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The value of hair in forensic toxicology has already been widely demonstrated. It offers a much wider detection window than other matrices, and its segmental analysis allows the documentation of a single, occasional or regular consumption of a large number of molecules. To date, considerable efforts are being made to achieve very high sensitivity using increasingly effective techniques in the forensic analysis of hair (gas chromatography with electron ionization mass spectrometry (GC--EI-MS); gas chromatography with chemical ionization mass spectrometry (GC--CI-MS); gas chromatography–tandem mass spectrometry (GC--MS-MS); high or ultra performance liquid chromatography–tandem mass spectrometry (HPLC–MS-MS and UPLC–MS-MS)). Since the early 2000s, work has been carried out to analyze hair using matrix-assisted laser desorption ionization (MALDI) coupled with imaging mass spectrometry (IMS). Intact, cut or pulverized, human head hairs are analyzed in all their forms. With a simplified and rapid sample preparation protocol, MALDI-IMS appears to be an attractive option for the forensic interpretation of hair analysis. The high spatial resolution clearly competes with conventional methods and strand segmentation. This article provides a complete overview on MALDI techniques used and its applications to better understand the pre-analytical and the analytical parts in the hair analysis.

Insulin aspart (NovoRapid®, NovoMix®, Novolog® and Fiasp®) is a fast-acting analog of human insulin, indicated in the treatment of type I and II diabetes. It is administered before meals to mimic the physiological insulin secretion that follows a rise in blood glucose. Its misuse for the purposes of suicide and murder and in the context of factitious order has often been described. In forensic medicine, the identification of insulin in biological samples has always been complex. In this paper, we present a case of suicide of a 64-year-old man who died after the injection of insulin aspart. He was suffering from terminal lung cancer and left a letter explaining the reasons for his suicide. Four empty NovoRapid® pens were found near the body. Body examination was unremarkable, and the femoral blood was collected in two dry Vacutainer™ tubes (red cap) and two sodium fluoride (NaF) tubes (gray cap). A liquid chromatography coupled to high-resolution mass spectrometry method was used to identify and discriminate insulin aspart from human insulin after immunopurification in the blood samples and in the pens. Blood specimens tested positive for insulin aspart with the concentrations of 36 and 37 ng/mL in dry tubes and 58 and 71 ng/mL in tubes containing NaF when tested ∼3 weeks after the collection of the specimens. The contents of the pens also matched with insulin aspart. The stability of insulin in blood is a critical point in the interpretation of the concentrations due to their rapid decrease caused by the activity of proteases in blood. During a degradation study implemented to compare three preservatives and dry tubes, suitable insulin aspart stability was observed with disodium salt of ethylenediaminetetracetic acid and NaF. Given that NaF is standard in forensic toxicology for measuring blood alcohol concentrations, the authors suggest its use for blood collection when insulin intoxication is suspected.

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 (

3-Methoxyphencyclidine (3-MeO-PCP) is a new psychoactive substance that belongs to the phencyclidines family, first identified in Europe in 2012. This drug presents a stronger binding to N-methyl-D-aspartate (NMDA) receptors when compared to phencyclidine, which results in more potent effects, even at low concentrations. Very few articles have been published regarding 3-MeO-PCP in forensic toxicology. In this paper, the authors present a fatal 3-MeO-PCP intoxication case. In addition to the detection of the parent drug, metabolites were investigated in urine and, for the first time in the scientific literature, in blood. 3-MeO-PCP and its metabolites were quantitated by liquid chromatography-tandem mass spectrometry system (LC-MS/MS). Identification was confirmed by liquid chromatography-high resolution mass spectrometry (LC-HRMS). 3-MeO-PCP tested positive in femoral blood (3 525 ng/mL) and urine (7 384 ng/mL). The femoral blood concentration was higher than the fatal concentrations range already reported in the literature (from 50 to 3 200 ng/mL). 3-MeO-PCP metabolites, including O-demethyl-3-MeO-PCP, piperidine-OH-3-MeO-PCP, O-demethyl-piperidine-di-OH-3-MeO-PCP and piperidine-di-OH-3-MeO-PCP, were detected in blood. In addition, two new metabolites, O-demethyl-piperidine-OH-3-MeO-PCP and O-demethyl-cyclohexyl-OH, were identified in both blood and urine. Unfortunately, due to the lack of reference material on the market, it was not possible to measure the concentration of these metabolites. However, the ratios between the metabolites and the parent drug were useful to estimate their analytical response and prevalence. At this time, considering the low ratios (

A 59-year-old male was found dead at home, with two empty vials of an oily preparation obtained from a manufacturer from East Europe. There was no label on the vial. The subject was a former weightlifter, also known as an anabolic steroids abuser. The local prosecutor ordered a body examination, which was unremarkable, and allowed collecting femoral blood, urine and scalp hair (6 cm, brown). He was treated for cardiac insufficiency with quinidine. Biological specimens were submitted not only to standard toxicological analyses including a screening with liquid chromatography (LC)–quadrupole time of flight, but also to a specific LC–tandem mass spectrometry method for anabolic steroids testing. Ethanol was not found in both blood and urine. Quinidine blood concentration (791 ng/mL) was therapeutic. No drug of abuse was identified. In blood, testosterone was less that 1 ng/mL and no other steroid was identified. In urine, testosterone/epitestosterone was 1.56 and boldenone was present at a concentration of 9 ng/mL. The hair test results, performed on the whole length, demonstrated repetitive steroids abuse, including not only testosterone (140 pg/mg), testosterone propionate (605 pg/mg) and testosterone decanoate (249 pg/mg), but also boldenone (160 pg/mg), trenbolone (143 pg/mg) and metandienone (60 pg/mg). Since forensic laboratories have limited access to steroid urinary metabolite reference material due to specific regulations (to avoid testing athletes before anti-doping verifications), hair analyses seem to be the best approach to document anabolic agents abuse. Indeed, in hair, the target drug is the parent compound; in addition, when compared to blood or urine, this matrix has a much larger window of detection. The pathologist concluded cardiac insufficiency in a context involving repetitive abuse of anabolic drugs. This case indicates that more attention should be paid to anabolic steroids, in a context of sudden cardiac death.

