Toxicologie Analytique et Clinique

Published by Elsevier
Print ISSN: 2352-0078
Aims After 11 years coding in the same data base, to make a descriptive analysis of 24 hours/24 7 days/7 calls managed by the poison center of Marseille (CAPM), to help complete the rare French epidemiological data in clinical toxicology. Methods Retrospective study of all cases treated by the CAPM from 01/01/2002 to 31/12/2012, all encoded in SICAP, the French poison centers’ common software. The analysis focused on the description and the evolution of enquirers, type of request and, if applicable, patients’ demographics, circumstances of exposure, and classes of implicated products. Results Two hundred and seventy-three thousand four hundred and nine files were collected (61% from public, 34% from health professionals), including 256,875 cases of exposure for 269,001 people (median age: 13 years old – 40% under 4 –; 53% women). Eighty-three percent were accidentally exposed, 12% in a suicidal situation. The most commonly found agents are pharmaceutical drugs (concerning 33% of accidentally exposed subjects, 80% of deliberately ones), followed by chemicals and household products. Three hundred and thirty-six fatal cases were collected (56% male, median age: 49 years), of which 67% in voluntary poisoning context. Concerning time trends, there is a decline in professionals’ requests vs. public ones, an increase in accidental exposures part, and a lower part of exposure to drugs, primarily anxiolytics, hypnotics and antidepressants; at the opposite, paracetamol poisonings are steadily rising. Conclusion This report highlights the richness and potential of data from poison centers to explain, treat and prevent poisonings. However, this efficient tool remains very fragile and requires a rapid and significant support.
Objective 4-Methyl-N-ethylcathinone (4-MEC) is a novel designer drug of the β-keto amphetamine family. A case of a mixed poisoning by 4-MEC and gamma-butyrolactone (GBL) is described. Two and half hours after an injected 0.25–0.5 g 4-MEC dose and thirty minutes after GBL ingestion, a 39-year-old man became unconscious and presented after admission to the hospital, transient apnea needing oxygenation and stimulation. After 30 min, he woke up, but remained confused for 4 hours. He was discharged 13 hours later without particular symptoms. For confirmation of the poisoning, different analytical procedures such as HPLC-UV/DAD, GC-MS, UPLC-MS/MS, and LC-HR-MS/MS were applied. Blood and urine levels (8 h after injection) were respectively 353 μg/L and 100 mg/L for 4-MEC and 300 mg/L and 1000 mg/L for gamma-hydroxybutyric acid. In urine, three metabolites (dihydro-, nor-, and nor-dihydro-4-MEC) could be identified for the first time and were similar to those described for other molecules of this class.
Objectives This paper has 2 main objectives: 1. to develop and validate a method for testing in whole blood for cannabinoids, including Δ9-Tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), and acid 11-nor-Δ9-tetrahydrocannabinol carboxylic acid (THC-COOH), according to NF EN ISO 17025 requests and taking into consideration the consensus of the SFTA 14 June 2013; and 2. to discuss the critical points that where addressed by the body of accreditation (COFRAC). Methods After liquid-liquid extraction of 1mL sample (hexane / ethyl acetate, 80/20, v/v), and derivatization of the dry extract by BSTFA-1% TMCS, cannabinoids and their corresponding deuterated standards (THC-d3, 11-OH-THC-d3, THC-COOH-d3) are injected into the analytical equipment. The system consists of a gas chromatograph Trace1310, a AI1310 autosampler and a tandem mass spectrometer TSQ8000. Results The French law n° 2003-87 of 3rd February 2003 states that THC must be detectable at 1ng/mL. The method was linear from 0.5 ng/mL to 25 ng/mL for THC and 11-OH-THC, and 0.5 ng/mL to 75 ng/mL for THC-COOH, respectively. The repeatability and reproducibility were established at two levels, a low concentration of 1.5 ng/mL for the 3 molecules and a high concentration of 18.5 ng/mL for THC and 11-OH-THC, and 40 ng/mL for THC-COOH. The method meets the criteria of the SFTA, with a LOQ of 0.5 ng/mL. The extraction efficiency ranged from 55% to 90% over three concentration levels: 1-10-25 ng/mL for THC and 11-OH-THC, and 1-20-75 ng/mL for THC -COOH. The carry-over was verified and calculated to be less than 5%. View Within Article Conclusion The validation of the method demonstrates that it respects predetermined performance criteria. However, during the COFRAC inspection, several issues were discussed, including the definition of the pre-analytical step (the laboratory has to inform in written the requesting authorities of all potential problems) and the set up of a back-up method on another machine in case this one would be out of order (use of a Trace1310/ITQ700, already available in the laboratory). Traceability of internal quality controls had to be redesigned with specific correction of the peak areas integration. The method is now accredited and used in routine after receiving a positive opinion from COFRAC. This presentation will mainly be focussed on the specific requests that were addressed during the inspection visit of the body of accreditation.
Aims In 2008, some synthetic cannabinoids named “Spice” were detected in herbal smoking mixtures and became popular owing to their strong cannabimimetic effects after smoking. Many other products have appeared since and are constantly changing in attempts by manufacturers to evade legislation. Hair samples, which generally contain the parent compounds, could be of interest to screen for these synthetic cannabinoids using a poor selective extraction method and a highly specific and sensitive analytical detection technique. A simple method was developed and validated for the quantitative detection of 18 synthetic cannabinoids, AM-694; AM- 2201;CP 47,497; HU-210; JWH-007; JWH-015; JWH-018 and its N-Pentanoic acid metabolite; JWH-019; JWH-020; JWH-073; JWH-081; JWH-122; JWH-200; JWH-203; JWH-210; JWH-250 and WIN 55, 212-2 in hair by HPLC-MS/MS and its application to authentic specimen. This work belong to a pilot study evaluating the prevalence of synthetic cannabinoids use in France and conducted with two other labs2,3. Methods After decontamination with methylene chloride, hair sections were cut into small pieces, weighed and incubated in methanol, overnight at 40 °C, in the presence of deuterated internal standards (JWH-018-d9, THC-d3, 11-OH-THC-d3, THC-COOH-d3). Finally, the organic phase was evaporated to dryness and reconstituted in mobile phase. The HPLC-MS/MS system consisted of an UFLC (Shimadzu Prominence) coupled with a triple-quadrupole ABSciex 5500 QTRAP instrument. Chromatographic separation was achieved on a Kinetex™ XB-C18 (2.1×100mm, 2.6μm) column. Tandem mass spectrometry was employed using an electrospray interface in positive and negative ionisation mode and acquisition was performed in scheduled MRM mode. Results The method was linear from LQ to 500pg/mg with quantification limits ranging from 0.5 to 5 pg/mg for all the targeted synthetic cannabinoids. Intra and inter-day precisions were below 20% at 0.5; 10; 50; 75 and 250 pg/mg. Intra and inter-day accuracies were established to be between 80% and 120% for the same concentration levels. There was no carry over and the absence of significative ion suppression phenomena was controlled. Sixty-five authentic hair specimens obtained from our routine activity (French forensic cases) were screened for synthetic cannabinoids. None of the specimen was found positive for the 18 targeted compounds. These preliminary results are in favour with a current very low prevalence of synthetic cannabinoids use in France, and are in accordance with those obtained by the two other labs which investigated urine specimens for the same 18 compounds. Conclusion Regarding the rising scientific interest about the synthetic cannabinoids, it appears necessary to develop an accurate, sensitive, but also evolutive method for the detection and quantification of synthetic cannabinoids in hair. Liquid chromatographymass spectrometry was deemed the most suitable analytical technique for its accuracy and sensitivity, particularly after a simple and unique direct extraction step from hair using methanol. This first method version was developed and fully validated for the large screening of 18 synthetic cannabinoids in hair.
Among the available neuroleptic drugs, amisulpride is increasingly prescribed. Thanks to its dose-dependent selective affinity for dopamine receptors, it is possible to modulate the effects of this substance showing antidepressant properties at low dose and acting as a powerful antipsychotic at high dose. However, amisulpride induces a dose-dependent prolongation of the QT interval which may potentiate the occurrence of ventricular arrhythmias, often fatal when its consumption is concomitant with those of other drugs that prolong the QT interval. However, the scientific literature remains poorly documented with regard to the determination of therapeutic, toxic and lethal blood concentrations. After presenting the pharmacodynamic and pharmacokinetic properties of amisulpride, we propose to define more precisely the blood levels of therapeutic, toxic and lethal concentrations for this molecule illustrated by a review of the fatal cases involving amisulpride in Western Switzerland since 2005.
Chloralose has a sedative and stimulating effect on the central nervous system. It is currently used as rat poison. Chloralose poisoning is responsible for seizures and sometimes death. The aim of our study was to evaluate the epidemiology of those poisonings in the emergency department of the university hospital of Pointe-à-Pitre to optimize the management of chloralose poisoning. Procedure This is a retrospective study between 01/01/2009 and 31/12/2012. The files were included after computer search from the emergency department software. Results Seventeen cases were included. No deaths were recorded. Seventeen percent of poisonings involved minors (3/17). The place of intoxication was mostly home (16/17). In three quarters of cases poisoning was voluntary (13/17). No pediatric poisoning was deliberate. The dominant symptoms were seizures and nicotinic signs. The hospitalization rate was 65% (11/17), and among its 27% (3/11) was in intensive care. Conclusion Chloralose poisoning is rare and not deadly. But increasing awareness among physicians to recognize signs of chloralose intoxication would allow earlier and a more appropriate care for these patients, including a wider and earlier use of benzodiazepines, thus preventing seizures and the use of intubation. The establishment of a register of intoxications would allow a better monitoring of the epidemiology of these intoxications.
Objective To achieve a annual report of calls received by the ten French poison centers about exposures of children. Method We analysed data of the French poison centers’ national poison data system (SICAP) for young people exposure occurred between 1 January 2012 and 31 December 2012. Results In 2012, 187,028 calls for human exposures were logged by SICAP – 86,478 calls (46%) involved children younger than 4 years of age and 15,157 (8%) children aged 10 to 19 years. The most common exposures were drugs (40%). The reason category for exposures was accidental in 90% of children aged 1 to 4 years and therapeutic error in 29% of children younger than 1 year. In the age range 10–19 years there were 46% intentional exposure. The mortality rate is 0.006% in children younger than 10 years and 0.07% in the age range 10–19 years. Conclusion The ten French poison centers automatically upload their cases in the national database “SICAP” and are able to produce, from a significant number of cases, comprehensive assessments on categories of population, circumstances, categories of agents, and to compare the data with those of other countries.
