Development of a microplate duplex immunoassay to simplify detection of growth hormone doping: Proof of concept

The World Anti-Doping Agency (WADA) prohibits athletes from using recombinant growth hormone (GH). The validated method used in antidoping laboratories for the direct detection of exogenous GH in serum requires two immunoluminometric assays (ILMAs): The first mainly measures the concentration of the full-length (22 kDa) form of GH (recGH), and the second measures concentrations of multiple GH fragments produced by the pituitary gland (22 kDa, 20 kDa and other forms) (pitGH). The tube-by-tube analysis is laborious. A recent development opened new possibilities to simplify the detection of recGH in serum: multiplexed immunoassays that detect multiple targets in a single well of a 96-well plate using an ELISA-like procedure with high sensitivity. Our aim was to evaluate this technology by developing a customized assay for GH detection. One pair of antibodies with specificities similar to those of the recGH assay and one pair of antibodies compatible with pitGH detection were selected for a single duplex assay. Forty-eight serum samples (negative athlete samples and positive samples following GH administration) were analyzed using the two methods. The microplate duplex assay discriminated between the negative athlete samples and the positive controls, although the rec/pit ratios from the duplex assay were lower than those obtained with the ILMAs. This new assay would offer a modern alternative to ILMAs, with fewer analytical steps and a smaller sample volume. However, an adaptation of the decision limits seems mandatory.     

Longitudinally monitoring of P‐III‐NP,IGF‐I,and GH‐2000 score increases the probability of detecting two weeks’ administration of low‐dose recombinant growth hormone compared to GH‐2000 decision limit and GH isoform test and micro RNA markers

To detect doping with growth hormone (GH), GH isoform and biomarkers tests are available. Both methods use population‐based decision limits. Future testing in anti‐doping is progressing toward individual‐based reference ranges, and it is possible that with such an approach the sensitivity to detect GH doping may increase. In addition to monitoring different proteins, the use of miRNAs as future GH biomarkers has been discussed. Here we have longitudinally studied the serum concentrations of IGF‐I, P‐III‐NP and the different GH isoforms in nine healthy men prior to, during and after two weeks’ administration with low doses (1 and 4 IU/day) of recGH. Moreover, three putative miRNAs were analyzed. The results show that 80% of the participants were identified as atypical findings using the GH isoform test. However, the participants were only positive 1.5–3 hours directly after an injection. Only one of the participants reached a GH‐2000 score indicative of doping when a population‐based decision limit was applied. When IGF‐I and P‐III‐NP were longitudinally monitored, 88% of the participants were identified above an individual upper threshold arbitrarily calculated as three standard deviations above the mean values of four baseline samples. The miRNA levels displayed large intra‐subject variations that did not change in relation to recGH administration. Our results show that the GH isoform test is very sensitive in detecting low doses of recGH but with a short detection window. Moreover, longitudinally monitoring of IGF‐I and P‐III‐NP may be a promising future approach to detect GH doping.     

Sensitivity and specificity of detection methods for erythropoietin doping in cyclists

Recombinant human erythropoietin (rHuEPO) is used as doping a substance. Anti‐doping efforts include urine and blood testing and monitoring the athlete biological passport (ABP). As data on the performance of these methods are incomplete, this study aimed to evaluate the performance of two common urine assays and the ABP. In a randomized, double‐blinded, placebo‐controlled trial, 48 trained cyclists received a mean dose of 6000 IU rHuEPO (epoetin β) or placebo by weekly injection for eight weeks. Seven timed urine and blood samples were collected per subject. Urine samples were analyzed by sarcosyl‐PAGE and isoelectric focusing methods in the accredited DoCoLab in Ghent. A selection of samples, including any with false presumptive findings, underwent a second sarcosyl‐PAGE confirmation analysis. Hematological parameters were used to construct a module similar to the ABP and analyzed by two evaluators from an Athlete Passport Management Unit. Sensitivity of the sarcosyl‐PAGE and isoelectric focusing assays for the detection of erythropoietin abuse were 63.8% and 58.6%, respectively, with a false presumptive finding rate of 4.3% and 6%. None of the false presumptive findings tested positive in the confirmation analysis. Sensitivity was highest between 2 and 6 days after dosing, and dropped rapidly outside this window. Sensitivity of the ABP was 91.3%. Specificity of the urine assays was high; however, the detection window of rHuEPO was narrow, leading to questionable sensitivity. The ABP, integrating longitudinal data, is more sensitive, but there are still subjects that evade detection. Combining these methods might improve performance, but will not resolve all observed shortcomings.     

