Minimum Required Performance Level Research Articles

The MAIIA EPO Purification Gel Kit, 7D3-An Alternative Purification Kit for Screening and Confirmation Procedures.

In this study, the performance of the new MAIIA EPO purification kit 7D3 was evaluated for human urine samples to confirm compliance with the WADA criteria of the WADA technical document 2024 (TD2024EPO). The kit was validated for selectivity, reliability at minimum required performance levels (MRPLs), limit of detection (LOD), carryover and recovery. A total of 81 urine samples were tested for selectivity, 29 samples for reliability at MRPL, LOD and 16 samples for recovery. The kit fulfils the criteria and the requirements of the upcoming WADA TD2024EPO and presents a valid solution for the purification of ERAs from human urine for doping analyses.

Alkanediol–hexafluoroisopropanol amphiphilic supramolecular solvent: Fabrication, characterization, and application potential for doping control of hormones and metabolic modulators in human urine

A novel amphiphilic supramolecular solvent (SUPRAS) was developed by hexafluoroisopropanol (HFIP)-induced self-assembly of 1,2-hexanediol in an aqueous matrix. Its application potential in multiclass determination was assessed by extracting from urine 10 prohibited hormones and metabolic modulators (log P from 2.38 to 7.57) listed by the World Anti-Doping Agency (WADA). This SUPRAS is stably formed across a wide pH range (2–12) and is unaffected by salt concentrations (<10 % g mL−1 of NaCl), and it has a higher density than water. The dehydration and hydrogen bonding ability of HFIP, as well as hydrophobic interactions between alkanediols, dominates the formation of the SUPRAS. The nanostructure of the SUPRAS, composed of 1,2-hexanediol, HFIP, and a high content of water (>25 %, w/w), forms large spherical micellar aggregates (10–100 μm) that exhibit amphiphilic properties. A 1,2-hexanediol-HFIP SUPRAS-based liquid-phase microextraction (LPME) method for detection of selected prohibited drugs in urine coupled with liquid chromatography–tandem mass spectrometry (LC-MS/MS) was established, optimized, and validated. The method showed fast extraction time (30 s of vortex) and low solvent consumption (160 μL of organic solvent). Under the optimal conditions (7 % g mL−1 of 1,2-hexanediol, 10 % v/v of HFIP), the SUPRAS achieved high extraction efficiencies (>80 %) and an enrichment factor of 6.0, with minimal matrix effects for all analytes due to its high amphiphilicity and large surface area. The method demonstrated excellent linearity (R ≥0.9909), with method limits of detection ranging from 0.018 to 0.18 ng mL−1 and method limits of quantitation ranging from 0.060 to 0.60 ng mL−1, which are well below the minimum required performance levels set by authorities. Furthermore, the intra- and inter-day relative recoveries for spiked human urine samples varied in the ranges of 90.3–121 % and 88.1–119 %, respectively, with relative standard deviations less than 13 % and 19 %, respectively. This SUPRAS-based LPME method proves to be simple, highly efficient, and eco-friendly, and it has promising potential for multiclass determination in doping control.

Chromatographic-mass spectrometric analysis of peptidic analytes (2-10kDa) in doping control urine samples.

Peptides with a molecular mass between 2 and 10kDa that are prohibited in elite sports usually require dedicated sample preparation and mass spectrometric detection that commonly cannot be combined with other (lower molecular mass) substances. In most instances, the physicochemical differences are too significant to allow for a generic analytical procedure. A simplification of established and comparably complex analytical approaches is therefore desirable and has been accomplished in the context of this study. With urine samples representing still the most frequently collected doping control specimens, efficient extraction of peptidic analytes from this matrix was a major goal of this method, as demonstrated for the included compounds such as insulins (human, lispro, aspart, glulisine, tresiba, glargine metabolite, bovine insulin, porcine insulin), growth hormone-releasing hormones (sermorelin, CJC-1295, tesamorelin) incl. their respective metabolites, insulin-like-growth factors (long-R3 -IGF-I, R3 -IGF-I, des1-3 -IGF-I), synacthen, gonadorelin and mechano growth factors (human MGF, MGF-Goldspink). Sample preparation and detection are controlled by five internal standards, covering all five included peptide drug categories. Nearly all requirements of the recent technical documents from the World Anti-Doping Agency (WADA) considering their minimum required performance levels (MRPL) are fulfilled, and the method was validated for its utilisation as initial testing procedure in doping controls. Finally, the approach was applied to authentic post-administration study urine samples (for insulins and gonadorelin) in order to provide proof of principle.

