Clostebol and sport: about controversies involving contamination vs. doping offence.

Clostebol, the 4-chloro derivative of testosterone, available as Over The Counter product in pharmacies and drugstores in several countries, is mostly commercialized as a cream or spray in the form of acetate ester. As other anabolic steroids, clostebol is listed as a prohibited substance by the World Anti-Doping Agency (WADA). Controlled transdermal application of clostebol acetate has been reported to produce detectable amounts of its metabolites in urine, even after a single exposure. Indeed, a low urine concentration can be interpreted as the tail of a drug voluntarily used to enhance performance or a direct consequence of a contamination. The increased number of adverse analytical findings (AAFs) involving clostebol reported in the last years should lead to highlight the need for athletes to be warned against personal and /or accidental use/exposure of dermal preparation containing this doping agent. Further discussion on possible threshold limits and laboratory testing on different matrices (e.g. hair) to better clarify the origin of minimal amounts of clostebol in urines is advisable.

Interest of hair tests to discriminate a tail end of a doping regimen from a possible contamination in case of challenging an anti-doping rule violation. V. Case reports involving trimetazidine, a drug where the concentration after a single 20 mg dose has been established.

The identification of trimetazidine, a medicine used for treating stable angina pectoris and for preventing angina attacks, has been recently observed in doping cases involving high profile athletes from various countries over the world. In all the files where the authors have been involved, the urine concentration of trimetazidine was low (<2 ng/mL), and the athletes argued that contamination was the source of their adverse analytical finding. It is possible to challenge imposed sanctions in relation to an adverse analytical finding, but it is the responsibility of the athlete to demonstrate he/she is innocent and can qualify for no fault or negligence. When the delay between the urine collection and the notification of the violation was not too long (less than 6 months), these athletes requested a head hair test. Trimetazidine was analyzed by an original LC-MS/MS method involving pH 9.5 borate buffer overnight incubation of 20 mg and subsequent solvents extraction in presence of trimetazidine-D8 used as internal standard. Linearity was verified from 1 to 200 pg/mg (R2 = 0.9987). Limit of detection of the method was 0.1 pg/mg. The hair specimen of a male subject, collected 4 weeks after single oral ingestion of 20 mg trimetazidine, tested positive at 146 pg/mg in the corresponding segment. Concentrations of trimetazidine measured in several hair specimens (n = 5) collected from athletes challenging their anti-doping rule violation were below 1 pg/mg, which is consistent with incidental exposure due to contamination. This is the first evidence that trimetazidine is incorporated in human hair after a single therapeutic dose administration.

First evidence in an oversea French department of the deadly risk of protonitazene use: about 5 post mortem cases.

Protonitazene is a synthetic benzoimidazole opioid of the nitazenes class, developed in the 1950s as an effective analgesic, but never released in the market due to severe side effects and major risk of dependence. The laboratory was involved in the determination of the cause of death for 5 subjects deceased in a French department of the Indian Ocean. The 5 victims were male, aged between 20 and 35 years. The first 2 victims were found dead in their prison cell and the 3 other victims were found dead in a squat. Therefore, we have developed and validated a specific procedure to identify and quantify the drug in post mortem specimens using LC-MS/MS. The procedure involves extraction of 0.5 mL fluid at pH 9.5 with a mixture of organic solvents in presence of 20 ng fentanyl-d5 used as internal standard. Linearity of the method was verified from 0.1 to 20 ng/mL in both whole blood and urine (r2 = 0.9983 and 0.9993, respectively). The limit of detection was estimated at 0.05 ng/mL in each matrix. Protonitazene was identified at < LOQ to 0.8 ng/mL, 0.4 to 2.9 ng/mL and 3.0 to 8.0 ng/mL in femoral blood, urine and bile, respectively. Post mortem concentrations were very low, which is consistent with reported high toxicity of protonitazene. As nitazenes represent a growing threat to public health in various parts of the world, this method seems to be a good response to the challenges posed by the identification of this class of substances.

Simultaneous detection of three hypoxia-inducible factor stabilizers-molidustat, roxadustat, and vadadustat-in multiple keratinized matrices and its application in a doping context.

