Toxicogenetic analysis of Δ9-THC-metabolizing enzymes.

While the impact of genetic polymorphisms on the metabolism of various pharmaceuticals is well known, more data are needed to better understand the specific influence of pharmacogenetics on the metabolism of delta 9-tetrahydocannabinol (Δ9-THC). Therefore, the aim of the study was to analyze the potential impact of variations in genes coding for phase I enzymes of the Δ9-THC metabolism. First, a multiplex assay for genotyping different variants of genes coding for phase I enzymes was developed and applied to 66 Δ9-THC-positive blood samples obtained in cases of driving under the influence of drugs (DUID). Genetic and demographic data as well as plasma concentrations of Δ9-THC, 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-Δ9-THC), and 11-nor-9-carboxy-Δ9-THC (Δ9-THC-COOH) were combined and statistically investigated. For cytochrome P450 2C19 (CYP2C19) variants, no differences in analyzed cannabinoid concentrations were found. There were also no differences in the concentrations of Δ9-THC and 11-OH-Δ9-THC for the different allelic CPY2C9 status. We recognized significantly lower Δ9-THC-COOH concentrations for CYP2C9*3 (p = 0.001) and a trend of lower Δ9-THC-COOH concentrations for CYP2C9*2 which did not reach statistical significance (p = 0.068). In addition, this study showed significantly higher values in the ratio of Δ9-THC/Δ9-THC-COOH for the carriers of the CYP2C9 variants CYP2C9*2 and CYP2C9*3 compared with the carriers of the corresponding wild-type alleles. Therefore, an impact of variations of the CYP2C9 gene on the interpretation of cannabinoid plasma concentrations in DUID cases should be considered.

Fatal poisoning with diethylene glycol in an unusual setting.

Morphological findings in cases of intoxication are relatively rare in forensic pathology. In this article we report on a 26-year-old man who drank clear fluid from a tequila bottle that was given to him by a friend. Afterwards, the clear fluid was assumed to be smoke fluid containing diethylene glycol (DEG). The man died eight days later. Macroscopic and microscopic examination of the kidneys and the liver at forensic autopsy revealed findings typical of a DEG intoxication. In addition, the clinical course showed the typical triphasic pattern of symptoms. Toxicological analysis confirmed the presence of DEG in both the original smoke fluid and the tequila bottle. In conclusion, death was due to fatal intoxication by DEG. While most DEG intoxications have been mass poisoning incidents attributed to pharmaceutical products, the present case describes an unusual example of a single decedent.

8ß-OH-THC and 8ß,11-diOH-THC-minor metabolites with major informative value?

The ∆9-tetrahydrocannabinol (THC) metabolites 8ß-hydroxy-THC and 8ß,11-dihydroxy-THC are mentioned in the literature as potential blood markers of recent cannabis use. However, the formation of these metabolites in in vivo detectable concentrations has been described controversially. Therefore, the aim of this study was to verify the in vivo metabolism of 8ß-hydroxy-THC and 8ß,11-dihydroxy-THC in order to evaluate their potential as blood markers of recent cannabis use. First, we developed and validated a solid-phase-extraction method coupled with gas chromatography-mass spectrometry in order to enable the selective and very sensitive determination of 8ß-hydroxy-THC and 8ß,11-dihydroxy-THC. The application of this method in the analysis of 70 authentic plasma samples of cannabis users revealed positive results for both analytes. We detected 8ß-hydroxy-THC in three and 8ß,11-dihydroxy-THC in 37 out of the 70 analyzed samples. For 8ß-hydroxy-THC, all of the three positive results were below the limit of quantification (LOQ; 0.3 ng/mL) but above the limit of detection (LOD; 0.2 ng/mL). For 8ß,11-dihydroxy-THC, only two positive results were below the LOQ (0.4 ng/mL) but above the LOD (0.3 ng/mL); the remaining 35 were quantified. Hence, we were able to prove the in vivo metabolism from THC to both 8ß-hydroxy-THC and 8ß,11-dihydroxy-THC in detectable concentrations. The quantitative comparison of 8ß-hydroxy-THC and 8ß,11-dihydroxy-THC with the main cannabinoids THC, 11-hydroxy-THC, and 11-nor-9-carboxy-THC revealed no further informative value for 8ß-hydroxy-THC regarding the last time of cannabis consumption. However, the detectability from 8ß,11-dihydroxy-THC compared to 11-hydroxy-THC suggests a shorter detection time for 8ß,11-dihydroxy-THC and thereby a promising application of this metabolite as a blood marker of recent cannabis use.

Development and validation of a solid-phase extraction method using anion exchange sorbent for the analysis of cannabinoids in plasma and serum by gas chromatography-mass spectrometry.

