Urinary excretion profile of prednisolone and prednisone after rectal administration: Significance in antidoping analysis.

The rectal administration of glucocorticoids, as well as any injectable, and oral ones, is currently prohibited by the World Anti-Doping Agency when occurs "in competition." A reporting level of 100 ng/ml for prednisolone and 300 ng/ml for prednisone was established to discriminate the allowed and the prohibited administration. Here, the urinary excretion profiles of prednisone and prednisolone were evaluated in five volunteers in therapy with glucocorticoid-based rectal formulations containing prednisone or prednisolone caproate. The urinary levels of the excreted target compounds were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) following the procedure validated and currently in use in our laboratory to detect and quantitate glucocorticoids in urine. Predictably, the excretion trend of the analytes of interest were generally comparable with those obtained after oral administration, even if the excretion profile showed a broad interindividual variability, with the absorption rate and the systemic bioavailability after rectal administration being strongly influenced by the type of formulations (suppository or rectal cream, in our case) as well as the physiological conditions of the absorption area. Results showed that the target compounds were detectable for at least 30 h after drug administration. After suppository administration, prednisolone levels reached the maximum after 3 h from drug administration and then dropped below the reporting level after 15-21 h; prednisone reached the maximum after 3 h from drug administration, and then dropped below the reporting level after 12-15 h. After cream administration, both prednisone and prednisolone levels remained in a concentration below the reporting level throughout the entire monitored period.

5α-reductase inhibitors: Evaluation of their potential confounding effect on GC-C-IRMS doping analysis.

5α-reductase inhibitors (5-ARIs) are considered by the World Anti-doping Agency as potential confounding factors in evaluating the athlete steroid profile, since they may interfere with the urinary excretion of several diagnostic compounds. We herein investigated 5α-reductase inhibitors from a different perspective, by verifying their influence on the carbon isotopic composition of 5α- and 5ß-reduced testosterone and nandrolone metabolites. The GC-C-IRMS analysis was performed on a set of urine samples collected from three male Caucasian volunteers after the acute and chronic administration of finasteride in combination with the intake of 19-norandrostenedione, a nandrolone precursor. The excretion and the isotopic profile of androsterone (A), etiocholanolone (Etio) 5α-androstane-3α,17ß-diol (5αAdiol), and 5ß-androstane-3α,17ß-diol (5ßAdiol) were determined as well as those of 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE). Pregnanediol (PD) and pregnanetriol (PT) were also measured as endogenous reference compounds to define the individual endogenous isotopic profile. Our results confirmed the impact of finasteride, especially if chronically administered, on the enzymatic pathway of testosterone and nandrolone, and pointed out the influence of 5-ARIs on δ13 C values of the selected target compounds determined in the IRMS confirmation analysis.

Arimistane: Degradation product or metabolite of 7-oxo-DHEA?

RATIONALE: The instability of androst-5-ene-3,7-dione structures under acidic conditions is known. The formation of arimistane from 7-oxo-DHEA, influenced by the conditions of sample extraction, and mainly derivatization reaction and gas chromatography (GC) injector temperature, was described earlier, potentially leading to misinterpretation of results. By using a liquid chromatography (LC)-mass spectrometry (MS) (LC-MS) we investigated the stability of the 7-oxo-DHEA in two different solvents (methanol and dimethyl sulfoxide [DMSO]), and the arimistane formation after the application common analytical procedures. Additionally, in vitro and in vivo studies of 7-oxo-DHEA were performed. METHODS: The stability of 7-oxo-DHEA was studied in solutions after 60 days storage at -20°C. In vitro studies were performed by incubating 7-oxo-DHEA with human liver microsomes (HLMs). Healthy volunteers collected urine samples before and after the administration of a single dose of 7-oxo-DHEA. Analyses were performed using high-performance LC (HPLC) coupled to a triple quadrupole mass spectrometer (MS/MS) and GC combustion isotope ratio mass spectrometry (GC-C-IRMS) following HPLC purification. RESULTS: 7-oxo-DHEA was stable after 60 days in DMSO while a protic solvent as methanol promotes the degradation of 7-oxo-DHEA to arimistane. HLM incubations showed no formation of arimistane and the sample preparation only influenced the degradation of 7-oxo-DHEA when solvolysis was applied. After the administration study the presence of arimistane also after the hydrolysis with ß-glucuronidase (Escherichia coli) was observed while using ß-glucuronidase/arylsulfatase (Helix pomatia) showed the presence of arimistane already in blank samples collected before administration. CONCLUSIONS: Our results confirm arimistane as a valuable diagnostic marker of 7-oxo-DHEA administration, but also indicate that its formation is due to degradation processes rather than to metabolic biotransformation reactions.

