Identifying N-nitrosofenfluramine in a nutrition supplement.
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The analytical data for identifying an unknown substance that was found in a nutrition supplement is presented. The unknown substance is purified using thin-layer chromatography and then measured using high-resolution mass spectrometry (HRMS) giving the exact mass from which the structure of the unknown substance was proposed. The procedure for synthesizing N-nitrosofenfluramine from fenfluramine is described. The extracted, synthesized, and standard N-nitrosofenfluramine are compared using HRMS, high-performance liquid chromatography (HPLC)-MS, HPLC-UV, Fourier transform IR spectroscopy, gas chromatography-MS, TLC, and NMR (1H NMR and 13C NMR). All analytical data obtained confirm that the unknown peak in the nutrition supplement is N-nitrosofenfluramine and that the synthetic procedure described can easily provide the N-nitrosofenfluramine reference substance for identification. (+info)
Validation of liquid chromatography-electrospray ionization ion trap mass spectrometry method for the determination of mesocarb in human plasma and urine.
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Mesocarb metabolism in humans is the target of this investigation. A high-performance liquid chromatographic (LC) method with electrospray ionization (ESI)-ion trap mass spectrometric (MS) detection ion trap "SL" for the simultaneous determination of mesocarb and its metabolites in plasma and urine is developed and validated. Ten metabolites and the parent drug are detected in human urine, and only four in human plasma, after the administration of a single oral dose of 10 mg of mesocarb (Sydnocarb, two 5-mg tablets). Seven of this metabolites have been found for the first time. The confirmation of the results and identification of all the metabolites except amphetamine is performed by LC-MS, LC-MS-MS, and LC-MS3. In the case of doping analysis, the reliable detection time for mesocarb (long-life dihydroxymesocarb metabolites of mesocarb) is approximately 10-11 days after the administration of the drug, which is a significant increase over the existing data. The detection of amphetamine in plasma and urine is made using simple flow-injection analysis without a chromatographic separation. The addition-calibration method is used with plasma and urine. The mean recoveries from plasma are 49.2% and 57.4% for mesocarb concentrations of 33.0 and 66.0 ng/mL, respectively, whereas the recoveries from human urine are 76.9% and 81.4% for concentrations of 1 and 2 ng/mL, respectively. Calibration curves (using an internal standard method) are linear (r2>0.9969) for concentrations 0.6 to 67 ng/mL and from 0.05 to 5 ng/mL in plasma and urine, respectively. Both intra- and interassay precision of plasma control samples at 3, 40, and 55 ng/mL are lower than 6.2%, and the concentrations do not deviate for more than -3.4% to 7.3% from their nominal values. In urine, intra- and interassay precision of control samples at 0.08, 1.5, and 3.0 ng/mL is lower than 14.1%, with concentrations not deviating for more than -11.3% to 13.7% from their nominal values. The plasma disappearance curve of the parent drug is obtained. The major pharmacokinetic parameters are calculated. (+info)
Examples of doping control analysis by liquid chromatography-tandem mass spectrometry: ephedrines, beta-receptor blocking agents, diuretics, sympathomimetics, and cross-linked hemoglobins.
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The application of modern and powerful analytical instruments consisting of liquid chromatographs (LCs), sophisticated atmospheric pressure ion sources, and sensitive mass analyzers has improved quality as well as speed of doping control analyses markedly during the last 5 years. Numerous compounds such as beta-receptor blocking agents or diuretics require derivatization prior to gas chromatographic (GC) and mass spectrometric (MS) measurement, which is the reason for extended sample preparation periods. In addition, several substances demonstrate poor GC-MS properties even after chemical modification, and peptide hormones such as cross-linked hemoglobins cannot be analyzed at all by means of GC-MS. With the availability of electrospray ionization and robust tandem MSs (e.g., triple-stage quadrupole or ion trap instruments) many new or complementary screening and confirmation assays have been developed, providing detailed qualitative and quantitative information on prohibited drugs. With selected categories of compounds (ephedrines, beta-blockers, b2-agonists, diuretics, and bovine hemoglobin-based oxygen therapeutics) that are banned according to the rules of the World Anti-Doping Agency and International Olympic Committee, the advantages of LC-MS-MS procedures over conventional GC-MS assays are demonstrated, such as enhanced separation of analytes, shorter sample pretreatment, and identification of substances that are not identified by GC-MS. (+info)
Isotopic fractionation of endogenous anabolic androgenic steroids and its relationship to doping control in sports.
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The use of gas chromatography (GC)-combustion (C)-isotope ratio mass spectrometry (IRMS) demonstrates that a single oral administration of dehydroepiandrosterone (DHEA, 100 mg) to a male subject significantly lowers the 13C content of etiocholanolone (Et) and androsterone (A) in the subject's urine. The difference in carbon isotope ratio (d13C per thousand) values between Et and A increases from 1.6 per thousand at the time of administration to 5.1 per thousand at 26 h post-administration, indicating preferential metabolism of administered DHEA to form Et in relation to A. Multiple oral administrations of DHEA to a male subject reveals lower d13C values during the excretion period of Et (-31.7 per thousand to -34.6 per thousand) and A (-31.4 per thousand to -33.0 per thousand) to that of the d13C value of the administered DHEA (-31.3 per thousand). Reference distributions of d13C Et and d13C A constructed from normal athlete populations within Australia and New Zealand show a small natural discrimination against 13C in the formation of Et relative to A (mean=0.3 per thousand, n=167, p=0.007). Amplified differences between d13C Et and d13C A, and in vivo 13C depletion measured by GC-C-IRMS are shown to be potentially useful for doping control. (+info)
Methodologies for detection of hemoglobin-based oxygen carriers.
