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OBJECTIVES: Heteroresistant infections are defined as infections in which a mixture of drug-resistant and drug-susceptible populations are present. In Mycobacterium tuberculosis (M. tb), heteroresistance poses a challenge in diagnosis and has been linked with poor treatment outcomes. We compared the analytical sensitivity of molecular methods, such as GeneXpert and whole genome sequencing (WGS) in detecting heteroresistance when compared with the 'gold standard' phenotypic assay: the agar proportion method (APM). METHODS: Using two rounds of proficiency surveys with defined monoresistant BCG strains and mixtures of susceptible/resistant M. tb, we determined the limit of detection (LOD) of known resistance associated mutations. RESULTS: The LOD for rifampin-R (RIF-R) detection was 1% using APM, 60% using GeneXpert MTB/RIF, 10% using GeneXpert MTB/RIF Ultra and 10% using WGS. While WGS could detect mutations beyond those associated with RIF resistance, the LOD for these other mutations was also 10%. Additionally, we observed instances where laboratories did not report resistance in the majority population, yet the mutations were present in the raw sequence data. CONCLUSION: The gold standard APM detects minority resistant populations at a lower proportion than molecular tests. Mycobacterium bovis BCG strains with defined resistance and extracted DNA from M. tb provided concordant results and can serve in quality control of laboratories offering molecular testing for resistance. Further research is required to determine whether the higher LOD of molecular tests is associated with negative treatment outcomes.
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Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Humanos , Secuenciación Completa del Genoma , Mutación , Farmacorresistencia Bacteriana/genética , Rifampin/farmacología , Rifampin/uso terapéutico , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Antituberculosos/farmacología , Antituberculosos/uso terapéuticoRESUMEN
Apprehensions about gene doping have grown consistently due to advancements in gene engineering techniques, particularly with the emergence of clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas)-based tools. These tools not only provide unprecedented possibilities for illicit performance enhancement by athletes but also offer new avenues for the detection of gene doping through biosensing of nucleic acids. Hence, pursuing on a previous study, an analytical method based on reverse transcriptase-recombinase polymerase amplification (RT-RPA) and subsequent qualitative nucleic acid detection by means of Specific High Sensitive Enzymatic Reporter UnLOCKing (SHERLOCK) was optimized for the direct detection of sgRNA associated with Streptococcus pyogenes in serum. Detection device, assay parameters, and sample handling were adjusted, to overcome previously determined assay limitations. The conducted method characterization confirmed the methods' specificity and increased detection sensitivity from 100 pM to 1 fM sgRNA in 100 µL of serum. Furthermore, reanalysis of in vivo mouse administration samples collected in a previous proof-of-concept study was conducted with successful identification of sgRNA in all anticipated postadministration samples within the 24-h collection period. Those findings support the applicability of the refined analytical procedure for the detection of illegal doping attempts via ribonucleoprotein-based CRISPR/Cas application through sgRNA identification, offering a new potential doping control strategy for CRISPR related gene doping.
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Sistemas CRISPR-Cas , Doping en los Deportes , Streptococcus pyogenes , Doping en los Deportes/prevención & control , Streptococcus pyogenes/genética , Animales , Ratones , Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/genética , Humanos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genéticaRESUMEN
PURPOSE: Development of a color scheme representation to facilitate the interpretation of tri-exponential DWI data from abdominal organs, where multi-exponential behavior is more pronounced. METHODS: Multi-exponential analysis of DWI data provides information about the microstructure of the tissue under study. The tri-exponential signal analysis generates numerous parameter images that are difficult to analyze individually. Summarized color images can simplify at-a-glance analysis. A color scheme was developed in which the slow, intermediate, and fast diffusion components were each assigned to a different red, green, and blue color channel. To improve the appearance of the image, histogram equalization, gamma correction, and white balance were used, and the processing parameters were adjusted. Examples of the resulting color maps of the diffusion fractions of healthy and pathological kidney and prostate are shown. RESULTS: The color maps obtained by the presented method show the merged information of the slow, intermediate, and fast diffusion components in a single view. A differentiation of the different fractions becomes clearly visible. Fast diffusion regimes, such as in the renal hilus, can be clearly distinguished from slow fractions, such as in dense tumor tissue. CONCLUSION: Combining the diffusion information from tri-exponential DWI analysis into a single color image allows for simplified interpretation of the diffusion fractions. In the future, such color images may provide additional information about the microstructural nature of the tissue under study.
