N-ethylhexedrone: a very long and bad trip! A Case series.

N-ethylhexedrone (NEH) is a new cathinone derivative with, currently, low toxicocokinetic, and toxicocodynamic knowledge. We present 3 documented clinical cases of NEH intoxication with plasma, and urine concentrations. A thorough search for metabolites was performed. The 3 patients were admitted to the emergency department, and 2 out of the 3 were hospitalized for an extended period. While recovering from the drug effects, 12 to 24 hours after nasal intake of New Psychoactive Substance (NPS), the patients described the following disorders: anxiety, feelings of persecution, asthenia, anhedonia, abulia, psychomotor slowing, and loss of consciousness. NEH was identified in all samples by liquid chromatography-high resolution mass spectrometry (LC-HRMS), and quantified by liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS). Quantitative analysis showed decreasing concentrations over time: for case 1, from 97.2 (Day 1, D1) to 0.7 (Day 7, D7) µg/L for plasma, and from 724 (D1) to 0.5 (D7) µg/L for urine. NEH concentration of 7.9 µg/L was found in the plasma collected at admission for case 2. For case 3: concentrations ranging from 49 (D1) to 1.8 (D7) µg/L in plasma, and from 327.3 (Day 5, D5) to 116.8 (D7) µg/L in urine were found. NEH was no longer detected in the urine sample at Day 10. Elimination half-life was estimated at 19, and 28 hours in patients 1 and 3, respectively. Four metabolites were identified in blood and urine: reduced NEH, dealkyl-NEH, reduced dealkyl-NEH, and hydroxy-NEH. The cases presented highlight the long detectable lifetime of NEH. Characterization of the metabolites will allow better identification of the consumption of this drug. Serious adverse events can be observed after NEH consumption, as 2 out of 3 patients required intubation and ventilation. A syndrome of inappropriate antidiuretic hormone secretion (SIADH) was also diagnosed. Two out of the three cases are notable because of the number of samples collected and because NEH was the only drug of abuse detected.

New psychoactive substances in oral fluid of drivers around a music festival in south-west France in 2017.

Driving under the influence of drugs (DUID) is a worldwide problem with potentially major forensic and life-threatening consequences. Although it is obvious that new psychoactive substances (NPS) could lead to impaireddriving, the prevalence of NPS use in a DUID context is unknown as the applied roadside screening tests for drugs of abuse (DOA) are not adapted for NPS detection. This works aims to tested oral fluid (OF) specimens for NPS in French drivers circulating around two music festivals (Artsenik 2017 and Garorock 2017) in order to assess the prevalence of consumption and the kind of used NPS in this particular population. OF samples consisted in dried saliva spots obtained from used Drugwipe-5S® tests (after a positive or negative roadside screening test for DOA). These OF were analyzed using a liquid chromatography coupled with tandem mass spectrometry or high-resolution mass spectrometry method. NPS were detected in 17 out of the 229 OF collected specimens (7.4%). Eleven various NPS were identified (number of identification): 5F-AKB48 (2), MAM2201 (1), JWH122 (1), 4F-PVP (1), 3- or 4-MMC (2), fluoromethamphetamine (1), ketamine (3), MXE (3), methoxyketamine (1), 6-APB (2) and 25C-NBOMe (1). There is an apparent effect of the music festival proximity on the prevalence of NPS in OF from this controlled driver population compared to that of 140 controlled drivers from Northern France analyzed in the same period (7.4% versus 3%). The variety of used NPS appears to be increasing (e.g. large proportion of cyclohexanones). In addition, 5% of drivers initially roadside-tested negative for DOA were in fact driving after NPS use in this specific population. From a forensic perspective, these results confirm the reality of driving after NPS use in French drivers, notably in those driving to or from a music festival.

A decontamination process adding a tensioactive agent and isopropanol to a closed-system drug transfer device for better control of isolator contamination. A prospective, parallel study.

