Assay of ethanol and congener alcohols in serum and beverages by headspace gas chromatography/mass spectrometry.

The analysis of ethanol and of its congeners in blood plays an important role in forensic cases, especially when allegations are made that alcohol has been consumed after an accident. In alcoholic beverages, congener alcohols are by-products and are generated during fermentation. The assay of these compounds in serum samples and beverages has been previously performed using headspace-gas chromatography-flame ionization detection methods (HS-GC-FID). As an alternative, a robust headspace-gas chromatography-mass spectrometry (HS-GC-MS) procedure was developed and validated, which has the following advantages:•Simultaneous determination of ethanol, congener alcohols and other endogenous substances.•Reduction of matrix interference by increasing selectivity and specificity.•Clear separation of the positional isomers 3-methyl-1-butanol and 2-methyl-1-butanol.

Endogenous formation of 1-propanol and methanol after consumption of alcoholic beverages.

INTRODUCTION: In cases of drunk-driving, allegations that alcohol has been consumed after the incident, are proved by analyzing congener alcohols in the blood sample. 1-Propanol, one of the main congener compounds, was tested, whether it is also endogenously formed when a person has consumed alcoholic beverages. METHODS: Eleven male and 13 female volunteers consumed congener-free vodka (37.5 vol% ethanol, individual doses: 0.15-0.32 l) within one hour. Blood samples were taken up to 10 h and analyzed for ethanol and congener alcohols by headspace gas chromatography-mass spectrometry. RESULTS: Ethanol concentrations reached in blood a maximum of 0.65-1.23 g/l and decreased by 0.18 g/l/h (median values). Of the congener alcohols analyzed, only methanol and 1-propanol were detected in the plasma samples of all subjects. The endogenous methanol concentration increased from 0.66 mg/l by 0.22 mg/l/h to 2.19 mg/l (medians). 1-Propanol was not detected prior to alcohol consumption. Maximum concentrations of 0.10-0.32 mg/L were measured after 1.0-4.5 h. A plateau of the 1-propanol concentration was observed in the plasma samples of the 18 subjects lasting for 0.5-4.0 h and this alcohol was completely eliminated at ethanol concentrations of 0.17 g/l (median, range 0.03-0.55 g/l). CONCLUSION: The results of the study confirm the formation of 1-propanol after consumption of 1-propanol-free beverages, which should be taken into account when evaluating its concentration.

Excretion of metabolites of the synthetic cannabinoid JWH-018 in urine after controlled inhalation.

Each year, synthetic cannabinoids are occurring in high numbers on the illicit drug market but data obtained after controlled application are rare. The present study on pharmacokinetics in urine is part of a pilot study on adverse effects of JWH-018, which is one of the oldest and best known synthetic cannabinoids. Six subjects inhaled smoke from 2 and 3mg JWH-018. The drug and ten potential metabolites were analyzed in urine samples collected during 12h after inhalation by liquid chromatography-mass spectrometry (LC-MS/MS) without and with conjugate cleavage. The parent compound was not detectable, but 13 of its metabolites, all of which were conjugated. Concentrations of the predominant metabolite, JWH-018 pentanoic acid, were less than 5ng/ml, but in two subjects it was still detected up to 4 weeks after ingestion. Other major metabolites were 5- and 4-HOpentyl-JWH-018, JWH-073 butanoic acid and a hypothetically dihydroxylated and dehydrogenated metabolite of JWH-018. Occasionally, further hydroxylated metabolites were found. Generally, hydroxylated metabolites were detected in concentrations lower than 1ng/ml already 10h after inhalation. All concentrations were much lower than reported for urine samples of authentic JWH-018 users. The formation of the metabolite JWH-018 pentanoic acid was found to be slightly delayed, but its rather high concentrations and detection over several weeks after single dosing makes it a useful target for urine analysis. The different excretion of carboxylic acid and hydroxylated metabolites may aid in evaluation of time of use.

Evading plant defence: Infestation of poisonous milkweed fruits (Asclepiadaceae) by the fruit fly Dacus siliqualactis (Diptera: Tephritidae).

To cope with toxic metabolites plants use for defence, herbivorous insects employ various adaptive strategies. For oviposition, the fruit fly Dacus siliqualactis (Tephritidae) uses milkweed plants of the genus Gomphocarpus (Asclepiadaceae) by circumventing the plant's physical (gluey latex) and chemical (toxic cadenolides) defence. With its long, telescope-like ovipositor, the fly penetrates the exo- and endocarp of the fruit and places the eggs on the unripe seeds located in the centre of the fruit. Whereas most plant parts contain high concentrations of cardenolides such as gomphoside, calotropin/calacatin and gomphogenin, only the seeds exhibit low cardenolide levels. By surmounting physical barriers (fruit membranes, latex), the fly secures a safe environment and a latex-free food source of low toxicity for the developing larvae. One amino acid substitution (Q111V) at the cardenolide binding site of the fly's Na+, K+-ATPase was detected, but the significance of that substitution: reducing cardenolide sensitivity or not, is unclear. However, poisoning of the larvae by low levels of cardenolides is assumed to be prevented by non-resorption and excretion of the polar cardenolides, which cannot passively permeate the midgut membrane. This example of an insect-plant interaction demonstrates that by morphological and behavioural adaptation, a fruit fly manages to overcome even highly effective defence mechanisms of its host plant.