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INTRODUCTION: The analysis of doping control samples is preferably performed by mass spectrometry, because obtained results meet the highest analytical standards and ensure an impressive degree of reliability. The advancement in mass spectrometry and all its associated technologies thus allow for continuous improvements in doping control analysis. AREAS COVERED: Modern mass spectrometric systems have reached a status of increased sensitivity, robustness, and specificity within the last decade. The improved sensitivity in particular has, on the other hand, also led to the detection of drug residues that were attributable to scenarios where the prohibited substances were not administered consciously but rather by the unconscious ingestion of or exposure to contaminated products. These scenarios and their doubtless clarification represent a great challenge. Here, too, modern MS systems and their applications can provide good insights in the interpretation of dose-related metabolism of prohibited substances. In addition to the development of new instruments itself, software-assisted analysis of the sometimes highly complex data is playing an increasingly important role and facilitating the work of doping control laboratories. EXPERT OPINION: The sensitive analysis and evaluation of a higher number of samples in a shorter time is made possible by the ongoing developments in mass spectrometry.
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Doping en los Deportes , Espectrometría de Masas en Tándem , Humanos , Espectrometría de Masas en Tándem/métodos , Detección de Abuso de Sustancias/métodos , Reproducibilidad de los Resultados , Estándares de ReferenciaRESUMEN
Caffeine is an ergogenic substance that is consumed globally in many forms. The use of buccally absorbable formulations instead of gastrointestinal uptake has become increasingly popular over the years, especially when accelerated absorption with minimal gastrointestinal stress is desired. This study investigated the impact of five different formulations and administration routes of caffeine on the whole blood concentrations of caffeine, paraxanthine, and theobromine: caffeinated capsules, tablets, shots, pouches, and chewing gums. A uniform dose of caffeine (200 mg) was administered to 16 healthy recreational athletes (26.0 ± 2.1 years) using a randomized crossover design. Samples were taken in the form of dried blood spots at 16 different time points in a 2-hr timeframe after drug administration. The samples were analyzed using a validated liquid chromatography-tandem mass spectrometry method. The results for caffeine showed no significant differences in the overall bioavailability (area under the concentration-time curve), maximal concentration, and time to maximum concentration. However, when analyzing the bioavailability of caffeine in the first 5, 10, and 15 min, the liquid caffeine formulation was superior to other administered forms (p < .05). This indicates that caffeine solubility has a major influence on its absorption rate. In sports, the rate of caffeine absorption must be considered, not only when ingesting anhydrous caffeine, but also when choosing buccal absorption. These findings imply that general guidelines for ergogenic caffeine use should consider the formulation used and, accordingly, the corresponding route of absorption.