Detecting the abuse of 19-norsteroids in doping controls: A new gas chromatography coupled to isotope ratio mass spectrometry method for the analysis of 19-norandrosterone and 19-noretiocholanolone.

The detection of 19-norsteroids abuse in doping controls currently relies on the determination of 19-norandrosterone (19-NA) by gas chromatography-tandem mass spectrometry (GC-MS/MS). An additional confirmatory analysis by gas chromatography coupled to isotope ratio mass spectrometry (GC-C-IRMS) is performed on samples showing 19-NA concentrations between 2.5 and 15 ng/ml and not originated from pregnant female athletes or female treated with 19-norethisterone. 19-Noretiocholanolone (19-NE) is typically produced to a lesser extent as a secondary metabolite. The aim of this work was to improve the GC-C-IRMS confirmation procedure for the detection of 19-norsteroids misuse. Both 19-NA and 19-NE were analyzed as target compounds (TCs), whereas androsterone (A), pregnanediol (PD), and pregnanetriol (PT) were selected as endogenous reference compounds (ERCs). The method was validated and applied to urine samples collected by three male volunteers after the administration of nandrolone-based formulations. Before the instrumental analysis, urine samples (<25 ml) were hydrolyzed with ß-glucuronidase from Escherichia coli and extracted with n-pentane. Compounds of interest were purified through a single (for PT) or double (for 19-NE, 19-NA, A, and PD) liquid chromatographic step, to reduce the background noise and eliminate interferences that could have affect the accuracy of δ13 C values. The limit of quantification (LOQ) of 2 ng/ml was ensured for both 19-NA and 19-NE. The 19-NE determination could be helpful in case of "unstable" urine samples, in late excretion phases or when coadministration with 5α-reductase inhibitors occur.

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.

Carbon isotopic characterization of prednisolone and prednisone pharmaceutical formulations: Implications in antidoping analysis.

Twenty-two pharmaceutical formulations containing prednisolone or prednisone commercially available in Italy, Belgium, Spain, Brazil, and India were analyzed through a specific gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) method. All of them showed typical non-endogenous δ13 C values, except for the Belgian nasal spray, Sofrasolone®, with a less depleted 13 C content (-17.84 ± 0.18‰). Observational studies were performed on two volunteers in therapy with Sofrasolone® to confirm the applicability of the method and to suggest adequate interpretation criteria also in the case of drugs with less negative δ13 C values. Urine samples were collected before, during, and within the 36 hours after the administration of the spray. Both δ13 C values and urinary concentrations of prednisolone and prednisone were evaluated. All samples were subjected to an adequate pre-treatment (enzymatic hydrolysis, liquid/liquid extraction, and two sequential HPLC steps) before injection to the GC-C-IRMS instrument, according to the method recently developed and validated in our laboratory. Pregnanediol (PD), tetrahydro-11-deoxycortisol (THS), and pregnanetriol (PT) were selected as endogenous reference compounds (ERC). The excretion profile was estimated through liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method used routinely for the quali-quantitative detection of glucocorticoids. δ13 C values and urinary levels of prednisolone and prednisone were also determined after the intake of one single vial of Sintredius®, a prednisolone oral formulation with a conventional more negative δ13 C value (-29.28 ± 0.25‰). Finally, the potential masking effect that combined therapy with Sofrasolone® and Sintredius® could induce on the IRMS findings was investigated.

