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.

Chronic alcohol abuse may lead to high skin iron content, but not to hepatic siderosis.

INTRODUCTION: In forensic cases, ante mortem chronic alcohol abuse can be of central importance in clarifying circumstances of death. However, reliable markers of alcohol consumption, which are still available postmortem, are needed. In addition to medical history data which may not be always authentic, the determination of ethyl glucuronide (EtG) in hair as a promising parameter is of no value in cases of missing or cosmetically treated hair. On the other hand, there exist reports that iron ions accumulate in liver tissue (siderosis) during chronic, excessive alcohol consumption, which, therefore, may be useful to serve as alcohol abuse correlate. However, the influence of ethanol on iron stored in the liver has not been adequately investigated and the study situation appears to be inconsistent. AIMS: The aim of the present study was to assess the suitability of assaying iron concentrations in liver and other tissues as postmortem alcoholism marker. METHODS: The iron concentration in tissue samples (liver, brain, skin, pancreas, spleen), vitreous fluid and blood taken during autopsy was analyzed by atomic absorption spectroscopy. The analytical method has been validated before. Cases were divided into two groups: chronic alcohol abusers and non-chronic alcohol consumers including total abstainers using ethyl glucuronide levels in hair as well as anamnestic data as criteria. RESULTS: No elevated iron concentrations in the liver of chronic alcohol abusers were detected. Surprisingly, the iron concentration in skin tissue was found to be significantly higher in cases of chronic alcohol abuse, independent on whether fatty liver or liver cirrhosis was present (as diagnosedduring autopsy). In 18.5% of the cases, chronic alcohol abuse was not confirmed by the EtG concentration in hair. Thus, anamnestic data should not be overestimated. CONCLUSION: The general assumption that chronic alcohol abuse induces hepatic siderosis, i.e. high iron concentrations in liver tissue, has not been supported by results of the present study. However, there seems to exist a correlation between chronic alcohol abuse and high iron concentrations in the skin.

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.

A Dual Modulator of Farnesoid X Receptor and Soluble Epoxide Hydrolase To Counter Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis arising from Western diet and lifestyle is characterized by accumulation of fat in liver causing inflammation and fibrosis. It evolves as serious health burden with alarming incidence, but there is no satisfying pharmacological therapy to date. Considering the disease's multifactorial nature, modulation of multiple targets might provide superior therapeutic efficacy. In particular, farnesoid X receptor (FXR) activation that revealed antisteatotic and antifibrotic effects in clinical trials combined with inhibition of soluble epoxide hydrolase (sEH) as anti-inflammatory strategy promises synergies. To exploit this dual concept, we developed agents exerting partial FXR agonism and sEH inhibitory activity. Merging known pharmacophores and systematic exploration of the structure-activity relationship on both targets produced dual modulators with low nanomolar potency. Extensive in vitro characterization confirmed high dual efficacy in cellular context combined with low toxicity, and pilot in vivo data revealed favorable pharmacokinetics as well as engagement on both targets in vivo.

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.

An extraordinary form of the Melkersson-Rosenthal syndrome successfully treated with the tumour necrosis factor-α blocker adalimumab.

Melkersson-Rosenthal syndrome (MRS) is a rare granulomatous inflammatory disease characterised by the triad of orofacial oedema, facial nerve palsy and furrowed tongue. We describe the case of a 29-year-old patient suffering from an oligosymptomatic form of the disease with orofacial oedema, cobblestone pattern on the buccal mucosa and swelling of the tongue, accompanied by intermittent fatigue, influenza-like symptoms, intermittent tinnitus and acute hearing loss. An increase of several autoimmune-associated antibodies was also detected. Treatment with prednisolone, azathioprine or methotrexate failed to adequately control all symptoms in the long term. In the absence of a specific and well-established therapy for MRS, treatment with adalimumab was administered. Under adalimumab, total remission of all symptoms was achieved, indicating that tumour necrosis factor-α blockers are a promising therapeutic option for patients with Melkersson-Rosenthal syndrome.

Carnosic acid and carnosol, phenolic diterpene compounds of the labiate herbs rosemary and sage, are activators of the human peroxisome proliferator-activated receptor gamma.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand activated transcription factor, belonging to the metazoan family of nuclear hormone receptors. Activation of PPARgamma increases the transcription of enzymes involved in primary metabolism, leading to lower blood levels of fatty acids and glucose. Hence, PPARgamma represents the major target for the glitazone type of drugs currently being used clinically for the treatment of type 2 diabetes. Furthermore, activators of PPARgamma show beneficial anti-inflammatory and anti-tumour effects. Utilizing a fusion receptor of the yeast Gal4-DNA binding domain joined to the hinge region and ligand binding domain of the human PPARgamma in combination with a Gal4-driven luciferase reporter gene, cotransfected into Cos7 cells, we tested sage and rosemary extracts prepared with 80 % aqueous ethanol for possible PPARgamma activation. This revealed that both extracts are capable of selectively activating Gal4-PPARgamma fusion receptor, in a concentration-dependent manner, with EC (50) values of 22.8 +/- 8.4 mg/L and 33.7 +/- 7.3 mg/L for rosemary and sage, respectively. Subsequent analysis of the characteristic constituents revealed the phenolic diterpene compounds carnosol, present in both herbs, and carnosic acid to be active principles of these extracts, showing EC (50) values of 41.2 +/- 5.9 microM and 19.6 +/- 2.0 microM, respectively. Thus it can be concluded that the glucose lowering effect reported recently for rosemary may be attributed to PPARgamma activation. Moreover, our observations may also explain the anti-inflammatory and antiproliferative effects of both compounds published previously.