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Cafeína , Deportes , Humanos , Administración Oral , Área Bajo la Curva , Disponibilidad Biológica , Estudios Cruzados , Adulto Joven , AdultoRESUMEN
AIMS/HYPOTHESIS: Post-bariatric hypoglycaemia is an increasingly recognised complication of bariatric surgery, manifesting particularly after Roux-en-Y gastric bypass. While hyperinsulinaemia is an established pathophysiological feature, the role of counter-regulation remains unclear. We aimed to assess counter-regulatory hormones and glucose fluxes during insulin-induced postprandial hypoglycaemia in patients with post-bariatric hypoglycaemia after Roux-en-Y gastric bypass vs surgical and non-surgical control individuals. METHODS: In this case-control study, 32 adults belonging to four groups with comparable age, sex and BMI (patients with post-bariatric hypoglycaemia, Roux-en-Y gastric bypass, sleeve gastrectomy and non-surgical control individuals) underwent a postprandial hypoglycaemic clamp in our clinical research unit to reach the glycaemic target of 2.5 mmol/l 150-170 min after ingesting 15 g of glucose. Glucose fluxes were assessed during the postprandial and hypoglycaemic period using a dual-tracer approach. The primary outcome was the incremental AUC of glucagon during hypoglycaemia. Catecholamines, cortisol, growth hormone, pancreatic polypeptide and endogenous glucose production were also analysed during hypoglycaemia. RESULTS: The rate of glucose appearance after oral administration, as well as the rates of total glucose appearance and glucose disappearance, were higher in both Roux-en-Y gastric bypass groups vs the non-surgical control group in the early postprandial period (all p<0.05). During hypoglycaemia, glucagon exposure was significantly lower in all surgical groups vs the non-surgical control group (all p<0.01). Pancreatic polypeptide levels were significantly lower in patients with post-bariatric hypoglycaemia vs the non-surgical control group (median [IQR]: 24.7 [10.9, 38.7] pmol/l vs 238.7 [186.3, 288.9] pmol/l) (p=0.005). Other hormonal responses to hypoglycaemia and endogenous glucose production did not significantly differ between the groups. CONCLUSIONS/INTERPRETATION: The glucagon response to insulin-induced postprandial hypoglycaemia is lower in post-bariatric surgery individuals compared with non-surgical control individuals, irrespective of the surgical modality. No significant differences were found between patients with post-bariatric hypoglycaemia and surgical control individuals, suggesting that impaired counter-regulation is not a root cause of post-bariatric hypoglycaemia. TRIAL REGISTRATION: ClinicalTrials.gov NCT04334161.
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Derivación Gástrica , Hipoglucemia , Obesidad Mórbida , Adulto , Humanos , Glucagón , Polipéptido Pancreático , Estudios de Casos y Controles , Hipoglucemia/complicaciones , Glucosa , Insulina , Hipoglucemiantes , Glucemia , Gastrectomía/efectos adversos , Obesidad Mórbida/cirugíaRESUMEN
A recent study addressed the possibility of unintentional ingestion of clomiphene through residues in chicken eggs. The method developed here helped distinguish between microdose intake of (E/Z)-clomiphene citrate and consumption of clomiphene-containing eggs by the urinary pattern of four mono-hydroxylated clomiphene metabolites. However, reanalyses of doping-control samples, which showed an adverse analytical finding for clomiphene, revealed a hydroxy clomiphene (HC) isomer that was not found after microdose intake or after consumption of clomiphene-containing eggs and could not be assigned to any of the available reference compounds. The aim of the present follow-up study was to identify this HC isomer and to characterize this metabolite with respect to its potential properties as long-term metabolite in doping controls. METHODS: (E/Z)-3'-HC and (E/Z)-4'-HC were synthesized involving the McMurry reaction. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and optimized after a derivatization step with dansyl chloride to separate eight HC isomers. Using this method, urine samples from a controlled clomiphene administration study were analyzed, in which male study participants received therapeutic doses of clomiphene for 30 days and collected urine samples for up to 8 months. Thus, isomer-specific HC elimination profiles could be monitored. RESULTS: The metabolite previously found in doping-control samples was identified as (Z)-3'-HC. The elimination profiles of the different HCs confirmed previous results, with (Z)-3-HC being the most abundant urinary hydroxy metabolite shortly after administration. A new finding was that the data suggest that (Z)-3'-HC is excreted at higher relative concentrations only several weeks after drug intake. CONCLUSION: These findings might be of particular importance in sport drug testing as they can assist in the decision-making process to distinguish between intentional doping and inadvertent exposure to clomiphene via food contamination.
