Small peptides with a molecular weight of <2 kDa represent a performance-enhancing substances. However, in vivo studies with human volunteers are limited because most of these peptides are not approved for human consumption. Thus, relevant in vitro models are a basic tool to study their metabolism for anti-doping purposes. To choose the best in vitro model the biotransformation of growth hormone releasing peptides (GHRPs), Desmopressin and TB-500 was investigated using various in vitro systems.High metabolic activity was observed during incubation of GHRPs and TB-500 with human kidney microsomes (HKM) and liver S9 fraction. Peptides degraded through cleavage of all bonds regardless protective modifications in primary structure. HKM and liver S9 fraction demonstrated enzymatic deamidation activity removing C-terminal amide group from all GHRPs. Fewer metabolites were produced during incubation with human serum. The metabolite pattern obtained with commercially available proteases was poor and included nonspecific hydrolyzed compounds. Thus, the maximum diversity of metabolites was achieved with HKM and liver S9 fraction which makes them the most efficient in vitro model systems for peptides biotransformation study.Biological significance: Currently, >60 peptide medicines are FDA approved and marketed in the United States as biopharmaceutical products. Approximately 140 peptide drugs are in clinical trials and about 500 therapeutic peptides in preclinical development. There is an emerging interest in small peptides with a molecular weight of <2 kDa, which can be used as doping in modern sport due a wide spectrum of their physiological activity. Most of peptide doping products are not yet approved for human use and some of them undergo preclinical or clinical trials, which complicates the study of metabolism in vivo. The investigation of the metabolism with in vitro methods is an alternative that does not require a human participation and an approval by the Ethics Committee. (C) 2016 Elsevier B.V. All rights reserved.

Growth hormone releasing peptides (GHRPs) could be widely used by cheating athletes because they produce growth hormone (GH) secretion, so may generate an ergogenic effect in the body. Knowledge of the essential amino acids needed in GHRP structure for interaction with the target biological receptor GHSR1a, the absorption through different administration routes, and the maintenance of pharmacological activity of potential biotransformation products may help in the fight against their abuse in sport. Several GHRPs and truncated analogues with the common core Ala-Trp-(D-Phe)-Lys have been studied with a radio-competitive assay for the GHSR1a receptor against the radioactive natural ligand ghrelin. Relevant chemical modifications influencing the activity for positions 1, 2, 3, and 7 based on the structure aa-aa-aa-Ala-Trp-(D-Phe)-Lys have been obtained. To test in vivo the applicability of the activities observed, the receptor assay activity in samples from excretion studies performed after nasal administration of GHRP-1, GHRP-2, GHRP-6, Hexarelin, and Ipamorelin was confirmed. Overall results obtained allow to infer structure-activity information for those GHRPs and to detect GHSR1a binding (intact GHRPs plus active metabolites) in excreted urines. Copyright © 2016 John Wiley & Sons, Ltd.

The design of new erythropoiesis-stimulating agents for clinical use necessitates constant development of methods for detecting the abuse of these substances, which are prohibited under the World Anti-Doping Code and are included in the World Anti-Doping Agency (WADA) prohibited list. This review integrates and describes systematically the published data on the key methods currently used by WADA-accredited anti-doping laboratories around the world to detect the abuse of erythropoiesis-stimulating agents, including direct methods (various polyacrylamide gel electrophoresis techniques, enzyme linked immunosorbent assay, membrane enzyme immunoassay and mass spectrometry) and indirect methods (athlete biological passport). Particular attention is given to promising approaches and investigations that can be used to control prohibited erythropoietins in the near future. The bibliography includes 122 references.

