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

Protonitazene is a synthetic benzoimidazole 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 LC-MS-MS. 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 a 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 at > 7600 pg/mg in the whole head hair specimens of two male subjects deceased from 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.

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.

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.

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.

Testing for clomiphene in keratinous matrices using LC-MS/MS in doping purpose: Is a single intake of clomiphene detectable in hair and nail clippings?

Clomiphene is a selective estrogen receptor modulator. It is indicated for the treatment of female infertility issues but in sport, it can be misused to stimulate endogenous testosterone secretion in men. Therefore, it has been prohibited at all times by the World Anti-doping Agency. The aim of this study was to get data to be able to interpret concentrations in athletes. A healthy volunteer (male, 62 years-old) ingested a single therapeutic dose of clomiphene (Clomid™, 50 mg). Strands of hair (blond, 4 cm) were collected one month after the ingestion. Body hair (beard, axillary, pubic and chest hair), and finger and toenails were collected over 4-5 months. A previous method was modified to identify and quantify clomiphene in keratinous matrices. 30 mg of specimen were sonicated and incubated in 1 mL of methanol, in presence of 200 pg of clomiphene-D5 (internal standard). After centrifugation and evaporation of the organic phase, the samples were analyzed using LC-MS/MS. Linearity was verified in hair and nail clippings between 1 and 500 pg/mg. The limits of detection and quantification were determined at 0.3 and 1 pg/mg respectively. The study demonstrated that clomiphene tested positive in all the analyzed specimens at 9 pg/mg in head hair, from 28 to 486 pg/mg (body hair) and from 4 to 57 pg/mg (nails). Clomiphene was identified for the first time in multiple keratinous matrices. This study demonstrated that a single oral therapeutic dose is detectable in keratinous matrices over a long period of time.

Testing for clomiphene in keratinous matrices (hair and nail).

Clomiphene or clomifene is a selective estrogen receptor modulator used to treat female fertility in case of ovulatory dysfunction. In sport, clomiphene is prohibited at all times for use by athletes and is listed in the section S4.2 (hormone and metabolic modulators) by the World Anti-Doping Agency. Indeed, clomiphene can indirectly increase testosterone levels in the body and can mitigate some side effects of synthetic steroid abuse. Despite its prescription to millions of subjects, its detection in human hair or nail clippings has never been reported. The aim of this study was to develop a specific method to identify clomiphene in hair and nail clippings by liquid chromatography-quadrupole tandem mass spectrometry. The procedure was then applied in a case of challenged doping results. The method involves sonication/incubation for 1 h of 30 mg of pulverized material in 1 mL of methanol in the presence of 2 ng diazepam-d5 used as internal standard. The chromatographic separation was performed using a HSS C18 column with a 15 min gradient elution. After spiking blank hair and nail with the corresponding amounts of clomiphene, linearity was verified from 1 to 500 pg/mg (r2 = 0.9994 and 0.9995 for hair and nail, respectively). The limit of detection was estimated at 0.3 pg/mg for both matrices. No interference was noted from endogenous compounds, particularly steroids. Clomiphene was identified at 85 and 20 pg/mg in the pubic hair and the fingernail clippings, respectively, of a male athlete challenging an adverse analytical finding.

The Power of Keratinous Matrices (Head Hair, Body Hair and Nail Clippings) Analysis in a Case of Death Involving Anabolic Agents.

A 29-year-old man with no previous medical history was found dead at home. Anabolic products (tablets and oily solutions) and syringes were found at the scene. The man was known to train regularly at a fitness club and to use anabolic drugs. Following an unremarkable autopsy with normal histology, toxicological analyses were requested by the local prosecutor to provide further information. Blood, head hair (5 cm, black), body hair (axillary and leg) and toe and finger nail clippings were submitted to liquid and gas chromatography coupled to tandem mass spectrometry (LC and GC-MS-MS) methods to test for anabolic steroids. Blood tested positive for testosterone (4 ng/mL), boldenone (26 ng/mL), stanozolol (3 ng/mL) and trenbolone (<1 ng/mL). Segmental head hair tests (2 × 2.5 cm) revealed a repeated consumption of testosterone (65-72 pg/mg), testosterone propionate (930-691 pg/mg), testosterone isocaproate (79 pg/mg to <5 pg/mg), nandrolone decanoate (202-64 pg/mg), boldenone (16 pg/mg), stanozolol (575-670 pg/mg), trenbolone (4 pg/mg-not detected), drostanolone (112-30 pg/mg), drostanolone enanthate (26-5 pg/mg) and drostanolone propionate (15-4 pg/mg). In addition to the substances identified in head hair, testosterone decanoate, testosterone cypionate and nandrolone were identified in both body hair and nails. The experts concluded that the manner of death can be listed as toxic due to massive repetitive use of anabolic steroids during the previous months. For anabolic agents, blood does not seem to be the best matrix to document a fatal intoxication. Indeed, these products are toxics when abused long term and are known to cause cardiac, hepatic and renal diseases. When compared to blood, hair and nails have a much larger window of detection. Therefore, keratinous matrices seem to be the best approach to test for anabolic steroids when a sudden death is observed in the context of possible abuse of steroids.