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Doping en los Deportes , Masculino , Animales , Clomifeno/orina , Espectrometría de Masas en Tándem/métodos , Cromatografía Liquida/métodos , Estudios de SeguimientoRESUMEN
Potential scenarios as to the origin of minute amounts of banned substances detected in doping control samples have been a much-discussed problem in anti-doping analysis in recent years. One such debated scenario has been the contamination of female athletes' urine with ejaculate containing doping agents and/or their metabolites. The aim of this work was to obtain complementary information on whether relevant concentration ranges of doping substances are excreted into the ejaculate and which metabolites can be detected in the seminal fluid (sf) and corresponding blood plasma (bp) samples. A method was established to study the concentration and metabolite profiles of stanozolol and LGD-4033-substances listed under anabolic substances (S1) on the World Anti-Doping Agency's Prohibited List-in bp and sf using liquid chromatography high-resolution mass spectrometry (LC-HRMS). For sf and bp, methods for detecting minute amounts of these substances were developed and tested for specificity, recovery, linearity, precision, and reliability. Subsequently, sf and bp samples from an animal administration study, where a boar orally received stanozolol at 0.33 mg/kg and LGD-4033 at 0.11 mg/kg, were measured. The developed assays proved appropriate for the detection of the target substances in both matrices with detection limits between 10 and 40 pg/mL for the unmetabolized drugs in sf and bp, allowing to estimate the concentration of stanozolol in bp (0.02-0.40 ng/mL) and in sf (0.01-0.25 ng/mL) as well as of LGD-4033 in bp (0.21-2.00 ng/mL) and in sf (0.03-0.68 ng/mL) post-administration. In addition, metabolites resulting from different metabolic pathways were identified in sf and bp, with sf resembling a composite of the metabolic profile of bp and urine.
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Anabolizantes , Doping en los Deportes , Masculino , Animales , Femenino , Porcinos , Estanozolol/metabolismo , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodos , Detección de Abuso de Sustancias/métodos , Cromatografía Liquida/métodos , Plasma/químicaRESUMEN
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 4 mm 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 4 h 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.
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Doping en los Deportes , Humanos , Pruebas con Sangre Seca/métodos , Automatización de Laboratorios/métodos , Manejo de Especímenes , Reproducibilidad de los ResultadosRESUMEN
Gene doping has been classified as a prohibited method by the World Anti-Doping Agency (WADA) and the International Olympic Committee (IOC) for over two decades. As gene therapeutic approaches improve and, concomitantly, safety concerns regarding clinical applications decline, apprehensions about their illicit use in elite sports continue to grow. Two products available via Internet-based providers and advertised as EPO-gene- and IGF1-gene-containing materials were analyzed for the presence of potential gene doping agents using a newly developed analytical approach, allowing for the detection of transgenic DNA corresponding to seven potential targets (EPO, FST, GH1, MSTN (Propeptide), IGF1, VEGFA, and VEGFD). Panel detection was based on a 20-plex polymerase chain reaction (PCR) followed by a single base extension (SBE) reaction and subsequent SBE product analyses via matrix-assisted time-of-flight laser desorption/ionization mass spectrometry (MALDI-TOF MS). Extracts of both products were found to contain transgenic EPO-DNA, while transgenic DNA for IGF-1 was not detected. The results were confirmed using SYBR Green qPCR with primer sets directed against EPO and IGF1 cDNA, and the CMV promotor sequence. In this case study, the detection of authentic (whilst low concentrated) transgenes, potentially intended for gene doping practices in readily available products, is reported for the first time.
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Doping en los Deportes , Deportes , Doping en los Deportes/métodos , Detección de Abuso de Sustancias/métodos , ADN/genética , TransgenesRESUMEN
BACKGROUND: The development of analytical approaches to help reduce the risk of growth hormone (GH) doping is important to fair competition and the health of athletes. However, the reliable detection of GH use remains challenging. The identification of novel biomarkers of GH administration could lead to a better understanding of the physiological response to GH, more sensitive detection of the illicit use of GH in sport, and better management of patients treated for GH disorders. METHODS: We developed a targeted liquid chromatography-tandem mass spectrometry method to simultaneously quantify the carboxyl-terminal propeptide of type III procollagen (P-III-CP) and type III collagen degradation products in human serum. Following proteolysis, we instituted a simple acid precipitation step to reduce digested sample complexity before peptide immunoenrichment, which improved the recovery of one target peptide from serum. We evaluated the concentration of each biomarker at different age ranges and after GH administration in healthy participants. RESULTS: The assay was linear over an estimated concentration range of 0.3 to1.0â nM and 0.1 to 0.4â nM for each surrogate peptide of P-III-CP and collagen fragments, respectively. Intra-day and inter-day coefficients of variation were ≤15%. Biomarker concentrations appeared to vary with age and to reflect age-specific collagen turnover. Moreover, their concentrations changed after GH administration. CONCLUSIONS: Our method quantifies the proteins belonging to the family of P-III-CP and type III collagen degradation products in human serum, which could be used to detect GH administration in athletes and better understand diseases involving GH therapy or altered type III collagen turnover.
