From population- to subject-based limits of T/E ratio to detect testosterone abuse in elite sports

In elite sports, indirect testing of testosterone abuse is mainly based on the testosterone over epitestosterone (T/E) ratio. Since this marker is characterized by a small ratio of intra- to inter-individual variation, it is surprising that current anti-doping strategy uses a screening test based on a population-based limit. From a database of more than 15,000 steroid profiles obtained from routine controls, the collection of steroids profiles of 11 elite athletes followed during 2 years, and a longitudinal study involving 17 amateur athletes, 8 of which were orally administrated testosterone undecanoate pills, we selected 12 case studies to represent the possible scenarios to which the anti-doping laboratories are confronted. Various detection strategies at the disposal of the laboratories are employed and discussed, including isotope ratio mass spectrometry (IRMS) analysis and a Bayesian interpretation of the T/E-time profile. The weak sensitivity versus specificity relation of a population-based limit for the T/E ratio is outlined. As a result, we propose a Bayesian screening test whose T/E threshold progressively evolves from a population basis to a subject basis as the number of individual test results increases. We found that this screening test heightens drastically the capacity to detect testosterone abuse, at no additional financial and administrative expenses for anti-doping authorities.     

Elevated and similar urinary testosterone/epitestosterone ratio in all samples of a competition testing: suspicion of a manipulation

The case of seven urine samples collected for anti-doping purposes during a cycling stage race with moderately elevated testosterone and epitestosterone ratio (T/E) is reported. The very low probability of having all seven urine samples with such similar elevated T/E ratio (from 3.2 to 4.7) was very suspicious. Different pattern classification tools were tested to categorize the most similar steroid profiles, but none of the models enabled a clear classification of the different urine samples. Subsequently, genetic profiling of all urine samples was performed and demonstrated that three of the seven samples were collected from the same cyclist. Finally, the International Federation confirmed DNA profiling results. This suggests that urinary steroid data using several methodologies are not appropriate for identification purposes and to an extent not unique to individuals.     

How to confirm C.E.R.A. doping in athletes' blood?

C.E.R.A. (Continuous Erythropoietin Receptor Activator) is a new third-generation erythropoiesis-stimulating agent that has recently been linked with abuse in endurance sports. The anti-doping community rapidly reacted by releasing a high-throughput screening ELISA allowing the detection of C.E.R.A. doping in athletes' blood. In order to return adverse analytical findings, anti-doping laboratories, however, need, as far as possible, to confirm the presence of the drug in athletes' samples through orthogonal methods. This article focuses on the comparison of 2 proposed confirmation assays based on gel electrophoresis that were coupled with a new sample immunopurification method. IEF, the classical method used to target erythropoietin (EPO) and its recombinant analogues in athletes' samples, and SARKOSYL-PAGE were applied to the plasma samples of subjects having received a single injection of C.E.R.A. It was demonstrated that SARKOSYL-PAGE was at least 6 times more sensitive than IEF, with comparable specificity. A longer detection window coupled with easier interpretation criteria led us to recommend the use of SARKOSYL-PAGE to confirm C.E.R.A. presence in athletes' blood.     

Involvement of the health industry in the fight against doping in sport

Most substances used for doping in sport are legitimate pharmaceutical products deviated from their intended therapeutic applications. One of the major challenges for anti-doping authorities, in anticipation of future doping trends, is to assess the doping potential of drugs in development by the health industry and to timely develop anti-doping analytical methods to detect their abuse before such drugs become available to athletes intending to use them as doping agents. In this regard, the World Anti-Doping Agency (WADA) has recently consolidated several agreements with representatives from the pharmaceutical sector in order to establish a framework of collaboration and to facilitate the identification and transfer of information on drugs in development. The context of the collaborative effort between WADA and the pharmaceutical and biotechnology industries, as well as the role of drug regulatory agencies in an integrated process in support of the fight against doping in sport are described in this article.     