7-keto-DHEAmetabolism in humans. Pitfalls in interpreting the analytical results in the antidoping field.

7-keto-DHEA (3ß-hydroxy-androst-5-ene-7,17-dione) is included in section S1 of the World Antidoping Agency (WADA) List of Prohibited Substances. The detection of its misuse in sports needs special attention, since it is naturally present in urine samples. The main goal of this study is to investigate the in vivo metabolism of 7-keto-DHEA after a single administration to healthy volunteers and to better describe the relationship between arimistane (androst-5-ene-7,17-dione) and 7-keto-DHEA after the application of the common routine procedures to detect anabolic steroids in WADA accredited antidoping laboratories. Free, glucuro-, and sulpho-conjugated steroids extracted from urine samples obtained before and after the administration of 7-keto-DHEA were analyzed by different gas chromatographic (GC)-mass spectrometric (MS) techniques. Gas chromatography coupled to tandem MS to study the effect on the endogenous steroid profile, coupled to isotope ratio mass spectrometry (IRMS) to investigate the potential formation of androgens derived from DHEA and coupled to high resolution accurate mass spectrometry (HRMS) to investigate new diagnostic metabolites. The analysis by IRMS confirmed that there is no formation of DHEA from 7-keto-DHEA. Ten proposed metabolites, not previously reported, were described. These include reduced and hydroxylated structures that are not considered part of the steroid profile in antidoping analyses. They showed considerable responses in all fractions analyzed. Some deoxidation reactions (including arimistane formation) were found and most probably can be linked to the sample preparation or instrumental analysis. This is important when interpreting the results after the application of procedures to detect steroids in urine currently used in antidoping laboratories. 7-keto-DHEA metabolism in humans for antidoping purposes was studied and unexpected results were found. This could lead to a misinterpretation of the data, depending on the procedure applied and the analytical instrumentation used.

Development and validation of a method to confirm the exogenous origin of prednisone and prednisolone by GC-C-IRMS.

Prednisone and prednisolone are two anti-inflammatory steroidal drugs listed by the World Anti-Doping Agency (WADA) within the class of glucocorticoids, which are prohibited "in competition" and when administered systemically. Their presence in collected urine samples may be attributed, if no exogenous administration has occurred, to an in situ microbial formation from endogenous steroids. In this work, a gas chromatography coupled to carbon isotope ratio mass spectrometry (GC-C-IRMS) method was developed and validated to distinguish an exogenous origin from an endogenous one. Eight prednisone/prednisolone pharmaceutical preparations commercially available in Italy were analysed to establish an exogenous δ13 C value reference range (-28.96 ± 0.39‰). No more than 25 mL of urine was processed and no derivatization nor intentional steroids structure modifications were performed before the GC-C-IRMS analysis. A first HPLC purification step was set up to isolate the three endogenous reference compounds (ERCs) selected (tetrahydro-11-deoxycortisol (THS), pregnanediol (PD), and pregnanetriol (PT)), while a second LC purification was necessary to separate prednisone from prednisolone. In the GC-C-IRMS analysis, two different GC run methods were set up to guarantee better sensitivity and selectivity for each compound. Both prednisone and prednisolone showed signals (m/z 44) with amplitudes within the method linearity range to a lower urinary concentration of 20 ng/mL (< WADA reporting level, 30 ng/mL). The method was fully validated according to WADA requirements. As a proof of concept, urine samples collected from two excretion studies in healthy male volunteers, after a prednisone or prednisolone administration, were analysed by the proposed method, demonstrating its applicability for the analysis of real samples.

Isotope ratio mass spectrometry in antidoping analysis: The use of endogenous reference compounds.