For several years, the misuse of stimulant substances is increasingly observed both in the field of sport, to improve the functions of the body and therefore to be more performant, and also by non-athletes to make life more tolerable on a daily basis. Adrafinil, 2-((diphenylmethyl)sulfinyl)-N-hydroxyacetamide, is a drug designed for the treatment of narcolepsy by promoting an awakened state, and to treat alertness and neurological symptoms in the elderly. It is primarily metabolized in vivo to an active form, i.e. modafinil, 2-((diphenylmethyl)sulfinyl)acetamide. The World Anti-Doping Agency (WADA) banned these two drugs in sports in 2004. The authors report an authentic case involving adrafinil and modafinil. The laboratory was requested to test for adrafinil in a hair strand collected from a woman found in possession of vials of adrafinil and suspected of trafficking. A specific method was developed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Unlike modafinil (varying from 6.8 to 13.9 ng/mg), adrafinil was not identified in the strand. The interpretation of the results was difficult because this is the first case describing human hair analysis. In order to be able to interpret the results, a self-administration study was conducted after an oral administration to a volunteer (200 mg) whose beard hair was collected 10 days after administration. The analysis of this specimen highlighted the presence of adrafinil at 0.8 ng/mg and modafinil at 0.5 ng/mg. These results demonstrate the dual identification of both compounds after a single consumption, even after administration of a low dose. According to these results, the analysis of the hair strand from the authentic case does not match with a consumption of adrafinil, in accordance with abuse of modafinil alone. Intelligence considered that this was a trafficking case of adrafinil, with no self-consumption.

Resume L’objectif de cet article est de presenter deux approches, l’une basee sur les analyses biochimiques et l’autre sur le dosage des oligo-elements dans les cheveux, afin d’explorer le statut nutritionnel d’enfants pour lesquels une denutrition est suspectee a l’autopsie. Nous presentons le cas d’une fillette de 9 mois decedee au domicile. Le corps est transporte a l’unite de mort inattendue du nourrisson le jour meme (j0) ou sont effectues differents prelevements. Des ecchymoses ayant ete constatees a l’examen du corps, un signalement est effectue et le corps transporte a l’institut medicolegal. L’autopsie effectuee a J1 revele un traumatisme crânien, cause du deces, et un retard staturoponderal majeur. Le magistrat ordonne, en plus de l’expertise toxicologique de reference, une evaluation du statut nutritionnel de l’enfant. Les analyses toxicologiques et biochimiques sont effectuees a partir des echantillons de serum preleve a J0 ainsi que de sang peripherique et de cheveux preleves a J1. Les analyses toxicologiques ne revelent aucune anomalie. Les parametres biochimiques doses dans le serum (albumine, pre-albumine, uree, creatinine, acide urique) et le sang total (β-hydroxybutyrate) sont normaux a l’exception d’une elevation de l’acide urique. Le dosage des oligo-elements dans les cheveux par ICP-MS montre des resultats comparables a ceux obtenus dans une population de sujets temoins et d’enfants de la meme tranche d’âge. L’examen anatomopathologique ne releve aucune lesion en faveur d’une denutrition. En conclusion, ces deux approches ont permis d’eliminer une denutrition. Bien que peu exploitee, l’approche par criblage des oligo-elements pourrait s’averer utile, notamment pour augmenter la fenetre de detection ou lorsque les prelevements necessaires aux analyses biochimiques ne sont pas disponibles. D’autres etudes de cas sont necessaires pour etayer cette hypothese.

During a party in another country, several adults sniffed a powder presented as being lysergic acid diethylamide (LSD). The next morning, two subjects, including a French citizen, were found dead. After a body examination that concluded that the death was due to respiratory and cardiac collapses, the French citizen’s corpse was returned to France and buried. Four years later, the body was exhumed, and an autopsy that did not reveal traumatic injury was performed. During the autopsy, biological specimens were collected. A comprehensive toxicological screening, followed by confirmation using ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS-MS) revealed the presence of 2,5-dimethoxy-4-chloamphetamine (DOC) in all specimens: liver (99 ng/g), spleen (28 ng/g), bone (14 ng/g), lung (15 ng/g) and pubic hair (32 pg/mg). No other drug, including pharmaceuticals and drugs of abuse were identified, but the circumstances of specimen collection can influence drug stability. Literature survey about DOC stability in biological material did not contribute in interpretation as there is no data dealing with storage for about 4 years before quantitative analysis. A stability study was performed at the laboratory. Blank blood was spiked with DOC at 1 mg/L, stored at + 4°C and −20°C and regularly tested over 6 months. The percentages of concentration remaining from the initial concentration of DOC stored for 6 months at + 4°C and −20°C were 53% and 59%, respectively. To characterize the metabolite(s) of DOC, the drug was incubated with a pool of human hepatic microsomes and the cofactors required to ensure the functioning of the main phase I enzymes. The incubation media were analyzed by liquid chromatography (LC) coupled to high-resolution mass spectrometry (MS), and the results showed hydroxy-DOC. However, the hydroxy-metabolite was not identified in the liver or spleen of the subject. Although the French pathologist considered that it was more likely than not a toxic death, it is difficult to attribute the death to DOC alone, as it was impossible to test for ethanol and other chemically instable drugs. This case presents original data, which can be useful to increase the knowledge in designer drug toxicity.