Detection of anabolic steroids in head hair

We developed a gas chromatography/mass spectrometry method for detection and quantitation of anabolic steroids in head hair. Following alkaline digestion and solid-phase extraction, the MO-TMS derivatives gave a specific fragmentation pattern with EI ionization. For stanozolol, the TMS-HFBA derivative showed several diagnostic ions. For androstanolone, mestanolone (methylandrostanolone), and oxymetholone two chromatographic peaks for cis and trans isomers of derivatives were seen. Recoveries were 35 to 45% for androstanolone, oxymetholone, chlorotestosterone-acetate, dehydromethyltestosterone, dehydrotestosterone, fluoxymesterone, mestanolone, methyltestosterone, and nandrolone; 52% for mesterolone, trenbolone; 65% for bolasterone; 24% for methenolone and 17% for stanozolol. Limits of detection were 0.002 to 0.05 ng/mg and of quantitation were 0.02 to 0.1 ng/mg. Seven white male steroid abusers provided head hair samples (10 to 63 mg) and urine. In the hair samples, methyltestosterone was detected in two (confirmed in urine); nandrolone in two (also confirmed in urine); dehydromethyltestosterone in four (but not found in urine); and clenbuterol in one (but not in urine). Oxymethalone was found in urine in one, but not in the hair. One abuser had high levels of testosterone: 0.15 ng/mg hair, and 1190 ng/mL urine. We conclude that head hair analysis has considerable potential for the detection and monitoring of steroid abuse.     

Searching for new markers of endogenous steroid administration in athletes: "looking outside the metabolic box"

A simple means of detecting the abuse of steroids that also occur naturally is a problem facing doping control laboratories. Specific markers are required to allow the detection of the administration of these steroids. These markers are commonly measured using a set of data obtained from the screening of samples by gas chromatography-mass spectrometry (GC-MS). Doping control laboratories further need to confirm identified abuse using techniques such as gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). An interesting urinary species was found while following the pharmacokinetics and changes to the steroid profile from single and multiple oral doses of the International Olympic Committee/World Anti Doping Agency (IOC/WADA) prohibited substance, dehydroepiandrosterone (DHEA). The urine samples collected from the administration studies were subject to GC-MS and GC-C-IRMS steroid analysis following cleanup by solid phase extraction techniques. A useful urinary product of DHEA administration was detected in the urine samples from each of the administration studies and was identified by GC-MS experiments to be 3alpha,5-cyclo-5alpha-androstan-6beta-ol-17-one (3alpha,5-cyclo). This compound occurs naturally but the concentrations of 3alpha,5-cyclo were elevated following both the single DHEA administration (up to 385 ng/mL) and multiple DHEA administrations (up to 1240 ng/mL), in relation to those observed prior to these administrations (70 and 80 ng/mL, respectively). A reference distribution of urine samples collected from elite athletes (n = 632) enabled the natural concentration range of 3alpha,5-cyclo to be established (0-280 ng/mL), with a mean concentration of 22 ng/mL. Based on this an upper 3alpha,5-cyclo concentration limit of 140 ng/mL is proposed as a GC-MS screening marker of DHEA abuse in athletes. GC-C-IRMS analysis revealed significant 13C depletion of 3alpha,5-cyclo following DHEA administration. In the single administration study, the delta13C value of 3alpha,5-cyclo changed from -24.3 per thousand to a minimum value of -31.1 per thousand at 9 h post-administration, before returning to its original value after 48 h. The multiple administration study had a minimum delta13C 3alpha,5-cyclo of -33.9 per thousand during the administration phase in contrast to the initial value of -24.2 per thousand. Preliminary studies have shown 3alpha,5-cyclo to most likely be produced from DHEA sulfate found at high levels in urine. The complementary use of GC-MS and GC-C-IRMS to identify new markers of steroid abuse and the application of screening criteria incorporating such markers could also be adapted by doping control laboratories to detect metabolites of androstenedione, testosterone and dihydrotestosterone abuse.     