Growth hormone releasing peptides (GHRPs) stimulate secretion of endogenous growth hormone and are listed on the World Anti-Doping Agency (WADA) Prohibited List. To develop an effective method for GHRPs anti-doping control we have investigated metabolites of GHRP-1, GHRP-2, GHRP-6, Hexarelin, and Ipamorelin in urine after nasal administration. Each compound was administrated to one volunteer. Samples were collected for 2 days after administration, processed by solid-phase extraction on weak cation exchange cartridges and analyzed by means of nano-liquid chromatography - high resolution mass spectrometry. Six metabolites of GHRP-1 were identified. GHRP-1 in the parent form was not detected. GHRP-1 (2-4) free acid was detected in urine up to 27 h. GHRP-2, GHRP-2 free acid and GHRP-2 (1-3) free acid were detected in urine up to 47 h after administration. GHRP-6 was mostly excreted unchanged and detected in urine 23 h after administration, its metabolites were detectable for 12 h only. Hexarelin and Ipamorelin metabolized intensively and were excreted as a set of parent compounds with metabolites. Hexarelin (1-3) free acid and Ipamorelin (1-4) free acid were detected in urine samples after complete withdrawal of parent substances. GHRPs and their most prominent metabolites were included into routine ultra-pressure liquid chromatography-tandem mass spectrometry procedure. The method was fully validated, calibration curves of targeted analytes were obtained and excretion curves of GHRPs and their metabolites were plotted. Our results confirm that the detection window after GHRPs administration depends on individual metabolism, drug preparation form and the way of administration.

Currently liquid chromatography - mass spectrometry (LC-MS) analysis after solid-phase extraction (SPE) on weak cation-exchange cartridges is a method of choice for anti-doping analysis of small bioactive peptides such as growth hormone releasing peptides (GHRPs), desmoporessin, LHRH, and TB-500 short fragment. Dilution of urine samples with phosphate buffer for pH adjustment and SPE on weak cation exchange microelution plates was tested as a means to increase throughput of this analysis. Dilution using 200 mM phosphate buffer provides good buffering capacity without affecting the peptides recoveries. SPE on microelution plates was performed on Waters Positive Pressure-96 Processor with subsequent evaporation of eluates in nitrogen flow. Though the use of smaller sample volume decreases the pre-concentration factor and increases the limits of detection of 5 out of 17 detected peptides, the recovery, linearity, and reproducibility of the microelution extraction were comparable with cartridge SPE. The effectiveness of protocols was confirmed by analysis of urine samples containing ipamorelin, and GHRP-6 and its metabolites. SPE after urine sample dilution with buffer can be used for faster sample preparation. The use of microelution plates decreases consumption of solvents and allows processing of up to 96 samples simultaneously. Cartridge SPE with manual рН adjustment remains the best option for confirmation. Copyright © 2015 John Wiley & Sons, Ltd.

EPO-Fc proteins have been under investigation as a potential drug for treating anaemia and have shown larger half-life values than other erythropoiesis-stimulating agents (ESAs). Sodium dodecyl sulfate/sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SDS/SAR-PAGE) methods and subsequent immunoblotting are used for routine anti-doping analysis. This paper reports that EPO-Fc fusion proteins can be detected in serum samples by isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) in carrier ampholyte-based gels with a pH 2-6 gradient after removing the Fc part via site-specific IdeS protease cleavage. The IdeS-digested EPO-Fc protein yields three fragments: two Fc fragments and one dimeric EPO-hinge fragment. After IEF-PAGE was followed by double Western blotting with chemiluminescent detection, the dimeric EPO-hinge fragment showed a unique isoelectric pattern, which differed from those of any other currently known analogue of EPO. We observed that the removal of the Fc fragment from EPO-Fc reduced the apparent molecular weight of entire fusion protein and increased its electrophoretic mobility. As a result, the band for the EPO-hinge fragment was located in a region between the rEPO and NESP standards, at which lower amounts of serum proteins are present. Simple and selective protocols for determining the EPO-Fc protein in human serum were developed to extend the methodological anti-doping arsenal. This protocol has been characterized. The limit of detection (LOD) of the IEF-PAGE method was 20 pg, and that of SDS/SAR-PAGE was 15 pg.

