Influence of exercise in normal and hot ambient conditions on the pharmacokinetics of inhaled terbutaline in trained men.

This study investigated the pharmacokinetics of inhaled terbutaline at rest and after exercise in normal and hot ambient conditions with respect to doping analysis. Thirteen trained young men participated in the study. Urine and blood samples were collected after inhalation of 4 mg terbutaline during three trials: exercise in hot ambient conditions (30-35 °C) (EXH), exercise in normal ambient conditions (20-25 °C) (EX), and rest (20-25 °C) (R). Exercise consisted of 130 min at various intensities. Adjustment of urine concentrations of terbutaline to a specific gravity (USG) of 1.02 g/mL was compared with no adjustment. Area under the serum concentration-time curve within the first 6 h was higher for EX (27 ± 3 ng/mL/h) (P ≤ 0.01) and EXH (25 ± 4 ng/mL/h) (P ≤ 0.05) than for R (20 ± 3 ng/mL/h). When unadjusted for USG, urine concentrations of terbutaline after 4 h were different in the order EXH > EX > R (P ≤ 0.01). When unadjusted for USG, urine concentrations of terbutaline were 299 ± 151 ng/mL higher (P ≤ 0.001) after 4 h compared with adjusted concentrations in EXH. Excretion rate of terbutaline was higher (P ≤ 0.001) for EX than for EXH and R within the first 0-1½ h. In conclusion, EXHs results in higher urine concentrations of terbutaline. This should be considered when evaluating doping cases of terbutaline.

Analysis of anabolic androgenic steroids as sulfate conjugates using high performance liquid chromatography coupled to tandem mass spectrometry.

Improvements in doping analysis can be effected by speeding up analysis time and extending the detection time. Therefore, direct detection of phase II conjugates of doping agents, especially anabolic androgenic steroids (AAS), is proposed. Besides direct detection of conjugates with glucuronic acid, the analysis of sulfate conjugates, which are usually not part of the routine doping control analysis, can be of high interest. Sulfate conjugates of methandienone and methyltestosterone metabolites have already been identified as long-term metabolites. This study presents the synthesis of sulfate conjugates of six commonly used AAS and their metabolites: trenbolone, nandrolone, boldenone, methenolone, mesterolone, and drostanolone. In the following these sulfate conjugates were used for development of a fast and easy analysis method based on sample preparation using solid phase extraction with a mixed-mode sorbent and detection by high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). Validation demonstrated the suitability of the method with regard to the criteria given by the technical documents of the World Anti-Doping Agency (WADA). In addition, suitability has been proven by successful detection of the synthesized sulfate conjugates in excretion urines and routine doping control samples.

Urine and serum concentrations of inhaled and oral terbutaline.

We examined urine and serum concentrations after therapeutic use of single and repetitive doses of inhaled and supratherapeutic oral use of terbutaline. We compared the concentrations in 10 asthmatics and 10 healthy subjects in an open-label, cross-over study with 2 mg inhaled and 10 mg oral terbutaline on 2 study days. Further, 10 healthy subjects were administrated 1 mg inhaled terbutaline in 4 repetive doses with total 4 mg. Blood samples were collected at baseline and during 6 h after the first inhalations. Urine samples were collected at baseline and during 12 h after the first inhalations. Median (IQR) urine concentrations peaked in the period 0-4 h after inhalation with Cmax 472 (324) ng/mL in asthmatics and 661 (517) ng/mL in healthy subjects, and 4-8 h after oral use with Cmax 666 (877) ng/mL in asthmatic and 402 (663) ng/mL in healthy subjects. In conclusion we found no significant differences in urine and serum concentrations between asthmatic and healthy subjects. We compared urine and serum concentrations after therapeutic inhaled doses and supratherapeutic oral doses and observed significant statistical differences in both groups but found it impossible to distinguish between therapeutic and prohibited use based on doping tests with urine and blood samples.

Urine concentrations of repetitive doses of inhaled salbutamol.

