Initial Testing Procedure Research Articles

Development and validation of general plasma screening method for performance enhancing drugs in racehorses utilizing liquid chromatography-high-resolution mass spectrometry (LC-HRMS).

The screening of drugs in plasma and urine often requires initial extraction (such as liquid-liquid extraction and solid-phase extraction) before the samples are submitted to instrumental analyses. These extraction procedures are often laborious and time-consuming. In this manuscript, a high-throughput automated assay based on liquid chromatography-high-resolution mass spectrometry (LC-HRMS) suitable for use as an initial testing procedure covering multiple classes of compounds prohibited in horse racing is described. The assay requires a 600-μL plasma aliquot, which is subjected to solid phase extraction (SPE) using OASIS HLB 96-well SPE with Biotage Extrahera system, evaporation, and reconstitution in a 96-well collection plate. LC-HRMS analyses were carried out on a Thermo Q-Exactive Mass spectrometer coupled with Thermo UHPLC system equipped with Thermo Accela ALS 2.4.0 autosampler linked to ACE Excel column. Drug targets were detected by retention time and accurate mass, with a mass tolerance window of 5ppm in positive and negative ionization mode. The screening method was validated for over 300 drug targets in a 13-min run. Validation data including sensitivity, specificity, extraction recovery, and precision are presented. As the method employs full-scan mass spectrometry, unlimited number of drug targets can theoretically be incorporated into this method.

Investigating the detection of the novel doping‐relevant peptide kisspeptin‐10 in urine using liquid chromatography high‐resolution mass spectrometry

Kisspeptin-10 is a peptide hormone capable of increasing circulating follicle-stimulating hormone, luteinizing hormone and testosterone levels in humans. Clinically, these effects suggest its use as a treatment for infertility. However, its testosterone-increasing effect indicates potential misuse in sports. As such, it is included in the 2024 World Anti-Doping Agency Prohibited List. This work describes the successful validation of an initial testing procedure (screening) and a confirmation procedure for kisspeptin-10 in urine using liquid chromatography-mass spectrometry. Additionally, kisspeptin-10 was incubated in human serum to mimic endogenous metabolism to improve method sensitivity, as previous research had demonstrated a rapid elimination time of only 30 min after injection (in rats). Four metabolites, corresponding to peptide fragments y9, y8, y7 and y5, were found and added to the ITP in full scan mode. A degradation product discovered during early experimentation was found to probably be caused by oxidation of the tryptophan residue into a kynurenine residue. Further research should elucidate the kinetic parameters of the reaction to improve product stability. Using the validated confirmation procedure, a black-market vial of kisspeptin-10 was analysed. The product contained no unexpected impurities, although it appeared to have undergone more degradation than the purchased reference standard.

Mechanical Characterization of Hybrid Steel Wire Mesh/Basalt/Epoxy Fiber-Reinforced Polymer Composite Laminates

Hybrid composite materials have been widely used to advance the mechanical responses of fiber-reinforced composites by utilizing different types of fibers and fillers in a single polymeric matrix. This study incorporated three types of fibers: basalt woven fiber and steel (AISI304) wire meshes with densities of 100 and 200. These fibers were mixed with epoxy resin to generate plain composite laminates. Three fundamental mechanical tests (tensile, compression, and shear) were conducted according to the corresponding ASTM standards to characterize the steel wire mesh/basalt/epoxy FRP composites used as plain composite laminates. To investigate the flexural behavior of the hybrid laminates, various layer configurations and thickness ratios were examined using a design of experiments (DoE) matrix. Hybrid samples were chosen for flexural testing, and the same procedure was employed to develop a finite element (FE) model. Material properties from the initial mechanical testing procedure were integrated into plain and hybrid composite laminate simulations. The second FE model simulated the behavior of hybrid laminates under flexural loading; this was validated through experimental data. The results underwent statistical analysis, highlighting the optimal configuration of hybrid composite laminates in terms of flexural strength and modulus; we found an increase of up to 25% in comparison with the plain composites. This research provides insights into the potential improvements offered by hybrid composite laminates, generating numerical models for predicting various laminate configurations produced using hybrid steel wire mesh/basalt/epoxy FRP composites.