A 23-month-old boy was brought to a medical center by his mother, as she noticed that the father has gripped him around the neck and this had left marks. As a result of this, a child protection medical examination was requested. However, there was a significant chronology of mental health issues in the mother. Among the mother’s medications, quetiapine and propranolol were the more active. Given a consultant pediatrician was concerned that the boy was vulnerable and potentially has experienced neglect and physical harm, the local authority instructed a hair test to document possible poisoning. However, this occurred several months later, due to court delays (postponed hearings and decisions) when the child was 32-month old. The laboratory received a strand of hair of the child (12 cm in length, light brown in color) and a strand of hair of the mother (>20 cm in length, dark in color) with the request to test both specimens by segmentation (12 x 1 cm) for quetiapine, an anti-psychotic drug and propranolol, a β-blocker agent. After decontamination and segmentation, the specimens were incubated in borate buffer pH 9.5 and extracted by a mixture of ether/dichloromethane/hexane/isoamyl alcohol to test for the drugs, including norquetiapine by a specific LC–MS-MS method. The first 3 cm segments of the child’s hair were free of drug, roughly corresponding to the period he was no more in contact with the mother. Propranolol tested positive in the other segments at 15–72 pg/mg, with a linear increase from the proximal to the distal end. This was also observed for quetiapine, with concentrations in the range 10–18 pg/mg. Norquetiapine was never identified in the child’s hair. The following concentrations were observed in the mother’s hair: 6028–10,284, 910–4576 and 1116–6956 pg/mg for propranolol, quetiapine and norquetiapine, respectively. This confirmed that the donor was a long-term repetitive user of propranolol and quetiapine. The hair test results have indicated that the child was in contact with propranolol and quetiapine for a long period. It is not possible to put a temporal period for each segment, as the hair growth at the age of 32 months is not the same as for an adult (difference in the duration of the anagen period), nor to put any quantitative dosage or frequency of exposure(s) when interpreting the data. An increase of concentrations from root to tip was observed which is considered highly indicative of external contamination, with the older hair segments (those which are the more concentrated) being in contact for a longer time with contaminated items (hands of the mother, home items such as furniture, dishes, beddings, etc.). Overinterpreting drug findings in hair can have very serious legal implications in child protection cases, particularly when no other toxicological test and no clinical report exist to support voluntary administration of drugs. Whatever the findings, a proper interpretation of hair test results is critical and should be done ideally with other information available, such as medical history, witness statements and the available circumstances of the matter. A single hair test should not be used to determine long-term exposure to a drug.

An athlete challenged the result from an in-competition doping test which returned with an adverse analytical finding for stanozolol, claiming it was due to supplement contamination. Her lawyer asked the laboratory to analyze several hair specimens simultaneously collected from five different anatomical regions, head, arm, leg, pubis and armpit, to document the pattern of drug exposure. A specific UPLC–MS-MS method was developed. After decontamination with dichloromethane, stanozolol was extracted from hair in the presence of stanozolol-d3 used as internal standard, under alkaline conditions, with diethyl ether. Linearity was observed for concentrations ranging from 5 pg/mg to 10 ng/mg. The method has been validated according to linearity, precision and matrix effect. Concentrations of stanozolol in head hair, pubic hair, arm hair, leg hair and axillary hair were 73, 454, 238, 244 and 7,100 pg/mg, respectively. The concentration of stanozolol in head hair is in accordance with data published in the literature. When comparing the concentrations, body hair concentrations were higher than the concentration found in head hair. These results are consistent with a better incorporation rate of stanozolol in body hair when compared to head hair. The simultaneous positive concentrations in different hair types confirm the adverse analytical finding in urine of the top athlete, as the measured concentrations do not support the theory of contamination. For the first time, an anabolic agent was simultaneously tested in hair collected from five different anatomical regions from the same subject, with a large distribution of concentrations, due to anatomical variations, and these findings will help interpretation in further doping cases when documented with hair.

Insulin aspart (Novorapid ®, NovoMix®, Novolog® and Fiasp®) is a fast-acting analog of human insulin, indicated in the treatment of type I and II diabetes. It is administered before meals to mimic the physiological insulin secretion that follows a rise in blood glucose. Its misuse for the purposes of suicide, murder and in the context of factitious order has often been described. In forensic medicine, the identification of insulin in biological samples has always been complex. In this paper, we present a case of suicide of a 64-year-old man who died after the injection of insulin aspart. He was suffering from terminal lung cancer and left a letter explaining the reasons of his suicide. Four empty Novorapid® pens were found near the body. Body examination was unremarkable and femoral blood was collected in 2 dry Vacutainer tubes (red cap) and 2 sodium fluoride tubed (grey cap). A LC-HRMS method was used to identify and discriminate aspart insulin from human insulin after immunopurification in the blood samples and in the pens. Blood aspart insulin tested positive at 36 and 37 ng/mL in dry tubes, and at 58 and 71 ng/mL in tubes containing sodium fluoride when tested about 3 weeks after collection of the specimens. The contents of the pens also matched with insulin aspart.The stability of insulin in blood is a critical point in the interpretation of the concentrations due to their rapid decrease caused by the activity of proteases in blood. During a degradation study implemented to compare 3 preservatives and dry tubes, suitable insulin aspart stability was observed with EDTA and NaF. Given NaF is standard in forensic toxicology for measuring blood alcohol concentrations, the authors suggest its use for blood collection when insulin intoxication is suspected.