The data are reported for an in vitro metabolism study of two novel synthetic cannabinoids, N-(1-adamantyl)-1-pentyl-1H-indole-3-carboxamide (APICA) and its fluorinated analog N-(1-adamantyl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (5F-APICA, STS-135), which are active ingredients of smoking mixtures sold in Russia since 2012. The cannabinoids were isolated from herbal mixtures using preparative liquid chromatography and then incubated with human liver microsomes (HLMs). The formed metabolites were characterized by liquid chromatography - triple quadrupole mass spectrometry and high-resolution mass spectrometry with electrospray ionization in positive ion mode. It was found that HLMs produce mono-, di-, and trihydroxylated metabolites, as well as N-desalkyl metabolites, which can be further hydroxylated; the amide bond resisted the metabolic cleavage. For 5F-APICA, a series of oxidative defluorination products formed as well. For in vivo confirmation of the formed in vitro metabolites, spot urine samples from drug users were analyzed with the created method. It was shown that for the detection of APICA abuse, the preferred metabolites are the di- and tri-hydroxylated species, while in case of 5F-APICA, a monohydroxy metabolite is a better target. The N-despentyl (desfluoropentyl) hydroxyadamantyl metabolite also provides good retrospectivity to confirm the administration of any of these cannabinoids.

The laboratory anti-doping services during XXII Winter Olympic and XI Paralympic games in Sochi in 2014 were provided by a satellite laboratory facility located within the strictly secured Olympic Park. This laboratory, established and operated by the personnel of Antidoping Center, Moscow, has been authorized by the World Anti-Doping Agency (WADA) to conduct doping control analyses. The 4-floor building accommodated the most advanced analytical instrumentation and became a place of attraction for more than 50 Russian specialists and 25 foreign experts, including independent observers. In total, 2134 urine and 479 blood samples were delivered to the laboratory and analyzed during the Olympic Games (OG), and 403 urine and 108 blood samples - during the Paralympic Games (PG). The number of erythropoietin tests requested in urine was 946 and 166 at the OG and PG, respectively. Though included in the test distribution plan, a growth hormone analysis was cancelled by the Organizing Committee just before the Games. Several adverse analytical findings have been reported including pseudoephedrine (1 case), methylhexaneamine (4 cases), trimetazidine (1 case), dehydrochloromethyltestosterone (1 case), clostebol (1 case), and a designer stimulant N-ethyl-1-phenylbutan-2-amine (1 case).

A medical and scientific multidisciplinary consensus meeting was held from 29 to 30 November 2013 on Anti-Doping in Sport at the Home of FIFA in Zurich, Switzerland, to create a roadmap for the implementation of the 2015 World Anti-Doping Code. The consensus statement and accompanying papers set out the priorities for the antidoping community in research, science and medicine. The participants achieved consensus on a strategy for the implementation of the 2015 World Anti-Doping Code. Key components of this strategy include: (1) sport-specific risk assessment, (2) prevalence measurement, (3) sport-specific test distribution plans, (4) storage and reanalysis, (5) analytical challenges, (6) forensic intelligence, (7) psychological approach to optimise the most deterrent effect, (8) the Athlete Biological Passport (ABP) and confounding factors, (9) data management system (Anti-Doping Administration & Management System (ADAMS), (10) education, (11) research needs and necessary advances, (12) inadvertent doping and (13) management and ethics: biological data. True implementation of the 2015 World Anti-Doping Code will depend largely on the ability to align thinking around these core concepts and strategies. FIFA, jointly with all other engaged International Federations of sports (Ifs), the International Olympic Committee (IOC) and World Anti-Doping Agency (WADA), are ideally placed to lead transformational change with the unwavering support of the wider antidoping community. The outcome of the consensus meeting was the creation of the ad hoc Working Group charged with the responsibility of moving this agenda forward.

Homologous blood transfusion is a prohibited method of blood manipulation that can be used to increase the number of erythrocytes circulating in the blood stream resulting in an increased oxygen transport capacity. In doping controls, homologous blood transfusions are determined by means of a procedure based on the detection of red blood cell phenotypes by flow cytometry. In the past six years, no adverse analytical findings concerning homologous blood transfusions were reported. One explanation for that phenomenon, assuming that athletes have not completely given up this kind of manipulation, would be a more careful selection of potential donors. If such a donor has the same set of minor erythrocyte antigens as the recipient, the established methodology to detect homologous transfusion would fail. We have hypothesized that any athlete can be a potential donor for teammates with the same RhD factor and AB0 blood group. Having analyzed the phenotype of erythrocytes of 535 Russian athletes in various endurance sports, several pairs of athletes with the same phenotype were observed. Based on the frequency of occurrence of red blood cell antigens, the theoretical probability of finding a donor within a team with exactly the same phenotype was calculated, and the existing number of occurrences where two individuals share the same phenotype in the same sport was in fact five times higher than the theoretical probability. Copyright © 2014 John Wiley & Sons, Ltd.

