[New biomarkers of alcohol use]. / Nouveaux marqueurs biologiquesde la consommation d'alcool.

Estimating alcohol consumption using biomarkers raises interpretation problems. The biomarkers currently used in clinical settings have limited performances to identify unhealthy alcohol use (e.g. CDT, AST, ALT). New direct biomarkers, ethylglucuronide (EtG) and phosphatydilethanol (PEth) are available and offer better sensitivity and specificity compared to indirect biomarkers. In forensic medicine, EtG and PEth are replacing indirect biomarkers. However, in clinical routine practice these markers are usually not considered. Still, for specific purposes such in pre-liver transplant evaluations, direct markers may help specialists in the decision process.

Estimer la consommation d'alcool en se basant sur des tests biologiques pose des problèmes d'interprétation. Les marqueurs actuellement utilisés en clinique (CDT et transaminases) présentent des performances limitées pour l'identification du mésusage d'alcool. Des nouveaux marqueurs directs, l'éthylglucuronide (EtG) et le phosphatidyléthanol (PEth), sont à disposition et offrent de meilleures performances en termes de sensibilité et spécificité que les marqueurs indirects. Ils sont principalement utilisés dans le cadre de suivis médico-légaux et remplacent les marqueurs indirects. En pratique clinique, l'EtG et le PEth ne sont que peu utilisés. On voit apparaître l'utilisation de ces tests dans le cadre d'évaluations spécifiques, par exemple avant transplantation hépatique.

Mouse alarm pheromone shares structural similarity with predator scents.

Sensing the chemical warnings present in the environment is essential for species survival. In mammals, this form of danger communication occurs via the release of natural predator scents that can involuntarily warn the prey or by the production of alarm pheromones by the stressed prey alerting its conspecifics. Although we previously identified the olfactory Grueneberg ganglion as the sensory organ through which mammalian alarm pheromones signal a threatening situation, the chemical nature of these cues remains elusive. We here identify, through chemical analysis in combination with a series of physiological and behavioral tests, the chemical structure of a mouse alarm pheromone. To successfully recognize the volatile cues that signal danger, we based our selection on their activation of the mouse olfactory Grueneberg ganglion and the concomitant display of innate fear reactions. Interestingly, we found that the chemical structure of the identified mouse alarm pheromone has similar features as the sulfur-containing volatiles that are released by predating carnivores. Our findings thus not only reveal a chemical Leitmotiv that underlies signaling of fear, but also point to a double role for the olfactory Grueneberg ganglion in intraspecies as well as interspecies communication of danger.

Elimination profile of low-dose chlortalidone and its detection in hair for doping analysis-Implication for unintentional non-therapeutic exposure.

Chlortalidone (CLT) is a thiazide-type diuretic with high affinity for the erythrocyte carbonic anhydrase. Therapeutically, it is mostly used to treat edema and hypertension due to liver cirrhosis, heart insufficiency, or renal dysfunction. Although diuretics and masking agents are prohibited by the World Anti-Doping Agency (WADA) at all times in sports, substances belonging to this category are constantly detected in athlete samples, according to WADA's annual testing figures. Within this group of structurally diverse compounds, a threshold of 20 ng/mL has been introduced for six substances solely due to their presence as contaminants in other permitted drugs because of pharmaceutical production processes. In a recent presumptive doping case with a low urinary CLT concentration, the question of unintentional doping, for example, by contaminated non-steroidal anti-inflammatory drug intake, arose. To examine this potential scenario, a co-elimination of low-dose CLT and hydrochlorothiazide (HCTA; 20 × 50 µg, 0.2 mg/day each) was conducted on five consecutive days in two volunteers. Urine samples were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Moreover, we examined the incorporation of CLT in scalp hair. HCTA is rapidly excreted renally in comparatively high concentrations. In contrast, the elimination of CLT is considerably slower (terminal elimination half-life extended by a factor of 12) and, consequently, much less concentrated in corresponding urine samples (45 and 53 ng/mL, respectively). Conversely, a higher hair incorporation of chlorthalidone was observed with simultaneous dosing of both. The results suggest that an unintentional intake of sub-therapeutic CLT doses due to contamination might result in an adverse analytical finding.

Diagnostic performance of ethyl glucuronide in hair for the investigation of alcohol drinking behavior: a comparison with traditional biomarkers.

