Urinary signature of anabolic steroids and glucocorticoids in humans by LC-MS.

A metabonomic strategy based on LC-MS was employed to investigate the metabolic profile of urine samples from 20 athletes who had been tested positive for corticoids and anabolic steroids and 29 controls. In this aim, different sample preparations and chromatographic conditions were compared. The acquired LC-MS data of doped athletes and controls were subjected to analysis of variance (ANOVA) and principal component analysis (PCA). Using this approach, molecular signature of human urine was obtained showing that metabonomics could be a complementary tool to discriminate different urinary profiles and to track down metabolic changes in humans.

Simultaneous quantification and qualification of synacthen in plasma.

Tetracosactide (Synacthen), a synthetic analogue of adrenocorticotropic hormone (ACTH), can be used as a doping agent to increase the secretion of glucocorticoids by adrenal glands. The only published method for anti-doping control of this drug in plasma relies on purification by immunoaffinity chromatography and LC/MS/MS analysis. Its limit of detection is 300 pg/mL, which corresponds to the peak value observed 12 h after 1 mg Synacthen IM administration. We report here a more sensitive method based on preparation of plasma by cation exchange chromatography and solid-phase extraction and analysis by LC/MS/MS with positive-mode electrospray ionization using 7-38 ACTH as internal standard. Identification of Synacthen was performed using two product ions, m/z 671.5 and m/z 223.0, from the parent [M + 5H](5+) ion, m/z 587.4. The recovery was estimated at 70%. A linear calibration curve was obtained from 25 to 600 pg/mL (R² > 0.99). The lower limit of detection was 8 pg/mL (S/N > 3). The lower limit of quantification was 15 pg/mL (S/N > 10; CV% < 20%). The performance of the method was illustrated by an 8-h kinetic analysis of plasma samples from nine subjects submitted to IM injections of either Synacthen® (five subjects) or Synacthen® Depot, the slow-release form of the drug (four subjects). Concentrations of Synacthen between 16 and 310 pg/mL were observed. A sensitive method for quantitation of Synacthen in plasma is proposed for anti-doping control analyses.

Short-term glucocorticoid intake improves exercise endurance in healthy recreationally trained women.

The present study investigated whether short-term oral administration of glucocorticoid would modify performance and selected hormonal and metabolic parameters during submaximal exercise in healthy women. Nine recreational female athletes completed cycling trials at 70-75% VO(2) max until exhaustion after either placebo (Pla, gelatin) or oral prednisone (Cor, Cortancyl, 50 mg per day for 1 week) treatment, according to a double-blind and randomized protocol. Blood samples were collected at rest; after 10, 20, and 30 min of exercise; at exhaustion; and after 10 and 20 min of passive recovery for adrenocorticotrophic hormone (ACTH), dehydroepiandrosterone (DHEA), prolactin (PRL), growth hormone (GH), insulin (Ins), blood glucose (Glu), and lactate (Lac) determination. Cycling time was significantly increased with short-term Cor intake (Cor: 66.4 +/- 8.4 vs. Pla: 47.9 +/- 6.7 min, P < 0.01). ACTH and DHEA remained completely blunted throughout the experiment with Cor versus Pla (P < 0.01), whereas GH and PRL were significantly decreased with Cor after, respectively, 20 and 30 min of exercise (P < 0.05). No significant difference in Ins or Glu values was found between the two treatments but Lac concentrations were significantly increased with Cor versus Pla between 10 and 30 min of exercise (P < 0.05). These data indicate that short-term glucocorticoid intake improved endurance performance in women, but further investigation is needed to determine whether these results are applicable to elite female athletes and, if so, current WADA legislation needs to be changed.

Urine ratio of tetrahydrocortisol to tetrahydrodeoxycortisol to screen for the systemic administration of cortisone and hydrocortisone.