BACKGROUND: Despite the use of closed system drug transfer devices (CSTD), residual contamination from antineoplastic drugs is still detected inside isolators. The aim of this study was to compare the decontamination level obtained using a CSTD + standard cleaning procedure with a CSTD + standard cleaning procedure + specific decontamination procedure. METHODS AND FINDINGS: A comparative and prospective study was carried out in a newly opened compounding unit. Compounding was performed with a CSTD (BD-Phaseal, Becton-Dickinson). In the Control isolator (C), the cleaning process was completed daily with a standard biocide solution (AnioxysprayTM, Anios, France). In the Intervention isolator (I), weekly decontamination with a homemade admixture of sodium dodecyl sulfate 10(-2) M/70% isopropanol (80/20, v/v) was added. Monitoring was performed via a validated LC-MS/MS method. Eight drugs (cyclophosphamide, cytarabine, dacarbazine, fluorouracile, gemcitabine, ifosfamide, irinotecan and methotrexate) were monitored daily over 14 consecutive weeks on three sites inside the isolators: gloves, workbench and window. Results are presented as the odds-ratio (OR) of contamination and as overall decontamination efficiency (EffQ, %). The proportion of EffQ ≥ 90% was assessed by a Fisher's exact test (p<0.05). Overall contamination rates (CR, %) were significantly different from one isolator to the other (CRC = 25.3% vs. CRI = 10.4%; OR = 0.341; p<0.0001). Overall EffQ values (median; 1st and 3rd quartiles) were higher in the intervention isolator (I: 78.3% [34.6%;92.6%] vs. C: 59.5% [-5.5%;72.6%]; p = 0.0015) as well as the proportion of days with an EffQ ≥ 90% (I: 42.9% vs. C: 7.1%; p = 0.077) but very variable depending on drugs. CONCLUSION: Adding a decontamination protocol with a tensioactive agent to a CSTD leads to better control of chemical contamination inside isolators. Improving decontamination by increasing decontamination frequency or modifying the protocol will be further studied.

Effectiveness of a Closed-System Transfer Device in Reducing Surface Contamination in a New Antineoplastic Drug-Compounding Unit: A Prospective, Controlled, Parallel Study.

BACKGROUND: The objective of this randomized, prospective and controlled study was to investigate the ability of a closed-system transfer device (CSTD; BD-Phaseal) to reduce the occupational exposure of two isolators to 10 cytotoxic drugs and compare to standard compounding devices. METHODS AND FINDINGS: The 6-month study started with the opening of a new compounding unit. Two isolators were set up with 2 workstations each, one to compound with standard devices (needles and spikes) and the other using the Phaseal system. Drugs were alternatively compounded in each isolator. Sampling involved wiping three surfaces (gloves, window, worktop), before and after a cleaning process. Exposure to ten antineoplastic drugs (cyclophosphamide, ifosfamide, dacarbazine, 5-FU, methotrexate, gemcitabine, cytarabine, irinotecan, doxorubicine and ganciclovir) was assessed on wipes by LC-MS/MS analysis. Contamination rates were compared using a Chi2 test and drug amounts by a Mann-Whitney test. Significance was defined for p<0.05. Overall contamination was lower in the "Phaseal" isolator than in the "Standard" isolator (12.24% vs. 26.39%; p < 0.0001) although it differed according to drug. Indeed, the contamination rates of gemcitabine were 49.3 and 43.4% (NS) for the Standard and Phaseal isolators, respectively, whereas for ganciclovir, they were 54.2 and 2.8% (p<0.0001). Gemcitabine amounts were 220.6 and 283.6 ng for the Standard and Phaseal isolators (NS), and ganciclovir amounts were 179.9 and 2.4 ng (p<0.0001). CONCLUSION: This study confirms that using a CSTD may significantly decrease the chemical contamination of barrier isolators compared to standard devices for some drugs, although it does not eliminate contamination totally.