Nuclear and mitochondrial DNA quantification of various forensic materials

Due to the different types and quality of forensic evidence materials, their DNA content can vary substantially, and particularly low quantities can impact the results in an identification analysis. In this study, the quantity of mitochondrial and nuclear DNA was determined in a variety of materials using a previously described real-time PCR method. DNA quantification in the roots and distal sections of plucked and shed head hairs revealed large variations in DNA content particularly between the root and the shaft of plucked hairs. Also large intra- and inter-individual variations were found among hairs. In addition, DNA content was estimated in samples collected from fingerprints and accessories. The quantification of DNA on various items also displayed large variations, with some materials containing large amounts of nuclear DNA while no detectable nuclear DNA and only limited amounts of mitochondrial DNA were seen in others. Using this sensitive real-time PCR quantification assay, a better understanding was obtained regarding DNA content and variation in commonly analysed forensic evidence materials and this may guide the forensic scientist as to the best molecular biology approach for analysing various forensic evidence materials.     

Hair and urine testing to assess drugs of abuse consumption in couples undergoing assisted reproductive technology (ART)

For the first time in Europe hair and urine testing have been applied to assess drugs of abuse consumption in couples undergoing assisted reproductive technology and the eventual association of toxic habits with other lifestyle, health status and sociodemographic factors was also investigated. Couples attending five assisted reproduction centers in Rome were invited to join the study. When they presented at the Centre for the visit, they were asked to answer a structured questionnaire concerning sociodemographic characteristics and lifestyle habits, and at the same time to provide hair and urine samples. Hair and urine testing for drugs of abuse, urinary profile of principal endogenous steroids involved in fertility process (testosterone, epitestosterone, androsterone, etiocholanolone and dehydroepiandrosterone) and of alcohol and tobacco smoke biomarkers were performed with validated methodologies. Of the 594 enrolled individuals (297 couples), 352 (164 couples and 24 single individuals from the couple) completed the questionnaire and gave both hair and urine samples, apart from 3 bald men, who only gave urine samples. Urine testing showed an overall 4.8% (17 individuals) positivity to drugs of abuse: 4.2% to cannabinoids, 1.4% to cocaine and 0.85% to both drugs. Results of 4cm segment hair samples testing matched those from urine samples. Thus, taking together, results of urine and hair testing confirmed repeated use of cannabis, cocaine and both drugs in 3.7, 0.85 and 0.57% examined individuals, respectively. Drug consumers were in a statistically higher percentage active smokers and alcohol drinkers, less prone to physical activity and with a trend towards higher weight than non consumers. Finally, repeated drug consumption was associated with significant lower concentration of urinary testosterone in males and of urinary dehydroepiandrosterone in females. The findings of the present study confirm the suitability of urine testing to disclose recent drugs of abuse consumption and of hair analysis to verify repeated consumption. Association between different toxic habits and sedentary lifestyle is also substantiated by the obtained results in our cohort of couples attending assisted reproduction centers.     

A fatal poisoning involving Bromo-Dragonfly

This paper reports a fatal overdose case involving the potent hallucinogenic drug Bromo-Dragonfly (1-(8-bromobenzo[1,2-b; 4,5-b′]difuran-4-yl)-2-aminopropane). In the present case, an 18-year-old woman was found dead after ingestion of a hallucinogenic liquid. A medico-legal autopsy was performed on the deceased, during which liver, blood, urine and vitreous humour were submitted for toxicological examination. Bromo-Dragonfly was identified in the liver blood using UPLC–TOFMS, and was subsequently quantified in femoral blood (0.0047 mg/kg), urine (0.033 mg/kg) and vitreous humour (0.0005 mg/kg) using LC–MS/MS. Calibration standards were prepared from Bromo-Dragonfly isolated from a bottle found next to the deceased. The structure and purity of the isolated compound were unambiguously determined from analysis of UPLC–TOFMS, GC–MS, HPLC–DAD, ¹H and ¹³C NMR data and by comparison to literature data.The autopsy findings were non-specific for acute poisoning. However, based on the toxicological findings, the cause of death was determined to be a fatal overdose of Bromo-Dragonfly, as no ethanol and no therapeutics or other drugs of abuse besides Bromo-Dragonfly were detected in the liver, blood or urine samples from the deceased. To our knowledge, this is the first report of quantification of Bromo-Dragonfly in a biological specimen from a deceased person. This case caused the drug to be classified as an illegal drug in Denmark on 5th December 2007.     

