Meldonium use by athletes at the Baku 2015 European Games.

BACKGROUND: The aim of this report was to estimate the prevalence of meldonium use in athletes competing in the Baku 2015 European Games to contribute to the surveillance of substances on the 2015 World Anti-Doping Agency (WADA) Monitoring Program. Meldonium is reported to be used by athletes to potentially enhance personal performance and shorten the recovery period after physical activity. METHODS: Three sources of data were reviewed to determine the prevalence of meldonium use during the Games including: (1) athlete self-reported declarations of drug and supplement use; (2) declarations from National Olympic Committee medical teams of the list of medicines that they imported into Azerbaijan as part of their stock of drugs for administration; (3) results from the antidoping laboratories reporting the detection of meldonium. RESULTS: Meldonium was declared as imported into Azerbaijan by 2 of 50 National Olympic Committee medical teams at the Games, but athletes from 6 countries declared the use of meldonium. Only 23 of the 662 (3.5%) athletes tested from 8 to 28 June 2015 declared the personal use of meldonium, which included 13 competition winners. However, 66 of the total 762 (8.7%) athlete urine samples analysed during the Games and during precompetition tested positive for meldonium. Meldonium use was detected in athletes competing in 15 of the 21 sports during the Games. CONCLUSIONS: This study highlights the widespread and inappropriate use and prescribing of this prescription drug in a generally healthy athlete population. Subsequent to these findings, WADA has included meldonium as a prohibited substance on the 2016 List of Prohibited Substances.

Evidence for acyl-iron ligation in the active site of [Fe]-hydrogenase provided by mass spectrometry and infrared spectroscopy.

[Fe]-hydrogenase catalyzes the reversible heterolytic cleavage of H(2) and stereo-specific hydride transfer to the substrate methenyltetrahydromethanopterin in methanogenic archaea. This enzyme contains a unique iron guanylylpyridinol (FeGP) cofactor as a prosthetic group. It has recently been proposed-on the basis of crystal structural analyses of the [Fe]-hydrogenase holoenzyme-that the FeGP cofactor contains an acyl-iron ligation, the first one reported in a biological system. We report here that the cofactor can be reversibly extracted with acids; its exact mass has been determined by electrospray ionization Fourier transform ion cyclotron resonance mass-spectrometry. The measured mass of the intact cofactor and its gas-phase fragments are consistent with the proposed structure. The mass of the light decomposition products of the cofactor support the presence of acyl-iron ligation. Attenuated total reflection infrared spectroscopy of the FeGP cofactor revealed a band near wave number 1700 cm(-1), which was assigned to the C=O (double bond) stretching mode of the acyl-iron ligand.

Formyl migration product of chanoclavine-I aldehyde in the presence of the old yellow enzyme FgaOx3 from Aspergillus fumigatus: a NMR structure elucidation.

A previous study showed that together with the festuclavine synthase FgaFS, the old yellow enzyme FgaOx3 from Aspergillus fumigatus catalyzed the conversion of chanoclavine-I aldehyde to festuclavine in the biosynthesis of ergot alkaloids. In the absence of FgaFS, a mixture containing two compounds with a ratio of 7:3 was detected in the enzyme assay of FgaOx3. NMR experiments including (DQF)-COSY, HSQC, HMBC and NOESY identified their structures as E/Z isomers of N-methyl-N-[(5R,10R)-10-(2-oxo-propyl)-2,4,5,10-tetrahydrobenzo[cd]indol-5-yl]formamide and proved the migration of the formyl group at C-8 in chanoclavine I-aldehyde to N-6 in the identified products.

Identification of highly polar nitroaromatic compounds in leachate and ground water samples from a TNT-contaminated waste site by LC-MS, LC-NMR, and off-line NMR and MS investigations.

Leachate and ground water samples from a trinitrotoluene-contaminated waste disposal site near a former ammunitions plant in Stadtallendorf, Germany, were analyzed by liquid chromatography (LC)-mass spectrometry (MS) and LC-NMR hyphenated techniques to comprehensively characterize the range of highly polar nitroaromatic compounds. Wherever unknown components could not be identified by comparison with a multistandard, the spectroscopic data obtained on-line were used to make initial structure proposals, which were later confirmed by comparison with authentic reference materials. In those cases where reference materials were not commercially available, unknown compounds were isolated by HPLC cuts and their structures were elucidated by off-line NMR and MS investigations. A variety of previously unknown compounds, including nitrophenols, nitrobenzyl alcohols, methylnitrobenzoic acids, and hydroxynitrobenzoic acids, could be identified. The NMR and MS data are presented here. The main polar compounds were additionally quantified.

Toxicological characterization of 2,4,6-trinitrotoluene, its transformation products, and two nitramine explosives.