Introduction Synthetic cannabinoids are the most common drugs among an expanding array of compounds that mimic the effects of traditional illicit psychoactive substances. A few methods have been developed for the determination of the parent synthetic cannabinoids in the keratin matrix, opening the scientific debate about how to interpret the quantitative results. In particular, the discrimination between (i) passive or active exposure, and (ii) chronic consumption and occasional use, appears to be essential within the forensic context. Just as occurred for the traditional drugs of abuse, the detection of drug metabolites is likely to represent the key factor to support the decision procedure. Therefore, we updated our existing method [A. Salomone et al., Drug Testing and Analysis, 2014, 6, 126–134] in order to complement the determination of 22 synthetic cannabinoids in human hair with 10 of their metabolites. Afterwards, we applied the new method to 15 real hair samples which previously tested positive for at least one synthetic cannabinoid [A. Salomone et al., cited], in order to verify the presence of metabolites. Methods The analytical method was validated in accordance with the criteria and recommendations of national and international guidelines. The following parameters were investigated: selectivity, specificity, linearity range, detection and quantification limits (LOD and LOQ), intra-assay and inter-assay precision and accuracy. Carry-over effect, recovery and matrix effects were also investigated. Two groups of subjects, namely driving re-licensing and drug abuse/withdrawal control subjects, provided hair samples that had previously resulted positive to at least one common drug of abuse and one synthetic cannabinoid. In this procedure, the samples were added with 1 mL NaOH 1N and subsequently incubated at 95 °C for 10 min. Immediately afterwards, the samples were extracted with 5 mL of n-hexane/ethylacetate 90:10 (v/v). The aqueous phase was newly added with glacial CH3COOH to reach a pH of 3.5–4.0 and again extracted with n-hexane/ethylacetate 90:10. The two organic phases were unified, dried under a nitrogen flow at 70 °C and reconstituted with 50 μL of methanol. An aliquot of 1 μL methanol solution was directly injected into the UHPLC-MS/MS system. Results The optimized UHPLC – MS/MS method allowed the simultaneous determination of 22 synthetic cannabinoids, 10 metabolites and 6 internal standards. The whole chromatographic run, comprehensive of the time required for column re-equilibration before the following injection, was completed in 8.0 min. Retention times ranged between 2.2 min (WIN 48,098) and 5.5 min (JWH- 020). Two samples out of 15 resulted positive to one metabolite, namely the N-(5-hydroxypentyl) metabolite of JWH-122. The calculated concentrations were 2.5 pg/mg and 0.72 pg/mg. In the same samples, the drug parent concentrations were, respectively, 2800 pg/mg and 760 pg/mg. Conclusion The present preliminary study describes for the first time an extensive search of metabolites of synthetic cannabinoids in human hair. Undoubtedly, the detection of metabolites in hair may represent the decisive factor to support the interpretation of hair analyses, particularly with respect to potential sources of external contamination. More samples will be analyzed in order to (i) verify to what extent the presence of metabolites can prove the active consumption, (ii) evaluate the concentration ratios between parent drugs and metabolites, and (iii) elaborate tentative cut-offs for parent drug and metabolites.
Introduction The human hairs occupy a prominent place as markers of exposure to xenobiotics in the domain of forensic toxicology. However, interest of using this matrix is a considerable improvement for the assay of metals, also, chromium has attracted the attention of toxicologists because the accidents observed in industrial settings using this metallic element (cement industry, paint, leather, automotive…). Objective This study related to the assessment of worker exposure in a tannery, located in Algiers, more accurately on the Rouiba-Reghaïa industrial estate, by measuring the capillary and urinary chromium of the population groups investigated, then study the correlation between total contents chromium in hair and urine. Patients and methods The study was carried in September 2012, and focused on 50 subjects exposed (49 men and one women) and 16 controls. It was preceded by the establishment analytical development and validation of an analytical method for the determination of chromium in hair and urine by graphite furnace atomic absorption spectrometry (GFAAS). Statistical calculations were performed using the software LXSTAT MS Excel 2012. Results and discussion The hair chromium average of the tanning workers are significantly higher (urinary Cr = 2.48 μg/L, Cr capillary = 4.93 ng/mg) than other groups. Washing the hair appears to be effective for decontaminating the exogenous chromium, this latter may reflect a recent exposure to chromium. Conclusion Human hair may offer the advantage for biological monitoring, first, to get information on the use of means of protection, and give some idea about the route of exposure (inhalation or ingestion).
Introduction The present study aimed to use the syringe exchange setting in order to facilitate transfer or heroin users into evidence-based substitution treatment with methadone or buprenorphinenaloxone. Out-of-treatment heroin-dependent patients were recruited from a syringe exchange program and actively referred to a research substitution treatment facility. Among 75 patients who were included and underwent baseline evaluation, 71 were successfully referred and started treatment. Segmental hair analysis was used to investigate previous drug abuse, abstinence from drugs of abuse during treatment as well as to confirm compliance to substitution treatment. Hair samples were taken at admission and then after approximately 3 months of treatment. Methods Hair samples were segmented into 5 segments (5, 5, 5, 10, 10 mm) and subjected to qualitative UHPLC-TOF screening for 30 drugs of abuse to elucidate previous drug use patterns. Hair samples were also segmented into 5 segments, washed and quantitation of mu-agonists and sedatives were performed using LC-MSMS including the substitution medications methadone and buprenorphine. Results Base line results from the UHPLC-TOF screening indicated an extensive poly-drug abuse of different opioids including heroin (74%), methadone (79%), fentanyl (16%), buprenorphine (16%) and tramadol (26%) as well as use of stimulants such as amphetamine (32%) together with sedatives (74%) of both the common benzodiazepine group and zopiclone and zolpidem. The segmental analysis showed even or decreasing concentrations of 6-acetylmorphine in the hair confirming continuous use of heroin but suggesting infrequent use or lower doses prior to inclusion. After the initiation of treatment the use of illicit opioids decreased dramatically and most subjects abstained from heroin when given methadone or buprenorphine. 50% had no positive segment during the treatment period and 15% were only positive in one or two of the distal segments which could be interpreted as remnants from dormant hair. However, the use of benzodiazepines and other sedatives (80%) was very common during treatment with alprazolam and clonazepam as the most frequent encounters. The mean methadone dose was 85 mg/d (N=12) and the mean concentrations of EDDP and methadone were 0.46 ng/mg and 8.8 ng/mg, respectively. The concentrations of methadone in hair did not correlate to the dose between subjects but there were clear relationships within patients when doses were escalated during titration. Three patients continued to use heroin and two continued their use of amphetamine. In two of these subjects the methadone concentrations from decreased to half the initial concentrations suggesting that the prescribed dose was diverted rather than taken. Buprenorphine was only detected in 1 patient and norbuprenorphine in 3 patients even though doses between 8 and 24 mg/d was given (N=7). Conclusion We conclude that segmental hair analysis was a good clinical tool to assess drug use histories prior to inclusion into substitution treatment and to prove abstinence from other drugs during treatment. The dose concentration relationship was weak but may be used within a patient to investigate dose changes or non-compliance for methadone. We believe that data from living subjects entering treatment may provide a basis for interpretation of acute heroin overdoses where abstinence have been suggested as a major factor contributing to death.
Aims Contrary to the illegal use of any form of manufactured cocaine, chewing of coca leaves and drinking of coca tea is allowed and is very common and socially integrated in several South American countries. Because of this different legal state, an analytical method for discrimination between use of coca leaves and abuse of artificial cocaine preparations is required. In this study, the applicability of hair analysis for this purpose was examined. Methods Hair samples from 26 Argentinean coca chewers and 22 German cocaine users were analysed for cocaine (COC), norcocaine (NC), benzoylecgonine (BE), ecgonine methyl ester (EME), cocaethylene (CE), cinnamoylcocaine (CIN), tropacocaine (TRO), cuscohygrine (CUS) and hygrine (HYG) by hydrophilic interaction liquid chromatography (HILIC) in combination with triplequad mass spectrometry (QQQ-MS) and hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS). Results The following concentrations (range, mean, median, ng/mg) were determined in hair of the coca chewers: COC 0.085–75.5, 22.7, 17.0; NC 0.03–1.15, 0.27, 0.12; BE 0.046–35.5, 10.1, 6.1; EME 0.014–6.0, 1.04, 0.66; CE 0.00–13.8, 3.06, 0.38; CIN 0.005–16.8, 2.82, 0.79; TRO 0.02–0.16, 0.035, 0.023; CUS 0.026–26.7, 2.45, 0.31. In lack of a reference substance, only qualitative data were obtained for HYG and two metabolites of CUS were detected which were not found in hair of the cocaine users. For interpretation, the concentrations of the metabolites and the coca alkaloids in relation to cocaine were statistically compared between coca chewers and cocaine users. By analysis of variance (ANOVA) significant differences were found for all analytes (α = 0.000 to 0.030) with the exception of TRO (α = 0.218). The ratios CUS/COC, CIN/COC and EME/COC appeared to be the most suitable criteria for discrimination between both groups with the means and medians 5-fold to 10-fold higher for coca chewers and a low overlap of the ranges between both groups. The same was qualitatively found for HYG. Screening for typical cutting agents led to detection of levamisole (21x), lidocaine (6x) and paracetamol (3x) in the 22 samples from German cocaine users whereas no levamisole, lidocaine (3x) and paracetamol (1x) was found in hair from the Argentinean coca chewers. Conclusion Hair analysis proved to be a suitable tool for discrimination between chewing of coca leaves and use of manufactured cocaine by detection of other coca alkaloids and their concentrations relative to cocaine, and by the absence of typical adulterants. However, these criteria have to be confirmed for South American cocaine consumers including smokers of coca paste because of possible different composition of illegal cocaine preparations and other use habits. They cannot exclude occasional cocaine use in addition to chewing of coca leaves.