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 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 x 2.5 cm) revealed a repeated consumption of testosterone (65-72 pg/mg), testosterone propionate (930-691 pg/mg), testosterone isocaproate (79 pg/mg-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 long term abused 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 seems to be the best approach to test for anabolic steroids when a sudden death is observed in a context of possible abuse of steroids.

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.

Although it has been published during the last 15 years that hallucinogens derived from new psychoactive substances are increasingly abused, the old psychedelic agents are still used. Among these vegetal compounds, magic mushrooms are frequently mentioned. The major ingredient of the Psilocybe species is psilocybin, which is rapidly converted into psilocin in the human body. Therefore, psilocin represents the key target to document magic mushrooms abuse. The aim of the present work was to develop a specific method to identify psilocin in human hair by LC-MS/MS and review potential pitfalls. The method involves incubation of 10 mg of cut hair in 1 mL of acid methanol in presence of psilocin-d10 for 2 h at 4 °C. The chromatographic separation was performed using a reverse phase column HSS C18 with a gradient elution of 8 min. Linearity was verified from 1 to 200 pg/mg (r2 = 0.9994), after spiking blank hair with the corresponding amounts of psilocin. The limit of detection was estimated at 0.4 pg/mg. The precision was lower than 20 % and there was no interference with any extractable material present in hair. Psilocin was identified in the hair of a repetitive magic mushrooms consumer at 2.5 pg/mg (segment 0–1 cm), 4.4 pg/mg (segment 1–2 cm) and 5.4 pg/mg (segment 2–3 cm). Cold conditions during hair preparation and protection from the light were found mandatory to avoid psilocin degradation.

A 43-year-old male, sport coach, presented him-self at the Emergency unit of a local hospital for epigastric pain, myalgia pain and severe headache. He claimed having used for some days a combination of GW1516 (cardarine), a peroxisome proliferator-activated receptor delta agonist (PPAR- δ) and MK2866 (ostarine), a selective androgen receptor modulator (SARM) to gain skeletal muscles. Cytolysis with marked increase of alanine aminotransferase or ALT (up to 922 UI/L) and aspartate aminotransferase or AST (up to 2558 UI/L) and massive rhabdomyolysis with elevated creatine phosphokinase or CPK (up to 86435 UI/L) were the main unusual biochemistry parameters. Using a specific liquid chromatography coupled to tandem mass spectrometry method, cardarine and ostarine tested positive in blood at 403 and 1 ng/mL, respectively. In urine, due to extensive metabolism, the parent GW1516 was not identified, while ostarine was at 88 ng/mL. Finally, both drugs were identified in hair (2 cm in length, brown in colour), at 146 and 1105 pg/mg for cardarine and ostarine, respectively. This clearly demonstrates repetitive abuse over the last 2 months. Asthenia was persistent for 2 weeks and 6 weeks after the admission, the subject fully recovered.

Promazine is one of the oldest phenothiazine derivatives that have been proposed for the treatment of various psychiatric disorders. The drug is available as tablets, as syrups and in injectable forms. Despite its prescription to millions of subjects, its detection in human hair has seldom been reported. The aim of the present work is to develop a specific method to identify promazine in human hair by liquid chromatography–tandem mass spectrometry and to apply it to a patient who was self-medicating. The method involves overnight incubation of 20 mg of cut hair in 1 mL of pH 9.5 borate buffer in the presence of amitriptyline-d3 at 40°C. The chromatographic separation was performed using a reverse phase column HSS C18 with a gradient elution for 15 min. Linearity was verified from 0.5 to 500 pg/mg (r2 = 0.9996), after spiking blank hair with the corresponding amounts of promazine. The limit of detection was estimated at 0.1 pg/mg. The precision was lower than 20%. Promazine was detected in the hair of a psychotic subject at 228–270 pg/mg in a 3 × 1 cm segment. Given this was a patient who was self-medicating, her physician requested an immediate drug discontinuation. In a fresh hair specimen collected 3 months later, the proximal segment (0–1 cm) tested positive at 0.9 pg/mg, clearly indicating that the time to obtain a negative result after promazine discontinuation is about 3–4 months.

The authors report the case of a 41-year-old man known as a polydrug addict and found dead at home. The initial toxicological screening was inconclusive for a toxic death. However, 7 small bags of unknown powders and crystals, supposed to be NPS, were found near the body and sent to the laboratory 3 weeks later. This article focuses on the interest of nuclear magnetic resonance (NMR) spectroscopy to solve this fatal case. The 7 items were identified by 1H NMR and their purities were measured by the quantitative NMR (qNMR) method using maleic acid as internal standard. The compounds were 4-chloro-α-pyrrolidinopentiophenone, dibutylone, 4-chloroethcathinone, 4-α-pyrrolidinohexuiphenone, 3,4-methylenedioxy-N-benzylcathinone, 3-methoxy-phencyclidine and α-pyrrolidinopropiophenone, with purities in the range 51–89%. In the biological specimens of the victim, only 3-methoxy-phencyclidine (3-MeO-PCP) was identified in cardiac blood, peripheral blood and urine at 743, 489 and 16,700 ng/mL. Hair (6 cm) concentration of the drug was 15,600 pg/mg. The other NPS were not detected. The peripheral blood concentration was higher than the values reported in previous intoxicated and fatal cases. The toxicological significance of the measured concentration in hair was difficult to establish because this is only the second case describing a test result for 3-MeO-PCP.