The soil and groundwater of former ordnance plants and their dumping sites have often been highly contaminated with the explosive 2,4,6-trinitrotoluene (2,4,6-TNT) leading to a potential hazard for humans and the environment. Further hazards can arise from metabolites of transformation, by-products of the manufacturing process, or incomplete combustion. This work examines the toxicity of polar nitro compounds relative to their parent compound 2,4,6-TNT using four different ecotoxicological bioassays (algae growth inhibition test, daphnids immobilization test, luminescence inhibition test, and cell growth inhibition test), three genotoxicological assays (umu test, NM2009 test, and SOS Chromotest), and the Ames fluctuation test for detection of mutagenicity. For this study, substances typical for certain steps of degradation/transformation of 2,4,6-TNT were chosen for investigation. This work determines that the parent compounds 2,4,6-TNT and 1,3,5-trinitrobenzene are the most toxic substances followed by 3,5-dinitrophenol, 3,5-dinitroaniline and 4-amino-2-nitrotoluene. Less toxic are the direct degradation products of 2,4,6-TNT like 2,4-dinitrotoluene, 2,6-dinitrotoluene, 2-amino-4,6-dinitrotoluene, and 4-amino-2,6-dinitrotoluene. A weak toxic potential was observed for 2,4,6-trinitrobenzoic acid, 2,4-diamino-6-nitrotoluene, 2,4-dinitrotoluene-5-sulfonic acid, and 2,6-diamino-4-nitrotoluene. Octahydro-l,3,5,7-tetranitro-l,3,5,7-tetrazocine and hexahydro-1,3,5-trinitro-l,3,5-triazine show no hint of acute toxicity. Based on the results of this study, we recommend expanding future monitoring programs of not only the parent substances but also potential metabolites based on conditions at the contaminated sites and to use bioassays as tools for estimating the toxicological potential directly by testing environmental samples. Site-specific protocols should be developed. If hazardous substances are found in relevant concentrations, action should be taken to prevent potential risks for humans and the environment. Analyses can then be used to prioritise reliable estimates of risk.

Determination of five nitrobenzoic acids in groundwater by solid-phase extraction and liquid chromatography-mass spectrometry.

A method involving solid-phase extraction (SPE) and reversed-phase liquid chromatography-mass spectrometry (LC-MS) has been developed for determination, in groundwater, of nitrobenzoic acids associated with 2,4,6-trinitrotoluene production. Pre-concentration on a co-polymer-based SPE cartridge enabled quantitative extraction of the analytes from water. Investigation of negative ion electrospray and atmospheric-pressure chemical ionization mass spectrometry indicated the sensitivity of APCI was more than twice that of ESI. An 15N-labeled internal standard was used to achieve more accurate quantitation and mass assignment. Recovery was better than 80% when 10 mL water was extracted with the SPE cartridge. Combination of SPE with LC-MS analysis resulted in method detection limits of less than 5 microg L-1. The method has been used for analysis of groundwater samples collected from a site of a former ammunition plant. Contamination with nitrobenzoic acids was determined at microg L-1 levels.

Predicting the failure of disc surgery by a hypofunctional HPA axis: evidence from a prospective study on patients undergoing disc surgery.

Patients with postoperative ongoing sciatic pain have been shown to exhibit reduced cortisol levels along with enhanced IL-6 levels. The aim of the present study was to clarify the relationship between a reduced cortisol secretion and enhanced cytokine levels by performing a prospective study on patients with disc herniation. Twenty-two patients were examined before and after their disc surgery. Twelve healthy, pain-free subjects matched for age, education and gender constituted the control group. The preoperative examinations included the assessment of the diurnal pattern of cortisol secretion and the feedback sensitivity of the hypothalamus-pituitary-adrenal (HPA) axis. Patients' subjective stress levels also were assessed during the preoperative examination. The diurnal pattern of cortisol secretion was again assessed during the postoperative examination. Furthermore, blood samples were collected to measure catecholamine, adrenocorticotropic hormone (ACTH)- and interleukin-6 (IL-6) levels before and after measuring the pressure pain thresholds (PPTs). An assessment of the sensitivity of circulating monocytes to the immunosuppressive effects of glucocorticoids was further included in the postoperative examinations. Failed back syndrome (FBS) patients (n=12) showed a reduced cortisol secretion in the morning hours and enhanced feedback sensitivity of the HPA axis. Furthermore, FBS patients displayed an increased in-vitro production of proinflammatory cytokines and a relative glucocorticoid resistance of pro-inflammatory cytokine producing monocytes as compared to non-FBS patients (n=10) and controls. After PPT measurement FBS patients exhibited an increased norepinephrine but decreased epinephrine response, together with lower ACTH levels and a four times higher plasma IL-6 response. These findings suggest that chronically stressed patients are at a higher risk for a poor surgical outcome as their reduced cortisol secretion promotes the postoperative ongoing synthesis of proinflammatory cytokines.

Analysis of dinitro- and amino-nitro-toluenesulfonic acids in groundwater by solid-phase extraction and liquid chromatography-mass spectrometry.