Pharmacoepigenetics of the role of DNA methylation in µ-opioid receptor expression in different human brain regions.

AIM: Exposure to opioids has been associated with epigenetic effects. Studies in rodents suggested a role of varying degrees of DNA methylation in the differential regulation of µ-opioid receptor expression across the brain. METHODS: In a translational investigation, using tissue acquired postmortem from 21 brain regions of former opiate addicts, representing a human cohort with chronic opioid exposure, µ-opioid receptor expression was analyzed at the level of DNA methylation, mRNA and protein. RESULTS & CONCLUSION: While high or low µ-opioid receptor expression significantly correlated with local OPRM1 mRNA levels, there was no corresponding association with OPRM1 methylation status. Additional experiments in human cell lines showed that changes in DNA methylation associated with changes in µ-opioid expression were an order of magnitude greater than differences in brain. Hence, different degrees of DNA methylation associated with chronic opioid exposure are unlikely to exert a major role in the region-specificity of µ-opioid receptor expression in the human brain.

Field study to detect illicit and medicinal drugs in car drivers in Southern and Western Hesse.

In the present study, immunochemical tests (Mahsan DrugInspector, DOA4, DOA8, DOA10, Protzek) as well as the detection rate of police checks were evaluated. Urine and blood samples of suspected car drivers were analysed by chromatography-mass spectrometry. Additionally, anonymised urine samples were analysed on a voluntary basis in cases where no legal proceedings were initiated. Toxicological analyses (total unknown screening) were performed using gas chromatography-mass spectrometry (GC-MS) after hydrolysis, acidic and alkaline extraction and derivatization. A data base for screening 9000 substance entries was applied. In addition, urine samples were analysed using liquid chromatography/ time-of-flight mass spectrometry (HPLC-ToF-MS) to screen psychiatric and narcotic drugs. In total, samples of 154 suspects were analysed, of these, 46 samples for no actual reason. In 5 of the latter samples, forensically relevant substances were detected; in two cases the consumption of illicit drugs, i. e. cannabis and methamphetamine, was proved. Of the 154 suspects, 108 were charged with driving under the influence of drugs; in samples of 103 of these cases, illicit drugs were found. Immunochemical pretesting showed posi- tive results in 97 of the 108 cases; in 6 samples, psychiatric drugs (citalopram, doxepin, promethazine, mirtazapine, fluoxetine, venlafaxine) were later identified, which are not detectable by ordinary pretesting systems. Police officers successfully identified 95.4 % of the suspects as drug consumers, which is an excellent result. In practice, pretesting of urine samples using immunochemical techniques proved to be very reliable. The Protzek system in particular corresponded well with the results of the chromatographic analyses. In conclusion, systematic chromatographic-mass spectrometric analysis of urine samples of suspects is recommended to identify car drivers consuming illicit drugs and to obtain data usable in legal proceedings (e. g. suspending of the driving license), which is not always possible when using blood samples in cases of drugs consumed some time ago.

Pitfall in cannabinoid analysis--detection of a previously unrecognized interfering compound in human serum.

In clinical and forensic toxicology, high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS) is increasingly used since it allows the development of sensitive and fast drug analysis procedures. During development of a LC-MS/MS method for determination of the psychoactive cannabinoid Δ(9)-tetrahydrocannabinol (THC) and of its two metabolites 11-hydroxy-THC (THCOH) and 11-nor-9-carboxy-THC (THCCOOH) in serum, a previously unrecognized interfering compound was detected. Extending the fast gradient elution program by an isocratic phase leads to sufficient separation of the interfering compound, initially co-eluting with THCCOOH and exhibiting the same fragments. For characterization, product ion scans and precursor ion scans were performed. Samples from cannabis users were analyzed to estimate the abundance of the interfering compound. The mass spectrometric experiments showed that the interfering compound exhibited the same molecular mass as THCCOOH and a similar fragmentation pattern except for relative fragment intensities. This compound was exclusively detectable in authentic samples. Concentrations were in the range of 4.5 to 51 % (median 14.6 %, n = 73) of those of THCCOOH. After further optimization of the gradient, the method was sufficiently selective and sensitive and validation parameters were within acceptance limits. A new compound related to cannabis use was detected in human serum, and data suggest an isomeric structure to THCCOOH. Considering the rather high amounts observed, it was surprising that this compound had not been detected previously. Further studies on its structure and origin are necessary.

Use of lidocaine in endotracheal intubation. Blood and urine concentrations in patients and deceased after unsuccessful resuscitation.