Temporal changes in physiology and haematology in response to high‐ and micro‐doses of recombinant human erythropoietin

There is evidence to suggest athletes have adopted recombinant human erythropoietin (rHuEPO) dosing regimens that diminish the likelihood of being caught by direct detection techniques. However, the temporal response in physiology, performance, and Athlete Biological Passport (ABP) parameters to such regimens is not clearly understood. Participants were assigned to a high‐dose only group (HIGH, n  = 8, six rHuEPO doses of 250 IU/kg over two weeks), a combined high micro‐dose group (COMB, n  = 8, high‐dose plus nine rHuEPO micro‐doses over a further three weeks), or one of two placebo control groups who received saline in the same pattern as the HIGH (HIGH‐PLACEBO, n  = 4) or COMB (COMB‐PLACEBO, n  = 4) groups. Temporal changes in physiology and performance were tracked by graded exercise test (GXT) and haemoglobin mass assessment at baseline, after high dose, after micro‐dose (COMB and COMB‐PLACEBO only) and after a four‐week washout. Venous blood samples were collected throughout the baseline, rHuEPO administration, and washout periods to determine the haematological and ABP response to each dosing regimen. Physiological adaptations induced by a two‐week rHuEPO high‐dose were maintained by rHuEPO micro‐dosing for at least three weeks. However, all participants administered rHuEPO registered at least one suspicious ABP value during the administration or washout periods. These results indicate there is sufficient sensitivity in the ABP to detect use of high rHuEPO doping regimens in athletic populations and they provide important empirical examples for use by anti‐doping experts. Copyright © 2017 John Wiley & Sons, Ltd.     

In vitro evaluation of the effects of anti‐fungals,benzodiazepines and non‐steroidal anti‐inflammatory drugs on the glucuronidation of 19‐norandrosterone: implications on doping control analysis

We have studied whether the phase II metabolism of 19‐norandrosterone, the most representative metabolite of 19‐nortestosterone (nandrolone), can be altered in the presence of other drugs that are not presently included on the Prohibited List of the World Anti‐Doping Agency. In detail, we have evaluated the effect of non‐prohibited drugs belonging to the classes of anti‐fungals, benzodiazepines, and non‐steroidal anti‐inflammatory drugs on the glucuronidation of 19‐norandrosterone. In vitro assays based on the use of either pooled human liver microsomes or specific recombinant isoforms of uridine diphosphoglucuronosyl‐transferase were designed and performed to monitor the formation of 19‐norandrosterone glucuronide from 19‐norandrosterone. Determination of 19‐norandrosterone (free and conjugated fraction) was performed by gas chromatography – mass spectrometry after sample pretreatment consisting of an enzymatic hydrolysis (performed only for the conjugated fraction), liquid/liquid extraction with tert‐butylmethyl ether, and derivatization to form the trimethylsilyl derivative. In parallel, a method based on reversed‐phase liquid chromatography coupled to tandem mass spectrometry in positive electrospray ionization with acquisition in selected reaction monitoring mode was also developed to identify the non‐prohibited drugs considered in this study. Incubation experiments have preliminarily shown that the glucuronidation of 19‐norandrosterone is principally carried out by UGT2B7 (39%) and UGT2B17 (31%). Inhibition studies have shown that the yield of the glucuronidation reaction is reduced in the presence of the anti‐fungals itraconazole, ketoconazole, and miconazole, of the benzodiazepine triazolam and of the non‐steroidal anti‐inflammatory drugs diclofenac and ibuprofen, while no alteration was recorded in the presence of all other compounds considered in this study. Copyright © 2015 John Wiley & Sons, Ltd.     