We use gas chromatography-mass spectrometry (GC-MS) to determine the urine peak area ratio of tetrahydrocortisol (THF) to tetrahydrodeoxycortisol (THS) in spot urine samples of eight male volunteers after a single intramuscular injection of 100 mg hydrocortisone (HC) and after a single oral administration of 10 mg HC at six different post-treatment times over 24 h with 1 week between the two treatments. Control spot urine samples were also obtained from a group of 100 volunteers of each sex for GC-MS analysis. In addition, one female volunteer was collected for GC-MS and isotope ratio mass spectrometry (IRMS) analysis after a single oral administration of 40 mg HC and 40 mg cortisone (C) at 15 and 10 different post-treatment times over 30 h, respectively. IRMS analysis focused on the acetylated derivative of 11-keto-etiocholanolone (11KE) and 11beta-hydroxy-etiocholanolone (11OHE) as target metabolites, and on androsterone (A) as an endogenous reference compound (ERC) for calculating the corresponding delta(13)C (per thousand) depletion values. There was a small but significant sex-related difference for the THF/THS ratio in the control group with mean THF/THS ratio values of 10 and 13.5 for women and men, respectively. A cut-off value of 28 (mean+2 S.D.) for the THF/THS ratio offered a narrow detection window with 39% of suspicious samples after HC-oral treatment, and a wide detection window with 94% of suspicious samples after HC-intramuscular administration in men. For the woman the same cut-off value offered a wide detection window after HC and C administration with 100% and 90% of suspicious samples, respectively. On the basis of a cut-off value of 3 per thousand for the delta(13)C (per thousand) depletion, the exogenous origin was widely evidenced for at least one target compound in 93% and 80% of the HC and C samples, respectively. We conclude by discussing the predictive ability of the urine THF/THS ratio and its usefulness in pointing out suspicious samples resulting from the systemic administration of HC and C.

Interest of molecularly imprinted polymers in the fight against doping. Extraction of tamoxifen and its main metabolite from urine followed by high-performance liquid chromatography with UV detection.

A molecular imprinted polymer (MIP) has been synthesized in order to specifically extract tamoxifen, a nonsteroidal antiestrogen, and its metabolites from urine by solid-phase extraction (SPE) before HPLC-UV analysis. Clomiphene, a chlorinated tamoxifen analogue, was selected as template for MIP synthesis. Polymerisation was achieved by thermal polymerisation of methacrylic acid (MAA) as functional monomer, ethylene glycol dimethacrylate (EDMA) as cross-linking agent and acetonitrile as porogen. The efficient elimination of the urinary matrix has been obtained by MIP-SPE but the elution recovery of tamoxifen was initially too low ( approximately 14%). This problem has been overcome following two ways. At first, a preliminary HLB-SPE of the urine has enabled to discard endogenous salts and to percolate an organic sample through the MIP cartridge. Extraction recoveries are equal to 56 and 74% for tamoxifen and 4-hydroxytamoxifen, respectively. Then, a second MIP has been prepared with styrene and MAA as functional co-monomers. Strong pi-pi interactions occurring between phenyl groups of styrene and tamoxifen promote rebinding of the analyte by the specific sites. The enhanced hydrophobic character of the imprinted polymer has enabled the direct percolation of urine through MIP-SPE and the easy elimination of endogenous salts from urine with only one aqueous washing step. HPLC-UV analysis has confirmed high extraction recoveries (85%) for tamoxifen and its metabolite with an enrichment factor of 8. This analytical protocol can selectively detect the presence of tamoxifen metabolites in urines and be useful as a proof of doping in competitive sports.

Effects of short-term prednisolone intake during submaximal exercise.

PURPOSE: To examine the prednisolone's ergogenic and metabolic effects during submaximal exercise. METHODS: Ten recreational male athletes completed two cycling trials at 70-75% peak O2 consumption until exhaustion after either placebo (Pla, lactose) or oral prednisolone (Pred, 60 mg.d(-1) for 1 wk) treatment, according to a double-blind and randomized protocol. Blood samples were collected at rest and during exercise and recovery to determine ACTH, growth hormone (GH), prolactin (PRL), DHEA, insulin, blood glucose, and blood lactate values. RESULTS: Time of cycling was significantly increased after chronic Pred treatment (Pred: 74.5+/-9.5 min; Pla: 46.1+/-3.3 min, P<0.01). Pred intake significantly lowered basal, exercise, and recovery ACTH, DHEA, and PRL concentrations, whereas GH concentrations were significantly lowered by Pred after 30 min of exercise. Blood glucose and insulin were significantly (P<0.05) increased by Pred during the whole experiment and until 30 min of exercise. Blood lactate concentrations were higher after Pred versus Pla at 10 min of exercise until 10 min of recovery (P<0.05). CONCLUSION: From these data, short-term Pred intake did seem to significantly improve performance during submaximal exercise, with concomitant alterations in hormonal and metabolic responses. Further studies will be necessary to elucidate the mechanisms of these hormonal and metabolic changes, and to determine whether the changes may be associated with the marked performance improvement obtained.