Intentional inhalation of volatile organic compounds (VOC) from deodorant or cigarette lighter refills causes euphoria or hallucinations and even death in adolescents. There seems to be a rise in this underestimated cause of death among adolescents. This rising cause of death goes by different mechanisms: suffocation, respiratory depression, trauma, transient increase in airways resistance, and even “sudden sniffing death syndrome, i.e. cardiac sudden death”. This is the case of a healthy young 15-year old boy who died secondary to inhalation of deodorant VOC in a friend's house. The patient was found with no respiratory and cardiac output by the French Urgent Resuscitation Medical Unit (SMUR). SMUR resuscitation manoeuvres were unsuccessful. Postmortem toxicological investigations revealed the presence of 4.7 μg/L isobutane in the blood thanks to head space – gaz chromatography (HS-GC). This very high concentration of isobutane explains the adolescent's death through a “sudden sniffing death syndrome”. This syndrome leads to hemodynamic failure due to different physiological complications. In some cases, coronary acute spasms or cardiac arrhythmia due to myocardial hypersensitivity to catecholamine (i.e. induced by isobutene) have been reported. In our case, the latter cardiac failure was confirmed according to the autopsy report. This case report alerts the public health care actors on the adolescent mortality risk associated with the misuse of isobutane, a VOC present in a range of readily everyday products. We suggest a dosage revising of such VOC utilisation in the prevention of adolescent-related mortality.
Introduction Among human remains from museum collections, mummies have intrigued researchers for a long time. There are subject to many researches in order to understand the mummification processes, to improve conservation and restoration protocols and to infer some interesting clues about the corresponding civilizations. We focus here on a corpus of Chilean mummies from the San Miguel de Azapa (Arica, Chile). The present work aims to study the conservation state of hair and to detect the heavy metal presence [Boston & al. (2009). Interciencia, 34:5, 338–343]. These aspects are related to the environmental conditions in which the individual lived, as well as various hair treatments performed. Furthermore, the molecular fingerprint of the material may have been modified by anthropic or natural alterations [Charrié-Duhaut & al. (2007). J. of Archaeological Science, 34, 957–967]. This complexity makes this research a true analytical challenge. Methods The hairs of mummies were first dated by measurements of carbon 14 using accelerator mass spectrometry (AMS) [Richardin & al. (2011). Archaeol. Anthropol. Sci., 3, 379–384; Richardin & al. (2013). Radiocarbon, 55:3–4, 345–352]. We undertook morphological and structural analyses (scanning electron microscopy – SEM – and infra-red spectroscopy), to obtain a global overview on the conservation state of hair. The heavy metals presence was studied by X-ray fluorescence and by SEM-EDS (energy dispersive X-ray spectroscopy). We are currently developing a proteomics approach (MALDI-MS and nanoLC-MS/MS analyses) in order to find out the amino acid sequences of keratin molecules and their alterations [Barthelemy (2011). PhD thesis, University of Strasbourg]. Results Radiocarbon dating has shown that the mummies are from the Archaic (8000–1000 BC) and Formative (1000 BC – 500 AD) periods. According to the first analyses, hair surfaces (cuticle) seem well preserved. On the surface and inside the hair fiber, lead, iron and bromine are detected in significant amounts, and arsenic is also present in some cases. Conclusion This work enables to better situate the corpus of mummies in their historical context, and to deny the fact that they came from the archaic Chinchorro. On the other hand, the detection of arsenic in some hair samples seems to suggest that some individuals suffered from chronic poisoning due to arsenic. The proteomics approach, currently in progress, should enable to confirm the first conclusions on the good preservation state of the hair and to study the interactions between heavy metals and keratin molecules. In this regard, it is crucial to develop as a first step an extraction protocol of the hair proteins, in order to preserve the non-covalent interactions.
Introduction If narcotics police officers or other persons handling drug material at work are suspected of consuming drugs, hair analysis may be used to prove or refute such suspicion. However, it is known for many drugs that differentiation of actual drug use from external contamination can be challenging or sometimes impossible. The aim of this study was to evaluate the extent of external contamination caused by handling of synthetic cannabinoid containing drug material under realistic conditions in a forensic laboratory. Methods Hair of laboratory workers was systematically analyzed for synthetic cannabinoids with a validated LC-MS/MS method after a big seizure of legal high products had to be analyzed in our laboratory. Hair samples were taken two days after the last exposure and again one week later. In addition, hair samples of laboratory staff not directly in contact with the drug material and close relatives of exposed subjects were analyzed to check for cross contamination. Results All samples of persons who were in direct contact with drug material were tested positive for at least one of the synthetic cannabinoids (JWH-018, JWH-073, JWH-081, JWH-122, JWH-210, JWH-307, JWH-368, AM-2201, AM-2201 indazole derivative, AM- 2232, MAM-2201, RCS-4, XLR-11, 5F-PB-22, RCS-4 ortho isomer). Concentrations ranged from trace amounts up to a maximum of 170 pg/mg (JWH-210) and roughly reflected duration and intensity of exposition. There was no significant decline in concentrations from sample 1 to sample 2 (one week later). Unexpectedly, also subjects without direct contact to drug material showed measurable hair concentrations. In one case, a hair sample (21 cm) was taken 10 weeks after the last exposition with plant material. In this case, relevant concentrations of 5F-PB-22 were detected with an increase of concentrations from distal to proximal segments (7.9 – 20 pg/mg). Conclusion Depending on duration and intensity of exposition, relevant concentrations of synthetic cannabinoids may be found in hair samples of persons exposed to these drugs at work. Unexpectedly, even cross contamination from an exposed person to a close relative may occur and lead to (false) positive hair findings. Concentrations caused by contamination are in the typical range found in known users of these drugs and could lead to wrong conclusions. In contrast, detection of metabolites could strongly suggest an actual consumption. However, we did not detect such metabolites so far even in samples of known consumers of synthetic cannabinoids showing extremely high concentrations of the unchanged compounds. Therefore, body fluids have to be analyzed to unambiguously prove use of these drugs.
In heavy alcohol consumption laboratory tests represent an objective evidence. In this study we compared older and newer biomarkers in blood and in hair. Carbohydrate-deficient transferrin (CDT), ethyl glucuronide (EtG), AST, ALT, GGT, MCV were measured in a large sample (n = 562). All people declared no alcohol consumption within the last 3 months. Serum CDT was measured by the candidate HPLC reference method and expressed as relative amount of disialotransferrin (%DST: cutoff 1.7%). EtG was measured in hair by a validated in-house method by LC-MS/MS (cutoff 30 pg/mg). Respectively, 42 (7.5%) and 76 (13.5%) subjects were positive to CDT and EtG. In particular, 30 (5.3%) subjects were positive to both tests, 12 (2.1%) only to CDT, while 46 (8.2%) only to EtG. The agreement (positive and negative pairs) between CDT and EtG was 89.7%. Interestingly, 6 out of 12 (50%) CDT-positive subjects had EtG < 15 pg/mg, whereas 27 out of 46 (59%) EtG-positive subjects had CDT < 1.1%. Forty-one out of 76 (54%) EtG-positive subjects display EtG values within 30-50 pg/mg. Large variability exists between CDT and EtG in detecting chronic alcohol consumption. We suggest to use CDT, or a combination of different biomarkers, to identify alcohol abuse in a forensic context. EtG results close to the cutoff (30-50 pg/mg) should be cautiously considered before any sanction is assigned. © The Author 2015. Medical Council on Alcohol and Oxford University Press. All rights reserved.
Introduction Ethyl glucuronide (EtG), a minor alcohol metabolite, accumulates in hair and is proposed as a stable marker for the detection of excessive alcohol consumption above a cut-off level of 30 pg/mg hair. A correlation between drinking behavior and EtG hair concentrations is observed, but variability exists, which could partially be explained by gender differences. Aims To investigate (1) the correlation between alcohol consumption and hair EtG in alcohol dependent patients, and (2) the effect of gender differences on this correlation. Methods The first 0–3 cm hair segments were analyzed using gas chromatography mass spectrometry (GC-MS) in negative ion chemical ionization (NICI) mode. Detailed retrospective alcohol consumption was obtained on a day per day basis over the last 3 months using the Timeline Follow Back Interview in alcohol dependent individuals starting an alcohol detoxification program. Results Thirty-six Caucasian alcohol dependent individuals (25 males/11 females; mean age 45 ± 8 years; mean BMI 25 ± 6) participated. Median alcohol consumption over the last three months (90 days) was 13050 g alcohol (range 65 – 650 g alcohol/ day). Hair EtG concentrations varied between 32 pg/mg hair and 662 pg/mg hair. There was a good linear and positive correlation between hair EtG and the amounts of alcohol consumed (Pearson r = 0.83; p < 0.001), which were observed both in males (Pearson r = 0.82; p < 0.001) and females (Pearson r = 0.78; p = 0.005). Conclusions Our data confirm the good linear correlation between hair EtG and amounts of alcohol consumed, extending findings to alcohol dependent individuals. Additionally, we show that gender differences do not play a crucial role in the interpretation of hair EtG values for evaluating the amounts of alcohol consumed. As a result, no separate cut-off values are necessary for male and female alcohol abusers. These findings should be replicated in recreational alcohol consumers to assess possible gender differences in the interpretation of lower hair EtG values.
Objective Binge drinking sometimes induces high blood alcohol levels requiring hospitalization. As ethanol was reported to produce respiratory depression, we aimed to characterize respiratory effects of intraperitoneal ethanol in young-adult rats. Method The respiratory parameters were measured by whole-body plethysmography; body temperatures were measured using telemetry devices, intraperitoneally implanted and blood-ethanol concentrations, from femoral artery samples, were assayed by GC/FID. Study took place over 240 minutes. Results Clinical signs: more the ethanol dose grown, more the animals were symptomatic. Higher doses rapidly induced coma and marked hypothermia. Respiratory effects: only rats in higher dose groups showed a trend to tachypnea without modification of minute volume, associated with acidemia. Blood studies: blood-ethanol levels were consistent with human exposure from moderate consumption to binge drinking. Non-compartmental analysis of kinetics showed a decrease in apparent clearance of ethanol with dose. Conclusion All observed effects occurred in higher dose groups. However, respiratory alterations are weak. Only a slight trend to tachypnea was observed which could be induced by action of ethanol or its metabolites. Major measured effects are metabolic and clinical. Acidemia was certainly caused by ethanol acid metabolites. Coma and hypothermia may be due to ethanol action on central system. Finally, origins of decrease in ethanol apparent clearance are less simple. At least three different causes can be listed: direct influence of hypothermia; physiological consequences of hypothermia or alcoholization; enzymatic inhibition. Results of this study did not confirm respiratory depressive action of ethanol at doses comparable to binge drinking in rats.