In a doping case, a top athlete challenged an anti-doping rule violation, involving molidustat. Molidustat is a stabilizing agent of the hypoxia-inducible factor (HIF) recently developed. It is currently undergoing clinical trials for anemia associated with chronic kidney disease. HIF stabilizers are banned at all times by the World Anti-Doping Agency (class S2). Because of their pharmacological proprieties, these new drugs can enhance athletic performance. The athlete's defense wanted to analyze multiple keratinized matrices as they allow long-term investigations. Requests concerning HIF stabilizers are constantly growing. We have therefore developed a liquid chromatography coupled with tandem mass spectrometry method to identify and quantify three molecules of this class: molidustat, vadadustat, and roxadustat. Thirty milligrams of keratinized matrices were incubated in 1 mL of pH 8.4 diammonium hydrogen phosphate buffer for 16 h at 40°C with 1 ng of testosterone-D3, used as internal standard. After extraction with ethyl acetate/diethyl ether (80/20), the organic phase was evaporated, and the dry residue was reconstituted in 30 µL of initial phase. The method was linear from 5 to 1000 pg/mg for the three analytes. Limits of quantification were 2, 0.5, and 5 pg/mg for molidustat, roxadustat, and vadadustat, respectively. The analysis of the athlete's head hair (collected 1 month after the urine test) showed a concentration of molidustat of 135 pg/mg, and his beard hair and his fingernails clippings contained 55 and 40 pg/mg, respectively.

Knowing the minimal detectable dose can facilitate the interpretation of a hair test result: Case example with chlortalidone.

In case of an adverse analytical finding, a low (estimate) urine concentration can be the consequence of 2 very different situations: it can be the tail end of a drug voluntarily consumed to enhance athletic performance, even by microdosing (which is not effective for all drugs), or it can be the result of a contamination, irrespective of its source. For numerous doping agents, a hair test can allow discriminating doping from contamination based on the measured concentration or even the absence of the target drug. Given hair produces incremental concentrations, its analysis offers the possibility of establishing a pattern of drug use and thus, verifying self-reported histories of exposure. In order to provide a retrospective calendar of drug use, segmental analysis of the hair strand can be performed. In doping, the usual practice is to test the substance in short segments, such as 1 cm to avoid drug dilution when using larger segments. During the last months, seven athletes have returned an adverse analytical finding for the diuretic chlortalidone, with reported urine concentrations in the range 20 to 50 ng/mL. All these athletes submitted, via their legal team, their hair for establishing a pattern of exposure. Results were always consistent with incidental contamination (hair concentration lower than 5 pg/mg), although the source of contamination was never identified. The interpretation of the findings was established in the light of the limited literature, including hair tests after microdosing and therapeutic use.

Testing for protonitazene in human hair using LC-MS-MS.

Protonitazene is a synthetic benzimidazole opioid of the nitazenes class, developed in the 1950s as an effective analgesic, but never released on the market due to severe side effects and possible dependence. Despite its increasing use as a new psychoactive substance starting in 2019, its detection in human hair of intoxicated and deceased consumers has never been reported. We present the development and validation of a specific procedure to identify protonitazene in hair by liquid chromatography with tandem mass spectrometry. Drugs were incubated overnight at 40°C in 1 mL borate buffer, pH 9.5 with 20 mg pulverized hair and 1 ng/mg fentanyl-d5 used as internal standard. Drugs were then extracted with a mixture of organic solvents. The chromatographic separation was performed using an HSS C18 column with a 15-min gradient elution. Linearity was verified from 1 to 100 pg/mg. The limit of detection was estimated at 0.1 pg/mg. No interference was noted from a large panel of natural and synthetic opioids, fentanyl derivatives, or other new synthetic opioids. Protonitazene was identified at 70 and >7600 pg/mg in the whole head hair specimens of two male subjects deceased from an acute drug overdose in jail. Protonitazene was also identified at 14 and 54 pg/mg in two living co-prisoners. As nitazenes represent a growing threat to public health in various parts of the world, this method was developed in response to the challenges posed by the identification of this class of substances.