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Hormona de Crecimiento Humana , Procolágeno , Biomarcadores , Cromatografía Liquida , Colágeno , Colágeno Tipo III , Hormona del Crecimiento , Humanos , Factor I del Crecimiento Similar a la Insulina/análisis , Fragmentos de Péptidos , Péptidos , Espectrometría de Masas en TándemRESUMEN
AIM: To obtain additional information on the incremental differences between using a sensor-augmented pump (SAP) without automated insulin delivery (AID), using it with predictive low-glucose management (PLGM) or as hybrid closed loop (HCL), in preschool and school children. METHODS: We conducted a monocentric, randomized, controlled, two-phase crossover study in 38 children aged 2-6 and 7-14 years. The primary endpoint was the percentage of time in range (TIR) of 70-180 mg/dl. Other continuous glucose sensor metrics, HbA1c, patient-related outcomes (DISABKIDS questionnaire, Fear of Hypoglycaemia Survey) and safety events were also assessed. Results from 2 weeks of SAP, 8 weeks of PLGM and 8 weeks of HCL were compared using a paired t-test or Wilcoxon signed-rank test. RESULTS: Overall, we found a high rate of TIR target (>70%) achievement with HCL in preschool (88%) and school children (50%), with average times in Auto Mode of 93% and 87%, respectively. Preschool children achieved a mean TIR of 73% ± 6% (+8% vs. SAP, +6% vs. PLGM) and school children 69% ± 8% (+15% vs. SAP and + 14% vs. PLGM). Overall, HbA1c improved from 7.4% ± 0.9% to 6.9% ± 0.5% (P = .0002). Diabetes burden and worries and fear of hypoglycaemia remained at low levels, without significant changes versus PLGM. No events of severe hypoglycaemia or diabetic ketoacidosis occurred. CONCLUSIONS: Preschool children profit from AID at least as much as those aged 7 years and older. To ensure safe use and prescribing modalities, regulatory approval is also required for young children.
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Diabetes Mellitus Tipo 1 , Hipoglucemia , Glucemia , Automonitorización de la Glucosa Sanguínea , Niño , Preescolar , Estudios Cruzados , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Hemoglobina Glucada , Humanos , Hipoglucemia/inducido químicamente , Hipoglucemia/epidemiología , Hipoglucemia/prevención & control , Hipoglucemiantes/efectos adversos , Insulina/efectos adversos , Sistemas de Infusión de Insulina/efectos adversos , Insulina Regular Humana/uso terapéuticoRESUMEN
RATIONALE: The synthetic ß-adrenoreceptor agonist zilpaterol is legitimately used as an animal feed supplement in selected countries due to its known effects on lipolysis and protein biosynthesis. These pharmacological characteristics of zilpaterol have contributed to its classification as doping agent in sport by the World Anti-Doping Agency. However, the use as a feed supplement can lead to residues of the drug in edible tissues and, possibly, also in the urine of consumers. METHODS: To provide urinary elimination profiles of microdosed zilpaterol and to determine whether the ingestion of zilpaterol below or at the acceptable daily intake level of 0.04 µg/kg bodyweight can result in an adverse analytical finding (AAF) in doping controls, healthy volunteers were administered single or multiple oral doses of 0.5 µg or 3 µg zilpaterol to mimic ingestion of contaminated cattle meat. Urine samples were collected and analyzed using a validated high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method and a newly developed chiral high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCI-MS/MS) method. RESULTS: Urinary peak concentrations of zilpaterol were observed for all volunteers 1.5-12.5 h after ingestion, and maximum levels >5 ng/mL, which would constitute an AAF in doping controls, were found after the intake of 3 µg of zilpaterol on five consecutive days in one out of five study participants. Noteworthy, the enantiomeric ratio of excreted zilpaterol remained constant over time. CONCLUSION: This study provides first insights into the urinary excretion of microdosed zilpaterol. Furthermore, a method was successfully developed and applied for the separation of the zilpaterol enantiomers with mass spectrometric detection.