Isoelectric profiles of human erythropoietin are different in serum and urine.

By adding a step of immunoaffinity to the method we had previously developed for analysing erythropoietin (EPO) in urine, we were able to study the isoelectric profiles of this hormone in human serum samples. This method was sensitive enough to investigate samples presenting physiological levels of this hormone. Comparison with the corresponding profiles in urine showed that natural EPO was systematically more acidic in urine. The acidification process, which was not patent in the non-human primate Cynomolgus macaque, clearly also affected recombinant EPO when injected into humans. This process was unrelated to any enzymatic activity in urine since the incubation of natural or recombinant EPO in urine induced no transformation of their isoelectric profiles. The nature and mechanism of the structural modifications occurring during the renal handling of this hormone remain to be investigated.

Detection of recombinant human erythropoietin in urine for doping analysis: interpretation of isoelectric profiles by discriminant analysis.

The detection in urine of recombinant human erythropoietin (rHuEPO), a hormone misused by endurance athletes as a doping agent, is based on the differentiation of its isoelectric pattern from that of the corresponding natural hormone. Different empirical criteria have been proposed for discriminating the images of the patterns but none of them have been elaborated from a rational statistical approach. Discriminant analysis was applied to a dataset of profiles defined as positive (116 profiles from 26 subjects) (presence of rHuEPO and possibly residual natural endogenous hormone) and negative (131 profiles from 131 subjects) (presence of natural endogenous hormone only). The different bands were numbered according to a template of 16 possible positions and their relative intensities constituted the 16 variables of the statistical analysis. This method was then tested with data from an administration trial of low doses (6.7-10 IU/kg) following high-dose (265 IU/kg) injections (71 profiles from one subject). The analysis of the dataset clearly separated the negative and positive profiles. A cross-validation procedure confirmed that the analysis was extremely stable: with ten-fold cross-validation, no false positives were observed even with 100,000 simulations. Furthermore, the detection of rHuEPO in the profiles from the low-dose trial was greatly improved in comparison with a previously validated empirical criterion.

Effects of acute salbutamol intake during supramaximal exercise in women.

OBJECTIVE: To study the effects of an acute therapeutic oral intake of beta(2) agonist on performance and substrate response during supramaximal exercise in women. METHODS: 12 healthy moderately trained female volunteers performed a Wingate test after ingestion of placebo (Pla) and salbutamol (Sal; 4 mg) according to a double-blind randomised crossover study. Blood samples were collected at rest, at the end of exercise and after 5 (r5), 10 (r10) and 15 (r15) min of passive recovery for adrenocorticotropic hormone (ACTH), growth hormone (GH), insulin, blood glucose and lactate measurements. RESULTS: Peak power (PP) and mean power (MP) significantly increased whereas time to peak power was significantly shorter with Sal than with Pla (p<0.05). No change was observed in the fatigue index. ACTH was not significantly modified but r15 growth hormone significantly decreased (p<0.05) after the intake of Sal. Both blood INS and blood glucose were significantly increased by the intake of Sal during all the experiments (p<0.01). Blood lactate was significantly increased by the intake of Sal compared with that of Pla (p<0.05) after 10 and 15 min of passive recovery. CONCLUSION: From these data, acute therapeutic oral intake of Sal seems to induce, irrespective of the subjects' gender, an improvement in performance during a supramaximal exercise--that is, increase in PP and MP. Further studies are necessary to clarify whether the mechanisms involved in the response to intake of Sal are linked to central and/or peripheral pathways.

Detection of recombinant epoetin and darbepoetin alpha after subcutaneous administration in the horse.

A direct detection method for anti-doping control of recombinant human erythropoietin (rHuEPO) abuse in racehorses is proposed. This method involves screening of plasma (or serum) by an enzyme-linked immunosorbent assay specific for human EPO and confirmation in urine samples by characterization of the urinary EPO isoelectric profile. This method was tested on horses that were administered epoetin alpha (rHuEPO) and the hyper-glycosylated form of this drug (darbepoetin alpha).

Detection of hemoglobin-based oxygen carriers in human serum for doping analysis: confirmation by size-exclusion HPLC.