1H NMR spectroscopic identification of a glue sniffing biomarker

Organic solvent abuse typically involves sniffing organic solvents to experience the mind-altering conditions they induce. In Republic of Korea, organic solvent abuse is a serious social problem, especially among teenagers. Several studies have addressed the effects of organic solvent abuse on mind and body, but there are no simple methods by which such abuse can be positively identified. In this report, we describe a method for analyzing toluene metabolites (toluene is the main ingredient of glue) in glue-sniffers' urine using (1)H NMR spectroscopy. Toluene is a commonly used solvent in the rubber, paint, plastics, leather, printing, and chemical industries. Inhaled toluene is metabolized to hippuric acid in the liver and excreted in the urine. Hippuric acid is known as a good biomarker for biological monitoring of toluene exposure. We have scanned hippuric acid and other toluene metabolites by NMR spectroscopy and performed statistical multivariate analysis of the data. Based on this analysis, we sought to determine parameters by which glue-sniffing (toluene inhalation) behavior may be verified. We also demonstrate the use of a pattern recognition method for accurate and efficient analysis of NMR data. In comparison to conventional methods, such as mass spectroscopy coupled with liquid chromatography or gas chromatography, nuclear magnetic resonance spectroscopy has several advantages, including simple sample preparation, non-destructive sampling, accuracy, short acquisition time, and reproducibility in the determination of urinary hippuric acid.     

One-step liquid-liquid extraction of cocaine from urine samples for gas chromatographic analysis

An improved technique for cocaine extraction from urine samples for gas chromatographic (GC) analysis is described. Employing a simple liquid-liquid extraction (LLE) of cocaine with a mixture of ethyl ether:isopropanol (9:1) the method presents a mean recovery of 74.49%. Limit of detection (LOD) and limit of quantification (LOQ) were 5 and 20 ng/ml, respectively. The method is highly precise (coefficient of variation (CV) <8%) and linear from 20 to 2000 ng/ml. It can he applied to detect the presence of cocaine in urine as a marker of its recent use in drug abuse treatment protocols.     

Fast quantification of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THCA) using microwave-accelerated derivatisation and gas chromatography–triple quadrupole mass spectrometry

A rapid and sensitive determination of cannabinoids in urine is important in many fields, from workplace drug testing over toxicology to the fight against doping. The detection of cannabis abuse is normally based on the quantification of the most important metabolite 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THCA) in urine. In most fields THCA needs to be present at a concentration of exceeding 15 ng/mL before a positive result can be reported.The method described in this paper, combines a 4 min GC–MS/MS method with a fast sample preparation procedure using microwave assisted derivatisation in order to complete the quantification of THCA in urine in 30 min, using only 1 mL of urine.The method is selective, linear over the range 5–100 ng/mL and shows excellent precision and trueness and hence, the estimated measurement uncertainty at the threshold level is small. The method also complies with applicable criteria for mass spectrometry and chromatography. Therefore the method can be used for rapid screening and confirmatory purposes.     