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Carne , Espectrometría de Masas en Tándem , Animales , Bovinos , Cromatografía Líquida de Alta Presión/métodos , Humanos , Carne/análisis , Estereoisomerismo , Espectrometría de Masas en Tándem/métodos , Compuestos de TrimetilsililoRESUMEN
RATIONALE: Exhaled breath (EB) was found to be a promising matrix in the field of sports drug testing due to the non-invasive and non-intrusive sampling procedure, but significant inter-individual variations regarding detected drug concentrations have been observed in previous studies. To investigate whether the detectability of doping agents in EB is affected by sex or tobacco smoking, two administration studies were conducted with male and female smokers and nonsmokers concerning the elimination of the beta blocker propranolol and the stimulant pseudoephedrine into EB. METHODS: Following the administration of 40 mg propranolol or 30 mg pseudoephedrine, a total of 19 participants, including female and male nonsmokers as well as female and male smokers, collected EB and dried blood spot (DBS) samples over a period of 24 h. Respective analyte concentrations were determined using liquid chromatography and high-resolution tandem mass spectrometry, and semi-quantitative assays were characterized with regard to selectivity, limit of detection and identification, precision, linearity, and carryover. RESULTS: Both propranolol and pseudoephedrine were identified in post-administration EB samples from female and male nonsmokers as well as female and male smokers, and the maximum detected drug levels ranged from 9 to 2847 pg/cartridge for propranolol and from 26 to 4805 pg/cartridge for pseudoephedrine. The corresponding DBS levels were in a range of 4-30 ng/mL for propranolol and 55-186 ng/mL for pseudoephedrine. CONCLUSIONS: Neither the consumption of cigarettes nor the sex appears to represent a decisive criterion as to the detectability of propranolol or pseudoephedrine in EB, but inter-individual variations regarding the detected drug levels were observed among all studied population groups.
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Nicotiana , Productos de Tabaco , Pruebas Respiratorias/métodos , Femenino , Hábitos , Humanos , Masculino , Proyectos Piloto , Fumar TabacoRESUMEN
The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) tool kit constitutes one of today's most frequently used gene editing techniques. Editing of virtually any DNA sequence can be realised, due to the quickly progressing research into different Cas effectors and their ever-expanding range of targets. Moreover, the simplicity and cost-effectiveness of those CRISPR tools can, unfortunately, also facilitate the illicit utilisation of CRISPR/Cas in order to achieve performance enhancements amongst athletes. Consequently, there is an urgent need for the direct detection of illegally applied CRISPR/Cas methods in doping control samples, for which a promising strategy is presented herein employing Specific High Sensitive Enzymatic Reporter UnLOCKing (SHERLOCK) for targeted nucleic acid detection. An analytical method was developed that enables the detection of sgRNA associated with Cas9 from Streptococcus pyogenes (SpCas9) in serum samples by means of reverse transcriptase-recombinase polymerase amplification (RT-RPA) and subsequent qualitative nucleic acid detection via SHERLOCK in combination with a complementary gel-based screening procedure in order to uncover doping attempts with lipid mediated CRISPR ribonucleoprotein (RNP) complexes. Initial qualitative method characterisation confirmed the specificity of both procedures and established a detection sensitivity of 10 nM uncomplexed target sequence and 100 pM sgRNA in the form of RNP complexes. Furthermore, a proof-of-concept in vivo adimistration study simulating a hypothetical gene doping scenario employing a mouse model revealed a detection window of 8 h after intravenous injection, supporting the principal applicability of the test strategy to authentic doping control samples in the future.