Pregnancy denial and unplanned home delivery: Considerations about fetal death causes and maternal drug use imputability.

Determining fetal death causes is a complex problem for the forensic pathologist. Beyond the medico-legal context, the expert must be able to evaluate the viability of the fetus at the time of death, to eliminate in-utero fetal death and to determine if the death is related to a fetal, a maternal, a placental cause, or simply related to obstetrical complications. The authors present the case of a 21-year-old woman who unexpectedly gave birth to a fetus during a party. As pregnancy was not acknowledged by the mother (regular menstrual cycles and use of hormonal contraception), no obstetrical check-up had been performed. She would have presented violent abdominal pain and expelled a mass in the toilet. The fetus body, enclosed in the amniotic pouch, and the placenta were found in the toilet. A forensic autopsy was performed jointly by a forensic pathologist and a specialist in fetal pathology. Histological, toxicological and genetic samples were collected. Body morphometry and bone maturation indicated a gestational age of 31-32 weeks of amenorrhea. A significant asphyxia syndrome and non-specific multi-visceral congestion were noted at autopsy. Histological analysis of the fetal tissues revealed a lung and skeletal muscle maturation in accordance with the estimated term. At the brain level, there were signs of anoxia and abnormal cortical development with periventricular nodular heterotopia areas. The placenta microscopic analysis revealed acute chorioamniotitis, the probable cause of the premature fetal expulsion. Toxicological analyses revealed the presence of ecstasy (48 ng/mL) and its metabolite MDA (2 ng/mL) in fetal blood. Although negative in blood, THC-COOH tested positive in urine (9 ng/mL). The fetus was repetitively exposed to cannabis, as Δ9-THC tested positive in hair (51 pg/mg). Maternal hair analysis on 4 × 3 cm evidenced a long-term use of cannabis, while results support single massive exposure to ecstasy. In this article, the authors try to explain the reflexive pathway carried out to establish death causes and the maternal toxic consumption imputability on the cerebral malformations and fetal death. This case illustrates both the interest of toxicological analyses in cases of fetal death and the importance of a collaborative work between forensic and fetal pathologists and toxicologists, which appeared critical to answer in the best conditions to the magistrates questions, as well as to the bereaved families.

Fingerprints: A New Specimen for Innovative Applications for the Detection of Xenobiotics.

Fingerprints are invisible traces that result from a deposition of sweat and sebum present on the papillary ridges. As sweat and sebum contain drugs, fingerprints are promising since collection is rapid, non-invasive and difficult to falsify. Very limited data are available in the literature, and therefore, it seems opportune to study the transfer of xenobiotics onto the items taken in hand via the fingerprints. Two studies were implemented using the ballpoint pen as a model. The objective of the first study was to compare the nicotine concentrations found on the pens of three smokers and three non-smokers. Five pens, belonging to each subject and used regularly, were rubbed with a cotton swab dipped in methanol and analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS). The second study was to analyze the transfer via fingerprints of four volunteers, after administration of 30 mg of codeine. The objective was to determine the feasibility of this study and the time corresponding to the highest concentration of codeine. Over a 24-h period, new pens were handled for 5 min by the four volunteers, rubbed with a cotton swab dipped in methanol, and then analyzed by LC-MS-MS. The nicotine study showed a major difference between the nicotine concentrations obtained from smokers (between 6 and 276 ng/pen) and non-smokers (between 2 and 4 ng/pen). After administration of 30 mg of codeine, the analysis of the pens of the four volunteers allowed to demonstrate the presence of codeine up to 24 h between 9 and 544 pg/pen. Normal hygiene practices did not influence the final result. The highest concentration was observed after 2 h. Morphine was also detected (between 19 and 33 pg/pen). These preliminary results should be considered a demonstration of the interest of fingerprints testing to document drug exposure.

Adverse analytical finding due to red blood cells transfusion: A rare case involving the diuretic dorzolamide.

Early November 2020 an Olympic gold medal winner returned during an out-competition control an adverse analytical finding for dorzolamide, a diuretic mostly used to treat glaucoma. Estimated urine concentrations were 2.2 and 1.6 ng/ml in the A and B specimens, respectively. As the athlete denied any use of dorzolamide, a complex forensic investigation was suggested. It revealed that the athlete was severely injured during a car crash 6 months before where he received 2× 500 ml of red blood cells transfusion. One of the blood donors declared using dorzolamide. A plasma aliquot, stored for legal purposes, was tested by LC-MS/MS several months later and contained 4.3 ng/ml of dorzolamide. Given the very long half-life of the drug, up to 150 days and its reported incorporation into erythrocyte, it was accepted by the French antidoping administration (AFLD) that the source of contamination was this blood transfusion and that the antidoping rule violation was unintended.

Fatal Rectal Injection of 3-MMC in a Sexual Context: Toxicological Investigations Including Metabolites Identification Using LC-HRMS.