Erythropoiesis‐stimulating agents: development,detection and dangers

Epoetin alfa, the first member of the family of erythropoiesis stimulating agents (ESAs), was introduced to the market in 1989. Since then development has progressed to epoetins of the third generation. Currently drugs that use alternative approaches to stimulate erythropoiesis are under development. Uptake of all available ESAs into doping has occurred rapidly after their introduction. A multitude of dangers to health are associated with the illicit use of these substances. Different approaches to detect ESAs in doping control have been developed to comply with the very diverse nature of the compounds used. Future developments in the field of ESA require the development of new techniques in doping analysis. This review gives an overview of the development of ESA and its detection methods as well as future developments. [Correction made here after initial online publication] Copyright © 2009 John Wiley & Sons, Ltd.     

The effect of a period of intense exercise on the marker approach to detect growth hormone doping in sports

The major objective of this study was to investigate the effects of several days of intense exercise on the growth hormone marker approach to detect doping with human growth hormone (hGH). In addition we investigated the effect of changes in plasma volume on the test. Fifteen male athletes performed a simulated nine‐day cycling stage race. Blood samples were collected twice daily over a period of 15 days (stage race + three days before and after). Plasma volumes were estimated by the optimized CO Rebreathing method. IGF‐1 and P‐III‐NP were analyzed by Siemens Immulite and Cisbio Assays, respectively. All measured GH 2000 scores were far below the published decision limits for an adverse analytical finding. The period of exercise did not increase the GH‐scores; however the accompanying effect of the increase in Plasma Volume yielded in essentially lower GH‐scores. We could demonstrate that a period of heavy, long‐term exercise with changes in plasma volume does not interfere with the decision limits for an adverse analytical finding. Copyright © 2014 John Wiley & Sons, Ltd.     

Research on spiking rat EPO as internal standard in doping control samples for detection of EPO using SAR‐PAGE analysis with biotinylated primary antibody

According to the current Technical Document (TD) for erythropoietin (EPO), SAR‐PAGE is the most commonly applied method for both screening and confirmation procedures. Although this method is effective and robust, it lacks an internal standard (IS) to monitor the efficiency of analysis for each sample covering every step of the whole procedure, including preparation, immunopurification, and western blotting. This internal standard needs to be recognized by both anti‐EPO antibodies used for immunopurification and western blotting, respectively. Besides that, the band of IS could not be allowed to interfere with the recognition of all types of targeted EPO and analogs. To meet these two principles, rat EPO was selected. In this study, rat EPO was used to spike both urine and blood samples at the beginning of analysis. After preparation and immunopurification, single blotting was performed with biotinylated AE7A5 as the primary antibody, followed by incubation with streptavidin‐coupled HRP. Based on the comparison of different immunopurification methods, the AB‐286‐NA antibody coupled to M‐280 magnetic beads was the better choice for urine samples, whereas the MAIIA column was suitable for blood samples. All these methods were validated for selectivity, repeatability, and sensitivity. The modified method in this study could not only eliminate the cross‐reactivity between antibodies but also monitor the whole procedure of the analysis of EPO with spiked rat EPO. Besides that, rat EPO could also be used as an indicator for monitoring the presence of protease(s) in urine samples.     