RATIONALE: Isotope ratio mass spectrometry (IRMS) is an analytical technique required by the World Antidoping Agency (WADA) before releasing of an adverse finding for the abuse of pseudoendogenous steroids (i.e. testosterone). For every single individual, the delta 13 C values (‰) of the selected target compounds (TCs, i.e. testosterone and/or its precursors/metabolites) are compared with those of endogenous reference compounds (ERCs). The aim of this work is to investigate the individual variation in the delta values of four different commonly used ERCs to establish the maximum acceptable variation, in order to detect potential outliers. METHODS: Routine urine samples collected for antidoping purposes were submitted to IRMS confirmation. After a specific liquid chromatographic purification of the analytes of interest, the final extracts were analyzed by gas chromatography/combustion (GC/C)-IRMS. The selected ERCs monitored were pregnanediol, pregnanetriol, 11-keto-etiocholanolone and 11ß-hydroxyandrosterone. The obtained 13 C delta values were statistically analyzed to evaluate their inter- and intra-individual distribution. RESULTS: The delta values of the ERCs studied showed a normal distribution and no major differences among genders were observed. As expected, there are differences depending on the geographical origin of the samples, reflecting different dietary habits and food sources. The intra-individual dispersion, expressed as the standard deviation (SD) of the values of the studied ERCs, did not greatly exceed the instrumental error (0.5‰), demonstrating the good preservation of the delta values along the metabolic pathway. CONCLUSIONS: For the selected ERCs of non-sporting volunteers and the urinary specimens from more than 1000 sportsmen, we can propose a maximum SD of 0.54‰ and range of 1.2‰ for delta 13 C values as acceptance criteria to detect potential outliers. These cases can be caused by the external masking effect of the administration of a substance modifying the delta values or outliers due to unforeseen procedural artifacts.

Fast IRMS screening of pseudoendogenous steroids in doping analyses.

The detection of the abuse of pseudoendogenous steroids (testosterone and/or its precursors) is currently based, when possible, on the application of the steroid module of the World Anti-Doping Agency (WADA), athlete biological passport (ABP), implemented through the global database, ADAMS. When a suspicious sample is detected, the confirmation by isotope ratio mass spectrometry (IRMS) is required. It is well known that this confirmation procedure is time consuming and expensive and can be only applied on a reduced number of samples. In previous studies we have demonstrated that the longitudinal evaluation of the IRMS data is able to detect positive samples that otherwise will be evaluated as negative, improving the efficacy of the fight against doping in sport. This would require the analysis of a much larger volume of samples by IRMS. The aim of the present work is to describe an IRMS screening method allowing to increase the throughput of samples that can be analyzed by IRMS. The detection efficacy of the method is compared with the confirmation method in use, and to assess its robustness and applicability, all the samples of a major cycling stage competition were analyzed, with the agreement of the testing authority, under routine conditions and response times. The results obtained permit to conclude that the IRMS screening method here proposed has adequate selectivity and produces results that overlap with the already validated method currently in use permitting to analyze a much higher volume of samples even during a major event without compromising the detection capacity. Copyright © 2017 John Wiley & Sons, Ltd.

Longitudinal evaluation of the isotope ratio mass spectrometric data: towards the 'isotopic module' of the athlete biological passport?

The detection of the abuse of pseudo-endogenous steroids (testosterone and/or its precursors) is currently based on the application of the steroid module of the World Anti-Doping Agency (WADA) Athletes' Biological Passport (ABP), implemented through ADAMS. Diagnostic metabolites are monitored for every athlete and statistically evaluated with a predictive Bayesian approach. In the case of suspicious samples, the data of the ABP are confirmed and the isotope ratio mass spectrometry (IRMS) test is activated. We have previously demonstrated that IRMS enables confirmation of the non-endogenous origin of pseudo-endogenous steroids in otherwise non-suspicious samples, after a longitudinal evaluation of the ABP, even after the inclusion of additional long-term diagnostic hydroxylated metabolites, and that the delta values of the parameters obtained during the IRMS confirmation process presented much less variability compared to the parameters of the ABP. The aim of the present work is to evaluate the application of the same methodology used for the evaluation of the ABP, on the delta values of the pseudo-endogenous steroids monitored. The effectiveness of the proposed model has been assessed on samples obtained after controlled administrations of oral androstenedione and transdermal testosterone. The results support the conclusion that, if applied, the longitudinal evaluation of the IRMS data allows the detection of positive samples that otherwise will be reported as atypical findings (ATF), improving the efficacy of the fight against doping in sport. This approach, by narrowing the individual acceptable range, could possibly improve the detection of the intake of preparations of synthetic origin with delta values close to or overlapping those of endogenously produced steroids. Copyright © 2016 John Wiley & Sons, Ltd.