In vitro characterization of protonitazene metabolites, using human liver microsomes, and first application to two urines collected from death cases.

Protonitazene, or N,N-diethyl-5-nitro-2-[(4-propoxyphenyl)methyl]-1H-benzimidazole-1-ethanamine, is a novel synthetic opioid, which belongs to the nitazene family. Over the last four years, nitazenes have re-emerged on the new psychoactive substances market and have been reported in several fatal intoxication cases. The metabolism of several nitazene analogues have already been studied, but to date, no data exists regarding protonitazene. The aim of the study was the detection of protonitazene and its metabolites in authentic human urine collected in two fatal intoxication cases, comparing the data after in vitro incubation with human liver microsomes, and subsequent analysis by ultra-performance liquid chromatography-tandem mass spectrometry and ultra-performance liquid chromatography-high-resolution mass spectrometry. Protonitazene metabolites, including N-desethyl-protonitazene, 5-amino-protonitazene and 4-hydroxy-nitazene, were characterized in vitro and were identified in the urine of both cases. The ratios between metabolites and parent protonitazene, higher than 1, were calculated to estimate the proportionality of metabolites. The results suggest that testing protonitazene metabolites should increase the window detection of exposure to protonitazene.

Evidence of ostarine cross-contamination via sweat in 2 athletes sharing the same neoprene hamstring sleeves. An original situation of drug transfer where the anti-doping rule violation was suspended by the sport authorities.

The presence of ostarine, a selective androgen receptor modulator (SARM) in an athlete's sample constitutes one of the most frequent anti-doping rules violation. It is possible to challenge this violation but it is the athlete who has to demonstrate he / she is innocent. The conditions to evidence no fault or negligence are mostly based on 2 points: 1. the athlete or his/her legal representative must present verified circumstances of contamination and the source of contamination must be identified; and 2. there must be verified claims by the athlete about the fact that he / she did not knowingly take the prohibited substance, i.e. that the violation was not intentional. During a 2-weeks period, a male athlete tested two times positive for ostarine in urine (<0.1 ng/ml) and he challenged these results. His hair and nail tests returned negative (LOQ at 0.5 pg/mg). He admitted using two neoprene hamstring sleeves of another athlete who confessed abusing ostarine. This was confirmed in his hair (190 pg/mg), his fingernail clippings (780 pg/mg) and his toenail clippings (45 pg/mg). To document the presence of ostarine in the hamstring sleeves and therefore possible drug transfer, the hamstring sleeves were analysed. Ostarine was identified in 12 different selected pieces (about 1 g) of the sleeves at concentrations ranging from 3 to 142 pg/g. Sport authorities (USADA) agreed that the most likely source of contamination was the hamstring sleeves, thus confirming the scenario of drug transfer and gave the athlete a no fault.

Is it really possible to kill with insulin without leaving traces? From lifesaver to killer, the issues surrounding the analytical characterization of postmortem insulin illustrated by an exemplary case.

Evidence of an insulin overdose is very complicated in the medico-legal field. The analysis and subsequent interpretation of results is complex, especially when treating postmortem blood samples. The instability of insulin, the special pre-analytical conditions and the absence of specific analytical methods has led most laboratories not to analyze insulin in their routine with a consequent underestimation of cases. This paper aims to assess the difficulties associated with the analytical characterization of insulin by describing a case that typically represents most of the inconveniences encountered following a suspected insulin overdose. The case concerns a man found dead at home by his brother. After an external examination, which did not reveal a specific cause of death, toxicological analysis was requested which did not reveal any substance of toxicological interest. Only 9 months later, it was reported to the toxicologist that the subject was diabetic, on insulin lispro treatment and that three empty syringes were found next to his body. Following analysis by LC-high-resolution mass spectrometry, the presence of insulin lispro at a concentration of 1.1 ng/mL, a therapeutic concentration, was evidenced. Despite the low concentration found, overdose cannot be excluded and this paper will describe the criteria evaluated to reach this conclusion. This case highlights that the interpretation of a postmortem insulin concentration is very complex and requires the evaluation of various elements including the circumstances of death, the subject's medical history, the interval between death and sampling and the sample storage.