We examined blood and urine concentrations of repetitive doses of inhaled salbutamol in relation to the existing cut-off value used in routine doping control. We compared the concentrations in asthmatics with regular use of beta2-agonists prior to study and healthy controls with no previous use of beta2-agonists. We enrolled 10 asthmatics and 10 controls in an open-label study in which subjects inhaled repetitive doses of 400 microgram salbutamol every second hour (total 1600 microgram), which is the permitted daily dose by the World Anti-Doping Agency (WADA). Blood samples were collected at baseline, 30 min, 1, 2, 3, 4, and 6 h after the first inhalations. Urine samples were collected at baseline, 0-4 h, 4-8 h, and 8-12 h after the first inhalations. Median urine concentrations peaked in the period 4-8 h after the first inhalations in the asthmatics and between 8-12 h in controls and the median ranged from 268 to 611 ng×mL (-1). No samples exceeded the WADA threshold value of 1000 ng×mL (-1) when corrected for the urine specific gravity. When not corrected one sample exceeded the cut-off value with urine concentration of 1082 ng×mL (-1). In conclusion we found no differences in blood and urine concentrations between asthmatic and healthy subjects. We found high variability in urine concentrations between subjects in both groups. The variability between subjects was still present after the samples were corrected for urine specific gravity.

A common deletion in the uridine diphosphate glucuronyltransferase (UGT) 2B17 gene is a strong determinant of androgen excretion in healthy pubertal boys.

BACKGROUND: Testosterone (T) is excreted in urine as water-soluble glucuronidated and sulfated conjugates. The ability to glucuronidate T and other steroids depends on a number of different glucuronidases (UGT) of which UGT2B17 is essential. The aim of the study was to evaluate the influence of UGT2B17 genotypes on urinary excretion of androgen metabolites in pubertal boys. STUDY DESIGN: A clinical study of 116 healthy boys aged 8-19 yr. UGT2B17 genotyping was performed using quantitative PCR. Serum FSH, LH, T, estradiol (E2), and SHBG were analyzed by immunoassays, and urinary levels of androgen metabolites were quantitated by gas chromatography/mass spectrometry in all subjects. RESULTS: Ten of 116 subjects (9%) presented with a homozygote deletion of the UGT2B17 gene (del/del), whereas 52 and 54 boys were hetero- and homozygous carriers of the UGT2B17 gene (del/ins and ins/ins), respectively. None of the reproductive hormones were affected by UGT2B17 genotype. In all subjects, mean urinary T/epitestosterone ratio was 1.56 [1.14 (SD); 0.1-6.9 (range)] and unaffected by age or pubertal stage. Subjects with homozygous deletions of UGT2B17 had significantly lower urinary levels of T and 5alpha- and 5beta-androstanediol. Mean urinary T/epitestosterone was significantly reduced in del/del subjects [0.29 (0.30); 0.1-1.0 (range), P < 0.0001]. CONCLUSION: In pubertal boys, a common homozygous deletion in the UGT2B17 gene strongly affected urinary excretion pattern of androgen metabolites but did not influence circulating androgen levels.

Determination of urinary norandrosterone excretion in females during one menstrual cycle by gas chromatography/mass spectrometry.

Conjugated norandrosterone is the main urinary metabolite of anabolic steroids like nandrolone, norandrostenedione and norandrostenediol. Nandrolone traces of endogenous origin have been identified in human follicular fluid, and further investigations revealed urinary excretion of norandrosterone in pregnant and non-pregnant females and even males. A threshold level for the norandrosterone concentration in urine has been established when controlling the administration of prohibited nandrolone or its precursors in human doping control. This level has been set to 2 ng/mL for males and females. To investigate the excretion of conjugated norandrosterone in females more systematically, we collected daily urine samples from 12 female volunteers during a whole menstrual cycle. These samples were analysed for norandrosterone down to a limit of quantification and identification of 0.05 ng/mL (180 pmol/L). The results clearly show that all the volunteers excreted norandrosterone glucuronide in a characteristic pattern during one menstrual cycle. Concentrations in urine were considerably lower at the beginning of the follicular and the end of the luteal phases than midcyclic. Peak concentrations up to 0.8 ng/mL (2.9 nmol/L) were recorded and they were three to four times higher than the values at the beginning and end of the cycle. The time of the peak concentration was clearly related to the increased excretion of luteinizing hormone. These results strongly support the possibility of endogenous nandrolone production as a side reaction to enzymatic aromatisation. However, a threshold value of 2 ng/mL for reporting adversed findings in doping control of females was never reached in any of the samples.

