steroidal module of the Athlete Biological Passport (ABP) aims to detect doping with endogenous steroids, e.g. testosterone (T), by longitudinally monitoring several biomarkers. These biomarkers are ratios combined into urinary concentrations of testosterone and metabolically related steroids. However, it is evident after 5 years of monitoring steroid passports that there are large variations in the steroid ratios complicating its interpretation. In this study, we used over 11000 urinary steroid profiles from Swedish and Norwegian athletes to determine both the inter‐ and intra‐individual variations of all steroids and ratios in the steroidal passport. Furthermore, we investigated if the inter‐individual variations could be associated with factors such as gender, type of sport, age, time of day, time of year, and if the urine was collected in or out of competition. We show that there are factors reported in today's doping tests that significantly affect the steroid profiles. The factors with the largest influence on the steroid profile were the type of sport classification that the athlete belonged to as well as whether the urine was collected in or out of competition. There were also significant differences based on what time of day and time of year the urine sample was collected. Whether these significant changes are relevant when longitudinally monitoring athletes in the steroidal module of the ABP should be evaluated further.     

Development and validation of a fast gas chromatography combustion isotope ratio mass spectrometry method for the detection of epiandrosterone sulfate in urine

In doping control, to confirm the exogenous origin of exogenously administered anabolic androgenic steroids (AAS), a gas chromatography combustion isotope ratio mass spectrometry (GC‐C‐IRMS) analysis is performed. Recently published work suggests that epiandrosterone sulfate (EpiAS) is a promising IRMS target compound for the detection of AAS, capable of prolonging the detection window. However, EpiAS is only excreted in urine in its sulfoconjugated form, while all other IRMS target compounds are excreted glucuronidated, meaning that EpiAS cannot be incorporated in the existing IRMS methods. A separate extensive sample preparation needs to be performed on this compound with a different hydrolysis and extraction procedure and a different liquid chromatography (LC) clean‐up. The current work presents a new, fast, and easy to implement EpiAS IRMS method. The approach was based on the direct GC analysis of non‐hydrolyzed EpiAS, making the solid phase extraction, hydrolysis, and acetylation step redundant. Sample preparation consisted of a simple liquid–liquid extraction, followed by LC fraction collection. A population study was performed to check compliance with the criteria drafted by the World Anti‐Doping Agency (WADA). To verify the applicability of the developed approach, the method was applied to the samples of four administration studies (i.e. dehydroepiandrosterone (DHEA), testosterone gel (T gel), androstenedione (ADION), and intramuscular testosterone undecanoate. In contrast to previously published data, the strength of EpiAS as the target compound and the prolongation of the detection window in comparison with the conventional IRMS target compounds was less pronounced.     

Long-term stability study and evaluation of intact steroid conjugate ratios after the administration of endogenous steroids

The most frequently detected substances prohibited by the World Anti-Doping Agency (WADA) belong to the anabolic steroids class. The most challenging compounds among this class are the endogenous anabolic steroids, which are detected by quantitative measurement of testosterone (T) and its metabolites with a so-called “steroid profiling” method. The current steroid profile is based on the concentrations and ratios of the sum of free and glucuronidated steroids. Recently, our group developed a steroid profiling method for the detection of three free steroids and 14 intact steroid conjugates, including both the glucuronic acid conjugated and sulfated fraction. The study aimed at evaluating the long-term stability of steroid conjugate concentrations and ratios, and the influence of different endogenous steroids on this extended steroid profile. A single dose of oral T undecanoate (TU), topical T gel, topical dihydrotestosterone (DHT) gel, and oral dehydroepiandrosterone (DHEA) was administered to six healthy male volunteers. One additional volunteer with a homozygote deletion of the UGT2B17 gene (del/del genotype) received a single topical dose of T gel. An intramuscular dose of TU was administered to another volunteer. To avoid fluctuation of steroid concentrations caused by variations in urinary flow rates, steroid ratios were calculated and evaluated as possible biomarkers for the detection of endogenous steroid abuse with low doses. Overall, sulfates do not have substantial additional value in prolonging detection times for the investigated endogenous steroids and administration doses. The already monitored glucuronides were overall the best markers and were sufficient to detect the administered steroids.     