A 23 year-old man, health care professional, was found dead in the toilets of a local hospital. Medical supplies for injection (syringe, needles) were found near the body at the scene, in a waste. External body examination revealed a single point of injection located at the left elbow crease and the lack of any traumatic injury. During examination, the pathologist collected cardiac blood and urine. These specimens were tested for ethanol, volatiles, pharmaceuticals and drugs of abuse, using headFspace GC/FID and GC/MS, Elisa, LC-DAD, GC/MS and LC/MS/MS. Ethanol tested positive in blood (0.99 g/L) and urine (0.19 g/L). Using a dedicated LC/MS/MS procedure, alfentanil was identified in both blood (19 ng/mL) and urine (25 ng/mL). Morphine was identified in blood, at 36 ng/mL (free morphine) and 39 ng/mL (total morphine). In urine, total morphine concentration was 81 ng/mL. No other drug was detected. Given the ratio (0.92) free morphine to total morphine in blood and the low concentrations of both alfentanil and morphine in urine, it was considered that the death occurred rapidly after drugs administration. The manner of death was considered as acute intoxication of both alfentanil and morphine, in presence of ethanol.
Introduction The quantitative determination of ethyl glucuronide (EtG) in scalp hair has become established in both clinical and forensic toxicology as a direct marker for alcohol intake. It is a useful tool in the retrospective assessment of alcohol consumption, providing a specific window of detection that depends on the length of the hair sample. However, scalp hair is not always available, e.g. due to cosmetic reasons or bodily changes like alopecia. The main focus of this study was on statistical evaluation of our routine cases and the practicability of non-head hair in the context of the recommended interpretation threshold of 7 pg/mg EtG in hair. Additionally, a comparison of hair from different alternative anatomical sites (chest, axilla, arm, leg, and beard) was made. Pubic hair samples were not investigated. Methods Two different analytical methods to analyze EtG in hair samples were used. One was a GC-NCI-MS/MS method with SPE clean up (OASIS Max) and PFPA-derivatisation and the other one a LC-MS/MS method; both were validated. Our routine work, e.g. during the assessment for re-granting the driver's license, includes several 1,000 cases per annum. Thereof, 9% were body hair samples, mainly chest and leg hair, and could be analyzed. In a recent paper, an intra-individual comparison of EtG quantified in scalp and nonhead hair was presented (A. Pianta, B Liniger, M. R. Baumgartner, Alcohol and Alcoholism, 48 (2013). 295 – 302). Additionally, we compared hair samples of one type (e.g. leg hair) collected from different regions (e.g. left and right leg, front and back side). Results and Discussion EtG concentration could be determined in arm hair samples (in 0.5% of all routine cases), in leg hair samples (4.4%), in chest hair samples (3.9%), and in beard hair samples (0.1%), respectively. The gender distribution of our overall case work is 87% men and 13% women. As far as body hair samples are concerned, only four arm hair samples from women (less than 0.1%) were tested during the last few years. From all the subjects that were tested for long term teetotalism (no alcohol intake during more than 6 months), the percentage of negative cases for head hair was 83%, for leg and chest hair 75%, for arm and beard hair 67% and 64%, respectively. Repeated analysis of body hair revealed no larger inhomogeneity than for head hair. Conclusion Chest, arm, leg, and beard hair samples may represent a suitable alternative to monitor alcohol intake, especially for assessment of long term teetotalism. Admittedly, due to today's beau ideal, body hair can usually be collected only from men.
Anserine (Chenopodium ambrosioides) is one of the medicinal plants with a widespread and unmarked consumption in Morocco. We compiled three new observations of liver toxicity after consumption of anserine during consultations within the Internal Medicine Department at the Instruction Military Mohammed V Hospital of Rabat. These observations incite to strengthen phytovigilance strategies and more awareness for the medical personnel to systematically integrate, in their diagnostic approach, the possibility of direct toxicity by a medicinal plant or its drug interaction effects. Raising public awareness against the irrational and abusive use of this plant is highly recommended.
We reported a case of a 6-month-old baby girl who was hospitalized in the pediatric emergency for central nervous system disorders then coma. Toxicology analysis showed the presence of amitriptyline (AMI) and its metabolite nortriptyline (NOR) in blood and urine of the baby. Additional investigations suggested a shaken baby syndrome. Given the family context, a judge ordered hair tests for both the child and his parents to document drug exposure.A liquid chromatography tandem mass spectrometric (LC-MS/MS) method was then developed to quantify AMI and NOR in hair. After decontamination and segmentation, 20. mg of hair was incubated overnight at 55. °C in methanol (MeOH). The LC-MS/MS method used an online solid phase extraction and the analysis was performed using two transitions per compound. The LOQ and LOD for the two compounds were estimated at 0.0075. ng/mg and 0.005. ng/mg respectively.All hair segments tested for both parents were negative. For the baby two strands of hair were collected one day after the acute intoxication for the first and 5 weeks later for the second. The first strand was not decontaminated before analysis to avoid losing specimen. The high and relatively homogenous concentrations of AMI (with a range of value from 6.65 to 9.69. ng/mg) and NOR (with a range of value from 7.12 to 8.96. ng/mg) measured suggested that contamination could have occurred. The analysis of the second strand after decontamination allowed to detect AMI and NOR in all hair segments. The obtained values varied between 0.54 and 1.41. ng/mg for AMI and between 1.26 and 4.00. ng/mg for NOR. These results supported the hypothesis of a chronic exposure during several months before hair collection with regular increase. However a single overdose could not be totally excluded. The interpretation of results must take into account the pharmacological and physiological parameters of hair of the children.
Introduction Amphetamine and derivatives are the second most commonly used group of illicit drugs worldwide. However, in the last years new psychoactive substances with stimulant effects have appeared in the illegal market, which are not detected with traditional analytical methods. The aim of this study was to develop and validate a method for the determination of amphetamine derivates (amphetamine (A), methamphetamine (M), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA)), synthetic cathinones (methylone, methedrone, mephedrone, methylenedioxypyrovalerone (MDPV), fluoromethcathinone and fluorometamphetamine), and piperazines (metachlorophenylpiperazine (mCPP) and trifluoromethylphenylpiperazine (TFMPP)) in hair using LC-MS/MS. Methods Thirty mg hair were incubated in 2 mL methanol with 0.1% HCl for 1 hour at 60°C. After centrifugation, the solvent was evaporated until dry, reconstituted in 2 mL 2% formic acid (FA) and submitted to solid phase extraction with Strata-XC cartridges. After cartridge conditioning, samples were loaded, and two washing steps were performed with 2 mL 2% FA and 2 mL methanol: water:FA (47.5:47.5:5). Analytes were eluted with 2 mL dichloromethane: isopropanol:ammonium hydroxide (47.5:47.5:5). Eluates were evaporated and reconstituted in 75 μL ammonium formate 2 mM pH 3, to inject 30 μL into the LC-MSMS. Chromatographic separation was performed using an Atlantis T3 (3 μm; 2.1×50 mm) analytical column in gradient mode with ammonium formate 2 mM pH 3 and acetonitrile as mobile phase. The method was validated, including specificity (endogenous and exogenous interferences); linearity (n=4); limit of detection (LOD) and quantification (LOQ); accuracy and imprecision (between and within-day) (n=20) at low, medium and high concentrations; extraction recovery (ER) (n=6), matrix effect (ME) (n=10), and process efficiency (PE) (n=6) at low and high concentrations; and stability after 72 hours in the autosampler. The method was applied to the analysis of 7 amphetamine derivatives positive hair specimens. Results All the analytes eluted within 9 min, with a total run time of 13 minutes. Validation parameters were within the accepted criteria. The method was specific (no endogenous or exogenous interferences were detected) and linearity (r2>0.99) was achieved between 2–4000 pg/mg for methylone, methedrone, mephedrone and MDPV; 10–4000 pg/mg for fluoromethcathinone, mCPP and TFMPP; 20–4000 pg/mg for A, M, MDMA and MDA; and 10–2000 pg/mg for fluoromethamphetamine. Accuracy was ±9.6% of target concentration; between and within-day imprecision were <15%, except for fluoromethamphetamine at medium concentration (20.6%). ER was 47.8–92.1%, and all the analytes showed ion enhancement up to 127.2%, except mCPP, MDMA and MDA (no matrix effect observed). PE was between 54.4–165.7%. All real specimens were positive for A and MDMA, 3 for M, 2 for MDA and 1 for mCPP, with concentrations ranging between 44.9–777.1, 98.7–3654.5, 120.4–1538.9, 27.8–135.4, and 6527.9 pg/mg, respectively. Conclusion A sensitive and specific method for the determination of ATS drugs in hair was developed and validated, achieving lower cut-offs than SoHT recommendations for amphetamine derivates. The method was successfully applied to the analysis of 7 real hair specimens.