BACKGROUND: Ethyl glucuronide (EtG) in hair has emerged as a useful biomarker for detecting alcohol abuse and monitoring abstinence. However, there is a need to establish a reliable cutoff value for the detection of chronic and excessive alcohol consumption. METHODS: One hundred and twenty-five subjects were classified as teetotalers, low-risk drinkers, at-risk drinkers, or heavy drinkers. The gold standard for subjects' classifications was based on a prospective daily alcohol self-monitoring log. Subjects were followed for a 3-month period. The EtG diagnostic performance was evaluated and compared with carbohydrate-deficient transferring (CDT) and the activities of aspartate aminotransferase, alanine aminotransferase, and γ-glutamyl-transferase (γGT). RESULTS: A cutoff of >9 pg/mg EtG in hair, suggesting an alcohol consumption of >20/30 g (at-risk drinkers), and a cutoff of >25 pg/mg, suggesting a consumption of >60 g (heavy drinkers), were determined by receiver operating characteristic analysis. The EtG diagnostic performance was significantly better (P < 0.05) than any of the traditional biomarkers alone. EtG, as a single biomarker, yielded a stronger or similar diagnostic performance in detecting at-risk or heavy drinkers, respectively, than the best combination of traditional biomarkers (CDT and γGT). The combination of EtG with traditional biomarkers did not improve the diagnostic performance of EtG alone. EtG demonstrated a strong potential to identify heavy alcohol consumption, whereas the traditional biomarkers failed to do so. EtG was not significantly influenced by gender, body mass index, or age. CONCLUSION: Hair EtG definitively provides an accurate and reliable diagnostic test for detecting chronic and excessive alcohol consumption. The proposed cutoff values can serve as reference for future cutoff recommendations for clinical and forensic use.

Is the formula of Traub still up to date in antemortem blood glucose level estimation?

According to the hypothesis of Traub, also known as the 'formula of Traub', postmortem values of glucose and lactate found in the cerebrospinal fluid or vitreous humor are considered indicators of antemortem blood glucose levels. However, because the lactate concentration increases in the vitreous and cerebrospinal fluid after death, some authors postulated that using the sum value to estimate antemortem blood glucose levels could lead to an overestimation of the cases of glucose metabolic disorders with fatal outcomes, such as diabetic ketoacidosis. The aim of our study, performed on 470 consecutive forensic cases, was to ascertain the advantages of the sum value to estimate antemortem blood glucose concentrations and, consequently, to rule out fatal diabetic ketoacidosis as the cause of death. Other biochemical parameters, such as blood 3-beta-hydroxybutyrate, acetoacetate, acetone, glycated haemoglobin and urine glucose levels, were also determined. In addition, postmortem native CT scan, autopsy, histology, neuropathology and toxicology were performed to confirm diabetic ketoacidosis as the cause of death. According to our results, the sum value does not add any further information for the estimation of antemortem blood glucose concentration. The vitreous glucose concentration appears to be the most reliable marker to estimate antemortem hyperglycaemia and, along with the determination of other biochemical markers (such as blood acetone and 3-beta-hydroxybutyrate, urine glucose and glycated haemoglobin), to confirm diabetic ketoacidosis as the cause of death.

Performance of self-reported measures of alcohol use and of harmful drinking patterns against ethyl glucuronide hair testing among young Swiss men.

BACKGROUND: There is a need for empirical studies assessing the psychometric properties of self-reported alcohol use as measures of excessive chronic drinking (ECD) compared to those of objective measures, such as ethyl glucuronide (EtG). OBJECTIVES: To test the quality of self-reported measures of alcohol use and of risky single-occasion drinking (RSOD) to detect ECD assessed by EtG. METHODS: A total of 227 samples of hair from young Swiss men were used for the determination of EtG. Self-reported measures of alcohol use (previous twelve-month and previous-week alcohol use) and RSOD were assessed. Using EtG (<30 pg/mg) as the gold standard of ECD assessment, the sensitivity and specificity were computed, and the AUROC were compared for alcohol use measures and RSOD. Logistic regressions were used to test the contribution of RSOD to the understanding of ECD after controlling for alcohol use. RESULTS: A total of 23.3% of participants presented with ECD. Previous twelve-month alcohol use with a cut-off of >15 drinks per week (sensitivity = 75.5%, specificity = 78.7%) and weekly RSOD (sensitivity = 75.5%, specificity = 70.1%) yielded acceptable psychometric properties. No cut-off for previous-week alcohol use gave acceptable results. In the multivariate logistic regression, after controlling for the previous twelve months of alcohol use, RSOD was still significantly associated with EtG (p = .016). CONCLUSION: Self-reported measures of the previous twelve months of alcohol use and RSOD were acceptable measures of ECD for population-based screening. Self-reported RSOD appeared to be an interesting screening measure, in addition to the previous twelve months of alcohol use, to understand ECD among young people.

