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 sulfate metabolites of the doping agents oxandrolone and danazol using high performance liquid chromatography coupled to tandem mass spectrometry.

The direct detection of sulfate conjugates of anabolic androgenic steroids (AAS) can be a powerful tool in doping control analysis. By skipping the solvolysis step analysis time can be reduced, and due to long term sulfate metabolites the detection time can be significantly extended as demonstrated for some AAS. This study presents the successful identification of sulfate metabolites of the doping agents oxandrolone and danazol in excretion urines by high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). The sulfate conjugate of 17ß-hydroxymethyl-17α-methyl-18-nor-2-oxa-5α-androsta-13-en-3-one could be identified as a new metabolite of oxandrolone. Sulfate conjugates of the danazol metabolites ethisterone and 2α-hydroxymethylethisterone were identified in an excretion urine for the first time. In addition, these sulfate conjugates were synthesized successfully. For a confirmation analysis, the number of analytes can be increased by additional sulfate conjugates of danazol metabolites (2-hydroxymethyl-1,2-dehydroethisterone and 6ß-hydroxy-2-hydroxymethylethisterone), which were also identified for the first time. The presented validation data underline the suitability of the identified sulfate conjugates for doping analysis with regard to the criteria given by the technical documents of the World Anti-Doping Agency (WADA).

Analysis of tolvaptan and its metabolites in sports drug testing by high-performance liquid chromatography coupled to tandem mass spectrometry.

Tolvaptan is prohibited by the World Anti-doping Agency (WADA) under class S5 - Diuretics and masking agents. Less than 1% of the administrated dose is excreted by humans in urine. Knowledge concerning the metabolism in humans, and especially the excretion of metabolites in human urine, is limited. An analysis method based on the dilute-and-shoot approach using high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for detection was developed and validated. Ion transitions, which are part of this method, can easily be included in already existing screening methods used in routine doping analysis for the detection of diuretics. After administration of a single dose of tolvaptan to one male subject, low concentrations of the drug itself could be detected in urine samples over a time period of 24 h. In addition, hydroxyl metabolites of tolvaptan and one carboxyl metabolite with a cleaved benzazepine ring system were identified. These metabolites showed detection times of up to 150 h. An inclusion of these metabolites in the methods used in doping control analysis seems therefore to be of value. Copyright © 2016 John Wiley & Sons, Ltd.

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.

ß2-adrenergic stimulation enhances Ca2+ release and contractile properties of skeletal muscles, and counteracts exercise-induced reductions in Na+-K+-ATPase Vmax in trained men.

The aim of the present study was to examine the effect of ß2-adrenergic stimulation on skeletal muscle contractile properties, sarcoplasmic reticulum (SR) rates of Ca(2+) release and uptake, and Na(+)-K(+)-ATPase activity before and after fatiguing exercise in trained men. The study consisted of two experiments (EXP1, n = 10 males, EXP2, n = 20 males), where ß2-adrenoceptor agonist (terbutaline) or placebo was randomly administered in double-blinded crossover designs. In EXP1, maximal voluntary isometric contraction (MVC) of m. quadriceps was measured, followed by exercise to fatigue at 120% of maximal oxygen uptake (V̇O2, max ). A muscle biopsy was taken after MVC (non-fatigue) and at time of fatigue. In EXP2, contractile properties of m. quadriceps were measured with electrical stimulations before (non-fatigue) and after two fatiguing 45 s sprints. Non-fatigued MVCs were 6 ± 3 and 6 ± 2% higher (P < 0.05) with terbutaline than placebo in EXP1 and EXP2, respectively. Furthermore, peak twitch force was 11 ± 7% higher (P < 0.01) with terbutaline than placebo at non-fatigue. After sprints, MVC declined (P < 0.05) to the same levels with terbutaline as placebo, whereas peak twitch force was lower (P < 0.05) and half-relaxation time was prolonged (P < 0.05) with terbutaline. Rates of SR Ca(2+) release and uptake at 400 nm [Ca(2+)] were 15 ± 5 and 14 ± 5% (P < 0.05) higher, respectively, with terbutaline than placebo at non-fatigue, but declined (P < 0.05) to similar levels at time of fatigue. Na(+)-K(+)-ATPase activity was unaffected by terbutaline compared with placebo at non-fatigue, but terbutaline counteracted exercise-induced reductions in maximum rate of activity (Vmax) at time of fatigue. In conclusion, increased contractile force induced by ß2-adrenergic stimulation is associated with enhanced rate of Ca(2+) release in humans. While ß2-adrenergic stimulation elicits positive inotropic and lusitropic effects on non-fatigued m. quadriceps, these effects are blunted when muscles fatigue.