Window of detection of cocaine related alkaloids in oral fluid collected with the FloqSwab™ after coca tea consumption.

Coca tea is a popular drink in some countries of South America where it is presented as a safe energy preparation, based on a limited total content of cocaine of about 3 to 5 mg. Tea bags can be bought with no legal considerations in these countries both by locals and tourists but its consumption can have consequences when consumed overseas. Driving under the influence of cocaine is banned in most places in the world and can be documented by oral fluid testing. A study was implemented with coca tea bags (Coca & Muna) purchased in Peru, after a French attorney at law contacted the laboratory to assess the involvement of coca tea in the positive oral fluid results of a driver. Ten healthy volunteers consumed 250 mL of coca tea containing 4.5 mg of cocaine. No volunteer reported any change in behavioral effects after consumption of the coca tea. Oral fluid was collected with a swab (FloqSwab™, Copan) over 8 hours to follow the elimination of cocaine and its major metabolites (benzoylecgonine and ecgonine methylester). This is the procedure used by the French police. All samples were analyzed by UHPLC-MS-MS after Quantisal™ buffer desorption. As the device does not allow measurement of the amount of collected fluid, the results are qualitative. This is in accordance with the French law that requires a yes or no response about the presence of cocaine, with a minimum required performance level of 10 ng/ml of cocaine or benzoylecgonine. Parent cocaine was identified for 30 to 120 min. Benzoylecgonine and ecgonine methylester were identified between 1 and 8 hours, with a large inter-individual variation. Although it is generally accepted that a 4 to 5 mg cocaine dose has no significant pharmacological effect, the consumption of coca tea can lead to the suspension of a person's driving license due to a positive oral fluid test.

QuEChERS as alternative extraction procedure in doping analyses.

QuEChERS is a dispersive solid phase extraction commonly applied in food analysis for residues, such as pesticides or mycotoxins for more than 20 years. Due to the quick and easy sample preparation procedure, a QuEChERS method based on ammonium acetate combined with formic acid in acetonitrile was tested for the preparation of urine samples for doping control purposes. Testing urine samples with different pH and specific gravity, using the combination of 10M ammonium acetate with 3% formic acid in acetonitrile, 312 out of 342 tested compounds could be extracted at their respective minimum required performance levels according to the World Anti-Doping Agency (WADA) technical documents. For nine selected analytes representing six categories of WADA's Prohibited List, we validated the QuEChERS extraction method fulfilling WADA's requirements for a confirmation procedure of the nonthreshold substances investigated. Especially for the intact stanozolol-glucuronides analyzed by high-resolution mass spectrometry, the described extraction method might be an alternative for confirmation procedures as it is time- and cost-saving compared with the commonly applied solid phase extraction.

Achieving routine application of dried blood spots for erythropoietin receptor agonist analysis in doping control: low-volume single-spot detection at minimum required performance level.

Background: Erythropoietin receptor agonists (ERAs) are substances prohibited in sports and currently monitored in urine and blood. There is a great interest in new matrices like dried blood spots (DBSs). Method: A direct method for the detection of ERAs in DBSs using one single spot of 25μl has been optimized and validated. Results: Limits of detection close or equal to those required by the World Anti-Doping Agency for serum/plasma samples were achieved, using a volume 20-times lower. All analytes were stable for at least 90days at room temperature. Conclusion: Method performance was comparable to the requirements established for blood samples and, thus, monitoring of ERAs is reliable in DBSs in the context of doping control.

Analysis of doping control samples using supercritical fluid chromatography-tandem mass spectrometry: Ready for routine use.