Screening for alcohol use disorder among individuals with comorbid psychiatric disorders: Diagnostic accuracy in a sample of young Swiss men.

Alcohol use disorder (AUD) is frequently comorbid with other psychiatric disorders. However, few studies investigated the psychometric properties of AUD screening tools in presence of co-occurring disorders. This study examined the diagnostic accuracy of a short AUD screening tool among young adults, in the presence of high vs. low or moderate symptomatology of other common psychiatric disorders. Data were collected among young Swiss men (n = 233) between 2016 and 2018. Measures included a diagnostic interview for AUD and screening tools for AUD and other psychiatric disorders (attention deficit hyperactivity disorder, antisocial personality disorder, bipolar disorder, borderline personality disorder, major depressive disorder, and social anxiety disorder). We computed receiver operating characteristic curves to test whether the AUD screening tool was an accurate indicator of AUD for groups with high vs. low or moderate symptomatology of each psychiatric disorder. The results showed that the optimal cut-off score was ≥3 (the original cut-off of the scale) for participants with a low or moderate symptomatology and ≥4 for participants with a high symptomatology. Our findings highlighted the urgent need for an integrated approach to screening. Psychiatric comorbidities should be included in the screen for AUD to obtain accurate results.

Identifying an accurate self-reported screening tool for alcohol use disorder: evidence from a Swiss, male population-based assessment.

BACKGROUND AND AIMS: Short screenings for alcohol use disorder (AUD) are crucial for public health purposes, but current self-reported measures have several pitfalls and may be unreliable. The main aim of our study was to provide empirical evidence on the psychometric performance of self-reports currently used. Our research questions were: compared with a gold standard clinical interview, how accurate are (1) self-reported AUD, (2) self-reported alcohol use over time and (3) biomarkers of alcohol use among Swiss men? Finally, we aimed to identify an alternative screening tool. DESIGN: A single-center study with a cross-sectional design and a stratified sample selection. SETTING: Lausanne University Hospital (Switzerland) from October 2017 to June 2018. PARTICIPANTS: We selected participants from the French-speaking participants of the ongoing Cohort Study on Substance Use and Risk Factors (n = 233). The sample included young men aged on average 27.0 years. MEASUREMENTS: We used the Diagnostic Interview for Genetic Studies as the gold standard for DSM-5 AUD. The self-reported measures included 11 criteria for AUD, nine alcohol-related consequences, and previous 12 months' alcohol use. We also assessed biomarkers of chronic excessive drinking (ethyl glucuronide and phosphatidylethanol). FINDINGS: None of the self-reported measures/biomarkers taken alone displayed both sensitivity and specificity close to 100% with respect to the gold standard (e.g. self-reported AUD: sensitivity = 92.3%, specificity = 45.8%). The best model combined eight self-reported criteria of AUD and four alcohol-related consequences. Using a cut-off of three, this screening tool yielded acceptable sensitivity (83.3%) and specificity (78.7%). CONCLUSIONS: Neither self-reported alcohol use disorder nor heavy alcohol use appear to be adequate to screen for alcohol use disorder among young men from the Swiss population. The best screening alternative for alcohol use disorder among young Swiss men appears to be a combination of eight symptoms of alcohol use disorder and four alcohol-related consequences.

Liquid chromatography-high resolution mass spectrometry for broad-spectrum drug screening of dried blood spot as microsampling procedure.