In toxicological analysis of postmortem samples the local anesthetic lidocaine is often identified. In most cases, lidocaine levels result from its use as aid in endotracheal intubation. The range of the drug's concentration in blood and urine was studied under controlled conditions from a cohort of cardiac surgery patients (n=35). Plasma concentrations 1 h after exposure to lidocaine in the range of the recommended 81 mg coating the endotracheal tube were less than 0.2 mg/l, its metabolite monoethylglycinxylidide (MEGX) less than 0.05 mg/l (median ratio 0.18, range 0.03-1.23). Also the concentrations of lidocaine and MEGX in urine samples were low (less than 1.2 and 0.1 mg/l, respectively) with MEGX/lidocaine ratios of 0.11 (median, range up to 1.2). These data were compared with results obtained by analyzing postmortem blood and urine samples of 18 deceased with a documented cardiopulmonary resuscitation attempt prior to death. Blood concentrations were in the same range (lidocaine median 0.07, range 0.02-1.07 mg/l; MEGX median 0.01, range <0.001-0.044 mg/l); besides low lidocaine concentrations in urine. MEGX was detected only in 2 out of 9 urine samples. The results of the present study confirm that lidocaine is absorbed in the trachea from the endotracheal tube coated with lidocaine containing gel. Postmortem quantitative results can be explained on the basis of the data obtained in the controlled study.

Influence of ethanol on the pharmacokinetic properties of Δ9-tetrahydrocannabinol in oral fluid.

Oral fluid (OF) tests aid in identifying drivers under the influence of drugs. In this study, 17 heavy cannabis users consumed alcohol to achieve steady blood alcohol concentrations of 0 to 0.7 g/L and smoked cannabis 3 h afterward. OF samples were obtained before and up to 4 h after smoking and on-site tests were performed (Dräger DrugTest 5000 and Securetec DrugWipe 5+). Maximum concentrations of tetrahydrocannabinol (THC) immediately after smoking (up to 44,412 ng/g) were below 4,300 (median 377) ng/g 1 h after smoking and less than 312 (median 88) ng/g 3 h later with 5 of 49 samples negative, suggesting that recent cannabis use might occasionally not be detectable. An influence of alcohol was not observed. Drinking 300 mL variably influenced THC concentrations (median only -29.6%), which suggests that drinking does not markedly affect on-site test performance. Many (92%) Dräger tests performed 4 h after smoking were still positive, indicating sufficient sensitivity for recent cannabis use. Differences in the results of a roadside study with DrugTest 5000 (sensitivity 84.8%, specificity 96.0%, accuracy 84.3%) could be explained by a higher number of true negatives, differences between OF and serum and differences between occasional and chronic users.

Influence of ethanol on cannabinoid pharmacokinetic parameters in chronic users.

Cannabis is not only the most widely used illicit drug worldwide but is also regularly consumed along with ethanol. In previous studies, it was assumed that cannabis users develop cross-tolerance to ethanol effects. The present study was designed to compare the effects of ethanol in comparison to and in combination with a cannabis joint and investigate changes in pharmacokinetics. In this study, 19 heavy cannabis users participated and received three alcohol dosing conditions that were calculated to achieve steady blood alcohol concentrations (BAC) of about 0, 0.5 and 0.7 g/l during a 5-h time window. Subjects smoked a Δ(9)-tetrahydrocannabinol (THC) cigarette (400 µg/kg) 3 h post-onset of alcohol dosing. Blood samples were taken between 0 and 4 h after smoking. During the first hour, samples were collected every 15 min and every 30 min thereafter. Mean steady-state BACs reached 0, 0.36 and 0.5 g/l. The apparent elimination half-life of THC was slightly prolonged (1.59 vs. 1.93 h, p < 0.05) and the concentration 1 h after smoking was slightly lower (24 vs. 17 ng/ml, p < 0.05) with the higher ethanol dose. The prolonged THC elimination might be explained by a small ethanol-mediated change in distribution to and from deep compartments. Concentrations and pharmacokinetics of 11-hydroxy-THC and 11-nor-9-carboxy-THC (THCA) were not significantly influenced by ethanol. However, THCA concentrations appeared lower in both ethanol conditions, which might also be attributable to changes in distribution. Though not significant in the present study, this might be relevant in the interpretation of cannabinoid concentrations in blood.

[Interpretation of blood analysis data found after passive exposure to cannabis]. / Interpretation analytischer Befunde in Blutproben bei passiver Cannabisexposition.

When defendants are confronted with evidence of cannabinoids in their blood suggesting consumption of cannabis they sometimes argue that this could only be due to a passive exposure. The small number of controlled studies available showed that tetrahydrocannabinol (THC), the active ingredient of cannabis, was actually found in the blood after passive exposure to cannabis smoke. The resulting blood concentrations were dependent on the applied THC doses and the size of the room in which the passive exposure occurred. However, the quantitative data indicated in the publications of the 1980s cannot be fully compared with the results of modern analytical methods. Due to the rapid distribution of THC in the body, which occurs also after passive exposure to low doses, the THC concentration in serum to be expected in a blood sample taken 1 hour after exposure is less than 1 ng/mL. For assessment of an alleged passive exposure, the metabolic THC-carboxylic acid, which is excreted more slowly, must also be taken into account. After passive exposure, similar and very low serum concentrations of THC and THC-carboxylic acid are to be expected (< 2 ng/mL), while higher blood levels suggest the deliberate consumption of a psychoactive dose.