RATIONALE:AICAR (5-aminoimidazole-4-carboxamide 1β-D-ribofuranoside) is prohibited in sport according to rules established by the World Anti-Doping Agency. Doping control laboratories identify samples where AICAR abuse is suspected by measuring its urinary concentration and comparing the observed level with naturally occurring concentrations. As the inter-individual variance of urinary AICAR concentrations is large, this approach requires a complementary method to unambiguously prove the exogenous origin of AICAR. Therefore, a method for the determination of carbon isotope ratios (CIRs) of urinary AICAR has been developed and validated.METHODS:Concentrated urine samples were fractionated by means of liquid chromatography for analyte cleanup. Derivatization of AICAR yielding the trimethylsilylated analog was necessary to enable CIR determinations by gas chromatography/combustion/isotope ratio mass spectrometry. The method was tested for its repeatability and stability over time and a linear mixing model was applied to test for possible isotopic discrimination. A reference population of n = 63 males and females was investigated to calculate appropriate reference limits to differentiate endogenous from exogenous urinary AICAR. These limits were tested by an AICAR elimination study.RESULTS:The developed method fulfills all the requirements for adequate sports drug testing and was found to be fit for purpose. The investigated reference population showed a larger variability in the CIR of AICAR than of the endogenous steroids. Nevertheless, the calculated thresholds for differences between AICAR and endogenous steroids can be applied straightforwardly to evaluate suspicious doping control samples with the same statistical confidence as established e.g. for testosterone misuse. These thresholds enabled the detection of a single oral AICAR administration for more than 40 h.CONCLUSIONS:Determination of thee CIRs is the method of choice to distinguish between an endogenous and an exogenous source of urinary AICAR. The developed method will enable investigations into doping control samples with elevated urinary concentrations of AICAR and clearly differentiate between naturally produced/elevated and illicitly administered AICAR.

After more than one year experience in the screening for recently reported dehydrochloromethyltestosterone (DHCMT) metabolites in our laboratory, it was demonstrated that the most long-term is a steroid tentatively characterized as 4-chloro-18-nor-17β-hydroxymethyl-17α-methyl-5β-androst-13-en-3α-ol (M3) and its 17-epimer, which is less abundant but normally always detectable alongside with M3. In the majority of cases, M3 was shown to be superior to the other known DHCMT metabolites, such as 4-chloro-18-nor-17β-hydroxymethyl-17α-methylandrosta-1,4,13-trien-3-one (“night watch” analog) and 4-chloro-3α,6β,17β-trihydroxy-17α-methyl-5β-androst-1-en-16-one (metabolite “656”). M3 is best detected by gas chromatography – tandem mass spectrometry (GC-MS/MS), whereas GC-MS could still be used but with much less confidence due to both sensitivity and selectivity issues. In 2011 Moscow Antidoping Centre reported twenty five adverse analytical findings (AAF) for DHCMT, of which only 4 would be declared positive based on the presence of 4-chloro-3α,6β,17β-trihydroxy-17α-methyl-5β-androst-1-en-16-one, and only one – if 6β-hydroxy-DHCMT would be used as the target. In 2012 twenty eight AAFs for DHCMT have already been reported by Moscow Antidoping Centre as of September 1, 2012, with most of them being solely relied on the detection of M3. Therefore, all antidoping laboratories that are capable of running GC-MS/MS technology are advised to include M3 in their screening methods.