Development and validation of a GC-C-IRMS method for the confirmation analysis of pseudo-endogenous glucocorticoids in doping control.

Glucocorticoids are included in the S9 section of the World Anti-doping Agency (WADA) prohibited list international standard. Some among them are pseudo-endogenous steroids, like cortisol and cortisone, which present the same chemical structure as endogenously produced steroids. We are proposing an analytical method based on gas chromatography coupled to isotope ratio mass spectrometry (GC-C-IRMS) which allows discrimination between endogenous and synthetic origin of the urinary metabolites of the pseudo-endogenous glucocorticoids. A preliminary purification treatment by high-performance liquid chromatography (HPLC) of the target compounds (TC) (i.e., cortisol, tetrahydrocortisone (THE) 5α-tetrahydrocortisone (aTHE), tetrahydrocortisol (THF), and 5α-tetrahydrocortisol (aTHF)) allows collection of extracts with adequate purity for the subsequent analysis by IRMS. A population of 40 urine samples was analyzed for the TC and for the endogenous reference compounds (ERC: i.e., 11-desoxy-tetrahydrocortisol (THS) or pregnanediol). For each sample, the difference between the delta values of the ERCs and TCs (Δδ values) were calculated and based on that, some decision limits for atypical findings are proposed. The limits are below 3% units except for cortisol. The fit to purpose of the method has been confirmed by the analysis of urine samples collected in two patients under treatment with 25 mg of cortisone acetate (p.o). The samples showed Δδ values higher than 3 for at least 24 h following administration depending on the TC considered. The method can easily be integrated into existing procedures already used for the HPLC purification and IRMS analysis of pseudo-endogenous steroids with androgenic/anabolic activity.

Detection of formestane abuse by mass spectrometric techniques.

Formestane (4-hydroxy-androstenedione) is an aromatase inhibitor prohibited in sports and included, since 2004, in the list of prohibited substances updated yearly by the World Anti-Doping Agency (WADA). Since the endogenous production of formestane has been described, it is mandatory for the anti-doping laboratories to use isotope ratio mass spectrometry (IRMS) to establish the exogenous origin before issuing an adverse analytical finding. The described IRMS methods for formestane detection are time-consuming, requiring usually two consecutive liquid chromatographic sample purifications in order to have final extracts of adequate purity before the mass spectrometric analysis. After establishing a procedure for the determination of the origin of formestane by IRMS without the need of derivatization, and integrated in the overall analytical strategy of the laboratory for pseudo-endogenous steroids, a mass spectrometric analysis by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) of formestane metabolites was carried out in order to investigate whether other biomarkers of formestane abuse could be integrated in order to avoid time-consuming and expensive IRMS confirmations for formestane. From the metabolic studies performed, the inclusion of 3ß,4α-dihydroxy-5α-androstan-17-one (4α-hydroxy-epiandosterone) in the routine GC-MS procedures has demonstrated to be diagnostic in order to reduce the number of unnecessary confirmations of the endogenous origin of formestane.

Metabolism of boldione in humans by mass spectrometric techniques: detection of pseudoendogenous metabolites.