High‐volume steroid isotopic standards developed as working standards for gas chromatography–combustion–isotope ratio mass spectrometry

High‐precision carbon isotope ratio analysis of urinary steroids by gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) is the official test to detect illicit doping of synthetic versions of endogenous steroids, such as testosterone. Our group created the first steroid isotopic standards (SIS) specifically for World Anti‐Doping Agency (WADA) accredited laboratories. The standards contain mixtures of steroids as acetates or free steroids at ~400 μg each per ampoule and have been widely distributed to anti‐doping laboratories to facilitate comparability of inter‐laboratory results. Here we report on the creation and characterization of 3 new high‐volume single component SIS suitable for use as working standards. They contain ~50 times more steroid mass per ampoule than previous SIS. The new SIS, coded CU/PCC 40‐1, CU/PCC 41‐1, & CU/PCC 42‐1, contain ~20 mg of androsterone, androsterone‐AC, and 5α‐cholestane, with determined isotopic values of ‐27.09 ± 0.07 mUr, ‐32.82 ± 0.01 mUr, ‐25.03 ± 0.01 mUr, respectively. We used our previously developed protocol to calibrate the isotopically uniform steroids against the isotopic standard gases methane and ethane in NIST RM 8559 that are traceable to the international standard Vienna PeeDee Belemnite (VPDB). Two sets of data, acquired 7 months apart, of absolute δ¹³C_VPDB and ?Δδ¹³C_VPDB values from 8 randomly selected ampoules of all 3 SIS indicate uniformity of steroid isotopic composition within measurement reproducibility, SD(δ¹³C) < 0.2 mUr Our results show that protocols for SIS extend to creation of high volume working standards that can also be used as internal standards under appropriate GC conditions.     

Stable carbon isotope ratio profiling of illicit testosterone preparations – domestic and international seizures

Gas chromatography‐combustion‐isotope ratio mass spectrometry (GC‐C‐IRMS) is now established as a robust and mature analytical technique for the doping control of endogenous anabolic androgenic steroids in human sport. It relies on the assumption that the carbon isotope ratios of naturally produced steroids are significantly different to synthetically manufactured testosterone or testosterone prohormones used in commercial medical or dietary supplement products. Recent publications in this journal have highlighted the existence of black market testosterone preparations with carbon isotope ratios within the range reported for endogenous steroids (i.e. δ¹³C ≥ ?25.8 ‰). In this study, we set out to profile domestic and international law enforcement seizures of illicit testosterone products to monitor the prevalence of ‘enriched’ substrates – which if administered to human subjects would be considered problematic for the use of current GC‐C‐IRMS methodologies for the doping control of testosterone in sport. The distribution of δ¹³C values for this illicit testosterone sample population (n = 283) ranged from ?23.4 ‰ to ?32.9 ‰ with mean and median of ?28.6 ‰ – comparable to previous work. However, only 13 out of 283 testosterone samples (4.6 %) were found to display δ¹³C values ≥ ?25.8 ‰, confirming that in the vast majority of cases of illicit testosterone administration, current GC‐C‐IRMS doping control procedures would be capable of confirming misuse. Copyright © 2014 John Wiley & Sons, Ltd.     

Studies of athlete biological passport biomarkers and clinical parameters in male and female users of anabolic androgenic steroids and other doping agents

The use of anabolic androgenic steroids (AAS) and other performance enhancing substances can change over time, so there is a need to constantly update what substances are used and can be detected. Six women and 30 men anabolic androgenic steroid users were recruited who filled out an anonymous questionnaire about their use of performance enhancing substances during the past year. Sampling took place on a single occasion and included blood and urine collection. Our aim was to identify which doping agents can be detected in men and women self‐reporting AAS use. The first choice of substances differed between men (testosterone) and women (oxandrolone). The use of growth hormones was reported among men (10%) and women (50%). Growth hormone releasing factors/secretagogs were reported by about ~ 20% in both genders. Nandrolone was the most frequently detected anabolic androgenic steroid even in those who did not report use in the past year. Of the current male testosterone users, 82% exhibited testosterone/epitestosterone (T/E) ratios of > 4. Men with current testosterone use displayed 4‐fold and 6‐fold higher median T/E, respectively, when compared with recent and previous testosterone users (P = 0.0001). Dermal testosterone use in women (n = 2) was not associated with a T/E ratio of > 4, but with supra‐physiological total serum testosterone concentrations. Changes in gonadotropins and hematological parameters were associated with the time of the last anabolic androgenic steroid intake in men, whereas in women these biomarkers were within the normal range. This highlights gender specific differences and indicates the need for additional biomarkers in female athletes.     