In contrast with urine, hair analysis has a wide window of detection, ranging from weeks to months, depending on the length of the hair shaft, and provides information concerning the pattern of an individual's drug abuse. The Society of Hair Testing has published on 16 June 1999, a consensus opinion on the use of hair in doping situations but hair analysis is not yet recognized by the World Anti-Doping Agency (WADA), although this technology is accepted in most courts of justice. While analysis of urine specimens cannot distinguish between chronic use or single exposure, hair analysis makes this distinction. This is very useful in case of identification in urine of restricted compounds. The major application may be in identifying false-negatives, since neither abstaining from a drug for a few days nor trying to “beat the test” by diluting urine, will alter the concentration in hair. Urine does not indicate the frequency of drug intake in subjects who might deliberately abstain for several days before screening. Doping during training and abstinence during the competition can therefore be detected, as athletes cannot evade the test. Hair testing should not be considered as an alternative to urinalysis but only as a complement in positive case to document the claim of the athlete, and it must be clear that in case of positive urine results, the negative hair result cannot exclude the administration of the detected drug and cannot overrule the positive urine result. In this compendium, 19 positives results from 51 requests for anabolic drugs testing in hair are presented. Boldenone (7–1270 pg/mg), stanozolol (85–881 pg/mg), methandienone (7–206 pg/mg), salbutamol (5–13 pg/mg), methyltestosterone (18 pg/mg), clenbuterol (2110 pg/mg), dihydrotestosterone (12 pg/mg), 19-norandrostenedione (8–165 pg/mg) and mesterolone (6 pg/mg) were identified during routine practice.
Formules chimiques des composés.  
A 53-year-old-man was hospitalized for a stroke. As usually, qualitative urine drug screenings were ordered in the emergency department. Urine immunoassay testing was negative for amphetamines, cannabis, cocaine and opiates, but was very positive for ecstasy using EXTC kit (Siemens®). Then chromatographic analyses were realized to confirm this result or identify a possible analytical interference. A specific assay of ecstasy performed using LC-MS/MS did not allowed confirming the presence of MDA and MDMA, the detection limit of this technique was 1 μg/L. Qualitative urine drug screens were also realized using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-diode array detection (LC-DAD). The screening of urine performed with GC-MS allowed for the identification of a piperazine, the (1,2-methoxyphenyl)-piperazine (2-MeOPP). Piperazine derivatives act as stimulants, these substances being classified as designer drugs. However, the screening performed using LC-DAD revealed the presence of urapidil, a sympathetic antihypertensive drug. More analyses were realized to control these different results. Urines of patients treated with urapidil induced positive results to EXTC kit and presented a signal identified as 2-MeOPP using GC-MS. In this article, two interferences associated with urapidil were shown.
Introduction Increasing interest is currently observed in hair analysis for the assessment of human exposure to organic pollutants. Nevertheless, only few studies have been published in this specific field so far, and several aspects still have to be investigated for optimum use of the information hair analysis can provide. In particular, relationships between individuals’ exposure and pollutants concentration in hair were only little investigated. The present work, based on animal experimentation, was therefore investigating to what extent hair concentration was associated with level of exposure to a series of pesticides. Methods Rats (Lister Hooded, bicolor animals) were administered pesticides by gavage over a 90 days-period, 3 times per week, at 7 different levels plus one control group (no exposure). Each level of exposure consisted of n=8 animals. Pesticide mix consisted of twenty different compounds (organochlorines, organophosphates, pyrethroids, carbamates and other pesticides). Animals’ hair was collected at the end of the experiment by shaving. Black hair and white hair were analyzed separately in order to assess any possible effect of melanin on the incorporation of chemicals in hair. After samples decontamination, pulverization, and extraction, both parent compounds and metabolites were analyzed by GC-MS/MS. Results Level of exposure was significantly associated with pesticide concentration in hair for most of the compounds (metabolites and parents) detected in animals’ hair, with coefficients of determination (R2) >0.9 for several compounds. The concentration detected in white hair was always significantly associated with the one quantified in black hair, but the slopes were varying for the different chemicals, demonstrating that incorporation was influenced by pigmentation only for some compounds. The levels of concentration detected in animals’ hair was in line with the ranges of concentration generally observed in human's hair. Conclusion The good matching between chemicals concentration observed in animals’ hair and concentration reported for the same compounds in humans’ hair suggests that the model was realistic and that levels of exposure animals were submitted to, were representative of human exposure. The significant association observed here between levels of exposure and chemicals concentration in hair supports the relevance of hair analysis for the assessment of human exposure to pollutants. (Abstract: Analytical Toxicology Meeting, Bordeaux, France, 10-13 June 2014)
Introduction Atractyloside (ATR) and carboxyatractyloside (CATR) are diterpene glycosides that are responsible for the toxicity of several Asteraceae plants around the world. Mediterranean gum thistle (Atractylis gummifera L.) and Zulu impila (Callilepis laureola DC.), in particular, are notoriously poisonous and the cause of many accidental deaths, some suicides and even some murders. When a case history is not available, it is not easy to determine ATR or CATR poisoning anatomically or histologically. There is no current method for measuring the two toxins in biological samples that meet the criteria of specificity required in forensic medicine. We have endeavoured to fill this analytical gap. Methods Analysis was carried out using an original technique of high-performance liquid chromatography coupled with highresolution mass spectrometry detection (HPLC-HRMS). Given the singular structure of ATR and CATR, it is difficult to achieve and maintain the conditions required for their chromatographic separation and perfect ionization for the mass spectrometry. Separation was thus performed using an XTerra® phenyl column (length: 150 mm; internal diameter: 2,1 mm; particle size: 3,5 μm) (Waters) [Steenkamp et al., Forensic Sci Int, 2006, 163: 81–92] with a gradient mobile phase composed of acetonitrile containing 10% of isopropyl alcohol and a 5 mM ammonium acetate buffer at pH = 4.5. The chromatographic run time was 12.5 min. Spectrometric detection was performed using a quadrupole-Orbitrap high-resolution detector (Q Exactive™; Thermo Scientific) after ionization by heated electrospray in negative-ion mode. The mass spectrometer operated in full-scan mode and targeted-MS2 mode alternately. MS scans (288 – 292, 723 – 727 and 767 – 771 amu) were acquired with a mass resolution of 140000. The [M. – H]− and [M – H+1]− ions of ATR (725.2154 and 726.2188 amu ± 5 ppm) and CATR (769.2053 and 770.2086 amu ± 5 ppm) were used for quantification. The fullscan product ion spectrum of the compounds (resolution of 17500) was used to confirm the identity of the toxins. The processing of the biological sample (1 mL) consisted of a protein precipitation followed by solid phase extraction on Oasis® HLB cartridges (Waters) at pH = 4.5. Results The method was validated in the whole blood with between- and within-day RSD (relative standard deviation) less than 5.8% and 5.2% for ATR (accuracy between 95.9% and 98.6%) and 5.4% and 9.8% for CATR (accuracy between 92.0% and 107.4%). The calibration curves were linear for concentrations ranging from 0.17 to 200 μg/L for ATR and 0.15 to 200 μg/L for CATR. The detection limits were 0.066 and 0,055 μg/L respectively. ATR and CATR were quantified in blood and urine samples from a non-fatal case of intoxication by A. gummifera. The concentrations were 883.1 and 119,0 μg/L respectively in peripheral blood and 230.4 and 140,3 mg/L in urine. ATR and CATR were quantified in dried A. gummifera roots by the standard addition method. The concentrations were 3.7 and 5,4 mg/g respectively. Conclusion We present the first validated method, applicable in forensic toxicology, for quantifying ATR and CATR in whole blood. The analysis is sensitive and quick.
Objective The present work is aimed to study blood lead level in Casablanca population exposed to lead particles from smelters in Ain Sebaa and Sidi Bernoussi districts and from companies manufacturing batteries in Sidi Bernoussi district. Blood lead level will be also studied in Rabat population not exposed to industrial pollution as control. Method This cross-sectional study exposed/unexposed was conducted in children and adults non-occupationally exposed. The study was based on blood lead level determination and on collecting socio-demographic parameters and lead exposure risk factors in 473 participants. Results Mean blood lead level (53.74 ± 42.08 μg/L) obtained in participants from the exposed area (Casablanca) was significantly higher than mean blood lead level (35.80 ± 34.15 μg/L) obtained in participants from the unexposed area (Rabat). Among the 473 participants in the study, 32 subjects had blood lead level higher than 100 μg/L, 25 cases (78.1%) were from the exposed area. Logistic regression analysis adjusted to the city showed that participants from the exposed area had a significantly higher likelihood to have high blood lead level than participants from unexposed area. Conclusion Increased blood lead levels in participants from Casablanca compared to those from Rabat could be attributed to the exposure to lead particles emitted by smelters in Ain Sebaa and Sidi Bernoussi districts and by companies manufacturing batteries in Sidi Bernoussi district.
Aluminium phosphide is a rodenticide widely used in agriculture for grain preservation. It is available as dark grey tablets of 3 gr. Each tablet contains two compounds: aluminium phosphide (56%) and aluminium carbonate (44%). Self-poisoning with this product represents a real problem in developing countries. It causes high mortality. The aluminium phosphide blocks the enzyme cytochrome-c oxidase causing cellular damage and multi-system organ failure. The confirmation of diagnosis is done by qualitative silver nitrate test on gastric content. In the absence of specific antidote, treatment is mainly based on gastric lavage as quickly as possible with potassium permanganate (1:10,000), and with sodium bicarbonate 7.5% solution, supplemental oxygen, maintaining a normal intravascular volume and correction of severe metabolic acidosis. Assisted ventilation is indicated in case there is persistence of hypoxemia or unconsciousness. For shock, the vasoactive agents could be used. The intravenous magnesium sulfate infusion decreases the incidence of cardiac arrhythmias and mortality, probably due to its membrane stabilizing and anti-peroxide effect. The prognostic factors were freshness of tablets, ingested dose, shock, elevated of Troponin, renal failure, metabolic acidosis, arrhythmias, need for mechanical ventilation and vasoactives drugs. Prevention remains the best treatment. The main preventive measures consist of restricting the sale and aluminium phosphide will not be available to general population.
Structure de l'amphétamine (a) et de la cathinone (b). 
Similarité de structure entre la méphédrone (a) et la méthamphétamine (b). 
Structure générale commune des cathinones de synthèse. 
Spectres de masse de la méphédrone (a), de la méthylone (b), de la MDPV (c) et de la butylone (d). 