Individual identification method using samples associated with doping tests: A comparison of mitochondrial and nuclear genetic data.

Doping offenses involve the use or attempted use of any prohibited method or substance as well as substituting samples. Consequently, it has been recommended that short tandem repeat (STR) analysis be used to determine if the doping control samples are from the same athlete. However, it has been recognized that it may be difficult to obtain full STR analysis using negligible amounts of DNA samples. Mitochondrial DNA (mtDNA) is characterized by its stability and high cellular copy number. Therefore, mtDNA testing in urine is expected to be used to analyze samples that cannot be analyzed using STR analysis. The objective of this study was to compare mtDNA testing with STR analysis by conducting sensitivity, concordance (whole blood, dried blood spot, and urine), and case-type studies. In sensitivity studies, mtDNA testing exhibited greater sensitivity compared with STR analysis. Concordance studies indicated that all samples were consistent with the mtDNA sequences and STR profiles. Allelic dropout occurred in some urine samples that were examined for STR analysis. Case-type sample studies demonstrated that mtDNA testing could be used to obtain DNA profiles of all the samples tested, including blood, dried blood spots, urine, blood residues on needles, and blood stains. In conclusion, mtDNA testing is valuable for analyzing highly degraded DNA samples, such as urine samples, compared with STR analysis. Urine testing should be performed for the initial testing procedure, because mtDNA is inherited maternally. In situations where the DNA match is detrimental to the athlete, additional blood STR analysis may be required.

Screening anabolic androgenic steroids in human urine: an application of the state-of-the-art gas chromatography-Orbitrap high-resolution mass spectrometry.

Over the past few decades, anabolic androgenic steroids (AASs) have been abused in and out of competition for their performance-enhancing and muscle-building properties. Traditionally, AASs were commonly detected using gas chromatography-mass spectrometry in the initial testing procedure for doping control purposes. Gas chromatography-Orbitrap high-resolution mass spectrometry (GC-Orbitrap-HRMS) is a new technology that has many advantages in comparison with GC-MS (e.g., a maximum resolving power of 240,000 (FWHM at m/z 200), excellent sub-ppm mass accuracy, and retrospective data analysis after data acquisition). Anti-doping practitioners are encouraged to take full advantage of the updated techniques of chromatography-mass spectrometry to develop sensitive, specific, and rapid screening methods for AASs. A new method for screening a wide range of AASs in human urine using GC-Orbitrap-HRMS was developed and validated. The method can qualitatively determine 70 anabolic androgenic steroids according to the minimum required performance limit of the World Anti-Doping Agency. Moreover, the validated method was successfully applied to detect six metabolites in urine after the oral administration of metandienone, and their excretion curves in vivo were studied. Metandienone M6 (17β-hydroxymethyl-17α-methyl-18-nor-androst-1,4,13-trien-3-one) has been identified as a long-term urinary metabolite which can be detected up to 7 weeks, thus providing a longer detection window compared with previous studies. This study provides a rationale for GC-Orbitrap-HRMS in drug metabolism and non-targeted screening.

Prevalence of nandrolone preparations with endogenous carbon isotope ratios in Australia.

Nandrolone and its prohormones, including 19-norandrost-4-ene-3,17-dione and 19-norandrost-4-ene-3β,17β-diol, are anabolic steroids forbidden at all times in sports according to the World Anti-Doping Code Prohibited List and its metabolite 19-norandrosterone (19NA) is the preferred urinary target compound to identify their abuse. In recent years, an increasing number of 19NA isotope ratio mass spectrometry (IRMS) cases have arisen that, based on the initial testing procedure, were likely to result in an adverse analytical finding but were concluded negative after IRMS analysis. The current study was therefore set up to gain a better insight on the prevalence of nandrolone preparations with endogenous carbon isotope ratio values in Australia. Suitable workplace (non-athlete) urine samples that had previously been reported positive for 19NA were identified and analysed on IRMS. A total of 82% of the samples that were analysed were reported with enriched carbon isotope ratios of 19NA (i.e., 19NA greater than -26‰). This indicates that there is a high prevalence of nandrolone-containing anabolic androgenic steroid preparations in Australia that have 'endogenous' carbon isotope ratios which reduces the ability to identify exogenous nandrolone.

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.