Influence of intravenous administration of growth hormone releasing peptide-2 (GHRP-2) on detection of growth hormone doping: growth hormone isoform profiles in Japanese male subjects

Administration of exogenous 22 kDa recombinant human growth hormone (rhGH) suppresses the non-22 kDa pituitary growth hormone (GH) secretion by negative feedback; then, the elevated 22 kDa GH to non-22 kDa GH ratio (Rec/Pit ratio) can be utilized to detect doping with rhGH (isoform differential immunoassay). The influence of intravenous administration of growth hormone releasing peptide GHRP-2 on the isoform differential immunoassay for detecting rhGH doping has been investigated.In this study, a reference population (n=100) was used, with 0.04 mg/kg rhGH subcutaneous administration (n=5), 100 μg of GHRP-2 intravenous administration (n=10) and 0.04 mg/kg rhGH combined with 100 μg of GHRP-2 (n=10) in Japanese male subjects. The results indicated that the low dose (0.04 mg/kg) of rhGH led to significantly increased Rec/Pit ratio compared with the Japanese reference limit (P < 0.001). Because GHRP-2 dose led to increases in concentrations of both recombinant GH (recGH) and pituitary GH (pit GH), no significant change in the Rec/Pit ratio was observed (P > 0.05). In a combined administration study, after GHRP-2 dose the Rec/Pit ratios decreased to 39.9-43.9% compared with the elevated ratio caused by the rhGH dose.The results indicated that GHRP-2 administration cannot only be detected by the isoform differential immunoassay but also masks rhGH doping. The analysis of GHRP-2 was found to be suitable for compensating for the disadvantages of the isoform differential immunoassay because GHRP-2 and its metabolite (AA-3) in urine could be detected during the periods of masking of the Rec/Pit ratio by means of liquid chromatography/tandem mass spectrometry.     

Quantification and validation of nine nonsteroidal anti‐inflammatory drugs in equine urine using gas chromatography–mass spectrometry for doping control

Nonsteroidal anti‐inflammatory drugs (NSAIDs) are commonly used in therapeutic doses in human and veterinary medicine for the treatment of inflammation, pain, and fever. A method for the simultaneous determination of nine NSAIDs, known as therapeutic prohibited substances, in equine urine was developed and fully validated according to the European Commission Decision 2002/657/EC and Association of Official Racing Chemists criteria. The validation was performed for naproxen, flunixin, ketoprofen, diclofenac, eltenac, meclofenamic acid, phenylbutazone, vedaprofen, and carprofen in equine urine in accordance with the International Screening Limits (ISL) regulated by International Federation of Horseracing Authorities. After basic hydrolysis, samples were extracted with a C18 cartridge using automated solid‐phase extraction. Several derivatization reagents were investigated, and trimethylphenylammonium hydroxide/methanol (20/80, v/v) was selected. Analyses were carried out using gas chromatography–mass spectrometry with selected ion monitoring mode, but the method can be applied to a large number of analytes. The within‐laboratory reproducibility was not more than 12.8% (≤15%), and mean relative recoveries ranged from 91.1% to 104.1% for inter‐day and intra‐day precision. The decision limits (CCα) and detection capabilities (CCβ) were evaluated at concentrations near the ISL for each therapeutic substance. The validation results demonstrated that the method is highly reproducible, easily applicable, and suitable for the analysis of some NSAIDs in equine urine that have not been previously published. Finally, the method was also applied to known positive samples.     

LC‐MS/MS method for the quantification of new psychoactive substances and evaluation of their urinary detection in humans for doping control analysis

The constant legal adaptation of new psychoactive substances (NPS), challenges their evaluation in different fields. In sports, NPS are prohibited in competition with a reporting limit (RL) of 50 ng/mL for the parent compound or a metabolite. However, there is a lack of comprehensive methodologies and excretion studies for monitoring NPS. This work aims to develop an analytical methodology for the NPS quantification and to evaluate the suitability of monitoring the urinary parent stimulants after NPS misuse. A method for the quantification of 14 common NPS was developed and validated. The method was found to be linear in the range 1–1000 ng/mL, and was shown to be accurate and precise. A lowest limit of quantification (LLOQ) of 1 ng/mL was established for all analytes except for benzylpiperazine (5 ng/mL). The method was able to confirm the identity of the analytes at the LLOQ for most NPS. The methodology was applied to the quantification of the parent compound in urine samples collected from an observational study where several healthy volunteers (n ≥ 6 per drug) ingested active doses of mephedrone (MEPH), methylone (MDMC), 2,5‐dimetoxy‐4‐ethylphenetylamine (2C‐E), or 6‐(2‐aminopropyl)benzofuran (6‐APB). It was observed that for MDMC and 6‐APB, the quantification of the urinary parent drug at the current RL is a proper strategy for detecting their misuse. However, this strategy seems to be insufficient for evaluating MEPH and 2C‐E misuse. Monitoring the most abundant metabolite of MEPH (4′‐carboxy‐MEPH) and the reduction of the RL to 10 ng/mL for the 2C‐E evaluation are proposed.     