The emergence of new psychoactive drugs is due to the supposed similar effects from ‘copied’ substances and also, in particular cases, to a legal alternative of controlled substances. This first paper deals with cathinones and synthetic cathinones. Different topics as structural properties, pharmacokinetics, modes of consumption, identification and quantification methods as well as legislation issues will be discussed. The scientific literature is reviewed until March 2013, resulting in a summary of the reported effects related to the use of those substances. In conclusion, risks involved by the use of synthetic cathinones are not yet well established. New synthetic cathinones launched on the market represent a potential public health problem and force the scientists to continually develop new sensitive analytical methods to assure their identification and quantification.
Introduction. - Baclofen has been used in alcohol withdrawal maintenance phase for several years and recently approved for this indication in France. Patient and methods. - We report a case of a chronic ethyl-addicted patient with severe neurological symptoms after self-induced intoxication with baclofen. Conclusion. - Symptomatic treatment with forced urinary output performed on this patient with normal renal function allowed a fast consciousness recovery. © 2014 Societe Francaise de Toxicologie Analytique. Published by Elsevier Masson SAS. All rights reserved.
Background Baclofen is a centrally acting gamma-aminobutyric acid agonist used for spasticity of brain or spinal origin. We report the case of a 60-year-old man with a chronic renal failure treated daily with 15 mg of baclofen for the treatment of a right spastic hemiplegia. Seven days after his hospitalization for an extended right nephrectomy, the patient presented an encephalopathy. The CT scan did not find any signs of stroke. A drug overdose was suspected. Materials and methods A double screening by GC/MS and HPLC/DAD was carried out on two blood samples taken before and after patient's dialysis. The concentration of baclofen was measured on the same samples by a validated method using liquid chromatography coupled to a mass spectrometer ion trap. Results The toxicology screening didn’t show the presence of baclofen. Serum concentrations of baclofen were measured at 2100 ng/mL in the sample taken before dialysis and 1150 ng/mL in the post-dialysis sampling. Conclusion Encephalopathy is most likely the result of baclofen overdose. Regression of symptoms after hemodialysis and the significant decrease in baclofen serum concentrations are in line with this hypothesis. Indeed, baclofen is mainly eliminated by glomerular filtration. So it was accumulated in this patient with impaired renal function. This observation highlights the importance of measuring baclofen trough concentrations in patients with renal impairment to avoid its accumulation by a dose adjustment.
Objective Studying stability of seven benzodiazepines (alprazolam, bromazepam, clobazam, clonazepam, diazepam, tetrazepam and triazolam) together with methadone and zolpidem in bloodstains for a sampling on crime scene. Methods Drops of 50 μL of blood overloaded with 10 ng/mL were deposited on glass slides and stored under various conditions of time (24 h, 48 h, 72 h, one week and one month) and environments (light and darkness at room temperature, (−) 20 °C, (+) 4 °C, (+) 35 °C and outside). Dried blood is then removed with a scalpel blade, weighed and rehydrated for 30 min with a pH 9.5 buffer. A liquid phase extraction was carried out and analysis was performed by UPLC-MS/MS. In parallel, dried blood was collected using swabs, previously impregnated with saline solution, then analyzed as previously described. Results The analytes tested were found stable as well as by scratching or swabbing, excepted for clonazepam (−) 20 °C and outside. After one month, losses up to 50 % could be observed at (+) 35 °C and in outside conditions for all drugs analyzed. Conclusion This study shows the stability of seven benzodiazepines together with methadone and zolpidem in dried blood spots with no major influence of exposure time and environmental conditions. In view of results, a new analytical approach could be considered, e.g. toxicological analysis of bloodstains on a crime scene.
Introduction In analytical toxicology, the extraction step is critical: the double aim is to separate xenobiotics from endogenous compounds and to clean-up the extract. Extraction processes such as liquid-liquid extraction and off-line solid-phase extraction are often efficient, but time consuming. Proteins precipitations with methanol or acetonitrile are quicker but only dedicated to whole blood or plasma-serum matrices. For more than ten years, another approach has been used for the determination of pesticides in food matrices (Anastassiades M et al. 2003). This method, named QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe), is based on a quick extraction with an acetonitrile and salt combination. The aim of this presentation is to report biological and forensic toxicological applications of this method initially dedicated to pesticides in food. Methods 200 to 500 μL of acetonitrile were added to the sample (100 μL of whole blood, urine, serum or plasma), followed by a small amount (i.e. 300 mg) of QuEChERS salts (4 g MgSO4/1 g NaCl/1 g Sodium citrate dehydrate/0,5 g Sodium citrate sesquihydrate). The mixture was vigorously shaken and then centrifuged at 13,000 rpm for 10 min. Before injection in the chromatographic system, the upper layer was diluted, or not, depending on the final application in LC-MS/MS. Results Some examples of QuEChERS method applications together with the comparison with our previous methods are presented in the following table. This approach was also successfully applied to the development of several new methods: antiepileptics, anticoagulants, curares, ivabradine… Despite the lower sample volume used and the simple sample preparation (coupled with a dilution of the sample), the obtained limits of detection that have been reached in LC-MS/MS were close or inferior to those obtained by GC-MS, using a time consuming extraction with a higher volume of sample. Among the previous methods cited, some of them have been successfully evaluated by the Comité Français d’Accréditation, according to the ISO 17025 standard. Conclusion In our opinion, the QuEChERS sample preparation is almost universal. The main benefit is the short time required for sample preparation and its efficiency in cleaning up the extract. The main limitation is the absence of a major concentration of the analytes. Nevertheless, in case of applications requiring a very low limit of detection, we can foresight that this problem could be solved with the next generation of mass spectrometers. Table Abstract O28.View Within Article
Due to lack of published pharmacokinetic and/or pharmacodynamic researches in children, therapeutic target levels in body fluids are not well known in this population, as underlined by a 2003 IGAS (General Inspectorate of Social Affairs, an inter-ministerial control institution specialized on social affairs) report. Pediatric values are often extrapolated from concentrations measured in adults, with correction factors based on age and maturity of the metabolic systems. Although clinical relevance is usually based on the physiological consequences induced by the treatment, the precise knowledge of some values is useful to evaluate the risk of toxic molecules, among others. Objective To collect pediatric therapeutic values published in refereed journals. Method To research on Pubmed literature and table of contents of pharmacology and/or pediatrics and/or specialties journals from the last 10 years, with the keywords “pharmacokinetics” and “children”. Results Although not exhaustive, several studies have revealed therapeutic values for 20 drugs, according to different routes of administration and age. Conclusion Few pharmacokinetic studies have been conducted in children, to suggest objective reference values and not only extrapolated from assays performed in adults.
Introduction A case report involving repeated intoxications investigated by segmented hair analysis is presented. A 41-year-old man intoxicated his 46-year-old wife several times over half a year. During this period of time the husband went away repeatedly for some days while the wife got sick. Often when he was away, she felt dizzy and nauseated and vomited. One night she was attacked by a man, who was later identified as the husband. The Police suspected the husband for several intoxication attempts on his wife. Methods Hair samples were collected from the top (5 cm long light brown hair with bleached stripes) and from the back of the head (2 cm long light brown unbleached hair) of the wife. Hair was sampled the 15-07-2013, so two of the suspected intoxication attempts (4-4 & 13-5-2013) are represented by segment 2 (1.5–4 cm). The segmented hair samples were washed, dissolved and analyzed for drugs and drugs of abuse by UPLC/TOF and quantified by UPLC/MS/MS (C. Montesano, SS. Johansen et al., JPBA 88 (2014) 295–306) Results Drug findings in head hair from the scalp and back (ng/mg) are shown in the Table. View Within Article Segment 2 has highest drug concentrations of opioids and zolpidem. O-desmethyltramadol is a metabolite from tramadol, while morphine due to the low morphine:codeine ratio probably derives from codeine metabolism. After the violent attack the wife took diazepam a few times and the trace of the metabolite desmethyl-diazepam in the inner segment from scalp hair confirmed the ingestion. In general the drug levels in hair were low indicating few intake(s) and not sustained therapeutic use. The drug findings in the inner hair segment (0–1,5 cm) were com-parable with the drug levels found in the back hair, although the back hair was not bleached. Bleaching is reported to reduce the opioid content in hair from 57 to 94%, however the scalp hair was only partly bleached suggesting a lower loss and high ability to retain the drugs. Conclusion The case illustrates that hair is a valuable forensic specimen when natural processes have eliminated the drug from typical biological specimens such as blood and urine. It was possible to verify suspected intoxications by segmented hair analysis. The husband admitted the intoxications by mixing opioids and zolpidem in the wife’ coffee. He was sentenced to prison for 6 years.
Introduction A study of 1383 drug users was undertaken in Brazil focusing on crack cocaine users. Detailed interviews were undertaken about their personal life history and psychoactive substance use and hair samples were taken to corroborate the interview data. The main objective of this paper is to evaluate hair drug testing as a biomarker capable of corroborating the interview data and history of drug use. Methods Head hair samples were collected from 333 known drug users, 298 males and 35 females, from various parts of Brazil, prior to their voluntary detoxification treatment. The samples were sent to ChromaTox for analysis. Head hair sections (N=327) measured from 0.5 to 4 cm. Body hair samples (N=6) were not sectioned and measured from 1.5 to 3 cm. The samples were extracted and analysed by LC-MS/MS using methods accredited to ISO/IEC 17025 standards. A cutoff of 0.5 ng/mg was used for cocaine and 0.2 ng/mg for AEME, cocaethylene, and norcocaine. The limit of quantitation (LOQ) was 0.1 ng/mg for all analytes (cocaine, benzoylecgonine, norcocaine, AEME, crack cocaine). Results Cocaine was detected above cutoff in 272 samples of hair with levels ranging from 0.5 ng/mg to 385.2 ng/mg. AEME was detected in 200 samples with levels from 0.3 to 16.6 ng/mg; benzoylecgonine was detected in 70 samples with levels from 0.2 to 242.8 ng/mg and norcocaine was detected in 207 samples with levels from 0.2 ng/mg to 8.2 ng/mg. Where current cocaine use was not detected (N=13) or was below cutoff but above LOQ (N=48), the period covered by the sample length corresponded with the period when crack and/or cocaine use ceased for the majority of the cases. Alprazolam, diazepam, nordiazepam and THC were also detected in various combinations in 89 samples. Levels detected in hair were not correlated with other data collected in the interviews such as type of abode, education, race, religion, income or parental drug use nor with the age people started using crack cocaine. Conclusions The results of the analysis of hair samples confirmed the usefulness of the detection of drugs in hair to assess drug consumption over a long period of time, and in this case to corroborate interview data. Samples where use of cocaine was not detected correlated with individuals who declared at their interviews that they had stopped using crack cocaine prior to the period covered by the sample. The results also showed no correlation between the declared amounts used of crack cocaine and/or of cocaine with the levels detected in hair. This is expected, since, whilst the hair results can be compared with the results previously analysed by the laboratory, it is well established that there are many factors that affect levels of drugs in hair and therefore it is not possible to infer from the levels detected in hair the amount of drug used. These factors are for example, inter-individual differences in metabolism, hair types and cosmetic treatments. Hair drug testing is an excellent tool for showing whether people used drugs or not over a long period of time. It is not possible therefore to establish the amount of drug used from the levels detected in hair. It is possible, however to compare the results with the results previously analysed by the laboratory and to use the levels of drugs in hair as a guide to the changes in pattern of use over time in the same individual.