Application of Skyline software for detecting prohibited substances in doping control analysis.

As the number of prohibited drugs has been progressively increasing and analytical methods for detecting such substances are renewed continuously for doping control, the need for more sensitive and accurate doping analysis has increased. To address the urgent need for high throughput and accurate analysis, liquid chromatography with tandem mass spectrometry is actively utilized in case of most of the newly designated prohibited substances. However, because all mass spectrometer vendors provide data processing software that is incapable of handling other instrumental data, it is difficult to cover all doping analysis procedures, from method development to result reporting, on one platform. Skyline is an open-source and vendor-neutral software program invented for the method development and data processing of targeted proteomics. Recently, the utilization of Skyline has been expanding for the quantitative analysis of small molecules and lipids. Herein, we demonstrated Skyline as a simple platform for unifying overall doping control, including the optimization of analytical methods, monitoring of data quality, discovery of suspected doping samples, and validation of analytical methods for detecting newly prohibited substances. For method optimization, we selected the optimal collision energies for 339 prohibited substances. Notably, 195 substances exhibited a signal intensity increase of >110% compared with the signal intensity of the original collision energy. All data related to method validation and quantitative analysis were efficiently visualized, extracted, or calculated using Skyline. Moreover, a comparison of the time consumed and the number of suspicious samples screened in the initial test procedure highlighted the advantages of using Skyline over the commercially available software TraceFinder in doping control.

Detection of doping peptides and basic drugs in equine urine using liquid chromatography-mass spectrometry.

The abuse of prohibited agents including peptides and basic small-molecule drugs is an area of great concern in horseracing due to their high potential to act as doping agents. These compound classes include agents such as growth hormone-releasing peptides, peptide analgesics, beta-2-adrenergic receptor agonists, and quaternary ammonium drugs that can be challenging to detect and regulate because of their chemical properties and potential rapid elimination following administration. The use of highly sensitive and selective analytical techniques such as liquid chromatography-mass spectrometry (LC-MS) is necessary to provide coverage of these substances and their potential metabolites. This study describes the development and validation of methodology capable of the detection of over 50 different peptide-based doping agents, related secretagogues, quaternary ammonium drugs, and other challenging small molecules in equine urine following solid-phase extraction using a mixed mode weak cation exchange sorbent. Following sample extraction, the compounds were analyzed using LC-MS with chromatographic separation via a reverse phase gradient and detection via selective reaction monitoring following introduction to a triple-stage quadrupole mass spectrometer using positive mode electrospray ionization. Validation parameters including limits of detection and quantitation, accuracy, precision, linear range, recovery, stability, and matrix effects were determined. Briefly, the limits of detection for most compounds were in the sub-ng/mL ranges with adequate precision and accuracy sufficient for an initial testing procedure. Stability studies indicated that most compounds were sufficiently stable to allow for effective screening using conditions commonly utilized in drug testing laboratories.

Dried blood spots for doping controls-Development of a comprehensive initial testing procedure with fully automated sample preparation.

Currently, primarily urine, whole blood and serum samples are analyzed for doping-relevant substances in professional sports, but recently dried blood spots (DBS) have been introduced as complementary matrix, offering advantageous features, e.g. a minimally invasive sampling procedure. In order to cope with the increased application of DBS, a comprehensive initial testing procedure (ITP) was developed, optimized and validated, comprising a total of 233 substances representing all groups on the World Anti-Doping Agency's (WADA's) Prohibited List. The sample preparation was conducted by employing a fully automated system using an efficient flow-through extraction of a 4mm diameter spot followed by LC-HRMS/MS analysis. The procedure was successfully validated in terms of selectivity, limit of detection, reproducibility, carryover and robustness with respect to an alternative manual sample preparation, an alternative dried blood collection device and the sample extract stability, and was thus found to meet the required criteria of the relevant guidelines published by WADA for routine application. As a proof-of-concept, DBS samples were analyzed after the administration of the glucocorticoids prednisone and dexamethasone, as well as the stimulant pseudoephedrine and the beta-blocker propranolol. All substances were detected in post-administration samples for at least 4h and up to 24 h after intake, depending on the collection time period, using the developed testing procedure. In particular, for substances that are only banned in-competition, data obtained from DBS samples can be useful for the interpretation of adverse analytical findings. In conclusion, the developed ITP accounts for the anticipated increasing relevance of DBS in anti-doping analysis in the future and provides a foundation for optimized approaches for specific substance classes.