The effects of a freeze-thaw cycle and pre-analytical storage temperature on the stability of insulin-like growth factor-I and pro-collagen type III N-terminal propeptide concentrations: Implications for the detection of growth hormone misuse in athletes

A method based on two serum biomarkers – insulin‐like growth factor‐I (IGF‐I) and pro‐collagen type III N‐terminal propeptide (P‐III‐NP) – has been devised to detect growth hormone (GH) misuse. The aims of this study were to determine the stability of IGF‐I and P‐III‐NP concentrations in serum stored at ?20°C and to establish the effects of one freeze‐thaw cycle. Blood was collected from 20 healthy volunteers. Serum aliquots were analyzed after storage for one day at 4°C and one day, one week, five weeks, and three months at ?20°C. IGF‐I and P‐III‐NP results were combined to calculate a GH‐2000 discriminant function score for each volunteer. Inter‐assay precision was determined by analysing one quality control sample at each time‐point. A single freeze‐thaw cycle, storage of serum at 4°C for one day and at ?20°C for up to three months had no significant effect on IGF‐I or P‐III‐NP concentration. Intra‐sample variability for IGF‐I was 6.8% (Immunotech assay) and 12.9% (DSL assay). Intra‐sample variability for P‐III‐NP was 10.9% (Cisbio assay) and 13.7% (Orion assay). When IGF‐I and P‐III‐NP results were combined, intra‐sample variability of the GH‐2000 score expressed as a standard deviation varied between 0.31 and 0.50 depending on the assay combination used. Variability in IGF‐I and P‐‐III‐NP results of stored samples is largely determined by the characteristics of the assays. A single freeze‐thaw cycle, storage of serum at 4°C for one day or at ?20°C for up to 3 months does not result in a significant change in GH‐2000 score. Copyright © 2012 John Wiley & Sons, Ltd.     

Detection of stanozolol O‐ and N‐sulfate metabolites and their evaluation as additional markers in doping control

Stanozolol (STAN) is one of the most frequently detected anabolic androgenic steroids in sports drug testing. STAN misuse is commonly detected by monitoring metabolites excreted conjugated with glucuronic acid after enzymatic hydrolysis or using direct detection by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). It is well known that some of the previously described metabolites are the result of the formation of sulfate conjugates in C17, which are converted to their 17‐epimers in urine. Therefore, sulfation is an important phase II metabolic pathway of STAN that has not been comprehensively studied. The aim of this work was to evaluate the sulfate fraction of STAN metabolism by LC‐MS/MS to establish potential long‐term metabolites valuable for doping control purposes. STAN was administered to six healthy male volunteers involving oral or intramuscular administration and urine samples were collected up to 31 days after administration. Sulfation of the phase I metabolites commercially available as standards was performed in order to obtain MS data useful to develop analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) to detect potential sulfate metabolites. Eleven sulfate metabolites (M‐I to M‐XI) were detected and characterized by LC‐MS/MS. This paper provides valuable data on the ionization and fragmentation of O‐ sulfates and N‐ sulfates. For STAN, results showed that sulfates do not improve the retrospectivity of the detection compared to the previously described long‐term metabolite (epistanozolol‐N ‐glucuronide). However, sulfate metabolites could be additional markers for the detection of STAN misuse. Copyright © 2016 John Wiley & Sons, Ltd.     