The identification and quantification of insulin and its analogs have always been a challenge in the forensic field. Murder, suicide attempts and induced hypoglycemia in the context of factitious disorders have been described with the use of synthetic analogs of human insulin. There is very less information in the literature about aspart insulin concentrations in overdose cases. In this paper, we present a case of a nurse who tried to murder her 10-year-old daughter by injecting her aspart insulin and who, later, tried to commit suicide by injecting herself the same hormone. Two empty syringes and a FIASP® Flextouch pen were found in the woman’s apartment. A liquid chromatography coupled to high resolution mass spectrometry (LC–HRMS) method was developed in order to identify and discriminate aspart insulin from human insulin in blood samples as well as in syringes and pen, while an liquid chromatography coupled to tandem mass spectrometry (LC–MS-MS) method was developed for the quantification of insulin in blood samples. Aspart insulin tested positive at 5.7 and 2.4 ng/mL in the blood specimens of the mother and the child, respectively. The substance contained in the syringes and pen also corresponded to aspart insulin. Although the mother claims to have injected an overdose of aspart insulin, the concentrations found were in the therapeutic range for subjects under therapy. Due to the high instability of insulin and the long time elapsed between sampling and forensic analysis (8 months) due to administrative reasons, the concentration at the time of collection was probably much higher. In this case, it was possible to identify aspart insulin and discriminate it from human insulin in a context of attempted murder and subsequent attempted suicide using high-resolution mass spectrometry, which is of paramount importance in forensic medicine.

A 60-year-old man was found dead at home by his wife. The man was known to be a regular trainer in a fitness center and was described as a long-term user of anabolic steroids. The autopsy revealed heart failure with cardiomegaly, visceral congestion, and pulmonary edema. The local prosecutor ordered toxicological analyses but no anatomic pathology test. Initial analyses by the liquid chromatography coupled to quadrupole time-of-flight mass spectrometry system (UPLC--q-TOF-MS) system indicated the presence in femoral blood of trenbolone, an anabolic steroid. Trenbolone was quantified with a liquid chromatography system coupled with a tandem mass spectrometer in various autopsy specimens, including hair. Concentrations of trenbolone were 3.9, 3.2, 231, and lower than 0.5 ng/mL in femoral blood, cardiac blood, bile, and vitreous humor, respectively. Moreover, in head hair, the concentration was 9 pg/mg. The presence of trenbolone in hair can be interpreted as repetitive exposures when compared to the limited literature data. Repetitive misuse of trenbolone can explain advanced heart failure. This paper highlights the importance of testing biological specimens for anabolic steroids in case of heart failure.

Hair analysis is very useful for toxicological investigations since, by providing a wider detection window, it gives the possibility to perform a retrospective study on the historical consumption of a substance. Unfortunately, there are no data available for hair concentrations in metformin-related deaths. In this study, the authors present 2 cases of fatal metformin intoxication in which, for the first time, hair analysis was performed using a specific GC–MS/MS method. Metformin was tested positive in femoral blood (112.3 mg/L and 64.7 mg/L respectively) and cardiac blood (226.9 and 203.2 mg/L) of the two subjects. For case 1, other samples were also tested positive, including vitreous humor (31.1 mg/L) and gastric contents (773.5 mg/L). In case 2, metformin was measured at 844.9 mg/L in urine. Metformin hair concentrations were 28.3–44.8 and 22.5 ng/mg for both cases, respectively. The concentrations found in the 2 fatal cases are clearly higher than those obtained in a previous study with subjects under treatment (0.3–3.8 ng/mg) or those found in 3 post-mortem cases where metformin death was excluded (0.6–1.4 ng/mg). Excessive sweating during the agonal phase due to fatal hypoglycemia could explain these elevated concentrations as sweat can have contaminated the hair.

International audience; Substandard/counterfeit drugs are a growing global problem. According to the World Health Organisation, counterfeit medicines are medicines that are mislabelled deliberately and fraudulently regarding their identity and/or source. In high income countries, drugs seized are mainly represented by performance and image enhancing drugs (PIEDs). The aim of this study was to present the qualitative and quantitative results of toxicological analyses of pharmaceutical and dietary supplements seized from the black market among bodybuilders in France. All dietary supplements and pharmaceuticals seized from the black market and addressed to the laboratory for a qualitative and quantitative analysis between January 2016 and December 2019 were included in the study. A screening was carried out by gas chromatography-mass spectrometry and liquid chromatography-high resolution mass spectrometry. Identified compounds were quantified by liquid chromatography-tandem mass spectrometry. One hundred and ten products were seized and submitted to the laboratory for identification of active compounds and quantification: 75 pharmaceuticals and 35 dietary supplements. This included 39 oily and 3 aqueous solutions for intramuscular injection, 34 tablets, 13 capsules, 14 powders, 4 liquids and 3 lyophilizates. Among the pharmaceuticals, 25/75 (33%) were substandard (dosage not on the acceptable range defined for original products), 24/75 (32%) were counterfeit (qualitative formulation does not match the label) and 14/75 (19%) were original (qualitative formulation and levels of active ingredients fully matches the declared formulation. The analysis of the 12 remaining products revealed a correct qualitative content for 11/75 (15%), but quantitation could not be carried out because of the lack of reference standards at the time of the analysis. Fifty-four pharmaceuticals contained anabolic-androgenic steroids (AAS). Four out of 54 (7.4%) AAS were found as original, 8/54 (15%) could not be quantified (one with wrong active ingredient), corresponding to 43/54 (80%) AAS being non-original. In contrast, only 1/35 dietary supplement (3%) was adulterated, with a doping substance (1,3-dimethylbutylamine, DMBA). This work allows to show that France is not spared by the trafficking of PIEDs. The use of counterfeit drugs in mainstream population is an underestimated public health issue.