Development of mass spectrometry-based methods for the detection of 11-ketotestosterone and 11-ketodihydrotestosterone.

The anabolic properties of 11-hydroxyandrostenedione (OHA4) and its physiologically active metabolites 11-ketotestosterone (KT) and 11-ketodihydrotestosterone (KDHT) have been discussed in several recent publications. Especially KT has become readily available via internet-based providers. No doping control methods for the detection of KT or KDHT exist, neither on the initial testing procedure level nor as confirmatory assay. Probing for the misuse of adrenosterone, the prohormone of OHA4, has already been addressed, and the suggested marker for its misuse was mainly the urinary concentration of 11-hydroxyandrosterone (OHA). In addition, for confirmation purposes, the carbon isotope ratios (CIR) were taken into consideration. The urinary concentration of OHA is highly variable, and the endogenous dilution after exogenous administration may therefore be considerable; hence, described approaches resulted in short detection times. In this study, the human metabolism of KT was investigated in order to provide additional means for the detection of KT and its prohormone OHA4. Two volunteers (one female and one male) orally administered 20 mg of KT each, and urine samples were collected for 5 days. Urinary concentrations of KT and its metabolites were investigated, and a reference population encompassing 220 male and female athletes was investigated in order to elucidate preliminary thresholds. As confirmation procedure, an isotope ratio mass spectrometry-based method was developed in order to determine the CIR of KT and relevant metabolites. The developed methods enabled the detection of exogenous KT for more than 20 h after a single oral administration, which is comparable to a single oral testosterone administration.

Detection of activin receptor type IIA and IIB-Fc fusion proteins by automated capillary immunoassay.

Activin receptor type IIA and type IIB fusion protein have been designed to sequester circulating molecules of the transforming growth factor-β (TGF-β) superfamily and inactivate their actions. Members of this superfamily have been reported as essential regulators of erythropoiesis by triggering the formation of activated ternary complexes containing different combinations of type I and type II receptors, which can limit RBC production by accelerating erythroid differentiation and inhibiting erythroid progenitor expansion. The recent approval of Luspatercept for the treatment of anemia associated to transfusion-dependent MDS and Beta-thalassemia in afflicted patients means that it can now pose a real threat of being abused in sport for its ability to stimulate erythropoiesis. Several methods for the detection of these molecules in blood have been proposed for the purpose of sport antidoping control. Here we propose the detection of the ActRIIA-Fc and ActRIIB-Fc fusion proteins by automated capillary Western immunoassay (Simple Western). The use of these immunoassays for the detection of protein targets has become widespread in the recent years. The work presented here demonstrates that this methodology enables a versatile, rapid, and sensitive detection of activin ligand traps in blood samples: plasma, serum, or dried blood spots (DBS). Preliminary results indicate that detection in urine samples is also possible. The option to use different antibodies allows the possibility to use this method as an initial testing procedure as well as a confirmation procedure. Finally, results coming from an administration study confirm that the method is suitable for routine analysis.

Probing for peptidic drugs (2-10kDa) in doping control blood samples.

Bioactive peptides with a molecular mass between 2 and 10kDa represent an important class of substances banned in elite sports, which has been recognized with an increasing number and variety of substances by anti-doping organizations. Also, the annually renewed list of prohibited substances of the World Anti-Doping Agency (WADA) explicitly mentions more and more of these peptides, and efficient testing procedures are required. Even under simplified sample preparation conditions, liquid chromatography coupled to high-resolution mass spectrometry (with resolution properties>100,000 full width at half maximum) offers suitable conditions for this task and can therefore be used as an initial testing procedure. In contrast to urine, blood analysis essentially relies on the detection of intact peptide hormones, and the expected concentrations are commonly higher in blood samples than in urine. This facilitates the analysis, and a generic sample preparation by means of mixed-mode solid-phase extraction could be realized in this study. Co-extraction and analysis of several different peptides such as insulins (human, lispro, aspart, glulisine, tresiba, detemir, glargine, bovine insulin and porcine insulin), growth hormone releasing hormones (sermorelin, CJC-1295 and tesamorelin), insulin-like growth factors (long-R3-IGF-I, R3-IGF-I and Des1-3-IGF-I) and mechano growth factors (human MGF and MGF-Goldspink) with criteria that fulfil the requirements of the WADA documents (TD2022 MRPL) for doping controls. The proof of principle was shown by the analysis of post administration samples after treatment with synthetic insulin analogues.