High sensitive analysis of steroids in doping control using gas chromatography/time-of-flight mass-spectrometry

The method of high sensitive gas chromatographic/time-of-flight mass-spectrometric (GC/TOF-MS) analysis of steroids was developed. Low-resolution TOF-MS instrument (with fast spectral acquisition rate) was used. This method is based on the formation of the silyl derivatives of steroids; exchange of the reagent mixture (pyridine and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)) for tert-butylmethylether; offline large sample volume injection of this solution based on sorption concentration of the respective derivatives from the vapour-gas mixture flow formed from the solution and inert gas flows; and entire analytes solvent-free concentrate transfer into the injector of the gas chromatograph. Detection limits for 100 μl sample solution volume were 0.5-2 pg/μl (depending on the component). Application of TOF-MS model 'TruTOF' (Leco, St Joseph, MO, USA) coupled with gas chromatograph and ChromaTOF software (Leco, St Joseph, MO, USA) allowed extraction of the full mass spectra and resolving coeluted peaks. Due to use of the proposed method (10 μl sample aliquot) and GC/TOF-MS, two times more steroid-like compounds were registered in the urine extract in comparison with the injection of 1 μl of the same sample solution.     

Studies on the minor metabolite 6a‐hydroxy‐androstenedione for doping control purposes and its contribution to the steroid profile

Recent publications have shown that the concentrations of minor metabolites such as formestane and 6a‐hydroxy‐androstenedione (6aOHADION) are import parameters, capable of increasing the specificity and efficiency of steroid abuse screening. The importance of such minor metabolites has been recognized for some time, but setting up concentration thresholds is not that straightforward with a single quadrupole gas chromatograph mass spectrometer (GC‐MS) because of the low concentrations; this is especially the case for 6aOH‐ADION. The main aim of this study was to propose a concentration threshold above which the detected 6aOH‐ADION is considered suspicious and isotope ratio mass spectrometry (IRMS) is recommended. Routine doping control samples (2128) from athletes that entered our lab and were not found suspicious for the intake of any doping substance were used to determine the baseline concentrations of 6a‐OH‐ADION. For this purpose, the more sensitive gas chromatography‐tandem mass spectrometry (GC‐MS/MS) was used, capable of quantifying these low concentrations with high reliability. A urinary concentration threshold of 5 ng/mL was set. Concentrations above this threshold are considered suspicious and are forwarded to IRMS for confirmation in routine practice. In addition, an IRMS method was developed, capable of determining the 13C value of 6aOH‐ADION. If a urine sample has an elevated 6aOH‐ADION concentration and normal 13C values for the traditional IRMS target compounds, we are still able to check the 13C value of 6aOH‐ADION. Six excretion studies were executed to stress the applicability of the threshold by visualizing the concentration and δ13C value time profiles of 6aOH‐ADION. Copyright © 2014 John Wiley & Sons, Ltd.     