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Doping en los Deportes , Ácidos Nucleicos , Ratones , Animales , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Streptococcus pyogenesRESUMEN
Test methods in anti-doping, most of which rely on the most modern mass spectrometric instrumentation, undergo continuous optimization in order to accommodate growing demands as to comprehensiveness, sensitivity, retrospectivity, cost-effectiveness, turnaround times, etc. While developing and improving analytical approaches is vital for appropriate sports drug testing programs, the combination of today's excellent analytical potential and the inevitable exposure of humans to complex environmental factors, specifically chemicals and drugs at the lowest levels, has necessitated dedicated research, particularly into the elite athlete's exposome. Being subjected to routine doping controls, athletes frequently undergo blood and/or urine tests for a plethora of drugs, chemicals, corresponding metabolic products, and various biomarkers. Due to the applicable anti-doping regulations, the presence of prohibited substances in an athlete's organism can constitute an anti-doping rule violation with severe consequences for the individual's career (in contrast to the general population), and frequently the question of whether the analytical data can assist in differentiating scenarios of 'doping' from 'contamination through inadvertent exposure' is raised. Hence, investigations into the athlete's exposome and how to distinguish between deliberate drug use and potential exposure scenarios have become a central topic of anti-doping research, aiming at supporting and consolidating the balance between essential analytical performance characteristics of doping control test methods and the mandate of protecting the clean athlete by exploiting new strategies in sampling and analyzing specimens for sports drug-testing purposes.
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The misuse of 2-phenylethylamine (PEA) in sporting competitions is prohibited by the World Anti-Doping Agency. As it is endogenously produced, a method is required to differentiate between naturally elevated levels of PEA and the illicit administration of the drug. In 2015, a sulfo-conjugated metabolite [2-(2-hydroxyphenyl)acetamide sulfate (M1)] was identified, and pilot study data suggested that the ratio M1/PEA could be used as a marker indicating the oral application of PEA. Within this project, the required reference material of M1 was synthesized, single and multiple dose elimination studies were conducted and 369 native urine samples of athletes were analyzed as a reference population. While the oral administration of only 100 mg PEA did not affect urinary PEA concentrations, an increase in urinary concentrations of M1 was observed for all volunteers. However, urinary concentrations of both PEA and M1 showed relatively large inter-individual differences and establishing a cut-off-level for M1/PEA proved difficult. Consequently, a second metabolite, phenylacetylglutamine, was considered. Binary logistic regression demonstrated a significant (P < 0.05) correlation of the urinary M1 and phenylacetylglutamine concentrations with an oral administration of PEA, suggesting that assessing both analytes can assist doping control laboratories in identifying PEA misuse.
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Doping en los Deportes , Fenetilaminas/farmacocinética , Fenetilaminas/orina , Detección de Abuso de Sustancias/métodos , Adulto , Biomarcadores/orina , Cromatografía Liquida , Femenino , Humanos , Límite de Detección , Modelos Lineales , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem , Adulto JovenRESUMEN
Based on in silico, in situ, and in vivo studies, this study aims to develop a new method for the quantitative chemical exchange saturation transfer (qCEST) technique considering multi-pool systems. To this end, we extended the state-of-the-art apparent exchange-dependent relaxation (AREX) method with a Lorentzian correction (LAREX). We then validated this new method with in situ and in vivo experiments on human intervertebral discs (IVDs) using the Kendall-Tau correlation coefficient. In the in silico experiments, we observed significant deviations of the AREX method as a function of the underlying exchange rate (kba) and fractional concentration (fb) compared to the ground truth due to the influence of other exchange pools. In comparison to AREX, the LAREX-based Ω-plot approach yielded a substantial improvement. In the subsequent in situ and in vivo experiments on human IVDs, no correlation to the histological reference standard or Pfirrmann classification could be found for the fb (in situ: τ = −0.17 p = 0.51; in vivo: τ = 0.13 p = 0.30) and kba (in situ: τ = 0.042 p = 0.87; in vivo: τ = −0.26 p = 0.04) of Glycosaminoglycan (GAG) with AREX. In contrast, the influence of interfering pools could be corrected by LAREX, and a moderate to strong correlation was observed for the fractional concentration of GAG for both in situ (τ = −0.71 p = 0.005) and in vivo (τ = −0.49 p < 0.001) experiments. The study presented here is the first to introduce a new qCEST method that enables qCEST imaging in systems with multiple proton pools.