Implementation of a marker substance for monitoring in situ 17-keto modifications in endogenous steroids caused by microbiological contamination.

The urinary steroid profile established for the monitoring of eventual testosterone or testosterone precursor application by athletes includes concentrations and ratios of various endogenously produced steroidal hormones and metabolites. Due to enzymatic activities in urine specimens, the concentrations of these endogenous steroids and consequently their ratios may alter, leading to potential misinterpretation of analytical results. Microbiological contamination in athletes' urine samples can occur due to urinary tract infections or due to contamination by the non-sterile sample collection conditions. Depending on the duration of transportation of urine samples, the transport and storage conditions may favour microorganisms' growth, and therefore, the enzymatic activity can be accelerated. Degradation effects on endogenous steroids caused by microorganisms have been observed, such as hydrolysis of steroid conjugates, increase of testosterone in the free fraction or modification of the steroid structure by oxidoreductive reactions. The World Anti-Doping Agency (WADA) implemented criteria to check for signs of microbial degradation in a technical document dealing with the detection, analysis and reporting of endogenous androgenic anabolic steroids (TD EAAS) in urine samples. During the endogenous steroid profile confirmation procedures (CPs) of the WADA accredited Seibersdorf Laboratory, significant differences in the concentrations of markers of the steroid profile were observed compared to the initial testing procedures (ITPs). The changes in concentrations of the urinary steroid profile were attributed to the reduction of the 17-keto group to a 17β-hydroxy group caused by increased enzymatic activity during the hydrolysis step. In order to monitor the 17-keto reduction activity in athletes' urine specimens, possible marker substances containing a 17-keto group were synthesised and added in the internal standards mixture (ISTD) of the ITP. The presence of the reduced 17β-hydroxy form of the marker substance indicated enzymatic activity leading to 17-keto reduction reactions. The substance 3β-ethoxy-5α-androstane-17-one was defined to be suitable to indicate 17-keto reduction reactions occurring during hydrolysis carried out at moderate temperatures.

Testing the Bromide penetration resistance of concrete: substitution of NaCl by NaBr in Rapide Chloride/Bromide Migration Test (RCM/RBM)

The chloride migration coefficient of concrete describes the resistance of concrete against chloride ingress. This important material variable can be determined with the rapid chloride migration test (RCM). During the test, chloride ions penetrate concrete by means of an applied electrical field. If the concrete has already been exposed to chlorides before the test starts, the test set up has to be adapted to achieve reliable results. Due to repeated exposure to the same salt (e.g. NaCl), there is no direct possibility to distinguish the chloride ions from the test procedure and the exposure before this, within the initial test procedure. Therefore, the RCM-test setup has been adapted by replacing NaCl through NaBr in the same molar amount. Consequently, this adapted test setup is called the rapid bromide migration test (RBM). The indicator silver nitrate AgNO3 forms sparingly soluble silver salts (AgCl, AgBr) in the presence of halogen ions (e.g. Cl-, Br-), which form a white precipitate in areas with Cl- or Br- ions. The penetration depths are measured at two split cylinder halves. In case the RBM-specimen has previously been exposed to NaCl, an analyzation technique has to be available for the two elements Cl and Br to enable a differentiation. As the white precipitate areas - a result of the formation of AgCl and AgBr - have a quite similar visual appearance, a differentiation cannot be achieved and other analytical techniques have to be considered. With laser-induced breakdown spectroscopy (LIBS), it is possible to determine the depth-dependent ion quantity (wt.-% of Cl or wt.-% of Br). In this study, it is shown by test results that it is possible to exchange NaCl by NaBr in the RCM test, since the penetration behavior of both ions (Cl-, Br-) is very similar in the migration test.

Introduction of a PEGylated EPO conjugate as internal standard for EPO analysis in doping controls.