Introduction Δ9-tetrahydrocannabinol (THC) can be measured in exhaled breath by using a simple aerosol particle collection device. The sampling procedure is simple, non-invasive and takes only 2–3 minutes. In the present study we measured the amount of THC in exhaled breath of cannabis users at specific time intervals up to three hours after smoking one cannabis cigarette. The breath concentration-effect relationship was studied by measuring the pulse rate and the pupil diameter to assess physiological changes. Methods Participants smoked self-supplied cannabis cigarettes, with an estimated content of 13 to 52 mg of THC, corresponding to 248 μg/kg to 693 μg/kg. They smoked a cigarette outside for 5–10 minutes and directly after smoking, were called inside for breath sampling and physiological tests. Breath sampling was performed before smoking, directly after smoking and 15, 30, 60, 120, 180 minutes after smoking, with a SensAbues DrugTrap® device (SensAbues AB, Huddinge, Sweden). The subjects were asked to breathe via a mouthpiece into a plastic bag for 2 to 3 minutes (± 20 L of exhaled breath). THC was analyzed in exhaled breath by liquid chromatography-tandem mass spectrometry (Beck O et al. Journal of breath research. 2013;7(2):026006.). Thirteen subjects (9 males and 4 females, aged 23–24 years) have participated. Results THC was detected in all breath samples but no THCCOOH (<LOD) was found in any sample. The mean THC concentrations were 1043, 27670, 14292, 10982, 4192, 3861 and 1479 pg/filter before, immediately after and 15, 30, 60, 120 and 180 minutes after smoking. THC remained detectable for over three hours after smoking. Pulse rate (p = 0.015) and pupil diameter (p = 0.011) were significantly altered up to 30 minutes after smoking. The mean THC breath concentration showed a counter-clock wise hysteresis relationship with the mean pulse rate. Conclusion The detection window of cannabis in breath after smoking one cannabis cigarette in occasional and chronic smokers was at least 3 hours. Only tetrahydrocannabinol, not the metabolite, was detected. The tetrahydrocannabinol concentration in exhaled breath was related to the physiological changes that occur over time by a counterclockwise hysteresis loop. Exhaled breath can be used to detect recent cannabis exposure.
Introduction Mass spectrometric (MS) methods are today widely used in analytical toxicology, but in comprehensive quantitative drug screening it is laborious to maintain quantitative calibration and use of historic calibration is not feasible. In non-MS gas chromatographic (GC) and liquid chromatographic (LC) methods, historic calibration is widely used. However, GC is not amenable to analysis of current polar drugs and traditional LC-UV methods do not have high enough identification power to meet the requirements of screening. Ultra-performance LC (UPLC) with two consecutive detectors appears to possess high potential for simultaneous multi-component screening and quantification. In this study, a comprehensive quantitative screening method for 170 basic drugs in blood samples was developed and validated using UPLC coupled with photodiode array (PDA) and corona charged aerosol detector (CAD). Methods Blood samples were extracted with organic solvent (ethyl acetate: butyl acetate, 25:75) in basic conditions. After extraction, the organic solvent was evaporated to dryness and samples were reconstituted with the UPLC mobile phase. Dibenzepine was used as an internal standard. Chromatographic separation was performed at 60 °C using a HSS C18 column (150 mm × 2,1 mm, particle size 1,8 μm), and the mobile phase consisted of 0.1% trifluoroacetic acid and methanol. After a three minute isocratic phase, a linear gradient from 5% to 95% methanol in 15 minutes followed. The flow rate was 0.4 mL/min. UV spectra were collected in the range of 210–400 nm, and wavelength of 230 nm was used for quantification. Substance identification was based on the UV spectrum, retention times on both detectors, and the response ratio of CAD and PDA at wavelength 230 nm. Calibration was carried out using a single calibration point at the vicinity of the upper limit of therapeutic range of each drug. Individual calibration curves were created for both detectors and the average result was used for quantification. Results The retention times were found be very stable, the relative standard deviation of retention times being <0.03% intraday and <0.3% interday. In order to simplify data processing and interpretation of the reports, the analytes were divided into five data processing methods, each containing every fifth analyte based the retention time. This approach provided a maximum of five candidates for each peak, thus simplifying manual interpretation even with more complicated samples. Linearity of one-point calibration was found to be acceptable within the therapeutic and toxic ranges of the drugs of interest, with linear ranges generally being 0.05–5,0 mg/L or 0.1–5,0 mg/L. Calibration was found to be stable over one month, the bias between old and new calibration being <15%. Conclusion The developed method allows the detection and quantification of 170 basic drugs in therapeutic and toxic concentrations in a single run. Due to the stability of calibration and good linearity, historic one-point calibration can be utilized. Adding the CAD detector with universal response after the PDA detector increases the reliability of both identification and quantification. In addition, drugs with poor UV absorption can be detected and quantitated. This method provides a tool for comprehensive quantitative screening for ordinary basic drugs in blood and leaves LC-MS target analysis to be applied to low-dose compounds.
Aims The differentiation between an active cannabis consumption and a passive drug exposure (i.e. through side stream marijuana smoke, or transfer from contaminated hands), remains an unsolved problem in hair analysis [Uhl M., Sachs H. Forensic Science International 2004;145:143–147]. The presence in hair of Δ9-tetrahydrocannabinolic acid A [THCA-A; a non-psychoactive precursor of Δ9-tetrahydrocannabinol (THC) and the main cannabinoid in a fresh plant] was recently proposed as a specific marker of external contamination [Roth N et al. Journal of Mass Spectrometry 2013;48:227–233]. However, the evidence of active consumption of cannabis (inhalated and/or oral) currently remains based on the detection of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol acid (THC-COOH) in hair. Nevertheless, the missing detection of this metabolite does not exclude an active consumption, as the low pg/mg range of THCCOOH concentrations in hair of cannabis users can unable to detect it despite using extremely sensitive analytical methods. In this context, the aim of this presentation is to highlight this problem in young children hair. Cases stories Twelve hair samples collected from 9 young children (4F/5M – 2 to 14 months old) were sent to the laboratory for cannabinoid determination in order to explore their cannabis exposure (their parents being regular cannabis users) after (or not) an acute cannabis intoxication (cannabis resin intake): overall eighteen hair segments were analysed. Methods A recently published liquid chromatography – tandem mass spectrometry (LC – MS/MS) method was used for the simultaneous quantitative determination in hair of THC-COOH, THC, Cannabinol (CBN) and Cannabidiol (CBD) [Dulaurent S et al. Forensic Science International 2014;236:151–156.]. Briefly, after a decontamination step, the sample preparation consisted of an alkaline hydrolysis of hair samples followed by a liquid-liquid extraction of compounds in acidic conditions. The chromatographic detection was performed by means of MRM and MS3 transitions using an API 5500 QTRAP mass spectrometer (AB Sciex, Courtaboeuf, France), with intra- and inter-assay accuracies and relative standard deviation below 9% and 15%, respectively, for the four compounds. LOQ (= LOD) was 0.2 pg/mg for THC-COOH and 50 pg/mg for THC, CBD and CBN. Results and conclusion Some selected results in hair segments (which do not correspond to a documented acute accidental intoxication period), are shown in the following table (concentration in ng/mg): View Within Article Interpretation of these results could be as follows: (case 1) low exposure in relation to low environmental exposure or in utero exposure, (case 2) low and (case 3) high exposure in relation to environmental exposure, and (case 4) high exposure in relation to environmental exposure with significant absorption by his organism. Nevertheless, in order to improve the interpretation of such results in this particular population and due to the tricky context, (i) many pitfalls have to be overcome, in particular ontogenic issues (i.e. CYP2C9 and UGT expression in infants), and (ii) additional clinical and analytical data are needed.
Introduction The detection of passive exposure to drugs present in the environment is often considered a critical pitfall in the use of hair analysis. This capability, however, of hair analysis to detect environmental exposures is of substantial benefit in identifying children passively exposed to drugs in their environment through direct exposure to drug smoke or ubiquitous presence of drug residues on surfaces in the home. In these two cases, children under the age of two years presented to hospital with clinical signs of possible drug intoxication. Child #1 has been removed from his biological home due to concerns about neglect and presented as excessively somnolent. Child #2 presented to hospital with signs of acute intoxication including emesis, sedation, impaired motor coordination, and produced a positive urine analysis for THC. Investigation for chronic environmental drug exposure was undertaken for both of these children and additional family members by collection of hair samples. Methods Hair samples were segmented to reflect the relevant time period of analysis applying a growth rate of one centimetre of hair per month. A minimum of 80 milligrams of hair was decontaminated with methlylene chloride, pulverized, extracted, and analyzed by liquid-chromatography tandem-mass spectrometry for the presence of THC-carboxylic acid. Additional analysis for exposure to cocaine and cocaine metabolites (benzoylecgonine, norcocaine, cocaethylene) was conducted by GC-MS analysis following segmentation of 10 milligrams of hair, overnight incubation in methanol, and derivatization by BSTFA. Results Child #1 tested positive for THC-carboxylic acid in a fivemonth section of hair at a concentration of 0.43 picograms per milligram, cocaine at a concentration above 16.00 ng/mg, benzoylecgonine at a concentration of 2.36 ng/mg, and trace amounts of norcocaine. An older sibling also tested positive for cocaine, benzoylecgonine, and THC-carboxylic acid. Sibling (n = 3) hair drug concentrations in this family were inversely proportional to age. Child #2 tested positive for THC-carboxylic acid in a six-month section of hair at a concentration of 0.22 picograms per milligram. Both parents tested negative for cannabinoids in hair, which was consistent will clinical suspicion that the exposure occurred outside of the family home. Conclusion Due to an absence of evidence that THC-carboxylic acid can be found in environmental cannabis smoke or in sweat analysis of cannabis users, coupled with the decontamination of these hair samples; the findings suggest that both children and one sibling have experienced chronic systemic cannabis exposure due to the presence of THC-carboxylic acid in their hair samples. Child #1 also presents with a high risk of systemic cocaine exposure due to the presence of norcocaine in their sample. These cases highlight the clinical value of hair analysis in determining chronic exposures in pre-verbal children, incapable to self-reporting their experiences to health-care providers.