Evidence of ostarine excretion in oral fluid after a single controlled oral administration.

The presence of ostarine, a selective androgen receptor modulator (SARM) in an athlete's urine specimen constitutes one of the most frequent anti-doping rules violation as the drug is listed as a member of the S1.2 class "other anabolic agents" of the World Anti-doping Agency Prohibited List, forbidden in- and out-competition. It is possible to challenge this violation but it is at the charge of the athlete to prove innocence. The conditions to evidence no fault or negligence are mostly based on 2 points: 1. the athlete must present verified circumstances of contamination and the source of contamination must be identified; and 2. there must be verified claims by the athlete that the violation was not intentional. Some months before the Olympic games, a female athlete was suspended by a national anti-doping agency because of an adverse analytical finding for ostarine. She claimed that her violation was due to drug transfer when kissing her boyfriend, who did not inform her about his ostarine daily intake. To document this claim (excretion of ostarine in oral fluid in sufficient amounts), a male volunteer ingested 17.3 mg of ostarine (dose verified by 1H NMR). Oral fluid was collected over 8 h using the NeoSal™ collection device and was tested by liquid chromatography coupled to tandem mass spectrometry. Maximal ostarine concentration was 468 ng/mL at T + 15 min, which can also be partially attributed to mouth contamination. Ostarine was detectable during the whole period of test, with concentrations at 1-2 ng/mL after T + 4 h. These results support drug transfer during kissing and subsequent possible contamination of the partner.

Testing for Kratom alkaloids in fingernail clippings - not only mitragynine.

Kratom (Mitragyna speciosa) is a species of large tree that grows in Southeast Asia and is part of the Rubiaceae family. Its fresh leaves are harvested for their medicinal properties and used for their psychoactive effects. Kratom contains many biologically active alkaloids, including mitragynine and 7-OH-mitragynine, which are considered the two most important psychoactive components and constitute approximately 66% and 2% of the total alkaloid content. Other alkaloids are present in the plant, such as speciogynine, speciociliatine and paynantheine, but have less psychoactive activity. Over the past decade, the sale of kratom powder has increased on the Internet. This led to a significant increase in forensic cases. Given the lack of data existing in the literature, and the total absence of data in nails, the authors report a study to determine the best target alkaloids for documenting kratom consumption in this matrix. Fingernail clippings from a supposed kratom powder user were analyzed after liquid-liquid extraction, chromatography separation using a HSS C18 column and performed on an ultra-high performance liquid chromatography coupled to a tandem mass spectrometer. In the specimen, mitragynine was quantified at 229 pg/mg, speciogynine and paynantheine were both quantified at 2 pg/mg, and speciociliatine was quantified at 19 pg/mg. 7-OH-mitragynine was not detected. The interpretation of these concentrations is complex, since there is currently no reference in the literature, as this is the first identification of mitragynine and other kratom alkaloids in nails. Nevertheless, in view of the high concentration of mitragynine, the subject seems to be a repetitive user of kratom. According to the measured concentrations, it seems that mitragynine remains the best target to document kratom consumption, but the identification of the other alkaloids would enhance the specificity of the test.

Hair analysis in postmortem investigations: Case of a skeletonized body.

Analysis of hair collected from putrefied or skeletal bodies is always complex and must take into account several pitfalls, such as external contamination and contamination by biological fluids. This work presents a case of particular complexity. A skeletonized body was discovered on a country road. A tuft of brown hair, detached from the scalp, irregular in length, non-oriented, in contact with soil and vegetation, was removed. An anthropological examination was carried out and genetic samples were taken from the right femoral shaft. After about 10 washes with warm water and dichloromethane, the tuft of hair was analyzed without segmentation. General unknown screening was performed by liquid chromatography system coupled to a high-resolution mass spectrometry (LC-HRMS) after incubation in pH 9.5 borate buffer and liquid-liquid extraction. Specific Multiple Reaction Monitoring (MRM) methods for date rape drugs were carried out by liquid chromatography system coupled to a tandem mass spectrometry (LC-MS/MS). The anthropological examination allowed to determine that the victim was a female individual, over 60 years old, the death dating from 3 months to 1 year. Comparison of the DNA results with the Missing Persons Index led to the identification, a 60-year-old woman who disappeared 5 months earlier. Hair analysis showed the presence of oxazepam (361 pg/mg), nordiazepam (54 pg/mg), and alimemazine (5 pg/mg). The interpretation of these concentrations is extremely difficult due to the risk of degradation of the hair cuticle during prolonged stay in the soil, as well as of contamination by putrefactive fluids. The authors discuss the value of using multiple biological and non-biological matrices in this context to improve the interpretation of the results.