BACKGROUND: Hemoglobin-based oxygen carriers (HBOCs) are being developed as potential substitutes for the oxygen-carrying functions of erythrocytes, but athletes may obtain and experiment with HBOCs as an illicit means of enhancing oxygen transport. An electrophoretic technique has been developed to screen for the presence of HBOCs in blood samples (Lasne et al. Clin Chem 2004;50:410-5). Interest has focused on complementary methods that can provide legally defensible scientific evidence for the presence of HBOCs in blood samples collected for doping control. METHODS: The aim of this research was to develop a size-exclusion SEC-HPLC technique to identify in plasma or serum samples the presence of HBOCs that are currently under development. This method was also used to detect a polymerized bovine hemoglobin (Hemopure) after infusion in 12 healthy males. RESULTS: The chromatograms of all HBOCs tested were clearly separated from the 54-min peak associated with human hemoglobin dimers. It was possible to differentiate between the different HBOC products based solely on their chromatographic profiles, provided they were at high concentrations. Differences were discernible not only based on the presence (or absence) of peaks, but also the separation between respective peaks. The profiles for serum samples collected from the men immediately after infusion of Hemopure showed a distinctive profile. The shape of the chromatographic profile remained consistent for at least 48 h. CONCLUSIONS: Under the analytical conditions reported here, SEC-HPLC was able to separate native hemoglobin from the modified hemoglobin molecules present in each of the HBOC products studied. In tandem with electrophoretic screening, SEC-HPLC provides evidence of the presence of HBOCs and can therefore be regarded as a method that satisfies the criteria for use in an antidoping control setting.

Detection of hemoglobin-based oxygen carriers in human serum for doping analysis: screening by electrophoresis.

BACKGROUND: Hemoglobin-based oxygen carriers (HBOCs) have recently been included in the International Olympic Committee and World Anti-Doping Agency lists of substances and methods prohibited in sports. To enforce this rule and deter abuse of HBOCs in elite sports, it is necessary to develop HBOC-specific screening and confirmation tests that are the usual steps in antidoping control analysis. METHODS: We developed a screening method based on electrophoresis of serum samples cleared of haptoglobin (Hp). Four successive steps (immunoprecipitation of Hp, electrophoresis of the cleared serum, Western blotting of the separated proteins, and detection of hemoglobin-related molecules based on the peroxidase properties of the heme moiety), provided electropherograms that could be easily interpreted in terms of the presence of HBOCs. This method was tested with serum samples enriched with various types of HBOCs: polymerized, conjugated, and cross-linked hemoglobins. It was also applied to blood samples collected from 12 healthy volunteers who had been infused with either 30 or 45 g of Hemopure, a glutaraldehyde-polymerized bovine hemoglobin. RESULTS: The method clearly detected the presence in serum of the various types of HBOCs tested and demonstrated no possible confusion with endogenous hemoglobin that may be present in cases of hemolysis. The test was able to detect Hemopure for 4-5 days after administration of 45 g to healthy individuals. CONCLUSIONS: The electrophoretic method is a simple, fast, and sensitive procedure that appears to fulfill the criteria of a screening test for the presence of HBOCs in antidoping control samples.

Detection of isoelectric profiles of erythropoietin in urine: differentiation of natural and administered recombinant hormones.

Erythropoietin (EPO) is normally present in urine at a low concentration (about 1IU/L, i.e., about 10ng/L) for a total protein concentration of at least 50mg/L. A method to study the isoelectric profile of this hormone from 20-ml urine aliquots without previous purification was developed. This method involves isoelectric focusing of the retentate from ultrafiltered urine. Both the ultrafiltration and the isoelectric focusing required precautionary measures to prevent EPO degradation by the proteases that are present in urine. Because classical immunoblotting gave rise to an unspecific detection of various urinary proteins in the focused retentate, it was essential to use the "double-blotting" process developed to solve this problem. Sufficient sensitivity was achieved using amplified chemiluminiscent detection after the blotting membrane was treated with dithiotreitol. The patterns that were revealed from various urinary samples proved to be highly heterogeneous as they were composed of more than 10 isoforms in a pI range of 3.7-4.7. Clear transformation of the patterns was observed in the case of treatment by the recombinant hormone, suggesting that this method can be regarded an efficient tool for indicating recombinant EPO misuse in sports. It may also open new investigations in the field of physiologic or pathologic exploration.