Supercritical fluid chromatography is proving to be a good separation and sample preparation tool for various analytical applications and, as such, has gained the attention of the anti-doping community. Here, the applicability of supercritical fluid chromatography hyphenated to tandem mass spectrometry for routine doping control analysis was tested. A multi-analyte method was developed to cover 197 drugs and metabolites that are prohibited in sport. More than 1000 samples were analyzed by applying a "dilute and inject" approach after hydrolysis of glucuronide metabolites. Additionally, a comparison with routinely used liquid chromatography-mass spectrometry was performed with 250 of the 1000 samples and a number of past positive anti-doping samples. It revealed some features where supercritical fluid chromatography-tandem mass spectrometry was found to be complementary or advantageous to liquid chromatography-mass spectrometry for anti-doping purposes, such as better retention of analytes that are poorly retained in reversed-phase liquid chromatography. Our results suggest that supercritical fluid chromatography-tandem mass spectrometry is sensitive (limit of detection <50% relevant minimum required performance level required by the World Anti-Doping Agency for anti-doping analysis), reproducible, robust, precise (analytes of interest area coefficient of variation <5%; retention time difference coefficient of variation <1%) and complementary to existing techniques currently used for routine analysis in the World Anti-Doping Agency accredited laboratories.

Doping control analysis of trimetazidine in dried blood spot.

Dried blood spot (DBS) analysis has been an inherent part of sports drug testing through the technological advancements of the past decade. Trimetazidine, a non-threshold banned substance, is excreted into urine after a dose of the permitted drug lomerizine. Therefore, a lomerizine-specific metabolite (M6) is analyzed to confirm the origin of trimetazidine in traditional urine analysis. Application studies were conducted to develop an analytical method for trimetazidine applicable to DBS. These studies comprise (1) the effect of different sampling sites on the detection of trimetazidine, (2) the determination of the appropriate trimetazidine level required for DBS analysis, and (3) differentiating between trimetazidine and lomerizine use. A high-resolution mass spectrometric method for detecting trimetazidine in DBS was validated. After oral administration of trimetazidine (n = 7), venous and capillary blood (fingertip and upper arm) were spotted on cellulose paper. Trimetazidine could be identified in DBS in all subjects up to 60 h after administration. The limit of detection was 0.05 ng/ml, and the limit of identification was 0.06 ng/ml, suggesting the minimum required performance level of 0.2 ng/ml. In the fingertip capillary blood, biases of 9.7% (vs. upper arm) and 13.0% (vs. vein) were observed in the trimetazidine intensity; however, there were no concerns in the qualitative analysis. After administering lomerizine (n = 10), the intact lomerizine has a strong peak intensity in blood compared to trimetazidine. Contrary to urine analysis, the M6 was less detectable in blood. Laboratories should confirm intact lomerizine whenever trimetazidine is identified in DBS.

Drugs of abuse in tap water from eight European countries: Determination by use of supramolecular solvents and tentative evaluation of risks to human health.

Recent research findings have confirmed the presence of illicit drugs in tap water from some European Union (UE) member states. Contaminants in tap water come directly from drinking water sources such as rivers or lakes owing to inefficient removal at wastewater treatment and water purification plants. This work was aimed at setting a starting point for assessing the health risks of exposure to twelve drugs of abuse through consumption of tap water in the European population. For this purpose, a method using supramolecular solvents (SUPRAS) was developed to extract drugs in the opioid, amphetamine, cocaine and cannabinoid groups from tap water for their determination by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 119 tap water samples were collected from eight EU countries for analysis. Seven drugs were found at concentrations from 0.3 to 340ng/L in 72 of the samples (60.5%). The mean exposure to the drugs through consumption of tap water was calculated to be 0.0064-3.531ng/kg·day for adults and 0.0247-6.7580ng/kg·day for children, whereas that resulting from dermal contact was estimated to be 4-7 orders of magnitude lower. Exposure values were compared with the minimum required performance levels (MRPL) for the drugs in urine set by the World Anti-Doping Agency (WADA). Based on the results, a need clearly exists for further research into the adverse effects on health of inadvertent, sustained exposure to low doses of drugs of abuse.

Optimizing detection of erythropoietin receptor agonists from dried blood spots for anti-doping application.