Determination of sulfur‐containing antibiotics using high‐performance liquid chromatography with integrated pulsed amperometric detection

Sulfur‐containing antibiotics (e.g., ampicillin, penicillin, lincomycin, cephalosporin, and sulfanilamide) are a general class of compounds that have antibacterial properties. Some of these antibiotics lack a strong chromophore, and consequently are poorly detected using absorbance detection. The presence of oxidizable sulfur within an antibiotic’s molecular structure allows its direct detection by pulsed amperometry. Pulsed amperometry is an electrochemical detection method that uses a pulsed potential‐time waveform at a noble metal electrode surface to electrocatalytically oxidize analytes in proportion to concentration. The current for sulfur compounds on a gold (Au) electrode is caused by the desorption of the analyte previously adsorbed at the oxide‐free Au surface. Current integration in conjunction with rapidly changed voltage potentials is termed integrated pulsed amperometric detection (IPAD). Recent publications using high‐performance liquid chromatography (HPLC) with IPAD have demonstrated the capability to measure sulfur antibiotics. Our investigations of HPLC‐IPAD showed poor long‐term peak area reproducibility for these drugs. In this article, we present a new IPAD waveform that we demonstrate has good long‐term (>2 months) peak area reproducibility (4–5% relative s.d.). We show that a broad spectrum of sulfur‐antibiotic chemical structures can be detected, and we compare IPAD to absorbance detection. For nonchromophoric sulfur‐containing antibiotics, IPAD showed greater sensitivity than absorbance detection. The lower limits of detection, linearities, and peak area precision are compared for both IPAD and absorbance detection methods using seven antibiotics representing a diversity of chemical structures, including one nonsulfur‐containing antibiotic. The stabilities of cephradine and cephapirin were monitored using both IPAD and absorbance detections. Cephradine showed high chemical stability in aqueous ambient conditions at 10 μg/ml, whereas cephapirin showed a decomposition rate of 10% per day by both detection techniques. Some decomposition products could only be detected by IPAD. High recoveries were demonstrated for two antibiotics (sulfamethoxazole and trimethoprim) in a pharmaceutical tablet formulation. For the same formulation, the feasibility of conducting a dissolution study was also demonstrated. IPAD is a good primary detection method to complement or replace absorbance detection for nonchromophoric sulfur‐containing antibiotics and their sulfur‐containing impurities and decomposition products. HPLC‐IPAD is also a good confirming technique for sulfur antibiotics with chromophoric properties, and their impurities and decomposition products. Drug Dev. Res. 53:268–280, 2001. © 2001 Wiley‐Liss, Inc.     

Determination of growth hormone releasing peptides metabolites in human urine after nasal administration of GHRP‐1, GHRP‐2, GHRP‐6, Hexarelin,and Ipamorelin

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. Copyright © 2015 John Wiley & Sons, Ltd.     

Anti‐doping analytes in serum: A comparison of SST and SST‐II Advance blood collection tubes

Serum insulin‐like growth factor‐1 (IGF‐1), procollagen type III N‐terminal peptide (PIIINP), and human growth hormone (hGH) isoforms were analyzed in identical serum samples collected into BD Vacutainer^® SST and BD Vacutainer^® SST‐II Advance serum separator tubes. Comparing the serum collected into each tube, measurement correlation was high (R² > 0.83) and percent bias was minimal (<|3.2%|) for all analytes measured using World Anti‐Doping Agency (WADA)‐approved tests. As such, it is recommended that both SST and SST‐II Advance tubes can be used interchangeably for anti‐doping purposes.     

Simultaneous detection of xenon and krypton in equine plasma by gas chromatography‐tandem mass spectrometry for doping control