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Disco Intervertebral , Imagen por Resonancia Magnética , Glicosaminoglicanos , Humanos , Imagen por Resonancia Magnética/métodos , ProtonesRESUMEN
RATIONALE: Exhaled breath (EB) has been demonstrated to be a promising alternative matrix in sports drug testing due to its non-invasive and non-intrusive nature compared with urine and blood collection protocols. In this study, a pilot-test system was employed to create drug-containing aerosols simulating EB in support of the analytical characterization of EB sampling procedures, and the used analytical method was extended to include a broad spectrum of prohibited substances. METHODS: Artificial and authentic EB samples were collected using sampling devices containing an electret filter, and doping agents were detected by means of liquid chromatography and tandem mass spectrometry with unispray ionization. The analytical approach was characterized with regard to specificity, limits of detection, carry-over, recovery and matrix effects, and the potential applicability to routine doping controls was shown using authentic EB samples collected after single oral dose applications of glucocorticoids and stimulants. RESULTS: The analytical method was found to be specific for a total of 49 model substances relevant in sports drug testing, with detection limits ranging from 1 to 500 pg per cartridge. Both ion suppression (-62%) and ion enhancement (+301%) effects were observed, and all model compounds applied to EB sampling devices were still detected after 28 days of storage at room temperature. Authentic EB samples collected after the oral administration of 10 mg of prednisolone resulted in prednisolone findings in specimens obtained from 3 out of 6 participants up to 2 h. In octodrine, dimethylamylamine (DMAA) and isopropylnorsynephrine post-administration EB samples, the drugs were detected over a period of 50, 48, and 8 h, respectively. CONCLUSIONS: With the analytical approach developed within this study, the identification of a broad spectrum of prohibited doping agents in EB samples was accomplished. Application studies and stability tests provided information to characterize EB as a potential matrix in sports drug testing.
Asunto(s)
Pruebas Respiratorias/métodos , Cromatografía Liquida/métodos , Doping en los Deportes , Espectrometría de Masas en Tándem/métodos , Adulto , Femenino , Humanos , Drogas Ilícitas/análisis , Límite de Detección , Modelos Lineales , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
Analytical methods to determine the potential misuse of the ghrelin mimetics capromorelin (CP-424,391), macimorelin (macrilen, EP-01572) and tabimorelin (NN703) in sports were developed. Therefore, different extraction strategies, i.e. solid-phase extraction, protein precipitation, as well as a "dilute-and-inject" approach, from urine and EDTA-plasma were assessed and comprehensive in vitro/in vivo experiments were conducted, enabling the identification of reliable target analytes by means of high resolution mass spectrometry. The drugs' biotransformation led to the preliminary identification of 51 metabolites of capromorelin, 12 metabolites of macimorelin and 13 metabolites of tabimorelin. Seven major metabolites detected in rat urine samples collected post-administration of 0.5-1.0 mg of a single oral dose underwent in-depth characterization, facilitating their implementation into future confirmatory test methods. In particular, two macimorelin metabolites exhibiting considerable abundances in post-administration rat urine samples were detected, which might contribute to an improved sensitivity, specificity, and detection window in case of human sports drug testing programs. Further, the intact drugs were implemented into World Anti-Doping Agency-compliant initial testing (limits of detection 0.02-0.60 ng/ml) and confirmation procedures (limits of identification 0.18-0.89 ng/ml) for human urine and blood matrices. The obtained results allow extension of the test spectrum of doping agents in multitarget screening assays for growth hormone-releasing factors from human urine.