Boldione is an anabolic androgenic steroid (AAS) related to boldenone, androstenedione, and testosterone bearing two double bonds in C1 and C4 positions. Boldione is rapidly transformed to the well-known AAS boldenone, being both compounds included in the list of prohibited substances and methods published yearly by the World Anti-Doping Agency (WADA). After the administration of boldione to a male volunteer, the already described urinary metabolites of boldenone produced after reduction in C4, oxydoreduction in C3 and C17, and hydroxylation have been detected. In addition, minor new metabolites have been detected and their structure postulated after mass spectrometric analyses. Finally, the reduction of the double bound in C1 produces metabolites identical to the endogenously produced ones. A method based on gas chromatography coupled to isotope ratio mass spectrometry (GC/C/IRMS) after a urine sample purification by high performance liquid chromatography (HPLC) permitted to confirm the main synthetic like boldione/boldenone metabolite (17ß-hydroxy-5ß-androst-1-en-3-one) and boldenone at trace levels (< 5 ng/mL) and then to establish its synthetic or endogenous origin, and to determine the exogenous origin of metabolites with the same chemical structure of the endogenous ones. The detection of pseudoendogenous androgens of synthetic origin partially overlapped boldenone and its main metabolite detection, being an additional proof of synthetic steroids misuse. By the use of IRMS, the correct evaluation of the modifications of the steroid profile after the administration of synthetic AAS that could be converted into endogenous like ones is possible.

A comprehensive procedure based on gas chromatography-isotope ratio mass spectrometry following high performance liquid chromatography purification for the analysis of underivatized testosterone and its analogues in human urine.

The confirmation by GC/C/IRMS of the exogenous origin of pseudo-endogenous steroids from human urine samples requires extracts of adequate purity. A strategy based on HPLC sample purification prior to the GC/C/IRMS analysis of human urinary endogenous androgens (i.e. testosterone, androsterone and/or androstenediols), is presented. A method without any additional derivatization step is proposed, allowing to simplify the urine pretreatment procedure, leading to extracts free of interferences permitting precise and accurate IRMS analysis, without the need of correcting the measured delta values for the contribution of the derivatizing agent. The HPLC extracts were adequately combined to both reduce the number of GC/C/IRMS runs and to have appropriate endogenous reference compounds (ERC; i.e. pregnanediol, 11-keto-etiocholanolone) on each GC-IRMS run. The purity of the extracts was assessed by their parallel analysis by gas chromatography coupled to mass spectrometry, with GC conditions identical to those of the GC/C/IRMS assay. The method has been validated according to ISO17025 requirements (within assay precision below 0.3‰(13)C delta units and between assay precision below 0.6‰(13)C delta units for most of the compounds investigated) fulfilling the World Anti-Doping Agency requirements.

A simplified procedure for GC/C/IRMS analysis of underivatized 19-norandrosterone in urine following HPLC purification.

Nandrolone and/or its precursors are included in the World Anti-doping Agency (WADA) list of forbidden substances and methods and as such their use is banned in sport. 19-Norandrosterone (19-NA) the main metabolite of these compounds can also be produced endogenously. The need to establish the origin of 19-NA in human urine samples obliges the antidoping laboratories to use isotope ratio mass spectrometry (IRMS) coupled to gas chromatography (GC/C/IRMS). In this work a simple liquid chromatographic method without any additional derivatization step is proposed, allowing to drastically simplify the urine pretreatment procedure, leading to extracts free of interferences permitting precise and accurate IRMS analysis. The purity of the extracts was verified by parallel analysis by gas chromatography coupled to mass spectrometry with GC conditions identical to those of the GC/C/IRMS assay. The method has been validated according to ISO17025 requirements (within assay precision of ±0.3‰ and between assay precision of ±0.4‰). The method has been tested with samples obtained after the administration of synthetic 19-norandrostenediol and samples collected during pregnancy where 19-NA is known to be produced endogenously. Twelve drugs and synthetic standards able to produce through metabolism 19-NA have shown to present δ(13)C values around -29‰ being quite homogeneous (-28.8±1.5; mean±standard deviation) while endogenously produced 19-NA has shown values comparable to other endogenous produced steroids in the range -21 to -24‰ as already reported. The efficacy of the method was tested on real samples from routine antidoping analyses.

Parallel analysis of stimulants in saliva and urine by gas chromatography/mass spectrometry: perspectives for "in competition" anti-doping analysis.