Hair and dietary supplements testing to identify contamination with roxadustat in an adverse analytical finding.

Roxadustat is an oral inhibitor of hypoxia-inducible factor (HIF) prolyl hydroxylase, which increases endogenous erythropoiesis. WADA has included roxadustat and other HIF stabilizers on its list of prohibited substances. We describe here the case of an elite athlete (female, 31 years old, 168 cm and 53 kg) with an adverse analytical finding (AAF) with concentration of roxadustat in her urine at 0.289 ng/mL in the A sample and 0.529 ng/mL in the B sample (83% higher than A). A stability study was carried out, showing total stability of roxadustat at this concentration in urine exposed to light for 50 h, so photoisomerization degradation cannot explain the difference in concentration. Her urine had been completely negative in a control test carried out three days previously, while roxadustat had been shown to be present in urine for at least 20 days after administration of pharmacologically effective doses to an athlete. Hair concentration was 0.39 and 0.35 pg/mg in the segments corresponding to the presumed period of intake, with few adjacent segments also positive (0.29-0.33 pg/mg), likely explained by cosmetic treatments. Concentrations found in a patient treated with a pharmacologically active dose (between 100 and 120 mg 3 days a week) were more than 100 times higher (between 41 and 57 pg/mg). Numerous supplements and pharmaceuticals taken by the athlete were analyzed. Only collagen powder showed the presence of roxadustat, at a very low but highly variable concentration (100 pg/g-1000 pg/g). A female volunteer (58 years old, 169 cm and 65 kg), taking this powder at the same doses as the athlete (10 g of powder 5 times for 6 days) presented 7 roxadustat-positive urine samples (although lower than those observed in the athlete) out of 34 sampled over 7 days, the difference in powder sampling location, age, weight, height, pharmacokinetic parameters variability and level of sporting activity between the athlete and the volunteer probably explaining the difference in concentrations observed. All these results could be consistent with an AAF due to contamination by dietary supplements, which are becoming increasingly common due to the current exposome of athletes in our society.

Evidence of exposure to flecainide in a newborn by keratinous matrices testing and interpretation of the findings.

Pediatric poisoning represents a serious problem all around the world. Abuse or neglect of children by adults must be highlighted in children exposed to drugs to which they would not normally have access. Usually, segmental hair analysis would allow in these contexts to determine whether the exposure was unique or repetitive. Hair and nail samples from a 9-month-old girl were received in our laboratory for analysis, after the child was hospitalized due to severe dehydration caused by her mother's neglect. At the admission, flecainide, an antiarrhythmic never prescribed to the child, was identified in the daughter urine. Using an LC-MS/MS method, flecainide tested positive in the child's hair at the following concentrations: 66 pg/mg (root to 1 cm), 61 pg/mg (1-2 cm), and 125 pg/mg (2-3 cm). Traces below the limit of quantification (1 pg/mg) were also present in the nail clippings. These concentrations are much lower than those obtained in adults under daily treatment. Given the different pharmacokinetic and dynamic parameters in children, the different rate of hair growth, and the greater porosity of the hair, which makes it more prone to external contamination, the interpretation of hair findings in children remains very complicated. In this case, it can be assumed that the presence of the drug in the urine indicates systemic incorporation and that administration had occurred for some months (three positive segments). The interpretation of hair tests from young children needs a global review of all the findings, as a positive result cannot stand alone to claim repetitive exposures.

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

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