BACKGROUND: Hyphenation of liquid chromatography (LC) with high-resolution mass spectrometry (HRMS) offers the potential to develop broad-spectrum screening procedures from low volumes of biological matrices. In parallel, dried blood spot (DBS) has become a valuable tool in the bioanalysis landscape to overcome conventional blood collection issues. Herein, we demonstrated the applicability of DBS as micro-sampling procedure for broad-spectrum toxicological screening. METHODS: A method was developed on a HRMS system in data dependant acquisition (DDA) mode using an extensive inclusion list to promote collection of relevant data. 104 real toxicology cases were analysed, and the results were cross-validated with one published and one commercial screening procedures. Quantitative MRM analyses were also performed on identified substances on a triple quadrupole instrument as a complementary confirmation procedure. RESULTS: The method showed limits of identification (LOIs) in appropriateness with therapeutic ranges for all the classes of interest. Applying the three screening approaches on 104 real cases, 271 identifications were performed including 14 and 6 classes of prescribed and illicit drugs, respectively. Among the detected substances, 23% were only detected by the proposed method. Based on confirmatory analyses, we demonstrated that the use of blood micro-samples did not impair the sensitivity allowing more identifications in the low concentration ranges. CONCLUSION: A LC-HRMS assay was successfully developed for toxicological screening of blood microsamples demonstrating a high identification power at low concentration ranges. The validation procedure and the analysis of real cases demonstrated the potential of this assay by supplementing screening approaches of reference.

Comparison of self-reported measures of alcohol-related dependence among young Swiss men: a study protocol for a cross-sectional controlled sample.

INTRODUCTION: Short screenings of alcohol-related dependence are needed for population-based assessments. A clinical interview constitutes a reliable diagnosis often seen as gold standard, but it is costly and time consuming and as such, not suitable for population-based assessments. Therefore, self-reported questionnaires are needed (eg, alcohol use disorder (AUD) as in the Diagnostic and Statistic Manual of Mental Disorders (DSM) 5), but their reliability is questionable. Recent studies called for more evidence-based measurements for population-based screening (eg, heavy alcohol use over time (HAU)). This study aims to test the reliability of different self-reported measures of alcohol use. METHODS AND ANALYSIS: Based on stratified random selection, 280 participants will be recruited from the French-speaking subgroup of the Swiss National Science Foundation-supported Cohort Study on Substance Use and Risk Factors (C-SURF). This cohort is a population-based sample of young Swiss men in their mid-20s (n=2668). The sample size calculation is based on a proportion non-inferiority test (alpha=5%, power=80%, margin of equivalence=10%, difference in sensitivity between self-reported AUD and HAU=5%, correlation between AUD and HAU=0.35, and drop-outs=15%). Assessment will include a clinical interview as the gold standard of alcohol-related dependence, self-reported alcohol measures (HAU, AUD and drinking patterns), biomarkers as gold standards of chronic excessive drinking, and health outcomes. To assess the validity of the self-reported alcohol measures, sensitivity analyses will be run. The associations between alcohol-related measures and health outcomes will be tested. A non-response analysis will be run using the previous waves of the C-SURF study using logistic regressions. ETHICS AND DISSEMINATION: The study protocol has been approved by the Human Research Ethics Committee of the Canton of Vaud, Switzerland (no. 2017-00776). The results will be submitted for publication in peer-reviewed journals and presented at national and international conferences.

Commentary on current changes of the SoHT 2016 consensus on alcohol markers in hair and further background information.

The consensus on alcohol markers in hair was revised for the fourth time by an expert group of the Society of Hair Testing based on current state of research. This revision was adopted by the members of the Society during the business meeting in Brisbane on August 29th 2016. For both markers, ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs), two cut-off values for discrimination between teetotalers or occasional low amount consumption and moderate alcohol drinking (low cut-off), and between non-excessive (abstinence up to moderate alcohol intake) and chronic excessive drinking (high cut-off value) were critically examined. For the current revision, the cut-off values for EtG (7pg/mg and 30pg/mg, respectively) remained unchanged despite different findings or discussions published in the meantime. This was mainly due to the lack of broader data collections from new studies with great numbers of volunteers following thorough study concepts. In contrast, an essential change of the consensus was accepted for the FAEEs, where the concentration of ethyl palmitate (E16:0) can be used autonomously for interpretation instead of the concentration sum (ΣFAEE) of the four esters ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate, as previously applied. After evaluation of the data from seven laboratories, the E16:0 cut-off for abstinence assessment was defined at 0.12ng/mg for the 0-3cm segment and at 0.15ng/mg for the 0-6cm segment. The cut-off for chronic excessive drinking was fixed at 0.35ng/mg for the 0-3cm segment and at 0.45ng/mg for the 0-6cm segment. The use of E16:0 with these cut-offs in place of ΣFAEE for alcohol intake assessment produces only a minor loss in discrimination power, leads to no essential difference in the interpretation concerning chronic excessive alcohol consumption and is suitable to confirm EtG results in abstinence assessment if ethanol containing hair sprays or lotions are excluded.