The urinary excretion of selective androgen receptor modulators (SARMs) andarine and ostarine after controlled administration study was investigated. It was demonstrated that andarine is subject to extensive metabolism by desacetylation, hydroxylation and dephenylation. Importantly, most of the andarine metabolites are excreted as glucuronide and sulfate conjugates. Andarine itself is also partly conjugated with glucuronic acid. Ostarine was shown to be much more stable metabolically as its major metabolites are ostarine glucuronide and hydroxyostarine glucuronide. For the detection of andarine, ostarine and their metabolites several methods such as gas and liquid chromatography coupled to tandem mass spectrometry (GCand LC-MS/MS) were compared. Of the analytical methods tested, LC-MS/MS of conjugated (total) fraction is preferred, though direct analysis of diluted urine is also suitable for determination of desacetylhydroxy-andarine sulfate and ostarine glucuronide. However, in the latter case the detection time window was not as long. Both SARMs were detectable in urine for about 2 weeks after a single oral dose of 60 mg of andarine and 30 mg of ostarine. Introduction Andarine (S-4) and ostarine (S-22) are the drug candidates belonging to the class of selective androgen receptor modulators which possess anabolic activity and promote muscle growth. Both compounds are currently advertised via the Internet [1] and could therefore be used by professional athletes. Thus, antidoping laboratories should have the analytical procedures in place to be capable of identifying these compounds. While the metabolism of andarine and ostarine has been reported earlier [2-4], the detection time window and what is the best target to detect their abuse is not yet clear enough. Therefore, the aim of present study was to investigate the urinary excretion of andarine and ostarine, compare different sample preparation protocols and select optimal detection methods. Experimental Three volunteers participated in this study. Single oral doses of 60 mg of andarine (male, age 53; female, age 26) and 30 mg of ostarine (female, age 33) were administered, and urine was collected up to 3 weeks. The excretion study was approved by local Ethics Committee at the Institute of Sport. Urine samples were processed as follows: (1) 200 μL of urine were diluted with 800 μL of solvent mixture (0.1% formic acid and 3% methanol in water with mefruside as internal standard); (2) 3 mL of urine were extracted with diethyl ether in the presence of Na2SO4 at pH 8.5-9.5, followed by evaporation of the extract and reconstitution in 100 μL of water/methanol (60/40); (3) 3 mL of urine were deconjugated with a solution of β-glucuronidase from E.coli in phosphate buffer containing methyltestosterone as internal standard (pH 6.4, 57°C, 1h), followed by extraction and reconstitution, as in (2); (4) similar to (3) but after evaporation of the organic extract the residue was derivatized with 50 μL of MSTFA/NH4I/dithiothreitol at 70°C for 30 min. LC–MS/MS analyses were accomplished on an Acquity LC (Waters, Milford, MA, USA) coupled to a TSQ Vantage (ThermoFisher Scientific, San Jose, CA, USA). A Waters Acquity BEH C18 column (100 mm × 2.1 mm, particle size 1.7 μm) maintained at 60°C and protected by a Vanguard BEH C18 column (20 mm × 2.1 mm) was used for separation.

An approach to determining the yield of derivatization reaction is based on a comparison of chromatographic peak areas of the derivative and native (underivatizied) compound. In contrast to the previous publication [J. Anal. Chem., 2011, vol. 66, no. 12, pp. 1186–1189], ratio of the sensitivity coefficients of the derivative and native forms of the analyte was calculated using only experimental data obtained upon varying the derivatization conditions (solution containing equal amounts of underivatized compound and a respective derivative was analyzed previously). The approach was used to investigate the influence of the reaction time and the type of an external action on the yield of the derivatization (silylation) reaction for some anabolic steroids (methyltestosterone, methandienone, oxandrolone and oral-turinabol) containing a hindered tertiary hydroxyl group at C17. The amounts of the derivatized steroids were equal to about 20–60 ng (depending on the component). Steroids were derivatized with a mixture of pyridine and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane. The derivatization reaction was carried out for 15, 30 or 45 min under conventional heating, sonication at room temperature, and sonication at elevated temperature.

The smoking of “herbal mixtures” as an alternative to the cannabis-based products has become an issue not only in the social context, but also in sport drug testing where reliable detection methods are demanded by antidoping laboratories. These herbal blends are legally sold via the Internet in many countries, and their composition is changed from time to time in response to the legislation bans. We performed an in vitro study on the metabolism of AM2233, 1-[(N-methylpiperidin-2-yl)methyl]-3-(2-iodobenzoyl)indole, and JWH-210, 4-ethylnaphthalen-1-yl-(1-pentylindol-3-yl)methanone, which were isolated using preparative liquid chromatography from the smoking mixtures sold in Russia. After incubation of pure fractions with human liver microsomes (HLM) as well as with CYP450 isoenzymes 3A4 and 2B6, the metabolic pathways were identified by means of liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in positive mode. It was found that in case of AM2233 the in vitro reactions mainly include monohydroxylation and N-demethylation, while JWH-210 formed a variety of products such as monohydroxy, dihydroxy, despentylhydroxy, oxo (or epoxy) and oxohydroxy metabolites. The HLM were found to be superior over the other two isoenzymes for generation of the metabolites of these cannabimimetics.