Driving under the influence of drugs -- evaluation of analytical data of drugs in oral fluid, serum and urine, and correlation with impairment symptoms

A study was performed to acquire urine, serum and oral fluid samples in cases of suspected driving under the influence of drugs of abuse. Oral fluid was collected using a novel sampling/testing device (Dr?ger DrugTest System). The aim of the study was to evaluate oral fluid and urine as a predictor of blood samples positive for drugs and impairment symptoms. Analysis for cannabinoids, amphetamine and its derivatives, opiates and cocaine was performed in urine using the Mahsan Kombi/DOA4-test, in serum using immunoassay and gas chromatography-mass spectrometry (GC-MS) confirmation and in oral fluid by GC-MS. Police and medical officer observations of impairment symptoms were rated and evaluated using a threshold value for the classification of driving inability. Accuracy in correlating drug detection in oral fluid and serum were >90% for all substances and also >90% in urine and serum except for THC (71.0%). Of the cases with oral fluid positive for any drug 97.1% of corresponding serum samples were also positive for at least one drug; of drug-positive urine samples this were only 82.4%. In 119 of 146 cases, impairment symptoms above threshold were observed (81.5%). Of the cases with drugs detected in serum, 19.1% appeared not impaired which were the same with drug-positive oral fluid while more persons with drug-positive urine samples appeared uninfluenced (32.7%). The data demonstrate that oral fluid is superior to urine in correlating with serum analytical data and impairment symptoms of drivers under the influence of drugs of abuse.     

Evaluating suspected co-proxamol overdose.

In three instances of suicidal poisoning by co-proxamol (paracetamol and dextropropoxyphene) blood samples were obtained from 11 sites together with eight tissue samples, bile, urine, gastric contents and duodenal contents. Site-dependent differences in blood propoxyphene concentration varied between the three cases but concentrations were consistently lowest in peripheral blood and highest in central sites: 3.9-5.5 (pulmonary vein) mg/l; 4.6-25 (pulmonary vein) mg/l; 3.2-40 (aorta) mg/l. There was a less than twofold variation in corresponding blood paracetamol concentrations. Reference data on fatal propoxyphene blood concentrations do not specify the blood sampling site and can be misleading. The intra-individual variability of propoxyphene concentrations in blood in these three cases underscores this problem. Tissue concentrations of propoxyphene showed considerable inter-individual variability in degree and pattern. Tissue concentrations of paracetamol showed a less than twofold intra-individual variation. Body drug loads were calculated by two methods: from organ weights and tissue concentrations; from published volume of distribution data (Vd). For paracetamol the body drug load is underestimated by the organ weight calculation but the Vd calculation approximates the suspected dose based on anamnestic information. For propoxyphene the body drug load is seriously underestimated by the organ weight calculation and overestimated up to 2.5 times by the Vd calculation. Since the two drugs have a fixed ratio in co-proxamol then the dose of propoxyphene (the effective lethal agent) can be inferred from the paracetamol dose calculated by Vd. This approach may be applicable to cases of overdose with other compounded drug preparations.     

Concurrent detection of heroin, fentanyl, and xylazine in seven drug-related deaths reported from the Philadelphia Medical Examiner's Office

Recreational drugs, such as cocaine and heroin, are often adulterated with other pharmacological agents to either enhance or diminish the drug effects. Between April 21, 2006 and August 8, 2006, the Philadelphia Medical Examiner's Office detected xylazine (a veterinary sedative) and fentanyl (a synthetic opioid) in specimens taken from seven cases. Initial immunoassay screening was performed on urine and blood for fentanyl, opiate, cocaine, phencyclidine (PCP), and benzodiazepines. All tests reported positive were confirmed by gas chromatography-mass spectrometry. All seven xylazine positive cases tested positive for fentanyl and six cases tested positive for 6-acetylmorphine (a metabolite and definitive marker for heroin). The seventh case was positive for morphine and had a history of heroin abuse. Xylazine was present in urine in all seven cases and blood levels were detected in three cases. The blood concentrations ranged from trace to 130 ng/mL. Fentanyl was present in the blood and urine in each case and blood concentrations ranged from 4.7 to 47 ng/mL. Adulteration of illicit drugs has become an epidemic health concern for drug users. Healthcare professionals need to be aware of this issue, so the patients can be treated in an effective, timely manner.     