A 36-year-old woman was tested to document excessive alcohol drinking behaviour. A hair strand and a blood specimen, both collected at the same time, were sent to a ISO 17025 accredited laboratory. This laboratory reported a negative ethyl glucuronide (EtG) result (over 0 to 3 cm segment, concentration lower than 2.4 pg/mg) and a positive fatty acid ethylesters (FAEE) result (over 0 to 6 cm segment, total concentrations at 1.82 ng/mg), based on the recommended SoHT cut-offs. In parallel, it was observed no alteration of the liver function (γGT at 18 UI) of the subject, and a specific excessive alcohol blood test (CDT at 0.5%) did not show any addiction to alcohol. Despite these biological findings, it was established by the laboratory expert, a statement of witness that concluded that the subject was a chronic excessive alcohol drinker, based on the FAEE results. The subject denied alcohol intake and I was asked to review the case. Given it is admitted by the scientific community that a positive FAEE test cannot overrule a negative EtG test, it is more likely that cosmetics have to be taken into consideration to explain the discrepancies of the hair results. It is therefore not possible to conclude from both the hair and blood tests that the subject was a chronic excessive alcohol drinker, based on the laboratory findings. In my opinion, there is no evidence to suggest from the subject excessive alcohol abuse.
Objectives A 44-year-old resident from Gironde died in a hospital from the island of Lombok, Indonesia, several hours after drinking a “homemade cocktail”. Potential poisoning with denatured alcohol was rapidly raised by friends and family. The body received embalmment treatments and was sent back to Bordeaux two weeks later. An autopsy and toxicological analyses were requested by the prosecutor. Methods Toxicological analyzes were performed with usual laboratory techniques and alcohols were analyzed by head space gas-chromatography with flame ionization detector in different fluids (abdominal liquid cavity, vitreous humor, gastric content, urine, bile). For formates, the capillary zone electrophoresis was performed with reverse UV detection at 254 nm. Results In different samples, ethanol concentrations ranged from 0.11 to 0.39 g/L, methanol from 1 to 5.1 g/L and formates from 0.42 to 2.5 g/L in urine. Formates assays were also performed on embalming fluids used in France as well as in formalin used in pathology. The results are below 0.015 g/L and 0.052 g/L, respectively. Acetates were found in significant concentrations in all studied liquids, documenting ethanol consumption. Discussion According to the scientific literature, concentrations of urinary formates are greater than 2 g/L in 90% proven methanol poisonings. Concentrations measured in the victim are compatible with methanol poisoning. Conclusion Unfortunately, we were not able to obtain the liquid used for embalming, so we cannot exclude contamination from the Indonesian fluid retention from which only an analysis would clear the doubt.
The exposure to xenobiotics, drugs or toxic molecules, depends on pharmacokinetic properties thereof at their phases of absorption, distribution, metabolism and elimination. The fate in the body of these compounds is not always superimposable between adults and children due to major physiological changes during the age that will affect their pharmacokinetics. This review summarizes the current knowledge of the major changes in the child in terms of (a) digestive physiology and hepato-biliary excretion (b) xenobiotic metabolism in the intestine and liver by phases 1 and 2 drug-metabolizing enzymes of (c) concentrations of plasma proteins, water and lipid body compositions and tissue partition and finally (d) renal excretion via glomerular filtration and tubular secretion via transport mechanisms.
Hair specimen is necessary to complement blood and/or urine analyses as it permits differentiation of a single exposure from chronic use of a drug by segmentation of the hair for a stated growth period. Moreover, due to a frequent long delay between event and police declaration, hair can be the only solution for lack of corroborative evidence of a committed crime. With the exception of lower amount of biological material in children versus adults, there is no specific analytical problem when processing samples from children. The issue is the interpretation of the findings, with respect to the different pharmacological parameters. In some very young children, the interpretation can be complicated by potential in utero exposure.
Introduction Methadone (MTD) is an analgesic compound used for treatment of severe pain or opioid addiction. MTD is therapeutically administered as racemic mixture although opioid activity is attributed to the R-enantiomer. In some countries the pure R-enantiomer is also used. Three cytochrome p450 (CYP) enzymes are involved in MTD metabolism, namely 2B6, 2C19, and 3A4 with different stereo selectivity and two of them (2B6, 2C19) being polymorphically expressed. Incorporation of a drug and its metabolite into hair matrix might be a mirror mimicking genetic polymorphism. The aim was to establish an enantioselective quantification of MTD and EDDP and to adopt this method for non-pigmented and pigmented hair samples of different colours. Methods After washing, the hair samples were cut into snippets and extracted in a single step with ultrasonication. These extracts were analysed without any purification. MTD and EDDP enantiomers were determined in hair samples by HPLC (CHIRALPAK AGP column, 100×4, 5um) coupled to a tandem mass spectrometer (ABSciex QTrap 3200, 2 MRMs) using deuterated standards of MTD and EDDP, respectively. The method was validated and applied to hair samples collected during driving licence assessments of 152 participants of a long term MTD maintenance program (16% women, 84% men). Unpigmented hair samples were obtained by manual separation of grey hair strands (n = 40) providing the possibility of a direct comparison of the incorporation rate of MTD and EDDP into unpigmented hair versus that of pigmented hair of different colours (blond, brown, dark brown/black). The incorporation rate was defined by the clearance index (CI) calculated as ratio of the self-declared monthly dose to the MTD concentration. Results and Discussion The ratio of MTD concentration in pigmented to MTD concentration in not-pigmented hair (mean value 5.5) was smaller than that for EDDP (8.5), indicating that the incorporation of EDDP is more affected by pigmentation. However, these ratios exhibited only slight enantioselectivity. For the pigmented samples (n=159), the metabolic ratio EDDP/MTD was always lower for the R-enantiomers (0.06 ± 0.025) than for the S-form (0.13 ± 0.061). This distribution pattern of these ratio values might be indicative for the phenotype of the subject. CI was calculated to be the lowest for participants with black hair and the highest for participants with non-pigmented hair (white hair); the pigmentationdependent incorporation factors ranged from 1 (non-pigmented) up to > 10 (black hair). Conclusion A chiral LC-MS/MS method for the quantification of methadone and EDDP in hair was established and validated. Incorporation factors for MTD were depending on the hair colour (white<blonde<brown<black). These factors range from 1 (white hair) up to > 10 for black hair samples; the clearance index shows an inverse order. The metabolic ratio for S-enantiomers was higher than for the R-enantiomers, indicating a stereo-selective biotransformation (CYP2B6) of MTD.
Introduction Disclosing chronic use of cannabis has always been a difficult task due to the fact that hair testing of THC alone is not conclusive of use and hair testing of THC-COOH require specific and sensitive instrumentation and laboratory skill, not available for many analytical laboratories. We hypothesized the presence of THC-COOH glucuronide in hair as possible alternative biomarker of repeated consumption of cannabis products. Methods We developed and validated an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to identify and quantify THC-COOH glucuronide in hair after applying one hour digestion of 25 mg keratin matrix with M3 (Comedical Spa, Italy) reagent at 100°C, using deuterated THC-COOH glucuronide as internal standard. An amount of 10 μl was injected in the ULPC-MS/MS system. Chromatographic separation was carried out on a Acquity UPLC HSS C18 column (2,1 mm × 150 mm, 1.8 μm) using a linear gradient elution with two solvents:0.1% formic acid in acetonitrile (solvent A) and 5 mM ammonium formate pH 3 (solvent B). The flow rate was kept constant at 0.40 mL/min during the analysis. The separated analytes were detected with a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode via positive electrospray ionization (ESI). The applied ESI conditions were the following: capillary voltage 1,3 kV, desolvation temperature 600 °C, source temperature 150 °C, cone gas flow rate 20 L/h, desolvation gas flow rate 1000 L/h and collision gas flow rate 0.13 mL/min, cone voltage of 65 V. Following transitions were considered: 521.2→345.03 (Collision energy:16), 521.2 →327.07 (Collision energy: 22),521.2 → 299.09 (Collision energy: 32), with the first one used for quantification. Results Linear calibration curves were obtained for THC-CCOH glucuronide with correlation coefficients (r2) of 0.99 and a LOQ of 0.1 pg/mg hair. Analytical recovery was between 70.9 and 100.7%. Intra and inter-assay imprecision and inaccuracy were always lower than 10%. No additional peaks due to endogenous substances which could have interfered with the detection of the analytes under investigation were observed in drug-free hair samples. No psychoactive drugs other than the compounds under investigation interfered with the assay. Blank hair samples injected after the highest point of the calibration curve did not present any traces of carryover. The matrix effect in quality control samples ranged from 80 to 101%. Preliminary analysis on 9 different hair samples of consumers disclosed the presence of THC-CCOH glucuronide in the range of 0.30–1.22 pg/mg with by median value of 0.58 pg/mg hair. Conclusions Simple extraction, identification and quantification of THC-COOH glucuronide in hair by UPLC-MS/MS was developed, validated and tested for its feasibility in clinical samples and provided a good start to consider THC-CCOH glucuronide as alternative hair biomarker of repeated cannabis consumption.
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