In vitro characterization of potential CYP- and UGT-derived metabolites of the psychoactive drug 25B-NBOMe using LC-high resolution MS.

One of the main challenges posed by the emergence of new psychoactive substances is their identification in human biological samples. Trying to detect the parent drug could lead to false-negative results when the delay between consumption and sampling has been too long. The identification of their metabolites could then improve their detection window in biological matrices. Oxidative metabolism by cytochromes P450 and glucuronidation are two major detoxification pathways in humans. In order to characterize possible CYP- and UGT-dependent metabolites of the 2-(4-bromo-2,5-dimethoxy-phenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25B-NBOMe), a synthetic psychoactive drug, analyses of human liver microsome (HLM) incubates were performed using an ultra-high performance liquid chromatography system coupled with a quadrupole-time of flight mass spectrometry detector (UHPLC-Q-TOF/MS). On-line analyses were performed using a Waters OASIS HLB column (30 x 2.1 mm, 20 µm) for the automatic sample loading and a Waters ACQUITY HSS C18 column (150 x 2 mm, 1.8 µm) for the chromatographic separation. Twenty-one metabolites, consisting of 12 CYP-derived and 9 UGT-derived metabolites, were identified. O-Desmethyl metabolites were the most abundant compounds after the phase I process, which appears to be in accordance with data from previously published NBOMe-intoxication case reports. Although other important metabolic transformations, such as sulfation, acetylation, methylation or glutathione conjugation, were not studied and artefactual metabolites might have been produced during the HLM incubation process, the record of all the metabolite MS spectra in our library should enable us to characterize relevant metabolites of 25B-NBOMe and allow us to detect 25B-MBOMe users.

UPLC-ESI-Q-TOF-MS(E) identification of urinary metabolites of the emerging sport nutrition supplement methoxyisoflavone in human subjects.

Methoxyisoflavone (5-methyl-7-methoxyisoflavone) is a synthetic isoflavone used by bodybuilders for its ergogenic properties. A recent study demonstrated that methoxyisoflavone metabolites can induce false-positive results in urinary immunoassay screening tests for cannabinoids, and only one metabolite has been identified. To improve the knowledge on the metabolic pathways of methoxyisoflavone, ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF) was applied. Urine samples were obtained from methoxyisoflavone regular users. After enzymatic hydrolysis and liquid-liquid extraction, the samples were analyzed by UPLC-Q-TOF fitted with an electrospray ionization source (ESI) operating under positive ion mode. Mass data were acquired with the MS(E) method. Five metabolites were identified. Those were divided into two metabolic pathways, depending on whether the B ring hydroxylation was preceded or not by the O-demethylation of the methoxy group. The MS(E) mass spectra of methoxyisoflavone and its metabolites are specific of isoflavones structures and revealed 1,3 retro Diels-Alder fragmentation and double CO loss. Losses of small neutral molecules CO and H2O, and radical CH3, typical of flavonoids, were also observed. This study illustrates the capacity of the sensitive UPLC-Q-TOF analytical system, combined with the MS(E) method of collection of fragmentation data, to rapidly elucidate the unknown xenobiotics metabolism.

Use of hair testing to determine methadone exposure in pediatric deaths.

A case of death attributed to methadone acute poisoning in an infant aged 11 months is reported. A sudden infant death syndrome (SIDS) was suspected, whereas a traumatic cause of death was excluded regarding autopsy findings. Specimens were submitted to a large toxicological analysis, which included ethanol measurement by HS-GC-FID, a targeted screening for drugs of abuse and various prescription drug classes followed by quantification using UPLC-MS/MS methods. Methadone and its metabolite (EDDP) were detected in all the tested fluids, as well as in hair, with a blood concentration of methadone considered as lethal for children (73 ng/mL). The cause of death was determined to be acute "methadone poisoning", and the manner of death was "accidental". A discussion of the case circumstances, the difficulties with the interpretation of toxicological findings in children (blood concentration and hair testing), and the origin of exposure are discussed.