Summary An unexpected death of a 46-year-old woman presenting a type II diabetes was observed and the Prosecutor requested an autopsy followed by toxicological investigations. Autopsy findings (labial ecchymosis, multiviscera congestion, asphyxia syndrome, moderate cerebral edema) were in accordance with a possible hypoglycemia death. Toxicological analyses revealed the presence of gliclazide in femoral blood at 2.2 mg/L, which is in the range of published therapeutic concentrations. During a meeting with the pathologist, 3 possible explanations were discussed: 1. unknown cause of death (other than a toxic death), 2. death due to gliclazide which could have been degraded due to chemical instability during the 6-weeks interval between death and analysis (at the time of death the blood concentration could have been much higher), and 3. blood concentration was enough to produce fatal hypoglycemia in a non-compliant patient. According to the pathologist, no traumatic cause of death could be established. Testing for gliclazide stability in whole blood over a period that matches the delay of the toxicological analyses demonstrated instability (loss of about 65% after 6 weeks storage at +4 °C), but this was found irrelevant for a massive overdose. Hair tested positive for gliclazide in the 3 × 2 cm segments, at 7, 8 and 3 pg/mg, and were highly indicative of non-compliant use of the medicine. It was therefore concluded by the pathologist that the cause of death of the subject was more likely than not an inappropriate use of gliclazide, which can be responsible of a fatal glycemic disorder.

Coca tea is a popular drink in some South American countries where it is reputed to have medicinal properties. This preparation is composed of natural cocaine 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, ecgonine methyl ester and cocaine (400 ng/mL, 180 ng/mL and 0.5 ng/mL, respectively). The athlete indicated that she had consumed a coca tea in the morning before the competition. As her lawyer contacted us to assess the scientific aspects of possible involvement of coca tea to explain the adverse analytical finding, a study was implemented with the same tea bags. Five volunteers from the laboratory consumed 250 mL of coca tea containing approximately 3.8 mg of cocaine. Urine (11 specimens for each subject) was collected over 3 days to follow the elimination of cocaine and metabolites (benzoylecgonine and ecgonine methyl ester). All samples were analyzed by UHPLC-MS/MS after alkaline extraction. Cocaine was identified for 20 hours, with concentrations ranging from 6 to 91 ng/mL. Benzoylecgonine and ecgonine methyl ester were identified for 70 hours and for 60 hours, respectively, with concentrations ranging from 6 to 3730 ng/mL and from 6 to 1738 ng/mL. The concentration profiles were identical for the five volunteers. This study supports the athlete's claims. In addition, the hair of the five subjects was collected a month later and all the hair tests were negative for cocaine using a limit of decision at 10 pg/mg. Although it is accepted that a 4 mg dose of cocaine has no significant pharmacological effect, the consummation of coca tea can lead to significant legal consequences since the measured urine concentrations sometimes cannot be considered incidental. Therefore, discrimination between coca tea consummation and recreational cocaine abuse relies primarily on hair analysis.

Among the hundred cannabinoids present in cannabis sativa indica, cannabidiol is a phytocannabinoid discovered in 1940, which can account for up to 40 % of the plant's extract. Medically, it has been proposed to treat convulsions, inflammation, anxiety and nausea. Contrary to the hallucinogenic ingredient of the plant, delta-9-tetrahydrocannabinol, cannabidiol does not seem to have a sedative effect, which can increase its popularity among users. The identification of cannabidiol in blood and urine has been widely described in the scientific literature for several years. Only few data after cannabis use has been reported regarding cannabidiol identification in alternative specimens, such as oral fluid, sweat, exhaled breath and hair. Cannabidiol capsules were purchased in the United States, from a grocery store and a green capsule containing 22 mg of cannabidiol was orally administered to a 59-year-old healthy man. Oral fluid was collected over 8 hours using the NeoSal™ device. Sweat was collected with PharmCheck™ sweat patch technology over 7 days. Exhaled breath was collected with the ExaBreath® DrugTrap device over 8 hours. Beard hair was collected 7 and 14 days after administration. Finally, urine specimens were collected over 48 hours in plastic tubes without preservative. Cannabidiol was only detected in oral fluid at 15 minutes, at 20 pg/mL. Increasing concentrations, up to 96 pg/patch of cannabidiol, were detected in the sweat patches. Cannabidiol was detected during 45 minutes in exhaled breath (Cmax 302 pg/filter at 30 minutes). Cannabidiol produced a very low but significant chromatographic signal in beard hair, with concentrations lower than 1 pg/mg. Finally, cannabidiol tested positive in urine after enzymatic hydrolysis with a Cmax at 70 ng/mL, after 6 hours.