Immunopurification of doping control samples is a mandatory necessity in erythropoietin (EPO) analysis during a confirmation procedure; moreover, it has become common practice to also immunopurify samples for the initial testing procedure. Typically used materials (e.g., Stemcell purification plate and MAIIA purification kit) rely on anti-EPO antibodies for purification. Also, the detection of EPO after electrophoretic separation and western blotting is based on a monoclonal anti-EPO antibody, clone AE7A5, directed against a 26 amino acid sequence of the N-terminal region of human EPO. While the electrophoretic separation and blot transfer efficiency can be monitored with reference standards and quality control samples, it is presently not possible to monitor the functionality of the entire sample preparation procedure. The reliance on antibodies for both purification and detection has complicated the implementation of an internal standard (ISTD). In this study, customized EPO-polyethylene glycol (PEG) conjugates were synthesized as potential ISTDs and assessed as to their compatibility with existing sample preparation procedures for urine and blood sample analysis using the most common immunopurification techniques. Moreover, probing for the impact of the ISTD on sodium N-lauroylsarcosinate ("sarcosyl") polyacrylamide gel electrophoresis (SAR-PAGE)-based EPO analysis concerning potential interference with target analytes was conducted. The presented data demonstrate that a 12-kDa PEG residue attached to human EPO represents a particularly useful construct to serve as ISTD for erythropoietin-receptor agonist (ERA) analysis. The conjugate is applicable to both urine and blood testing using the commonly employed purification techniques, supporting and improving result interpretations especially concerning specimens where the natural abundance of human EPO is low.

Emergence of the less common cannabinoid Δ8 -Tetrahydrocannabinol in a doping sample.

Δ8 -Tetrahydrocannabinol (Δ8 -THC) as isomer of the well-known Δ9 -THC has a similar mode of action, and the potency was estimated to be two thirds compared with Δ9 -THC. Content of Δ8 -THC in plant material is low, but formulations containing Δ8 -THC in high concentrations are gaining popularity. Δ8 -THC is to be regarded as prohibited substance according to the Prohibited List of the World Anti-Doping Agency (WADA). Contradictory results between initial testing procedure and confirmatory quantitation for 11-Nor-9-carboxy-Δ9 -tetrahydrocannabinol (Δ9 -THC-COOH) of a doping control sample gave rise for follow-up testing procedures. After alkaline hydrolysis and liquid-liquid extraction, the sample was analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) using isocratic elution instead of gradient elution, which is used for standard procedure. Isocratic elution resulted in two peaks instead of one using gradient elution. Both peaks showed same fragmentation. Using certified reference materials, one peak could be assigned to Δ9 -THC-COOH and the other one with higher intensity to the less common 11-Nor-9-carboxy-Δ8 -Tetrahydrocannabinol (Δ8 -THC-COOH) in a concentration of approximately 1200 ng/ml. As complementary method, gas chromatography tandem mass spectrometry (GC-MS/MS) can also be used for identification. Here Δ8 - and Δ9 -THC-COOH can be distinguished by chromatography and by fragmentation. Additional investigations of doping control samples containing Δ9 -THC-COOH revealed the simultaneous presence of Δ8 -THC-COOH in low concentrations (0.22-8.91 ng/ml) presumably due to plant origin. Percentage of Δ8 -THC-COOH varies from 0.05 to 2.83%. In vitro experiments using human liver microsomes showed that Δ8 -THC is metabolized in the same way as Δ9 -THC.

Multiplexed detection of agents affecting erythropoiesis (AAEs) and overall strategy for optimizing initial testing procedure.

