Gonadotropin-releasing hormone (GnRH) and its small peptide synthetic analogues are included in Section S2 of the World Anti-Doping Agency (WADA) Prohibited List as they stimulate pituitary luteinizing hormone (LH) and testicular testosterone (T) secretion. Both the following approaches can be applied for determination of abuse of these peptides: direct identification of intact compounds and their metabolites in athletes' biofluids and evaluation of LH and T concentrations as mediate markers of drug intake. To develop an effective concept for GnRH and its analogues determination in anti-doping control, in vitro and in vivo studies were conducted. A new method was applied to the evaluation of the slow-release profile of buserelin, goserelin, and leuprolide biodegradable microspheres after the intramuscular injection in male volunteers. Eight metabolites of 10 GnRH analogues were identified after incubation with human kidney microsomes, most of them were leuprolide degradation products. Obtained data were added into ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for GnRH analogues determination. The detection time windows for administered peptides and their metabolites in urine samples were evaluated with 2 sample preparation techniques: dilute-and-shoot and solid-phase extraction. To support the second hypothesis, the measurement of LH and the main parameters of the steroid profile were performed in urine samples. Just 1 compound among those investigated resulted in the LH concentration dropping to non-physiological levels. Thus, for doping-control purposes, monitoring of hormone levels fluctuations could be applied only together with longitudinal passport steroid profile data.
The quantification of the low levels of catecholamines and metanephrines in biological fluids is important for clinical screening of pheochromocytoma/paraganglioma and diagnosis of overtraining syndrome in athletes. We introduce a novel, accurate and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of these biogenic amines in human plasma. Simple protein precipitation combined with rapid 2-aminoethyl diphenylborinateassisted liquid-liquid extraction allow us to quantify catecholamines and metanephrines over broad concentration ranges. Target analytes were monitored in positive electrospray ionization mode by multiple reaction monitoring. Method performance was validated for linearity, lower limit of quantification, limit of detection, intra-day and inter-assay precision, carry-over, recovery, and matrix effect. The assay was linear within analytical range 25-1000 pg mL −1 for epinephrine, 30-2500 pg mL −1 for norepinephrine, 15-1000 pg mL −1 for dopamine, 25-2000 pg mL −1 for metanephrine and 50-10000 pg mL −1 for normetanephrine, with lower limits of quantification of 15, 20, 10, 15 and 30 pg mL −1 , respectively. The intra-and inter-day precisions for all compounds ranged from 0.4 to 6.9% and from 0.9 to 6.6%, respectively. The efficiency of novel method was confirmed by assaying external quality control samples which demonstrated consistent and accurate results.
The very high intensity of exercise accompanied by mental stress triggers adaptive mechanisms associated with adrenocortical steroidogenesis. However, the association between adrenocortical steroidogenesis and the high intensity of exercise in elite athletes is poorly studied. A significant obstacle to solving this complex task is the wide range (4–5 orders) of steroid concentrations in serum and limitations related to the amount of biological samples taken from professional athletes. To solve this task, we have developed and validated a non-trivial approach for targeted serum metabolic profiling based on the use of LC-MS/MS with dual-polarity electrospray ionization. The developed method based on the proposed approach allows for the quantitative determination of 14 stress resistance biomarkers in elite athletes using a small amount of specimen within 8.5 min.
In the present study, a rapid, sensitive, and selective method for determination of several synthetic analogues of gonadotropin-releasing hormone in human urine by solid-phase extraction and ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) was developed. Various parameters affecting sample preparation, LC separation, and MS/MS detection were investigated, and optimized conditions were identified. The UPLC-MS/MS system was equipped with an electrospray ion source operating in positive ion mode with selected reaction monitoring. Leuprolide-13С6 was used as internal standard for analytes quantitative assessment. The proposed method was validated considering the parameters specificity, linearity (0.1-10 ng/ml), recovery (52-98%), limit of detection (0.1 ng/ml), matrix effects and stability.
Aim of the study: to identify mutual interaction between the reaction to a moving object with functional state of the central nervous system and kinematic-dynamic parameters of complex coordination movement.Materials and methods: 9 elite alpine skiers were participated in this study. Visual-motor coordination variables were assessed by computer complex for psychophysiological testing NS-Psychotest (Neurosoft, Russia). Dynamic parameters of complex coordination movement during counter movement jump were registered on the MuscleLab Force Plate (Ergotest Innovation A.S., Norway). Quantitation of hormones — adrenaline and noradrenaline as well as neurotransmitters — dopamine and serotonin in blood samples was performed using ultra-high performance liquid chromatograph combined with triple quadrupole mass analyzer LCMS-8060 (Shimadzu, Japan).Results: a significant negative relationship between the maximum output of motor efforts during counter movement jump, mean reaction time and the number of negative reactions recorded within visual-motor coordination testing was documented. A reliable positive relationship between excitation processes, jump power and jump time was established. Increases in noradrenaline and serotonin concentrations are positively associated with the number of accurate reactions, whereas dopamine level was positively correlated with jump altitude.Conclusion: the predominance of excitation over inhibition processes in the central nervous system had a positive effect on reducing the time spent on counter moving and increasing the maximum power of movement. As applied to alpine skiers we registered the following relationship: the higher the speeds of signal perception and muscle activation when solving a visual-motor task, the higher the power of working efforts, the shorter the time of the eccentric phase and total time spent on performing counter movement jump.
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