The aim of this work was to develop and validate a solid-phase extraction (SPE) method for the analysis of cannabinoids with emphasis on a very extensive and effective matrix reduction in order to ensure constant good results in selectivity and sensitivity regardless of the applied measuring technology. This was obtained by the use of an anion exchange sorbent (AXS) and the purposive ionic interaction between matrix components and this sorbent material. In a first step, the neutral cannabinoids ∆9-tetrahydrocannabinol (THC) and 11-hydroxy-∆9-tetrahydrocannabinol (11-OH-THC) were eluted, leaving 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THC-COOH) and the main interfering matrix components bound to the AXS. In a second step, exploiting differences in pH and polarity, it was possible to separate matrix components and THC-COOH, thereby yielding a clean elution of THC-COOH into the same collecting tube as THC and 11-OH-THC. Even when using a simple measuring technology like gas chromatography with single quadrupole mass spectrometry, this two-step elution allows for an obvious decrease in number and intensity of matrix interference in the chromatogram. Hence, in both plasma and serum, the AXS extracts resulted in very good selectivity. Limits of detection and limits of quantification were below 0.25 and 0.35 ng/mL for the neutral cannabinoids in both matrices, 2.0 and 3.0 ng/mL in plasma and 1.6 and 3.3 ng/mL in serum for THC-COOH. The recoveries were ≥79.8 % for all analytes. Interday and intraday imprecisions ranged from 0.8 to 6.1 % relative standard deviation, and accuracy bias ranged from -12.6 to 3.6 %.

Determination of psilocin, bufotenine, LSD and its metabolites in serum, plasma and urine by SPE-LC-MS/MS.

A validated method for the simultaneous determination of psilocin, bufotenine, lysergic acid diethylamide and its metabolites in serum, plasma and urine using liquid chromatography-electrospray ionization/tandem mass spectrometry was developed. During the solid-phase extraction procedure with polymeric mixed-mode cation exchange columns, the unstable analytes were protected by ascorbic acid, drying with nitrogen and exclusion of light. The limits of detection and quantitation for all analytes were low. Recovery was ≥86 % for all analytes and no significant matrix effects were observed. Interday and intraday imprecisions at different concentrations ranged from 1.1 to 8.2 % relative standard deviation, bias was within ±5.3 %. Processed samples were stable in the autosampler for at least 2 days. Furthermore, freeze/thaw and long-term stability were investigated. The method was successfully applied to authentic serum and urine samples.

A validated method for quantitation of psilocin in plasma by LC-MS/MS and study of stability.

A liquid chromatography-electrospray ionization/tandem mass spectrometry method for the quantitation of psilocin in plasma is presented. Sample workup was performed with mixed-mode solid-phase extraction using ascorbic acid and nitrogen for drying to protect the unstable analyte. Calibration curves were linear from 2 to 100 ng/mL, and no selectivity problems occurred. The limit of detection was 0.1 ng/mL, and the limit of quantitation was 0.34 ng/mL. Recovery was >86% and matrix effects were <110%. Both were reproducible. Interday and intraday precisions at different concentrations were 1.5-4.3% relative standard deviation, bias within ±9%. Processed samples were stable in the autosampler for at least 26 h. Furthermore, the stability of psilocin in blood stored at different temperatures over various periods of time was investigated. Samples stored at room temperature showed a continuous decrease of analyte leading to a loss of about 90% after 1 week. Storage in the fridge improved sample stability significantly. Freezing of blood samples led to a not reproducible loss of psilocin.

Separation of positional CPP isomers by chiral HPLC-DAD of seized tablets.

Meta-chlorophenylpiperazine, one of the synthetic piperazine-derived designer drugs, is to date controlled as an illicit substance in five European member states. Depending on the position of the chlorine atom, different positional isomers of CPP (ortho-, meta- and para-) are possible. Therefore, there is a need to develop an analytical method for the separation and identification of the three 1-chlorophenylpiperazines in tablets containing CPP. In this work, the position isomers o-, m- and p-CPP were separated by liquid chromatography (HPLC) on a reversed-phase chiral column. Different mobile phase compositions and pH ranges were systematically studied to find optimum chromatographic conditions. Best results were achieved with isocratic mobile phase of triethyl amine buffer and methanol (V/V = 70/30) at pH 9 with a flow rate of 0.8 ml/min. The method was validated in terms of selectivity, linearity, limit of detection and quantification and precision. At last, the developed method was successfully applied on seized ecstasy tablets.

Immunohistochemical expression of fibronectin and C5b-9 in the myocardium in cases of carbon monoxide poisoning.

Even if there is clinical evidence that carbon monoxide poisoning determines cardiac damage, the literature on the cardiac pathomorphology in such cases is scarce. We investigated the immunohistochemical expression of two known markers of fresh cardiac damage, fibronectin and the terminal complement complex C5b-9, in both cardiac ventricles in 26 cases of CO intoxication (study group, 15 ♀, 11 ♂, mean age 47 years, mean COHb level 65.9%, min. 51%, max. 85%) compared to a group of 23 cases of hanging (n = 23, 4♀, 19♂, mean age 42 years) as well as to 25 cases of myocardial infarction (n = 25, 13♀, 12♂, mean age 64 years). Fresh cardiac damage was detected with the antibody fibronectin in cases of CO poisoning and was prevalently localised at the right ventricle.