Formation of 19-norsteroids by in situ demethylation of endogenous steroids in stored urine samples.

The formation of 19-norsteroids by demethylation of endogenous steroids in stored urine samples was observed. Suspicious urine samples (i.e. containing trace amounts of 19-norandrosterone and 19-noretiocholanolone) were selected and spiked with deuterated analogues of androsterone and etiocholanolone at concentrations corresponding to high endogenous levels (4 microg/mL). After incubation, respective 19-norsteroids (19-norandrosterone-d4 and 19-noretiocholanolone-d5) were identified in these samples by high-resolution mass spectrometry. The transformation of the 5 beta-isomer (etiocholanolone) yields about three-fold higher concentrations, compared to the 5 alpha-isomer. A significant temperature dependence was observed by comparison of reaction kinetics at room temperature (23+/-2 degrees C) and 37 degrees C. Concentrations of 19-norandrosterone-d4 and 19-noretiocholanolone-d5, respectively, were 2.7 and 3.6 times higher at elevated temperature. The conversion of androsterone-d4 to 19-norandrosterone-d4 did not exceed a relative amount of 0.1%. Incubation of the urine samples with androsterone-d4-glucuronide led to the production of 19-norandrosterone-d4-glucuronoide. A partial stabilization was observed after addition of metabolic inhibitors (e.g. EDTA). The application of the incubation experiments described may contribute to the clarification of adverse analytical findings regarding low levels of 19-norsteroid metabolites.

Effect of rhEPO administration on serum levels of sTfR and cycling performance.

PURPOSE: We assessed the possibility of using soluble transferrin receptor (sTfR) as an indicator of doping with recombinant erythropoietin (rhEPO). METHODS: A double-blind, placebo-controlled study was conducted with the administration of 5,000 U of rhEPO (N = 10) or placebo (N = 10) three times weekly (181-232 U x kg(-1) x wk-1) for 4 wk to male athletes. We measured hematocrit and the concentration of hemoglobin, sTfR, ferritin, EPO, and quantified the effects on performance by measuring time to exhaustion and maximal oxygen uptake (VO2max) on a cycle ergometer. RESULTS: Hematocrit increased from 42.7 +/- 1.6% to 50.8 +/- 2.0% in the EPO group, and peaked 1 d after treatment was stopped. In the EPO group, there was an increase in sTfR (from 3.1 +/- 0.9 to 6.3 +/- 2.3 mg x L(-1) , P < 0.001) and in the ratio between sTfR and ferritin (sTfR-ferritin(-1)) (from 3.2 +/- 1.6 to 11.8 +/- 5.1, P < 0.001). The sTfR increase was significant after 1 wk of treatment and remained so for 1 wk posttreatment. Individual values for sTfR throughout the study period showed that 8 of 10 subjects receiving rhEPO, but none receiving placebo, had sTfR levels that exceeded the 95% confidence interval for all subjects at baseline (= 4.6 mg x L(-1)). VO2max increased from 63.6 +/- 4.5 mL x kg(-1) x min(-1) before to 68.1 +/- 5.4 mL x kg(-1) x min(-1) 2 d post rhEPO administration (7% increase, P = 0.001) in the EPO group. Hematocrit, sTfR, sTfR-ferritin(-1), and VO2max did not change in the placebo group. CONCLUSION: Serum levels of sTfR may be used as an indirect marker of supranormal erythropoiesis up to 1 wk after the administration of rhEPO, but the effects on endurance performance outlast the increase in sTfR.

The future of doping control in athletes. Issues related to blood sampling.