Reference ranges for the urinary steroid profile in a Latin‐American population

The urinary steroid profile has been used in clinical endocrinology for the early detection of enzyme deficiencies. In the field of doping, its evaluation in urine samples is used to diagnose the abuse of substances prohibited in sport. This profile is influenced by sex, age, exercise, diet, and ethnicity, among others; laboratories own reference ranges might compensate for ethnic differences among population and inter‐laboratory biases. This paper shows the reference ranges obtained in the Antidoping Laboratory of Havana for the following steroid profile parameters: ten androgens (testosterone, epitestosterone, androsterone, etiocholanolone, 5α‐androstan‐3α,17β‐diol, 5β‐androstan‐3α,17β‐diol, dehydroepiandrosterone, epiandrosterone, 11β‐hydroxyandrosterone and 11β‐hydroxyetiocholanolone), three estrogens (estradiol, estriol and estrone), two pregnanes (pregnanediol and pregnanetriol) and two corticosteroids (cortisol and tetrahydrocortisol). The urine samples (male: n = 2454 and female: n = 1181) and data obtained are representative of population from Latin‐American countries like Cuba, Venezuela, Mexico, Dominican Republic, Guatemala and Chile. Urine samples were prepared by solid‐phase extraction followed by enzymatic hydrolysis and liquid‐liquid extraction with an organic solvent in basic conditions. Trimethylsilyl derivatives were analyzed by gas chromatography coupled to mass spectrometry. Reference ranges were established for each sex, allowing the determination of abnormal profiles as a first diagnostic tool for the detection of the abuse of androgenic anabolic steroids. The comparison with the Caucasian population confirms that the urinary steroid profile is influenced by ethnicity. Copyright © 2013 John Wiley & Sons, Ltd.     

Effect of non‐prohibited drugs on the phase II metabolic profile of morphine. An in vitro investigation for doping control purposes

The potential consequences of drug‐drug interaction on the strategies adopted by anti‐doping laboratories to report an adverse analytical finding for morphine were investigated. We evaluated in vitro the effects of 14 drugs on the principal metabolic pathways of morphine. The selected drugs are among those most commonly used by the athletes, none of them presently included in the World Anti‐Doping Agency (WADA) Prohibited List. The non‐prohibited drugs included 4 antifungals (fluconazole, itraconazole, ketoconazole, and miconazole), 6 benzodiazepines (alprazolam, bromazepam, clonazepam, lorazepam, lormetazepam, and triazolam), and 4 non‐steroidal anti‐inflammatory drugs (diclofenac, ibuprofen, ketoprofen, and nimesulide). The in vitro assays were based on the use of either human liver microsomes or uridine 5’‐diphospho‐glucuronosyl‐transferases. Morphine and its glucuronides were determined by developed liquid chromatography–mass spectrometry procedure after dilution with an aqueous solution containing their deuterated isotopologues as internal standards. Morphine is mainly excreted as phase II metabolites: about 70% of the parent compound is found to be biotransformed by UGT2B7 to morphine‐3‐glucuronide (6065%) and morphine‐6‐glucuronide (5‐10%). A reduction of the enzymatic activity of the UGT2B7 was recorded in the presence of 9 of the 14 drugs under investigation (ketoconazole, miconazole, itraconazole, diclofenac, ibuprofen, clonazepam, lorazepam, lormetazepam, and triazolam), with a consequent significant reduction of the levels of the glucuronide metabolites. This phenomenon in vivo may affect the rate of the urinary excretion of morphine with the risk of reporting “false negative” results, especially in case of results close to the decision limit value set by WADA.     

Sulfate metabolites as alternative markers for the detection of 4‐chlorometandienone misuse in doping control

Sulfate metabolites have been described as long‐term metabolites for some anabolic androgenic steroids (AAS). 4‐chlorometandienone (4Cl‐MTD) is one of the most frequently detected AAS in sports drug testing and it is commonly detected by monitoring metabolites excreted free or conjugated with glucuronic acid. Sulfation reactions of 4Cl‐MTD have not been studied. The aim of this work was to evaluate the sulfate fraction of 4Cl‐MTD metabolism by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) to establish potential long‐term metabolites valuable for doping control purposes. 4Cl‐MTD was administered to two healthy male volunteers and urine samples were collected up to 8 days after administration. A theoretical selected reaction monitoring (SRM) method working in negative mode was developed. Ion transitions were based on ionization and fragmentation behaviour of sulfate metabolites as well as specific neutral losses (NL of 15 Da and NL of 36 Da) of compounds with related chemical structure. Six sulfate metabolites were detected after the analysis of excretion study samples. Three of the identified metabolites were characterized by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) and gas chromatography‐tandem mass spectrometry (GC‐MS/MS). Results showed that five out of the six identified sulfate metabolites were detected in urine up to the last collected samples from both excretion studies. Copyright © 2016 John Wiley & Sons, Ltd.