Acute arsenic poisoning: clinical, toxicological, histopathological, and forensic features.

This report describes a suicide case by acute arsenic intoxication via intravenous injection. A 30-year-old woman injected arsenic As (V) (sodium arseniate disodique: Disodium Hydrogena Arsenik RP) in a successful suicide attempt. Three hours following administration, the woman developed severe digestive symptoms. She was admitted to a hospital and transferred to the intensive care unit within 12 h of the massive administration of arsenic. Despite therapeutic efforts, over the next 2 h she developed multiorgan failure and died. A postmortem examination was performed. Pulmonary edema and congestion of liver were apparent. As (V) and As (III) were determined by high performance liquid chromatography and inductively coupled plasma mass spectrometry after mineralization of samples by concentrated nitric acid. Toxicological analysis revealed high concentrations of arsenic in biological fluids as well as in organs. Histopathological examination showed a typical indication of myocarditis. These findings were in agreement with acute arsenic poisoning. The symptoms developed by this young woman (intoxication by intravenous administration) were comparable to oral intoxication. The clinical signs, survival time, and administration type are discussed in light of the literature on acute and chronic arsenic poisoning.

Screening of xenobiotics by ultra-performance liquid chromatography-mass spectrometry using in-source fragmentation at increasing cone voltages: library constitution and an evaluation of spectral stability.

In clinical and forensic toxicology, general unknown screening is used to detect and identify exogenous compounds. In this study, we aimed to develop a fast (15 min) comprehensive screening method for 500 toxicologically relevant analytes based on ultra-performance liquid chromatography (UPLC) coupled with a quadrupole mass spectrometer (MS) system operated in full scan mode. Data were acquired using both positive and negative electrospray ionization by scanning across the range m/z 80-650. For each ionization mode, data were also collected under multiple fragmentation conditions (i.e., at six different cone voltages). Consequently, each molecule could be characterized by a combination of retention time and up to a maximum of 12 individual spectra. Investigation of the 500 analytes resulted in the compilation of a library containing 2975 spectra. An assessment of the stability of these spectra was evaluated under various conditions, that is, the impact of increasing drug concentration and the presence of biological matrix. In addition, the transferability of the spectral library was assessed by comparison with data acquired using several other instruments of same model and from the same manufacturer. These data are presented in addition to the utility of the method for the analysis of routine clinical and forensic samples. Following extraction, identified compounds were compared to those found with two other techniques, one based on immunoassay and the other, on high-performance liquid chromatography-photodiode-array.

Major metabolites of zolpidem: expeditious synthesis and mass spectra.

An expeditious route to the two major metabolites of Zolpidem-and readily applicable to the synthesis of the drug-was established via a cyclization reaction between a 2-aminopyridine and a suitable alpha-bromoacetophenone. The structures of the target compounds were confirmed from a 2D (1)H-(15)N NMR correlation. Their mass spectra contribute to a reliable toxicological identification of the drug in the case of drug-facilitated crimes.

Neonatal hair analysis contribution to establishing a gestational drug exposure profile and predicting a withdrawal syndrome.

Recently, interest in hair analysis in such fields as drug abuse, driving, or for clinical purposes (determination of drug-exposed neonates especially) has grown because of the highly sensitive method of detection (GC-MS) that can now be applied. Neonates born to drug-addicted mothers can suffer from neonatal withdrawal syndrome (NWS), which requires morphine treatment in its severe forms. To assess and measure toxicologic factors predicting the appearance and the severity of this syndrome, matrices such as urine, meconium, and hair are necessary. Cannabinoids, opiates, cocaine (and its metabolites), and methadone in particular were determined in the various matrices collected in 17 mother/neonate pairs. An immunologic screening method was used, and quantification was achieved with GC-MS. In spite of some bias (color, length, race) that might hinder an accurate interpretation, the results of hair analysis makes it possible to confirm a fetal drug exposure and to reinforce the diagnosis of the NWS observed, particularly when results obtained in other matrices are negative. Hair analysis contributes to our ability to predict a NWS.