When current antidoping programmes were developed, the most frequently used doping agents were xenobiotics, such as stimulants and anabolic steroids, that are readily detectable in urine with the use of gas chromatography and mass spectrometry. As control of traditional doping agents became effective, some athletes turned to other means to improve performance, including blood doping and the application of recombinant peptide hormones such as erythropoietin and growth hormone. Doping with these agents is not easily detected in urine samples, and therefore new strategies must be developed as a supplement to those already in use. Such strategies will probably include analysing blood samples, as several of the most promising methods that are able to detect modern doping agents use blood as the analytical matrix. Non-autologous blood doping results in an admixture of self and foreign red blood cells that can be detected in a blood sample with the methods available. Methods to indicate doping with erythropoietin include the indirect finding of an elevated level of soluble transferrin receptor in serum, or a direct demonstration of a shift from the normal to an abnormal spectrum of erythropoietin isoforms. To indicate doping with growth hormone, a set of serum parameters including insulin growth factors and their binding proteins are under investigation as indirect evidence. A direct method using isotopic differences between endogenous and recombinant growth hormones is being investigated. A similar method has been established to detect the administration of testosterone esters. Several legal and ethical questions must be solved before blood sampling can become a part of routine doping control, but the major ethical question is whether sport can continue as today without proper methods to detect many modern doping agents.

Bone mineral density and menstrual irregularities. A comparative study on cortical and trabecular bone structures in runners with alleged normal eating behavior.

Bone mineral density (BMD), and associated biochemical and endocrine markers were compared in a group of runners with menstrual dysfunction (IR, n=13), and a group of performance matched eumenorrheic runners (R, n=15). All subjects claimed to have normal eating habits. Body height and weight, body mass index, and amount of body fat were similar. The IR group consisted of 5 presently oligomenorrheic (O) and 8 presently amenorrheic (A) runners. The BMD values of the athletes were additionally compared with corresponding values in a reference group (C) of healthy age matched controls (n=54). BMD values were significantly lower in IR compared with R on all measuring sites: Total body (-9%, p=0.03), femoral neck (-11%, p=0.01), lumbar spine (-12%, p=0.001), lower leg (-6.5%, p=0.03) and arms (-7%, p=0.01). In addition, IR athletes had lower total body (-5%, p=0.01), and lumbar spine BMD (-10%, p=0.001) than C. No differences were observed in serum IGF-1, SHBG, testosterone and cortisol, or in the biochemical marker of bone formation (osteocalcin) and bone resorption (1 CTP). Values of serum E2, FSH and LH were low in IR and normal in R. TSH was in the normal range in both groups, but f-T4 was significantly lower in IR than in R. The athletes were furthermore grouped according to past and present menstrual dysfunction severity. At all measuring sites, with the exception of the lower leg, increasing menstrual dysfunction severity was linearly associated with declining BMD values (p<0.05). In conclusion, even highly conditioned cortical bone tissue seems to be negatively related to menstrual disorders, which may serve to explain the high incidence of stress fractures in athletes with menstrual disorders. Single measurements of biochemical markers of bone resorption and formation may not reflect the current bone status.

Determination of benzodiazepines in human urine and plasma with solvent modified solid phase micro extraction and gas chromatography; rationalisation of method development using experimental design strategies.

Solid phase micro extraction (SPME) and gas chromatographic analysis was used for the analysis of several benzodiazepines (oxazepam, diazepam, nordiazepam, flunitrazepam and alprazolam) in human urine and plasma. Several factors likely to affect the analyte recovery were screened in a fractional factorial design in order to examine their effect on the extraction recovery. Parameters found significant in the screening were further investigated with the use of response surface methodology. The final conditions for extraction of benzodiazepines were as follows: Octanol was immobilised on a polyacrylate fibre for 4 min. The fibre was placed in the sample and extraction took place at pH 6.0 for 15 min. Urine samples were added to 0.3 g ml(-1) sodium chloride. In plasma, the extraction recovery was less than in urine and releasing the benzodiazepines from plasma proteins followed by protein precipitation was found necessary prior to sampling. The method was validated and found linear over the range of samples. The limits of detection in urine were determined to be in the range 0.01-0.45 micromol l(-1). The corresponding limits of detection in plasma were in the range 0.01-0.48 micromol l(-1). Finally, the method developed was applied to determine some benzodiazepines after administration of a single dose. This method offers sufficient enrichment for bioanalysis after a single dose of high dose benzodiazepines as diazepam, but for low dose benzodiazepines as flunitrazepam, further sensitivity is needed.

Blood sampling in doping control. First experiences from regular testing in athletics.