Selective androgen receptor modulators (SARMs) are a class of drugs presenting identical anabolic properties to anabolic steroids in addition to marked reduced androgenic effects. These drugs have emerged in the doping area within the early 2000′s. Ligandrol, ostarine, RAD-140 and andarine are the most popular agents belonging to this class. According to the world anti-doping agency (WADA) prohibited list, SARMs are prohibited at all times (i.e. in and out-of-competition) and are listed under the section S1.2 (other anabolic agents). The compilation of the WADA testing figures reports from 2015 to 2019 has indicated a regular increase of adverse analytical findings (AAF) due to SARMs, particularly with ostarine and ligandrol. The implementation of highly sensitive chromatographic anti-doping analyses has induced high-profile challenges of anti-doping rules violations as athletes have claimed in numerous occasions that contamination was the reason for their AAF. Since the early 2000′s, it has been accepted by the Court of Arbitration for Sports (CAS) in Lausanne (Switzerland) that, under some specific circumstances, unusual explanations can be provided to the Panel to explain an AAF. This was the open door for forensic investigations, as it is done in criminal Courts. A forensic approach can include testing for SARMs in food, drinks, but mostly in dietary supplements. As most anti-doping rules violations are only known several weeks after urine collection, this biological matrix is seldom use for further tests, despite the fact that most SARMs can be detected for several weeks in urine. Luckily, hair or nail testing can be a complement to document the claim of the athlete but of course, it cannot be considered as an alternative to urinalysis. This is because a negative hair or nail result cannot exclude the use of the detected drug and cannot overrule the urine result. To date, all methods for SARMs identification in various matrices involve liquid chromatography coupled to tandem mass spectrometry or high-resolution mass spectrometry. The aim of this paper is to review the scientific literature on the analytical possibilities of testing SARMs in dietary supplements, urine and hair or nail clippings after an AAF to document the claims of an athlete or his/her legal team.

The retrospective calendar of an individual's drug use requires a multisectional analysis in which the length of hair, corresponding to the full temporal window available, is cut into shorter sections to measure drug use during shorter periods of time (generally 1 cm corresponds to ~1 month). Segmental hair analysis is used to verify both previous drug history and recent enforced abstinence. However, after drug discontinuation, the fresh new hair growth segment cannot be immediately negative, due to the contribution of dormant hair. The objective of the study was to test hair samples from chronic tramadol and cannabis users after the discontinuation of both drugs and to evaluate the delay to wait until the hair will become negative. Hair specimens were obtained from eight subjects with a known history of tramadol abuse. Hair was collected 3-6 months after tramadol discontinuation. Tramadol was tested by liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS) with a LOQ at 5 pg/mg. A second set of hair specimens were obtained from 15 subjects with a known history of cannabis abuse. Hair was collected 6-9 months after cannabis discontinuation. THC-COOH was tested by LC-MS-MS with a LOQ at 0.2 pg/mg. The hair stands were cut into L × 1 cm segments, according to their length (L), and tested for the respective drug. It was asked to each subject to clearly indicate the date of drug discontinuation. Assuming a rate of hair growth of 1 cm/month, the segment corresponding to the time of last drug use was calculated. The older segment just before this one was considered as the 100% of the response. THC-COOH and tramadol concentrations in this segment ranged from 2.3 to 8.9 and 895 to 21,010 pg/mg, respectively. After cessation of drug consumption, the presence of both drugs in new growing hair segments continued for a certain period with a more or less broad transition zone. Negative hair results were obtained ~3-4 and 6-7 months after cessation of tramadol and cannabis abuse.

Summary The authors describe the identification of 7 powders, supposed to be NPS, involved in a complex toxicological fatal case. The identification of these substances requires the development of a specific, quality and comprehensive method. However, it is difficult to obtain and purchase the reference standards of all substances. Some are excessively expensive, and some, even, do not exist in the catalogue of referenced suppliers. In addition to the difficulties associated with molecule characterization (identification, quantification, purity), analytical methods used routinely, such as LC-MS/MS, are not always sufficient to firmly identify NPS, for example in case of positional isomers. It is therefore necessary to use another method, such as NMR spectroscopy, to discriminate the species. The analytical strategy and the performance of NMR are developed in this article. The 7 powders were 4-chloro-α-pyrrolidinopentiophenone, dibutylone, 4-chloroethcathinone, 4-fluoro-α-pyrrolidinohexanophenone, 3,4-methylenedioxy-N-benzylcathinone, 3-methoxy-phencyclidine and α-pyrrolidinopropiophenone, with purity between 50.8 and 89.5%. There was no matching between the label and the active ingredient contained in the dispatched bags. This clearly demonstrates the danger in consuming substances bought on the internet, as both identity of the substance and its purity are missing.

Summary We report five cases involving use of 3 Meo-PCP (3 methoxyphencyclidine) with 2 fatalities and 3 non-fatal cases seen in France in 2018. PCP (Phencyclidine) is an arylcyclohexanexylamine, synthesised by Maddox in 1956, and has a potent dissociative anaesthetic drug. One derivative 3 Meo-PCCP appeared on the street market in 2012 with potency slightly lower than PCP. The three non-fatal cases were a 36 Y.O. man presenting alternatively confusion, aggressiveness, logorrhoea and calmness. He was positive for alcohol, buprenorphine and tramadol. Others cases were a man admitted for hyperemesis syndrome and a methadone user during laboratory survey. CG-MS (qualitative) identified 3 Meo-PCP in urine. The two deaths were forensic cases. A 39 Y.O. woman was found murdered by ligature strangulation. UPLC-MS-MS (with quantitative) on blood samples showed cocaine, benzo-ecgonine; diazepam, nordiazepam and alcohol. 3 MeO-PCP was found on femoral blood 63 ng/mL, 94 ng/mL in urine and also in hair segments. The last case was a 41 y.o. male found dead at home without traumatic lesions, femoral blood level was 498 ng/mL and urines levels were 16, 700 ng/mL. Forensic data reported in literature have shown that fatal values for 3 Meo-PCP in blood ranged from 50 to 3,200 ng/mL and in urine from 0,4 to 120 ng/mL. There are no clear toxic and fatal concentrations established for 3-MeO-PCP, mainly because the drug is often associated to other recreative substances. In Europe during years 2017-8, 18 published related cases with 3 Meo-PCP have been shown with 10 fatalities and 8 non-fatal.