Deaths Involving Methylenedioxypyrovalerone (MDPV) in Upper East Tennessee

Two deaths involving 3, 4‐methylenedioxypyrovalerone (MDPV) are reported. MDPV is a synthetic cathinone stimulant found in “bath salts” with neurological and cardiovascular toxicity. Biological specimens were analyzed for MDPV by GC/MS and LC/MS. A White man was found dead with signs of nausea and vomiting after repeatedly abusing bath salts during a weekend binge. Femoral venous blood and urine had MDPV concentrations of 39 ng/mL and 760 ng/mL. The second fatality was a White man with a history of drug and bath salt abuse found dead at a scene in total disarray after exhibiting fits of anger and psychotic behavior. Femoral venous blood and urine had MDPV concentrations of 130 ng/mL and 3800 ng/mL. The blood and urine MDPV concentrations are within the reported recreational concentration ranges (blood 24–241 ng/mL and urine 34–3900 ng/mL). Both decedents’ deaths were attributed to relevant natural causes in a setting of MDPV abuse.     

Methamphetamine and amphetamine concentrations in postmortem rabbit tissues

The feasibility of detecting methamphetamine and its major metabolite, amphetamine, in postmortem tissues over a 2-year period was examined. It is important to determine if the abuse and toxic effects of drugs can be proved from evidence found in decayed, submerged, or stained tissue materials. The blood, urine, liver, skeletal muscle, skin and extremity bones from rabbits given methamphetamine intravenously were kept at room temperature, under 4 different conditions: sealed in a test tube, dried in the open air, submerged in tap water and stained on gauze. Methamphetamine was present in all the samples, with slight change in concentration in case of sealed and air dried tissues. Changes varied in bones kept in water. There were considerable decreases in methamphetamine in blood and urine stains. Despite long term storage, drug abuse and/or toxicity could be determined, in all tissues examined.     

唾液检材在毒品检测中的应用

近年来,毒品滥用问题日益突出,提高生物样品中毒品检测技术的性能是法庭毒物学研究的重点。相比于血液和尿液样品,唾液在的样品采集和毒品检测中具有诸多优势,因而逐渐受到重视。本文对近年来国内外唾液样本用于毒品检测方面的研究成果进行综述,介绍唾液毒品检测的发展情况以及相关的代谢动力学研究状况,旨在为相关研究和实践提供参考。     

Analysis of cannabis in oral fluid specimens by GC-MS with automatic SPE

Methamphetamine (MA) is the most commonly abused drug in Korea, followed by cannabis. Traditionally, MA analysis is carried out on both urine and hair samples and cannabis analysis in urine samples only. Despite the fact that oral fluid has become increasingly popular as an alternative specimen in the field of driving under the influence of drugs (DUID) and work place drug testing, its application has not been expanded to drug analysis in Korea. Oral fluid is easy to collect and handle and can provide an indication of recent drug abuse.In this study, we present an analytical method using GC–MS to determine tetrahydrocannabinol (THC) and its main metabolite 11-nor-Δ⁹-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in oral fluid. The validated method was applied to oral fluid samples collected from drug abuse suspects and the results were compared with those in urine. The stability of THC and THC-COOH in oral fluid stored in different containers was also investigated.Oral fluid specimens from 12 drug abuse suspects, submitted by the police, were collected by direct expectoration. The samples were screened with microplate ELISA. For confirmation they were extracted using automated SPE with mixed-mode cation exchange cartridge, derivatized and analyzed by GC-MS using selective ion monitoring (SIM).The concentrations of THC and THC-COOH in oral fluid showed a large variation and the results from oral fluid and urine samples from cannabis abusers did not show any correlation. Thus, detailed information about time interval between drug use and sample collection is needed to interpret the oral fluid results properly. In addition, further investigation about the detection time window of THC and THC-COOH in oral fluid is required to substitute oral fluid for urine in drug testing.