Xenon can activate the hypoxia‐inducible factors (HIFs). As such, it has been allegedly used in human sports for increasing erythropoiesis. Krypton, another noble gas with reported narcosis effect, can also be expected to be a potential and less expensive erythropoiesis stimulating agent. This has raised concern about the misuse of noble gases as doping agents in equine sports. The aim of the present study is to establish a method for the simultaneous detection of xenon and krypton in equine plasma for the purpose of doping control. Xenon‐ or krypton‐fortified equine plasma samples were prepared according to reported protocols. The target noble gases were simultaneously detected by gas chromatography‐triple quadrupole mass spectrometry using headspace injection. Three xenon isotopes at m /z 129, 131, and 132, and four krypton isotopes at m /z 82, 83, 84, and 86 were targeted in selected reaction monitoring mode (with the precursor ions and product ions at identical mass settings), allowing unambiguous identification of the target analytes. Limits of detection for xenon and krypton were about 19 pmol/mL and 98 pmol/mL, respectively. Precision for both analytes was less than 15%. The method has good specificity as background analyte signals were not observed in negative equine plasma samples (n = 73). Loss of analytes under different storage temperatures has also been evaluated. Copyright © 2016 John Wiley & Sons, Ltd.     

The conformational and biological analysis of a cyclic anti‐obesity peptide from the C‐terminal domain of human growth hormone

Abstract: The three‐dimensional solution structure of anti‐obesity drug (AOD), a 15‐residue, disulfide‐bonded, cyclic peptide, cyclo(6,13)‐H₂N‐Leu‐Arg‐Ile‐Val‐Gln‐Cys‐Arg‐Ser‐Val‐Glu‐Gly‐Ser‐Cys‐Gly‐Phe‐OH, derived from the C‐terminal domain of the human growth hormone (hGH) (residues 177–191) was determined using two‐dimensional ¹H NMR spectroscopy. AOD stimulates lipolysis and inhibits lipogenesis, in vitro, in rodent, porcine and human adipose tissues. These biological effects suggest that AOD is a potential therapeutic candidate for the treatment of obesity. Conformational studies of AOD were conducted in aqueous solution and in water/dimethylsulfoxide mixtures. In general, spectral quality was superior in the water/dimethylsulfoxide mixtures. The cyclic region of AOD in water/dimethylsulfoxide adopts type I β‐turns at residues Ser⁸‐Val⁹‐Glu¹⁰‐Gly¹¹ and Ser¹²‐Cys¹³‐Gly¹⁴‐Phe¹⁵, each preceded by loop‐like structures. Comparison of the conformation of this peptide with residues 177–191 in the native hGH protein X‐ray crystal structure indicates that the synthetic peptide retains some structural similarity to the intact protein. This study provides evidence that the C‐terminal region of hGH is a specific functional domain of the multifunctional hGH protein.     

Evaluation of R‐ (−) and S‐ (+) Clenbuterol enantiomers during a doping cycle or continuous ingestion of contaminated meat using chiral liquid chromatography by LC‐TQ‐MS

Clenbuterol is known to improve competition resistance and muscular growth in athletes. Although it is an illegal drug, its use by farmers is widely spread to induce growth of their cattle. Thus, when clenbuterol is found in the urine of an athlete, there is doubt whether it was consumed with doping purposes or if it is due to the consumption of meat from a clenbuterol‐fed animal. Previous studies suggest that enantiomeric relationship of clenbuterol may be different according to the intake source. However, the enantiomeric relationship throughout a doping cycle or a continuous intake of contaminated meat has not yet been explored. In this first approximation, our aim was the development and validation of a sensitive and rapid method for the determination of S‐ (+) and R‐ (─) clenbuterol enantiomers to be used in a controlled study in rats fed for one week with contaminated meat or simulating a doping cycle. Enantiomers were measured using liquid chromatography coupled to mass spectrometry with a triple quadrupole analyzer (LC‐TQ‐MS) and were separated on an AGP Chiralpak column. The method was fully validated following the VICH (Veterinary International Conference on Harmonization guidelines) and was linear in the range of 12.5–800 pg/mL with a correlation coefficient of ≥0.98 for each enantiomer, and with a limit of quantitation and detection (LOQ and LOD) of 12.5 pg/mL and 6.5 pg/mL, respectively, for both enantiomers. The application of this method pointed out the shift of the enantiomeric relationship in urine from rats during the first five days of the doping cycle compared to those fed with contaminated meat. This finding can be of substantial importance in further doping studies.