The body of a 61-year-old man was found at his home by his wife, lying on the floor, near the bathroom, around midnight. He was known to be training for bodybuilding, using anabolic steroids. Police investigations revealed the presence of two types of tablets at home, one supposed to contain clenbuterol (0.040 mg) and the other stanozolol (10 mg). Testing the tablets revealed different dosages from what was expected, i.e., 0.073 and 11.5 mg/tablet, for clenbuterol and stanozolol, respectively. External body examination and autopsy, which was performed the next day, revealed generalized organ congestion and lack of any traumatic injury (confirmed by radiology). Cardiomegaly, with a heart weighing 692 g, was obvious. Anatomic pathology tests did not reveal evidence of malformations, but atheromatous plaque was identified in the coronaries during complete histology investigations. Femoral blood, urine, bile, gastric contents and two strands of hair (6 cm) were collected for toxicology. These specimens were submitted to standard analyses, but also to a specific LC–MS-MS method for clenbuterol and stanozolol testing. Clenbuterol was identified in all the tissues, including femoral blood (1.1 ng/mL), urine (7.2 ng/mL), bile (2.4 ng/mL), gastric content (3.2 ng/mL) and hair (23 pg/mg). Stanozolol only tested positive in hair (11 pg/mg). All other analyses were negative, including blood alcohol and drugs of abuse. The pathologists concluded to cardiac insufficiency with support of cardiomegaly, in a context involving repetitive abuse of anabolic drugs. This case indicates that more attention should be paid to clenbuterol, a drug widely used as a stimulant by people who want to lose weight, athletes and bodybuilding practitioners.

Betel nut is the fruit of Areca palm, growing in Papua New Guinea. Mixed with limestone and stick mustard, arecoline and guvacoline, which are present in betel nut, are hydrolyzed into arecaidine and guvacine, respectively. As part of the study on dietary habits of Papuans residents, our laboratory was asked to analyze the four alkaloids in hair to document long-term exposure. Hair samples were collected from 19 adult subjects (males = 11; females = 8), by some of the authors, and were sent to the laboratory for analysis. The four alkaloids have very similar chemical structures. In order to accurately identify the drugs, two methods were developed. First, the compounds were identified using an ultra-high-performance liquid chromatography system coupled to time-of-flight mass spectrometry. Then, they were quantified by an ultra-high-performance liquid chromatography system coupled to tandem mass spectrometry. After decontamination with dichloromethane, hair samples were cut into very small segments and 20 mg were incubated in methanol for 2 h 30 min in an ultrasound bath. After cooling, the methanol was evaporated to dryness in presence of 20-μL octanol to prevent volatilization. Nicotine-d4 was used as an internal standard. Linearity was observed for concentrations ranging from the limit of quantification to 20 ng/mg for arecoline, arecaidine, guvacine and guvacoline. Measured concentrations were in the range 60 pg/mg to 18 ng/mg for arecoline (n = 19), 14 pg/mg to 2.5 ng/mg for guvacoline (n = 11), 63 pg/mg to 3.8 ng/mg for arecaidine (n = 11) and 100 pg/mg to 3.2 ng/mg for guvacine (n = 6). There was no correlation between concentrations of arecoline and arecaidine (ratio from 0.01 to 0.18) and guvacoline and guvacine (ratio from 0.06 to 3.50). However, the identification of these substances in hair is a good marker of consumption of betel nut and allows us to document a local practice that remains difficult to evaluate just by questioning.

Resume Lors de l’analyse toxicologique de certaines expertises medicolegales, dans de rares cas, aucun milieu biologique conventionnel n’est disponible en post-mortem et seuls les vetements peuvent etre utilises. Nous presentons le cas d’un squelette retrouve partiellement devetu sous une dalle de beton. Le magistrat ayant refuse l’analyse des restes osseux, les vetements ont ete transmis au laboratoire pour expertise toxicologique. Les scelles contenaient un blouson, un reste de pantalon avec une ceinture et deux morceaux de tissu non identifies. Des pieces de 3 × 3 cm ont ete decoupees au niveau des zones susceptibles d’avoir ete en contact avec le revetement cutane ou les fluides corporels : une au niveau du col de la veste, deux au niveau de la braguette du pantalon, une au niveau de l’entre-jambe. Enfin, trois pieces ont ete decoupees dans les morceaux de tissu non identifies. Un screening est effectue par UPLC-MS/MS apres extraction liquide-liquide en milieu alcalin. Les analyses ont montre la presence de tiapride, venlafaxine et tramadol au niveau du col de la veste, et de tiapride et tramadol au niveau de la braguette. L’analyse des autres pieces de tissus s’est averee negative. Selon les informations recueillies par les enqueteurs, la victime ne suivait pas de traitement particulier et les molecules identifiees pourraient avoir contribue au deces. En conclusion, dans le cas present, l’analyse des vetements a permis de documenter une absorption ante-mortem de medicaments psychoactifs. Le screening realise dans les pieces de tissu peut donc permettre, lorsqu’aucun autre milieu biologique n’est disponible, de recueillir des informations necessaires au bon deroulement de l’enquete, et les textiles peuvent etre consideres comme une matrice alternative.