Erythropoietin receptor agonists (ERAs) are drugs acting on the early erythropoietic stages developed to treat anemia and other erythropoiesis disease and are prohibited by the World Anti-Doping Agency (WADA). As an alternative to ERAs, a new drug, belonging to the transforming growth factor-b inhibitors family, was recently developed to treat diseases linked to ineffective erythropoiesis. This drug, named as Luspatercept (Reblozyl®), is acting on the later stages of erythropoiesis to promote erythrocytes. This drug might be used by cheating athletes either independently or in combination with ERAs. Indeed, it was shown that Luspatercept and recombinant erythropoietin (rEPO) can act synergistically to increase red blood cells production, potentially allowing the use of lower doses for an efficient effect. Our aim was to find a way to combine the detection of ERAs and Luspatercept without impacting the sensitivity and specificity of ERAs detection from the current techniques implemented in antidoping laboratories and to reduce the time of analysis and total sample volume needed. Magnetic beads coated with antibodies were preferred for IP of samples for its potential multiplexing. Then, the following steps of the method were selected considering that SAR/SDS-PAGE are the electrophoretic methods authorized for initial testing procedure by WADA and that biotinylated primary antibodies used for the immunodetection results in the best sensitivity and specificity and is time saving. The method developed in this work for the combined detection of agents affecting erythropoiesis (AAEs) showed specificity, sensitivity, and robustness and is easily and quickly implementable to all antidoping laboratories.

A comprehensive study on the performance of different retention mechanisms in sport drug testing by liquid chromatography tandem mass spectrometry

Anti-doping substances listed by the World Anti-Doping Agency (WADA) include hundreds of compounds of very different physico-chemical properties. Anti-doping control laboratories need to screen all these substances in the so-called Initial Testing Procedures (ITPs) what is very challenging from an analytical point of view. ITPs are mostly based on reversed-phase (RP) liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using C18 columns, which feature poor retention and peak tailing for polar and basic compounds, respectively. While studies on this field dealing with the comparison of stationary phases are focused on certain chemical classes, this research provides a wide multi-target approach. For this purpose, a representative group of 93 anti-doping agents (log P from −2.4 to 9.2) included in ten different classes of prohibited substances was selected. A comprehensive study on the performance of six columns and four eluents on different separation parameters (retention factors, asymmetry factors, co-elutions, total run times) and matrix effects (signal enhancement or suppression) was performed for LC-MS/MS-based ITPs. Columns working in both RP [C18, C8, phenyl hexyl (PH), pentafluorophenyl (PFP) and mixed-mode hydrophilic/RP (HILIC-RP)) and hydrophilic (HILIC)] modes were investigated. Eluents contained methanol or acetonitrile as organic modifiers, with or without the addition of ammonium acetate. The best column-mobile phase binomial for ITPs was PFP using water-methanol (0.1% formic acid) as eluent, while HILIC was the best option for highly polar non-aromatic anti-doping agents, which were poorly addressed by PFP. Excellent good peak shapes and relative acceptable matrix interferences were obtained for HILIC-RP, which was tested for the first time for the analysis of anti-doping agents, although the number of compounds eluting too fast was too high. On the whole, the alkyl phase C18 showed the worst performance and although C8 and PH were better, their performance did not surpass that of PFP. Possible retention mechanisms underlying separation in the different stationary phases were discussed. This research provides valuable information to anti-doping control labs for improving LC-MS/MS-based ITPs and it proposes PFP as a suitable alternative to the already established C18.

EPO transgene detection in dried blood spots for antidoping application

The modification of gene expression to treat diseases is a field of research with exponential growth. As doping in sport closely follows emerging therapies, a surveillance of the modification of gene expression to enhance performance is needed. The gene coding for erythropoietin (EPO) is one target of interest. Since 2010, several protocols have been proposed to identify EPO gene doping by focusing on the presence in blood of a transgene that differ in size from the endogenous gene sequence, normally found in the human DNA. In this work, our aim was to validate an easily applicable method for EPO gene doping detection in dried blood spots (DBS). We evaluated the detection of EPO transgene in 20-μl DBS after the spike of a plasmid carrying the EPO transgene in whole blood. Three different DBS were compared: Nucleic-Card™, Whatman® 903, and the volumetric 20-μl VAMS™. Detection was performed with real-time polymerase chain reaction (PCR) and validated with two Taqman assays (one commercial and one custom) specific for the EPO transgene. The initial testing procedure could be done using one assay (custom) and the confirmation using the second one (commercial Taqman) with a final check of the size of the PCR product. Starting from 20-μl dried blood, 1000 copies of EPO transgene could efficiently be detected with the three types of DBS, VAMS showing a slightly better sensitivity. No loss of sensitivity was observed after 1-month storage of DBS at room temperature. This method could be applied to DBS collected during doping controls and allows reanalysis.