The World Anti‐Doping Agency (WADA) has recently implemented dried blood spots (DBSs) as a matrix for doping control. However, specifications regarding the analysis of the class of prohibited substances called erythropoietin (EPO) receptor agonists (ERAs) from DBSs are not yet described. The aim of this study was to find optimal conditions (sample volume and storage) to sensitively detect endogenous erythropoietin (hEPO) and prohibited ERAs from DBSs and compare detection limits to WADA‐stipulated minimum required performance levels (MRPLs) for ERAs in serum/plasma samples. Venous whole blood was spotted onto Whatman 903 DBS cards with primarily 60 μl of blood, but various volumes from 20 to75 μl were tested. All samples were immunopurified with MAIIA EPO Purification Gel kit (EPGK) and analysed with sodium N‐lauroylsarcosinate polyacrylamide gel electrophoresis (SAR‐PAGE) and Western blot. Sixty‐microliter DBSs allowed the detection of the four main ERAs (BRP, NESP, CERA and EPO‐Fc) at concentrations close to WADA's MRPLs described for 500 μl of serum/plasma. Different storage temperatures, from −20°C to 37°C, were evaluated and did not affect ERA detection. A comparison of the detection of endogenous EPO from the different anti‐doping matrices (urine, serum and DBSs produced from upper arm capillary blood) from five participants for 6 weeks was performed. Endogenous EPO extracted from DBSs showed intra‐individual variations in male and female subjects, but less than in urine. Doping controls would benefit from the stability of ERAs on DBSs: It can be a complementary matrix for ERA analysis, particularly in the absence of EPO signals in urine.

Determination of ghrelin and desacyl ghrelin in human plasma and urine by means of LC-MS/MS for doping controls.

The hunger hormone ghrelin (G) is classified as prohibited substance in professional sport by the World Anti-Doping Agency (WADA), due to its known growth hormone releasing properties. The endogenous bioactive peptide consists of 28 amino acids with a caprylic acid attached to serine at position 3. Within this study, it was aimed to develop methods to determine G and desacyl ghrelin (DAG) in plasma and urine by means of LC-MS/MS. Two strategies were applied with a bottom-up approach for plasma and top-down analyses for urine. Both sample preparation procedures were based on solid-phase extraction for enrichment and sample clean-up. Method validation showed good results for plasma and urine with limits of detection (LODs) for G and DAG between 30 and 50 pg/ml, recoveries between 45-50%, and imprecisions (intra- and inter-day) between 3% and 24%. Plasma analysis was also valid for quantification with accuracies determined with ~100% for G and ~106% for DAG. The minimum required performance level for doping control laboratories is set to 2ng/ml in urine, and the herein established method yielded acceptable results even at 5% of this level. As proof-of-concept, plasma levels (G and DAG) of healthy volunteers were determined and ranged between 30 and 100 pg/ml for G and 100-1200 pg/ml for DAG. In contrast to earlier reported studies using ligand binding assays for urinary G and DAG, in this mass spectrometry-based study, no endogenous urinary G and DAG were found, although the LODs should enable this.

Combining direct urinary injection with automated filtration and nanoflow LC-MS for the confirmatory analysis of doping-relevant small peptide hormones

Nano-liquid chromatography (nanoLC) has proven itself as a powerful tool and its scope entails various applications in (bio)analytical fields. Operation at low (nL/min) flow rates in combination with reduced inner dimensions (ID < 100 µm), leads to significantly enhanced sensitivity when coupled with electrospray ionization-mass spectrometry (ESI-MS). Challenges that remain for the routine implementation of such miniaturized setups are related to clogging of the system and robustness in general, and thus the application of tedious sample preparation steps. To improve ruggedness, a filter placed upstream in the LC prevents particles from entering and clogging the system. This so-called online automatic filtration and filter back-flush (AFFL) system was combined with nanoLC and the direct injection principle for the sensitive confirmatory analysis of fifty different doping-relevant peptides in urine.The presented assay was fully validated for routine purposes according to selectivity and matrix interference, limit of identification (LOI), carryover, matrix effect, sample extract stability, analysis of educational external quality assessment (EQAS) samples, robustness of the online AFFL-setup and retention time stability. It was also fully compliant with the most recent minimum required performance levels (MRPL) and chromatographic/mass spectrometric identification criteria (IDCR), as imposed by the World Anti-Doping Agency (WADA).In the absence of labor-intensive sample preparation, the application of AFFL allowed for the injection of diluted urine samples without any noticeable pressure buildup in the nanoLC system. Contrary to earlier observations by our group and others, the addition of dimethylsulfoxide (DMSO) to the mobile phase did not enhance sensitivity in the presented nanoflow setup, yet was beneficial to reduce carry over. Although the robustness of the presented setup was evaluated only for the analysis of diluted urine samples, it is entirely conceivable that routine applications employing other matrices and currently running on analytical scale LC instruments could be transferred to micro/nanoLC scale systems to reach lower detection limits.