The dead body of a 59-year-old man was found at his home by his father. The subject was naked in the corridor, wearing a black hood and a collar around the neck where a dog leash was attached. An empty syringe was discovered in the decedent's rectal vein. The autopsy revealed marked asphyxia signs with no indication of violence or trauma. Femoral blood, urine and hair (4 cm, brown) were collected and submitted for comprehensive toxicological investigation. Initial screening did not indicate the presence of ethanol or any other over-the-counter or prescription pharmaceuticals. Routine toxicology screening by liquid chromatography-tandem mass spectrometry (LC-MS-MS) tentatively identified only the cathinone stereoisomer(s), 3-methylmethcathinone (3-MMC) or mephedrone. Analysis by gas chromatography-MS to distinguish between the isomers revealed the presence of 3-MMC, which was subsequently quantified by LC-MS-MS. Femoral blood and urine concentrations were 1,437 and 16,733 ng/mL, respectively. In 4 × 1-cm hair segments, 3-MMC was detected at <10 pg/mg (limit of quantification). Further analysis by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) allowed the identification of two metabolites in both blood and urine: desmethyl-3-MMC and hydroxyl-3-MMC. The pathologist established the cause of death in this case as acute 3-MMC poisoning in the context of ChemSex.

In vitro characterization of S-23 metabolites produced by human liver microsomes, and subsequent application to urine after a controlled oral administration.

The selective androgen receptor modulators are a recent class of anabolic agents, used to improve athletic performance. Among these molecules, there is (2 S)-N-(4-cyano-3-trifluoromethylphenyl)- 3-(3-fluoro-4-chlorophenoxyl)2-hydroxy-2-methyl-propanamide, commonly known as S-23. This molecule appeared very recently on the doping market. As a result, very few data are available in the literature, and nothing has been published about long-term effects of S-23. The authors focused on the detection of S-23 and its metabolites in human urine, following a single oral administration of approx. 8 mg to a volunteer, using standard ultra-performance liquid chromatography-triple quadrupole-mass spectrometry (UPLC-MS/MS), and ultra-performance liquid chromatography-quadrupole time of flight-mass spectrometry (UPLC-Q-TOF-MS). To the best of the authors knowledge, this seems to be the first study ever achieved on S-23. In vitro experiment was performed, using human liver microsomes, in order to investigate the potential CYP- and UGT-dependent S-23 metabolites. Four metabolites were produced, which were identified as hydroxy-S-23 (C18H12O4N2ClF4: m/z [M-H-] 431.0423); O-dephenylate-S-23 (C12H10O3N2F3: m/z [M-H-] 287.0647); S-23-glucuronide (C24H20O9N2ClF4: m/z [M-H-] 591.0794) and hydroxy-S-23-glucuronide (C24H20O10N2ClF4: m/z [M-H-] 607.0743). After consumption of S-23, the parent drug was detectable in hydrolyzed urine from 2 h post administration up to 28 days, with concentrations ranging between 0.5 and 93 ng/mL. In the urine, only one of the four metabolites identified in vitro was detected, hydroxy-S-23. This metabolite was detected up to 28 days. It does not seem to increase the window of detection of S-23 as the ratio between hydroxy-S-23 and the parent drug was always lower than 1. Another metabolite, dihydroxy-S-23, not identified in vitro, was identified in the urine of the volunteer. Hair sample, collected one month after the consumption of a single tablet, was negative for S-23 and hydroxy-S-23, with a LOQ at 0.1 pg/mg.

Analysis of Cocaine and Its Metabolites in Urine after Consumption of Coca Tea by Five Subjects and Subsequent Hair Testing.

Coca tea is a popular drink in some countries of South America where it is reputed to have medicinal properties. This preparation is composed of natural cocaine (COC) alkaloids and therefore can be banned in some countries. During an anti-doping control in Peru the urine of an athlete tested positive for benzoylecgonine (BZE) ecgonine methyl ester (EME) and COC (400 180 and 0.5 ng/mL respectively). The athlete indicated that she had consumed coca tea in the morning before the competition. As her lawyer contacted us to assess the scientific aspects of the possible involvement of coca tea to explain the adverse analytical finding a study was implemented with similar tea bags urine specimens were collected for each subject for 3 days to follow the elimination of COC and metabolites (BZE and EME). All samples were analyzed byultra high performance liquid chromatography tandem mass spectrometry after alkaline extraction. Maximum detection times for COC was 20 h with concentrations ranging from 6 to 91 ng/mL. Maximum detection times for BZE and EME were 70 h and 60 h respectively with concentrations ranging from 6 to 3,730 ng/mL and from 6 to 1,738 ng/mL. The concentration profiles were identical for the five volunteers. This study supports the athlete's claims. In addition, the sample of hair strands of the five subjects was collected a month later and all the hair tests showed a negative result for COC with a limit of decision of 10 pg/mg. Although it is accepted that a 4 mg dose of COC has no significant pharmacological effect the consumption of coca tea can lead to significant legal consequences since the measured urine concentrations sometimes cannot be considered incidental. Therefore, discrimination between coca tea consumption and recreational COC abuse relies primarily on hair analysis.