Hair analysis of opiates in mothers and newborns for evaluating opiate exposure during pregnancy.

The increasing interest in toxicological hair analysis as a marker of human exposure to xenobiotics such as illicit substances or therapeutic drugs, has been made feasible by the extension of mass spectrometry, a highly sensitive method of detection. A newborn exposed to drugs in utero can suffer from a varying degree of withdrawal syndrome, a few days after birth. If of opiate origin, the withdrawal syndrome can be treated with morphine, among other therapeutics, but it is not easy to diagnose because of atypical symptoms presented by neonates and especially when maternal drug addiction has not been revealed. To assess and measure toxicological factors linked with the appearance and the severity of this syndrome, maternal and neonatal matrices such as urine, meconium and hair were collected during a protocol approved by the ethical committee. Opiates in particular were measured with GC-MS and potential combined dependences (cannabis, cocaine, amphetamine, LSD and benzodiazepines) and/or substitutive therapeutics (methadone or buprenorphine) were also assessed in 17 mother/neonate couples. Gestational opiate exposure profiles were drawn up and linked with the observed withdrawal syndromes. A withdrawal syndrome seems to appear more frequently after foetal exposure to an association of opiates/substitutive molecules (8 out of 10 withdrawal syndromes observed in this study), although the impact of cocaine and benzodiazepines must also be taken into account. The results obtained in neonatal hair make it possible to affirm foetal drug exposure and are in accordance, for the majority, with the appearance of a neonatal withdrawal syndrome (NWS). Neonatal hair analysis could contribute to assess in utero exposure to opiates, particularly when results in urine and meconium are negative or when these matrices are not available.

1H NMR spectroscopy and GC-MS analysis of alpha-chloralose. Application to two poisoning cases.

Alpha-chloralose, a compound widely used as a rodenticide and in the control of bird pests, is readily available. Two cases of intentional poisoning are reported. Both patients became comatose and presented hypersialorrhea and myoclonal crises in the legs. They were discharged from hospital after several days. As clinical signs of alpha-chloralose poisoning lack specificity, anamnesis might be difficult, particularly in the case of delayed diagnosis. Toxicological analysis is therefore critical, and this article reports the investigation of serum and urine samples by gas chromatography-mass spectrometry (GC-MS) in the electron-impact mode, and by 1H nuclear magnetic resonance (1H NMR) spectroscopy. Non-hydrolyzed urinary samples and those hydrolyzed by beta-glucuronidase were taken into consideration. After acetylation, GC-MS analysis was based on characteristic mass-to-charge ratio values of 272 for alpha-chloralose and 206 for beta-hydroxyethyltheophylline, which was used as internal standard. Characterization of alpha-chloralose species by 1H NMR spectroscopy was performed taking two parameters into account: chemical shift and coupling-constant values. Without any pretreatment, 1H NMR spectroscopy revealed the presence of free (5.50 and 6.15 ppm) and conjugated forms of alpha-chloralose by characteristic resonances of H1 and chloral-type protons, respectively. Quantitative analysis was performed by relative integration of peak areas. Serum alpha-chloralose showed concentrations below the quantitation limit of both methods. In urine samples, the free chemical species rapidly decreased. GC-MS analysis revealed the predominence of conjugation after a beta-glucuronidase hydrolysis step. 1H NMR analysis directly showed that on admission of the first patient, average urinary concentrations were 1.73 mmol/L (535 mg/L) for the free form and 13.72 and 6.25 mmol/L for the two conjugated forms. A later enzymatic treatment confirmed the total concentration of alpha-chloralose chemical species. Analysis of alpha-chloralose in urine by either GC-MS or 1H NMR spectroscopy methods proved to be comparable.