Ion mobility-high resolution mass spectrometry in doping control analysis. Part II: Comparison of acquisition modes with and without ion mobility

In the second part of this study, a systematic comparison was made between two ion fragmentation acquisition modes, namely data-independent acquisition (DIA) and DIA with ion mobility spectrometry (IMS) technology. These two approaches were applied to the analysis of 192 doping agents in urine. Group I included 102 compounds such as stimulants, diuretics, narcotics, and β2-agonists, while Group II contained 90 compounds included steroids, glucocorticoids, and hormone and metabolic modulators. Important method parameters were examined and compared, including the fragmentation, sensitivity, and assignment capability with the minimum occurrence of false positive hits.The results differed between Group I and II in number of detected fragments when exploring the MS/MS spectra. In Group I only 13%, while in the Group II 64% of the substances had a higher number of fragments in DIA-IMS mode vs. DIA. In terms of sensitivity, the performance of the two modes with and without activated IMS dimension was identical for about 50% of the doping agents. The sensitivity was higher without IMS, i.e. in simple DIA mode, for 20–40% of remaining doping agents. Despite this sensitivity reduction with IMS, 82% of compounds from both Groups met the minimum required performance level (MRPL) criteria of the World Anti-Doping Agency (WADA) when the DIA-IMS mode was applied.Automated data processing is important in routine doping analysis. Therefore, processing methods were optimized and evaluated for the prevalence of false peak assignments by analysing the target substances at different concentrations in urine samples. Overall, a significantly higher number of misidentified compounds was observed in Group II, with an almost 2-fold higher number of misidentifications in DIA compared to DIA-IMS. This result highlights the benefit of the IMS dimension to reduce the rate of false positive in screening analysis. The optimized UHPLC-IM-HRMS method was finally applied to the analysis of urine samples from administration studies including nine doping agents from both Groups. However, to limit the number of interferences from the biological matrix, an emphasis is needed on the adequate settings of the data processing method.

Facilitated Qualitative Determination of Insulin, Its Synthetic Analogs, and C-Peptide in Human Urine by Means of LC-HRMS.

The increasing importance to determine bioactive peptide hormones such as insulin, its synthetic analogs, and C-peptide in urine samples represents an analytical challenge. The physiological concentrations of insulin in urine are commonly found at sub-ng/mL levels and thus represent a complex analytical task. C-peptide concentrations, on the other hand, tend to be in the moderate ng/mL range and are hence much easier to determine. Insulin and C-peptide are important in the diagnostics and management of metabolic disorders such as diabetes mellitus and are also particularly relevant target analytes in professional sports and forensics. All insulins are classified on the World Anti-Doping Agency’s (WADA) list of prohibited substances and methods in sports with a minimum required performance level (MRPL) of 50 pg/mL. Until now, methods combining immunoextraction and subsequent mass spectrometric detection have mostly been used for this purpose. With the method developed here, sample preparation has been simplified considerably and does not require an antibody-based sample purification. This was achieved by a sophisticated mixed-mode solid-phase extraction and subsequent separation with liquid chromatography coupled to high-resolution mass spectrometry. Included target insulins were human, lispro, glulisine, aspart, glargine metabolite, degludec, and additionally, human C-peptide. The method was validated for the synthetic insulin analogs considering WADA requirements including specificity, limit of detection (10–25 pg/mL), limit of identification, recovery (25–100%), robustness, carry over (<2%), and matrix effects. All sample preparation steps were controlled by two stable isotope-labeled internal standards, namely, [[2H10] LeuB6, B11, B15, B17]-insulin and [[13C6] Leu26, 30] C-peptide. Finally, the method was applied to samples from patients with diabetes mellitus treated with synthetic insulins.