Представлена ВЭЖХ-МС/МС-методика селективного и надежного скринингового определения кортикостероидов и диуретиков в моче человека. Процедура пробоподготовки заключается в проведении экстракции, упаривании органического экстракта в токе азота и перерастворении сухого остатка. Экстракт проанализирован методом высокоэффективной жидкостной хроматографии в сочетании с тандемной масс-спектрометрией с использованием электрораспылительной ионизации при атмосферной ионизации с регистрацией отрицательных ионов. Для всех определяемых соединений получены масс-спектры и определены характеристичные ионы, времена удерживания и пределы детектирования, при валидации процедуры проведена оценка степени подавления ионизации матрицей и извлечения аналитов из биологической жидкости человека, селективности и специфичности определения соединений.

The presence of a large number of endogenous steroids and corticosteroids with similar structures in a urinary matrix can hamper the specific detection of exogenous steroids using LC–MS with reversed-phase columns. Therefore, the development of LC–MS methods using alternative columns is of great interest. Porous graphitized carbon is a unique stationary phase for HPLC, with properties differing from traditional silica-based and polymeric stationary phases. Non-derivatized porous graphitic carbon surface allows unique retention and separation of geometric isomers. This study demonstrates the application of a porous graphitized carbon column for selective separation of steroids. A screening method for the detection of 56 exogenous steroids has been developed. The method involves enzymatic hydrolysis, liquid–liquid extraction, and determination by high-temperature liquid chromatography–Orbitrap mass spectrometry with atmospheric pressure photoionization. For all of the analytes, the relative retention times proved to be stable between days, with RSDs below than 0.3%. The matrix effect for the 56 analytes varied between 5% and 11.0%. Of the 56 steroids studied, 53 showed limit of detection smaller than 1 ng/mL-. The proposed method meets the general criteria for all methods used to analyze drugs or metabolites in an antidoping laboratory, i.e., sensitivity, selectivity, and specificity. Introduction The routine LC–MS methods are applied for the detection of a limited number of steroids, normally those that exhibited poor detection by GC–MS. Recently, an LC–MS/MS method was developed for the detection of 44 anabolic steroids and metabolites [1]. However, the presence in a urinary matrix of a large number of endogenous steroids and corticosteroids with similar structures can hamper the specific detection of the exogenous steroids by LC–MS. Therefore, up to now the development of selective LC–MS methods is of great interest. Because the most important issue in qualitative steroid analysis is the selectivity, we developed in this study a sensitive and selective high–temperature LC–Orbitrap MS (HTLC–Orbitrap MS) method with a porous graphitized carbon column for screening anabolic steroids. Experimental Chemicals. Anabolic steroids were purchased from Sigma, LGC Standards (formerly LGC Promochem, Wesel, Germany), and Steraloids (Newport, RI, USA). The β-glucuronidase preparation (from Escherichia coli) was purchased from Roche (Mannheim, Germany). Analytical-grade potassium carbonate, sodium hydrogen carbonate, ammonium hydroxide, trifluoroacetic acid, diethyl ether, 2-propanol, ethanol, and methanol were obtained from Merck (Darmstadt, Germany). The HPLC-grade water and acetonitrile were purchased from Biosolve (Valkenswaard, The Netherlands). Standard stock solutions of the analytes (concentration 1 mg/mL) were individually prepared in methanol. For validation purposes, working standard solution were prepared in methanol by subsequent dilution of the stock solution. Instrumentation. The experiments were performed using an Accela HPLC system interfaced to an Exactive mass spectrometer (Thermo Scientific, Bremen, Germany) with an APPI ion source. The mass spectrometer was operated in the positive ion mode. The desolvation temperature was 230 °C. The mass spectrometer was operated at a resolution of 50000