A reversed-phase LC-MS method with quadrupole-time of flight (QTOF) detection has been developed for the determination of four dinitro-toluenesulfonic acids and two amino-nitro-toluenesulfonic acids in groundwater. The analytes were separated by HPLC with 0.1% ( v/ v) formic acid as mobile phase modifier compatible with mass spectrometric detection. QTOF-MS analysis with negative ion electrospray ionization afforded good selectivity and sensitivity for analysis of the dinitro- and amino-nitro-toluenesulfonic acids. Structure elucidation and confirmation were accomplished by tandem mass spectrometry. Characteristic ions resulting from the loss of NO, NO(2), and SO(2) from the [M-H](-) ions were detected. An intense fragment ion at m/ z 80 representing the [SO(3)](-) ion was detected for all dinitro- and amino-nitro-toluenesulfonic acids. Solid-phase extraction using a co-polymer cartridge was developed for preconcentration of the analytes from water. Good recovery (>85%) was achieved when 0.1% formic acid was added into the water samples before extraction. Method detection limits ranged from 10 to 76 ng L(-1) for the targeted compounds when 10 mL water was analyzed. Groundwater samples collected from wells close to a former ammunition plant in Stadtallendorf, Germany, were analyzed for the dinitro- and amino-nitro-toluenesulfonic acids.

UV-A/blue-light inactivation of the 'metal-free' hydrogenase (Hmd) from methanogenic archaea.

H2-forming methylenetetrahydromethanopterin dehydrogenase (Hmd) is an unusual hydrogenase present in many methanogenic archaea. The homodimeric enzyme dubbed 'metal-free' hydrogenase does not contain iron-sulfur clusters or nickel and thus differs from [Ni-Fe] and [Fe-Fe] hydrogenases, which are all iron-sulfur proteins. Hmd preparations were found to contain up to 1 mol iron per 40 kDa subunit, but the iron was considered to be a contaminant as none of the catalytic and spectroscopic properties of the enzyme indicated that it was an essential component. Hmd does, however, harbour a low molecular mass cofactor of yet unknown structure. We report here that the iron found in Hmd is most probably functional after all. Further investigation was initiated by the discovery that Hmd is inactivated upon exposure to UV-A (320-400 nm) or blue-light (400-500 nm). Enzyme purified in the dark exhibited an absorption spectrum with a maximum at approximately 360 nm and which mirrored its sensitivity towards light. In UV-A/blue-light the enzyme was bleached. The cofactor extracted from active Hmd was also light sensitive. It showed an UV/visible spectrum similar to that of the active enzyme and was bleached upon exposure to light. Photobleached cofactor no longer had the ability to reconstitute active Hmd from the apoenzyme. When purified in the dark, Hmd consistently contained per monomer about one Fe, which was tightly bound to the cofactor. The iron was released from the enzyme and from the cofactor upon light inactivation. Hmd activity was inhibited by high concentrations of CO and CO protected the enzyme from light inactivation indicating that the iron in Hmd is of functional importance. Therefore, reference to Hmd as 'metal-free' hydrogenase is no longer appropriate.

HPLC-MS investigations of acidic contaminants in ammunition wastes using volatile ion-pairing reagents (VIP-LC-MS).

In order to hyphenate ion pairing chromatography and MS detection we used several types of formates as volatile ion pairing reagents (IPRs) instead of common tetraalkylammonium salts, as these salts tend to precipitate in the ion source. The formates were prepared by mixing formic acid with the corresponding amine. Both tributyl- and trihexylammonium formate proved to be valuable IPRs for the separation of acidic compounds like nitrobenzoic acids, nitrobenzenesulfonic acids and nitrated phenols. Due to the weaker retention of the ion-pairs with trialkylammonium formates compared with tetraalkylammonium compounds, either less organic modifier or a higher concentration of the IPR had to be used. With negative atmospheric pressure chemical ionization mass spectrometry and electrospray ionization mass spectrometry it was possible to unambiguously identify several acidic oxidation products of 2,4,6-trinitrotoluene (TNT) in ammunition wastewater and soil extracts. 2-amino-4,6-dinitrobenzoic acid was often found to be the main metabolite of TNT in such water samples.

Analysis of polynitrophenols and hexyl by liquid chromatography-mass spectrometry using atmospheric pressure ionisation methods and a volatile ion-pairing reagent.

An LC-MS method for the determination of picric acid (2,4,6-trinitrophenol), its reductive transformation products picramic acid (2-amino-4,6-dinitrophenol) and iso-picramic acid (4-amino-2,6-dinitrophenol) and hexyl (2,2',4,4',6,6'-hexanitrodiphenylamine) has been developed. The analytes were separated using ion-pairing chromatography with a volatile ion-pairing reagent suitable for subsequent MS detection. The performance of an atmospheric pressure chemical ionisation (APCI) and an electrospray ionisation (ESI) interface was compared. ESI-MS is more sensitive for the analytes, especially for hexyl and picric acid, APCI-MS delivered more fragments necessary for unequivocal identification. With LC-ESI-MS limits of detection using single ion monitoring (SIM) mode are 4 ng (iso-picramic acid), 800 pg (picramic acid), 400 pg (picric acid) and 80 pg (hexyl). For quantification, 15N-picric acid was used as an internal standard. Using this new method, the degradation of picric acid in soil was monitored in a laboratory study. Furthermore, the presence of picramic acid was for the first time verified in soil samples from a former ammunition plant.