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Blood substitutes based on hemoglobin or hemoglobin-based oxygen carriers (HBOCs) are oxygen-carrying therapeutic agents developed for use in operations and emergencies in place of donated blood. Increased oxygen-carrying capacity through the use of blood substitutes could help elite athletes to lengthen endurance capacity and improve their performance. As blood substitutes become more readily available, it is essential that a qualitative detection method for their abuse in sport is available. Ideally, such a method would be simple and inexpensive. This study investigates methods that could be used as screening procedures to easily detect HBOCs in plasma and develops tests that can unequivocally confirm their presence. The investigation into the screening method indicates that the direct visual screening of plasma discoloration is the most appropriate with detection limits of less than 1% HBOC in plasma. Two methods are shown to confirm the presence of exogenous hemoglobin in plasma samples, size-exclusion chromatography with photodiode array detection and high-performance liquid chromatography analysis of enzymatic digests with detection by electrospray mass spectrometry. This work emphasizes the need for cooperation between drug developers and sports testing laboratories to ensure that methods for the detection of putative doping agents are available prior to product release. (+info)
Applications of liquid chromatography-mass spectrometry in doping control.
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This paper reviews liquid chromatographic-mass spectrometric (LC-MS) procedures for the screening, identification and quantification of doping agents in urine and other biological samples and devoted to drug testing in sports. Reviewed methods published approximately within the last five years and cited in the PubMed database have been divided into groups using the same classification of the 2004 World Anti-Doping Agency (WADA) Prohibited List. Together with procedures specifically developed for anti-doping analysis, LC-MS applications used in other fields (e.g., therapeutic drug monitoring, clinical and forensic toxicology, and detection of drugs illicitly used in livestock production) have been included when considered as potentially extensible to doping control. Information on the reasons for potential abuse by athletes, on the requirements established by WADA for analysis, and on the WADA rules for the interpretation of analytical findings are provided for the different classes of drugs. (+info)
Quantification and profiling of 19-norandrosterone and 19-noretiocholanolone in human urine after consumption of a nutritional supplement and norsteroids.
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Nandrolone is one of the synthetic anabolic steroids banned in sports and has been a popular substance abused by athletes in recent years. One of its major metabolites, 19-norandrosterone (19-NA), has been used as a determinant for drug violations in sports. Current reports regarding nandrolone-positive cases have been related to intake of some nandrolone-free nutritional supplements. The aim of this study was to learn whether if a nutritional supplement sold by over-the-counter (OTC) nutritional stores could yield the same metabolic products as that of nandrolone. If so, what is (are) the substance(s) that contributed to the nandrolone metabolites? To determine the content of an OTC nutritional supplement, a tablet was dissolved in methanol, followed by N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA)-trimethyliodosilane (TMIS) derivatization prior to gas chromatography-mass spectrometry (GC-MS) analysis. The collected urine samples underwent extraction, enzymatic hydrolysis, and derivatization before the analyses of GC-MS. The results showed that seven anabolic steroids were found as contaminants in the nutritional supplement, in addition to six that were listed in the ingredients by the manufacturer. We confirmed previous reports that administration of the OTC supplement could produce a positive urine test for nandrolone metabolites. Furthermore, the results from excretion studies showed that 19-NA and 19-noretiocholanolone (19-NE) were present in urine after consuming the nutritional supplement, nandrolone, 19-nor-4-androsten-3,17-dione, 19-nor-4-androsten-3beta,17beta-diol, and 19-nor-5-androsten-3beta,17beta-diol. The 19-NA concentrations in urine were generally higher than that of 19-NE (19-NA/19-NE ratio > 1.0) especially during the early stage of excretion, that is, before 6 h post-administration. After this period of time, the concentrations of 19-NA and 19-NE fluctuated and might even have reversed (19-NA/19-NE ratio < 1.0) in their ratio, that is, higher yield in 19-NE than that in 19-NA. On the basis of this study, we postulate that some doping violations of nandrolone could be attributed by indiscriminate administration of the OTC nutritional supplements that contained 19-norsteroids. (+info)
Method development for cortisol and cortisone by micellar liquid chromatography using sodium dodecyl sulphate: application to urine samples of rugby players.
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The chromatographic behavior of cortisol and cortisone using a micellar medium of sodium dodecyl sulphate (SDS) as surfactant, a Hypersil C18 (150- x 3.2-mm i.d., 5 microm) column, a flow rate of 0.5 mL/min, and UV absorbance detection at 245 nm is described. The effect of several organic modifiers and the surfactant concentration on the separation is studied. A mobile phase of 18 mM SDS and 8.3% tetrahydrofuran allows for the separation of cortisol and cortisone up to baseline. These results are also achieved by applying a bivariant optimization method. The proposed method is sensitive, reproducible, and selective. In addition, it is less expensive than conventional high-performance liquid chromatography methods for cortisol and cortisone. The method is applied to the determination of cortisol and cortisone in urine samples of rugby players before and after stress for doping control purposes. (+info)