We report the results from blood sampling taken for the first time during doping control in athletics. The study includes samples from 99 athletes tested during IAAF-meetings in 1993-94. Blood doping with allogenic blood was not detected. The distribution of haemoglobin levels in athletes did not differ markedly from that found in controls. Erythropoietin (EPO) values were markedly lower in athletes than in controls, and 58% had EPO lower than the detection limit for the assay. This may be due to high-altitude residence prior to testing. Measurements of growth hormone (GH) and insulin-like growth factor 1 did not suggest GH-misuse in any athlete tested. One third of the male athletes had testosterone levels that were lower than the normal reference interval. This may at least partly be due to the combination of sampling at night and after strenuous exercise. One female athlete was found to have a grossly elevated testosterone level. In conclusion, the present results show the importance of taking into account the special circumstances during sampling when interpreting results from blood testing in athletes. Future research should focus on developing more sensitive and specific tests to detect doping with endogenous substances such as GH and EPO.

Stimulants, narcotics and beta-blockers: 25 years of development in analytical techniques for doping control.

More than 25 years of developing doping control methods have led to comprehensive screening and confirmation procedures for stimulants, narcotics and beta-blockers. Much of this work has been initiated and/or improved by the late Prof. Dr. Manfred Donike. The methodological approach covered in this overview was applied to doping control procedures during the XXV Summer Olympics in Barcelona, Spain, in 1992 and the XVII Winter Olympics in Lillehammer, Norway, in 1994. Urine samples are screened through a combination of two analytical methods that are complementary: (a) gas chromatographic analysis of the parent compound and unconjugated metabolites, following single-step sample extraction and detection by a nitrogen-specific detector based on a retention index identification system and (b) gas chromatographic analysis including also conjugated drugs and metabolites after hydrolysis, solid-phase extraction, derivatisation and mass spectrometric detection. Confirmation and identification is always performed by gas chromatographic separation and full scan mass spectrometric detection. These methods facilitate the rapid screening and confirmation of more than 100 stimulants, narcotic analgesics and beta-blockers in urine for at least 24 h after the intake of a pharmaceutical dose. Application of the methods ensures high quality standards for the unequivocal identification of doping agents as well as a rapid turnaround time for sample analyses.

Chemometric evaluation of urinary steroid profiles in doping control.

Ten endogenous steroid hormones and metabolites were determined according to the screening procedure for anabolic steroids in spot urine samples from 105 healthy young male athletes (control samples) and 23 males that tested positive for anabolic steroids in the doping control (positive samples). The GC-MS peak areas for each sample were normalized to total area. Multivariate data analysis by Partial Least Square Regression (PLSR) and using a coded Y-variable (positive samples: +1 and control samples -1) allows projection of the most systematic profile structures into a 2D plot revealing a clear distinction between the control and misuser groups. The most important determinants of the location in the loading plot were the ratios of testosterone to epitestosterone and androsterone to etiocholanolone. The ratio between 11-beta-hydroxyandrosterone and 11-beta-hydroxy-etiocholanolone was less important, in accordance with the fact that anabolic-androgenic steroid intake primarily affects the excretion of testosterone from the testis and to a much lesser degree adrenal steroid genesis. We present a preliminary validation of this model (PLS1-DISCRIM) for analysing steroid profiles in doping control samples from several categories of athletes, some of which are suspected for drug misuse, and results from a one dose excretion study in healthy volunteers. Our findings suggest that use of multivariate PLS-regression may give valuable information about anabolic androgenic steroid misuse in sport. When appropriately calibrated, this methodology may delineate drug misusers directly from the screening procedure for anabolic steroids in spot urine tests.

[Endocrine effects of doping with androgenic anabolic steroids]. / Endokrine effekter av doping med androgene anabole steroider.

We describe two case histories that highlight some of the endocrine effects of doping with androgenic anabolic steroids. The main endocrine effect observed after use of androgenic anabolic steroids is the development of hypogonadotrope hypogonadism, characterized by low levels of gonadotrophins, suppression of testosterone production and azoospermia. If testosterone is used alone, or in combination with synthetic anabolic steroids, the circulating levels of testosterone are normal or high. Oestrogen levels may be elevated owing to aromatization of testosterone. The level of sex hormone binding globulin is suppressed. These endocrine parameters are of practical use in evaluating patients misusing androgenic anabolic steroids.