Stimulants are banned by the World Anti-Doping Agency (WADA) if used "in competition". Being the analysis of stimulants presently carried out on urine samples only, it might be useful, for a better interpretation of analytical data, to discriminate between an early intake of the substance and an administration specifically aimed to improve the sport performance. The purpose of the study was to investigate the differences, in terms of excretion/disappearance of drugs, between urine and oral fluid, a sample that can reflect plasmatic concentrations. Oral fluid and urine samples were collected following oral administration of the following stimulants: modafinil (100 mg), selegiline (10 mg), crotetamide/cropropamide (50 mg each), pentetrazol (100 mg), ephedrine (12 mg), sibutramine (10 mg), mate de coca (a dose containing about 3mg of cocaine); analysis of drugs/metabolites was carried out by gas chromatography/mass spectrometry (GC/MS) in both body fluids. Our results show that both the absolute concentrations and their variation as a function of time, in urine and in oral fluid, are generally markedly different, being the drugs eliminated from urine much more slowly than from oral fluid. Our results also suggest that the analysis of oral fluid could be used to successfully complement the data obtained from urine for "in competition" anti-doping tests; in all those cases in which the metabolite(s) concentration of a substance in urine is very low and the parent compound is not detected, it is indeed impossible, relying on urinary data only, to discriminate between recent administrations of small doses and remote administrations of higher doses.

Detection of sibutramine administration: a gas chromatography/mass spectrometry study of the main urinary metabolites.

A gas chromatographic/mass spectrometric (GC/MS) study aimed at identifying the metabolites of sibutramine (1-(4-chlorophenyl)-N,N-dimethyl-alpha-(2-methylpropyl)cyclobutanemethanamine) in urine is described. Urinary excretion of sibutramine metabolites following the oral administration of a single dose of sibutramine was followed by GC/MS analysis. After identification of the chromatographic signals corresponding to the six main urinary metabolites, the fragmentation pattern was studied in electron ionization (EI) mode after derivatization to the corresponding methyl and trimethylsilyl derivatives. Urine samples were pretreated according to a reference procedure (liquid/liquid separation, enzymatic hydrolysis, pre-concentration under a stream of nitrogen and derivatization, either under thermal incubation and by microwave irradiation). All sibutramine metabolites were excreted as glucuroconjugates, and retain the chiral carbon present in the sibutramine skeleton. The metabolites identified included mono-desmethylsibutramine (nor-sibutramine), bi-desmethylsibutramine (nor-nor-sibutramine), and the corresponding hydroxylated compounds, the hydroxylation taking place either on the cyclobutane or on the isopropyl chain. The excretion profiles of the different metabolites were also evaluated. From an analytical point of view, the method can be applied to different fields of forensic analytical toxicology, including anti-doping analysis. Although the lack of certified reference materials does not allow a precise determination of the limits of detection (LODs) of all the sibutramine metabolites, an estimation taking into account the response factor of similar compounds ensures that all metabolites are still clearly detectable in a range of concentrations between 10 and 50 ng/mL, thus satisfying the minimum required performance limits (MRPLs) of the World Anti-Doping Agency (WADA).

Determination of clenbuterol in human urine by GC-MS-MS-MS: confirmation analysis in antidoping control.

This work presents a GC-MS-MS-MS method for the direct determination of clenbuterol in human urine. The method comprises a pretreatment procedure and the instrumental analysis of the derivatives performed by GC-MS(3) (ion trap) with electron impact ionization. The GC-MS(3) analysis allows isolation and characterization of specific fragments from the original (MS(1)) molecular structure, and in particular, those fragments originating from the precursor ion cluster (m/z=335-337) characteristic of clenbuterol. The MS(2) product fragment m/z=300 is in turn used as a further precursor fragment giving rise to a MS(3) spectrum specific for clenbuterol. MS(4) fragmentation spectra were also investigated. However, further fragmentation of MS(3) product ions does not lead to functional MS(4) spectra nor to any significant increase in the signal-to-noise ratio. The sensitivity limit of the MS(3) technique is lower than 0.2 microg/l, with a linear range between 0.5 and 5 microg/l, thus matching the basic requirements for antidoping analysis according to the guidelines of the International Olympic Committee. Due to its overall analytical performance, the method is presently being evaluated as a confirmation protocol to be followed to detect illicit clenbuterol administration to the athletes, and compared with reference GC-MS and GC-MS-MS techniques.