Application of headspace solid phase microextraction to qualitative and quantitative analysis of tobacco additives in cigarettes.

Cigarettes may contain up to 10% by weight additives which are intended to make them more attractive. A fast and rugged method for a cigarette-screening for additives with medium volatility was developed using automatic headspace solid phase microextraction (HS-SPME) with a 65 microm carbowax-divinylbenzene fiber and gas chromatography-mass spectrometry (GC-MS) with standard electron impact ionisation. In three runs, each cigarette sample was extracted in closed headspace vials using basic, acidic and neutral medium containing 0.5 g NaCl or Na2SO4. Furthermore, the method was optimized for quantitative determination of 17 frequently occurring additives. The practical applicability of the method was demonstrated for cigarettes from 32 brands.

A systematic review of passive exposure to cannabis.

Passive exposure to cannabis smoke may induce effects on behavior and psychomotor skills, and have legal consequences, including the risk of being falsely considered as a cannabis user. This can become a concern, especially in occupational contexts or when driving vehicles. In order to enable a differentiation between a passive and an active exposure to cannabis and to limit the likeliness to be detected positive following passive exposure, this review identified specific biomarkers of passive exposure in urine, blood, oral fluid, hair, and sebum. Out of 958 papers identified on passive exposure to cannabis, 21 were selected. Although positive tests had been observed in all matrices following extremely high passive exposure, some distinctive features were observed in each matrix compared to cannabis active use. More specifically, in everyday life conditions, 11-nor-delta-9-THC-carboxylic acid (THC-COOH) urinary level should be detected below the positivity threshold used to confirm active smoking of cannabis, especially after normalization to creatinine level. Measuring delta-9-tetrahydrocannabinol (THC) and THC-COOH in blood is an appropriate alternative for appraising passive exposure as low and very low concentrations of THC and THC-COOH, respectively, should be measured. In hair, oral fluid (OF) and sweat/sebum emulsion, no THCCOOH should be detected. Its presence in hair argues for regular cannabis consumption and in OF or sweat for recent consumption. The experts should recommend to persons who have to demonstrate abstinence from cannabis to avoid heavily smoky and unventilated environments.

Concentration of drugs in blood of suspected impaired drivers.

Analytical records concerning 440 living drivers suspected of driving under the influence of drug (DUID) were collected and examined during a 2 years period ranging from 2002 to 2003 in canton de Vaud, Valais, Jura and Fribourg (Switzerland). This study included 400 men (91%) and 40 women (9%). The average age of the drivers was 28+/-10 years (minimum 16 and maximum 81). One or more psychoactive drugs were found in 89% of blood samples. Half of cases (223 of 440, 50.7%) involved consumption of mixtures (from 2 to 6) of psychoactive drugs. The most commonly detected drugs in whole blood were cannabinoids (59%), ethanol (46%), benzodiazepines (13%), cocaine (13%), amphetamines (9%), opiates (9%) and methadone (7%). Among these 440 cases, 11-carboxy-THC (THCCOOH) was found in 59% (median 25 ng/ml (1-215 ng/ml)), Delta(9)-tetrahydrocannabinol (THC) in 53% (median 3 ng/ml (1-35 ng/ml)), ethanol in 46% (median 1.19 g/kg (0.14-2.95 g/kg)), benzoylecgonine in 13% (median 250 ng/ml (29-2430 ng/ml)), free morphine in 7% (median 10 ng/ml (1-111 ng/ml)), methadone in 7% (median 110 ng/ml (27-850 ng/ml)), 3,4-methylenedioxymethamphetamine (MDMA) in 6% (median 218 ng/ml (10-2480 ng/ml)), nordiazepam in 5% (median 305 ng/ml (30-1560 ng/ml)), free codeine in 5% (median 5 ng/ml (1-13 ng/ml)), midazolam in 5% (median 44 ng/ml (20-250 ng/ml)), cocaine in 5% (median 50 ng/ml (15-560 ng/ml)), amphetamine in 4% (median 54 ng/ml (10-183 ng/ml)), diazepam in 2% (median 200 ng/ml (80-630 ng/ml)) and oxazepam in 2% (median 230 ng/ml (165-3830 ng/ml)). Other drugs, such as lorazepam, zolpidem, mirtazapine, methaqualone, were found in less than 1% of the cases.