Immunohistochemical expression of fibronectin and C5b-9 in the myocardium in cases of fatal ethanol intoxication.

Data from the literature indicate that the pulmonary pressure rises in cases of ethanol intake. We have recently proposed a method for the detection of prevalent right ventricular damage in cases of fatal pulmonary thromboembolism and pulmonary fat embolism. In the present study, we compared the expression of the antibodies against fibronectin and C5b-9 in 19 cases of lethal alcohol intoxications (study group: 5 females, 14 males, mean age 46 years, mean blood ethanol concentration 3.5‰, min. 2.11‰, max. 5.31‰) to a group of 26 cases of fatal pulmonary thromboembolism (PE; group 2: 16 females, 10 males, mean age 56 years). Moreover, a group of 15 cases of hanging (group 3: 5 females, 10 males, mean age 50 years) as well as a group of 18 cases of myocardial infarction (group 4: 5 females, 13 males, mean age 61 years) were investigated as examples of typical cardiac damage due to global hypoxia during agony and ischemic damage, respectively. The results of this study show that fresh cardiac damage can be detected at both ventricles in cases of fatal ethanol intoxication with the antibody against fibronectin. The damage is prevalently localised at the right ventricle (RV), as already observed in cases of acute pulmonary hypertension determining right heart failure. The degree of damage at the RV in cases of ethanol intoxications is lower than the one observed in cases of fatal PE.

Analysis of psilocin, bufotenine and LSD in hair.

A method for the simultaneous extraction of the hallucinogens psilocin, bufotenine, lysergic acid diethylamide (LSD) as well as iso-LSD, nor-LSD and O-H-LSD from hair with hydrochloride acid and methanol is presented. Clean-up of the hair extracts is performed with solid phase extraction using a mixed-mode cation exchanger. Extracts are measured with liquid chromatography coupled with electrospray tandem mass spectrometry. The method was successfully validated according to the guidelines of the 'Society of Toxicological and Forensic Chemistry' (GTFCh). To obtain reference material hair was soaked in a solution of the analytes in dimethyl sulfoxide/methanol to allow incorporation into the hair. These fortified hair samples were used for method development and can be employed as quality controls.

Incidence of alcohol dependence among drunken drivers.

To discriminate 'alcoholics' and 'non-alcoholics', individual Alc-Indices (determined by methanol, acetone, 2-propanol, gamma-GT and CDT-concentrations) were calculated in a collective of 327 alcohol-impaired drivers with regard to the blood alcohol concentration, the time of the event and the age of the drivers. Applying this new defined Alc-Index, 48% of the drivers investigated could be characterised as alcohol dependent. The prevalence of alcoholics among individuals with blood alcohol concentrations (BAC) higher than 1.9per thousand was more than 80%. The diagnostic value of alcohol concentrations for the recognition of 'alcoholics', considering the legal limit in Germany (1.1per thousand) as well as statistically calculated limits, were compared to the Alc-Index.

Synthesis of a psilocin hapten and a protein-hapten conjugate.

Derivatives of psilocin with omega-functionalized alkyl spacers in position 1 of the indole ring were synthesized as haptens for use in a radioimmunoassay. Whereas the psilocin analogues with a 3-aminopropyl and a 4-aminobutyl moiety at the indole nitrogen decomposed during synthesis, the analogous 3-carboxypropyl psilocin derivative proved to be stable. This compound was coupled to bovine serum albumin (BSA) using the N-hydroxysuccinimide ester-mediated conjugation. The protein-hapten conjugate was characterized by matrix-assisted laser desorption ionization mass spectrometry. The mass spectrometry data indicated an average incorporation ratio of 4-5 molecules of psilocin hapten per molecule of BSA.

Synthesis, hydrolysis and stability of psilocin glucuronide.

A two-step synthesis of psilocin glucuronide (PCG), the main metabolite of psilocin, with methyl 2,3,4-tri-O-isobutyryl-1-O-trichloroacetimidoyl-α-d-glucopyranuronate is reported. With the synthesized PCG, hydrolysis conditions in serum and urine were optimized. Escherichia coli proved to be a better enzyme source for ß-glucuronidase than Helix pomatia. It was essential to add ascorbic acid to serum samples to protect psilocin during incubation. Furthermore the stability of PCG and psilocin was compared as stability data are the basis for forensic interpretation of measurements. PCG showed a greater long-term stability after six months in deep frozen serum and urine samples than psilocin. The short-term stability of PCG for one week in whole blood at room temperature and in deep frozen samples was also better than that of psilocin. Therefore, PCG can be considered to be more stable than the labile psilocin and should always be included if psilocin is analyzed in samples.