Ion mobility-high resolution mass spectrometry in anti-doping analysis. Part I: Implementation of a screening method with the assessment of a library of substances prohibited in sports

In this series of two papers, 192 doping agents belonging to the classes of stimulants, narcotics, cannabinoids, diuretics, β2-agonists, β-blockers, anabolic agents, and hormone and metabolic modulators were investigated, with the aim to assess the benefits and limitations of ion mobility spectrometry (IMS) in combination with ultra-high performance liquid chromatography (UHPLC) and high resolution mass spectrometry (HRMS) in anti-doping analysis.In this first part, a generic UHPLC-IM-HRMS method was successfully developed to analyze these 192 doping agents in standard solutions and urine samples, and an exhaustive database including retention times, TWCCSN2 values, and m/z ratios was constructed. Urine samples were analyzed using either a simple “dilute and shoot” procedure or a supported liquid-liquid extraction (SLE) procedure, depending on the physicochemical properties of the compounds and sensitivity criteria established by the World Anti-Doping Agency (WADA) as the minimum required performance levels (MRPL). Then, the precision of the generic UHPLC-IM-HRMS method was assessed as intraday, interday as well as interweek variation of UHPLC retention times and TWCCSN2 values, for which RSD the values were always lower than 2% in urine samples. The possibility to filter MS data using IMS dimension was also investigated, and in average, the application of IMS filtration provided low energy MS spectra with 86% less interfering peaks in both standard and urine samples. Therefore, the filtered MS spectra allowed for an easier interpretation and a lower risk of false positive result interpretations. Finally, IMS also offers additional selectivity to the UHPLC-HRMS enabling to separate isobaric and isomeric substances. Among the selected set of 192 doping agents, there were 30 pairs of isobaric or isomeric compounds, and only two pairs could not be resolved under the developed conditions. This illustrates the potential of adding ion mobility to UHPLC-HRMS in anti-doping analyses.

Preliminary data on the potential for unintentional antidoping rule violations by permitted cannabidiol (CBD) use.

According to the World Anti-Doping Agency (WADA) regulations, cannabinoids use is prohibited in competition except for cannabidiol (CBD) use. For an adverse analytical finding (AAF) in doping control, cannabinoid misuse is based on identification of the pharmacologically inactive metabolite 11-nor-delta-9-carboxy-tetrahydrocannabinol-9-carboxylic acid (carboxy-THC) in urine at a concentration greater than 180 ng/ml. All other (minor) cannabinoids are reported as AAF when identified, except for CBD that has been explicitly excluded from the class of cannabinoids on WADA's Prohibited List since 2018. However, due to the fact that CBD isolated from cannabis plants may contain additional minor cannabinoids, the permissible use of CBD can lead to unintentional violations of antidoping regulations. An assay for the detection of 16 cannabinoids in human urine was established. The sample preparation consisted of enzymatic hydrolysis of glucuronide conjugates, liquid-liquid extraction, trimethylsilylation, and analysis by gas chromatography/tandem mass spectrometry (GC-MS/MS). Spot urine samples from CBD users, as well as specimens obtained from CBD administration studies conducted with 15 commercially available CBD products, were analyzed, and assay characteristics such as selectivity, reproducibility of detection at the minimum required performance level, limit of detection, and limit of identification were determined. An ethical committee approved controlled single dose commercially available CBD products administration study was conducted to identify 16 cannabinoids in urine samples collected after ingestion or application of the CBD products as well as their presence in spot urine samples of habitual CBD users. Variable patterns of cannabinoids or their metabolites were observed in the urine samples, especially when full spectrum CBD products were consumed. The presence of minor cannabinoids or their metabolites in an athlete's in-competition urine sample represents a substantial risk of an antidoping rule violation.

High-throughput liquid chromatography tandem mass spectrometry assay as initial testing procedure for analysis of total urinary fraction.

In the recent years, a lot of effort was put into the development of multiclass initial testing procedures (ITP) to streamline analytical workflow in antidoping laboratories. Here, a high-throughput assay based on liquid chromatography-triple quadrupole mass spectrometry suitable for use as initial testing procedure covering multiple classes of compounds prohibited in sports is described. Employing a 96-well plate packed with 10 mg of weak cation exchange polymeric sorbent, up to 94 urine samples and their associated positive and negative controls can be processed in less than 3 h with minimal labor. The assay requires a 0.5-ml urine aliquot, which is subjected to enzymatic hydrolysis followed by solid phase extraction, evaporation, and reconstitution in a 96-well collection plate. With a 10-min run time, more than 100 analytes can be detected using electrospray ionization with polarity switching. The assay can be run nearly 24/7 with minimal downtime for instrument maintenance while detecting picogram amounts for the majority of analytes. Having analyzed approximately 28,000 samples, nearly 400 adverse analytical findings were found of which only one tenth were at or above 50% of the minimum required performance level established by the World Anti-Doping Agency. Compounds most often identified were stanozolol, GW1516, ostarine, LGD4033, and clomiphene, with median estimated concentrations in the range of 0.02-0.09 ng/ml (either as parent drug or a metabolite). Our data demonstrate the importance of using a highly sensitive ITP to ensure efficient antidoping testing.