Simultaneous and sensitive analysis of THC, 11-OH-THC, THC-COOH, CBD, and CBN by GC-MS in plasma after oral application of small doses of THC and cannabis extract.

Besides the psychoactive Delta(9)-tetrahydrocannabinol (THC), hashish and marijuana as well as cannabis-based medicine extracts contain varying amounts of cannabidiol (CBD) and of the degradation product cannabinol (CBN). The additional determination of these compounds is interesting from forensic and medical points of view because it can be used for further proof of cannabis exposure and because CBD is known to modify the effects of THC. Therefore, a method for the simultaneous quantitative determination of THC, its metabolites 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH), CBD and CBN from plasma was developed. The method was based on automatic solid-phase extraction with C(18) ec columns, derivatization with N,O-bistrimethylsilyltrifluoroacetamide (BSTFA), and gas chromatography-electron impact ionization-mass spectrometry (GC-EI-MS) with deuterated standards. The limits of detection were between 0.15 and 0.29 ng/mL for THC, 11-OH-THC, THC-COOH, and CBD and 1.1 ng/mL for CBN. The method was applied in a prospective pharmacokinetic study after single oral administration of 10 mg THC alone or together with 5.4 mg CBD in cannabis extract. The maximum plasma concentrations after cannabis extract administration ranged between 1.2 and 10.3 ng/mL (mean 4.05 ng/mL) for THC, 1.8 and 12.3 ng/mL (mean 4.9 ng/mL) for 11-OH-THC, 19 and 71 ng/mL (mean 35 ng/mL) for THC-COOH, and 0.2 and 2.6 ng/mL (mean 0.95 ng/mg) for CBD. The peak concentrations (mean values) of THC, 11-OH-THC, THC-COOH, and CBD were observed at 56, 82, 115, and 60 min, respectively, after intake. CBN was not detected. Caused by the strong first-pass metabolism, the concentrations of the metabolites were increased during the first hours after drug administration when compared to literature data for smoking. Therefore, the concentration ratio 11-OH-THC/THC was discussed as a criterion for distinguishing oral from inhalative cannabis consumption.

Identification of pyridine analogs as new predator-derived kairomones.

In the wild, animals have developed survival strategies relying on their senses. The individual ability to identify threatening situations is crucial and leads to increase in the overall fitness of the species. Rodents, for example have developed in their nasal cavities specialized olfactory neurons implicated in the detection of volatile cues encoding for impending danger such as predator scents or alarm pheromones. In particular, the neurons of the Grueneberg ganglion (GG), an olfactory subsystem, are implicated in the detection of danger cues sharing a similar chemical signature, a heterocyclic sulfur- or nitrogen-containing motif. Here we used a "from the wild to the lab" approach to identify new molecules that are involuntarily emitted by predators and that initiate fear-related responses in the recipient animal, the putative prey. We collected urines from carnivores as sources of predator scents and first verified their impact on the blood pressure of the mice. With this approach, the urine of the mountain lion emerged as the most potent source of chemical stress. We then identified in this biological fluid, new volatile cues with characteristic GG-related fingerprints, in particular the methylated pyridine structures, 2,4-lutidine and its analogs. We finally verified their encoded danger quality and demonstrated their ability to mimic the effects of the predator urine on GG neurons, on mice blood pressure and in behavioral experiments. In summary, we were able to identify here, with the use of an integrative approach, new relevant molecules, the pyridine analogs, implicated in interspecies danger communication.

Headspace solid-phase microextraction with 1-pyrenyldiazomethane on-fibre derivatisation for analysis of fluoroacetic acid in biological samples.

A new and in part automated headspace solid-phase microextraction method for quantitative determination of the highly toxic rodenticide fluoroacetic acid (FAA) in serum and other biological samples has been developed. FAA and deuterated acetic acid (internal standard) were extracted from acidified samples by a StableFlex divinylbenzene-Carboxen on polydimethylsiloxane fibre. The acids were derivatised on the fibre in-situ with 1-pyrenyldiazomethane and detected using gas chromatography-mass spectrometry with electron impact ionisation and selected ion monitoring. The calibration curve for FAA in serum was linear over the range from 0.02 to 5 microg/ml, with limits of detection and quantification of 0.02 and 0.07 microg/ml, respectively. The method was also tested with spiked whole blood, urine, stomach contents and kidney samples. It was sufficiently reliable, reproducible and sensitive for use in routine forensic toxicology applications.