The salmeterol anomaly and the need for a urine threshold.

Salmeterol is a long acting beta2-agonist (LABA) used widely for the treatment of airways disease. There is evidence that beta2-agonists, including salmeterol, have the potential for performance enhancing effects when delivered at supratherapeutic doses. For this reason, all beta2-agonists are currently on the Prohibited List issued by the World Anti-Doping Agency (WADA), regardless of dosing route with some exemptions for inhaled salbutamol, formoterol, and salmeterol when used at therapeutic inhaled doses. For 2020, salmeterol use is permitted up to a therapeutic dosing threshold of 200 μg daily, but unlike salbutamol and formoterol, there is an anomaly; currently there is no urine threshold to control for supratherapeutic dosing beyond this dosing threshold. Salmeterol, however, is reportable as an adverse analytical finding (AAF) at levels above 10 ng/mL. Complicating matters is that following inhalation, salmeterol parent drug is present at relatively low levels compared with other beta2-agonists due to rapid metabolism to the metabolite, alpha-hydroxysalmeterol, which is typically present at higher levels than the parent drug. Moreover, peak parent drug levels following permitted therapeutic dosing are below the minimum required performance level (MRPL) of 10 ng/mL for salmeterol (50% of the MRPL that analytical laboratories are required to meet for non-threshold beta2-agonists), hence the presence of salmeterol may be unreported. For consistency, a urine threshold should be introduced for salmeterol as a matter of priority, to balance the needs of athletes who use salmeterol therapeutically up to the agreed dosing threshold, with the need to control supratherapeutic dosing for doping intentions and athlete harm minimization.

Determination of anabolic androgenic steroids as imidazole carbamate derivatives in human urine using liquid chromatography-tandem mass spectrometry.

Anabolic androgenic steroids are widely abused substances in sports doping. Their detection present limitations regarding the use of soft ion sources such as electrospray or atmospheric pressure chemical ionization by liquid chromatography-tandem mass spectrometry. In the current study, a novel derivatization method was developed for the ionization enhancement of selected anabolic androgenic steroids. The proposed method aims at the introduction of an easily ionizable moiety into the steroid molecule by converting the hydroxyl groups into imidazole carbamates using 1,1'-carbonyldiimidazole as derivatization reagent. The proposed method was applied to water and urine samples spiked with exogenous anabolic androgenic steroids in various concentration levels. Steroid imidazole carbamate derivatives have shown intensive [M+H]+ signals under electrospray ionization and common fragmentation patterns in tandem mass spectrometry mode with [M-CO2 +H]+ and [M-ΙmCO2 +H]+ as major ions with low collision energy. The obtained results showed that the majority of steroids were detectable at concentrations equal or lower to their minimum required performance level according to the World Anti-Doping Agency technical document. The proposed method is sensitive with a preparation procedure that could be easily applied to the analysis of doping control samples.

The use of dietary supplements among soccer referees: How much do they know?

Referees are essential for the correct execution of each soccer match and national soccer associations support them with physical training guidelines in order to improve agility, aerobic and sprint capacity, to reduce the risk of injury and to meet the minimum required performance levels. Despite the importance given to performance skills no specific nutritional guideline is provided to support the performance required. This study was aimed to investigate soccer field and assistant referees knowledge and consumption of dietary supplements. Seventy-one European referees participated to this survey by filling a short and quick anonymous questionnaire designed to collect information about their refereeing category, educational level, frequency of supplement consumption, type of supplement consumed. Moreover, a sub-group of 20 participants wore a training watch to record energy expenditure, distance, and heart rate during training sessions and matches. Our data show that referees frequently consume supplements especially those refereeing in lower categories, moreover they often follow a friend’s suggestion when deciding to take a supplement instead of trusting a professional prescription. According to our survey, national and international soccer federations should care more about their referee nutritional education.