Determination of cathinone, cathine and norephedrine in hair of Yemenite khat chewers.

A sensitive and reproducible method for the quantitative determination of cathinone (CTN), norpseudoephedrine (NPE, cathine) and norephedrine (NE) from hair was developed. The compounds were extracted for 4 hours with phosphate buffer pH 2.0, followed by a standard solid phase extraction procedure on a mixed phase column, derivatization with heptafluorobutyric anhydride (HFBA) and GC-MS separation and quantification using D(3)-ephedrine (D(3)-E) and alpha-aminoacetophenone (AAP) as the internal standards. The diastereomers NPE and NE were satisfactorily separated. In the validation, the limits of detection and of quantification were determined at 0.03-0.08 ng/mg and 0.10-0.24 ng/mg, respectively and the interday standard deviation was between 10 and 15%. The method was applied to hair samples of 24 Yemenite khat chewers. All three compounds were detected in 23 of these cases. The concentrations ranged from 0.57 to 23.9 ng/mg for NPE, 0.19-25.0 ng/mg for NE and 0.11-22.7ng/mg for CTN. A highly significant correlation was found between the self-reported data about the khat consumption habits of the volunteers (4-56h chewing per week) and the concentrations of norephedrine and norpseudoephedrine in hair.

A fatal overdose of cocaine associated with coingestion of marijuana, buprenorphine, and fluoxetine. Body fluid and tissue distribution of cocaine and its metabolites determined by hydrophilic interaction chromatography-mass spectrometry(HILIC-MS).

Chromatographic separation of highly polar basic drugs with ideal ionspray mass spectrometry volatile mobile phases is a difficult challenge. A new quantification procedure was developed using hydrophilic interaction chromatography-mass spectrometry with turbo-ionspray ionization in the positive mode. After addition of deuterated internal standards and simple clean-up liquid extraction, the dried extracts were reconstituted in 500 microL pure acetonitrile and 5 microL was directly injected onto a Waters Atlantis HILIC 150- x 2.1-mm, 3-microm column. Chromatographic separations of cocaine, seven metabolites, and anhydroecgonine were obtained by linear gradient-elution with decreasing high concentrations of acetonitrile (80-56% in 18 min). This high proportion of organic solvent makes it easier to be coupled with MS. The eluent was buffered with 2 mM ammonium acetate at pH 4.5. Except for m-hydroxy-benzoylecgonine, the within-day and between-day precisions at 20, 100, and 500 ng/mL were below 7 and 19.1%, respectively. Accuracy was also below +/- 13.5% at all tested concentrations. The limit of quantification was 5 ng/mL (%Diff < 16.1, %RSD < 4.3) and the limit of detection below 0.5 ng/mL. This method was successfully applied to a fatal overdose. In Switzerland, cocaine abuse has dramatically increased in the last few years. A 45-year-old man, a known HIV-positive drug user, was found dead at home. According to relatives, cocaine was self-injected about 10 times during the evening before death. A low amount of cocaine (0.45 mg) was detected in the bloody fluid taken from a syringe discovered near the corpse. Besides injection marks, no significant lesions were detected during the forensic autopsy. Toxicological investigations showed high cocaine concentrations in all body fluids and tissues. The peripheral blood concentrations of cocaine, benzoylecgonine, and methylecgonine were 5.0, 10.4, and 4.1 mg/L, respectively. The brain concentrations of cocaine, benzoylecgonine, and methylecgonine were 21.2, 3.8, and 3.3 mg/kg, respectively. The highest concentrations of norcocaine (about 1 mg/L) were measured in bile and urine. Very high levels of cocaine were determined in hair (160 ng/mg), indicating chronic cocaine use. A low concentration of anhydroecgonine methylester was also found in urine (0.65 mg/L) suggesting recent cocaine inhalation. Therapeutic blood concentrations of fluoxetine (0.15 mg/L) and buprenorphine (0.1 microg/L) were also discovered. A relatively high concentration of Delta(9)-THC was measured both in peripheral blood (8.2 microg/L) and brain cortex (13.5 microg/kg), suggesting that the victim was under the influence of cannabis at the time of death. In addition, fluoxetine might have enhanced the toxic effects of cocaine because of its weak pro-arrhythmogenic properties. Likewise, combination of cannabinoids and cocaine might have increase detrimental cardiovascular effects. Altogether, these results indicate a lethal cocaine overdose with a minor contribution of fluoxetine and cannabinoids.