Evidence for the transfer of methadone and EDDP by sweat to children's hair.

In cases where there is a question as to whether children have come into contact with drugs, examinations of their scalp hair are frequently carried out. Positive test results are often discussed in the forensic community due to the various possible modes via which drugs and their metabolites can be incorporated into the hair. These include drug uptake by the child (e.g. oral ingestion or inhalation), but also contamination of hair via contact with the sweat from drug users. In this study, the possibility of methadone and its metabolite EDDP being incorporated into children's hair by contact with sweat from persons undergoing opiate maintenance therapy (methadone) was examined. The transfer of methadone and EDDP via sweat from methadone patients (n = 15) to children's hair was simulated by close skin contact of drug-free children's hair, encased in mesh-pouches, for 5 days. Sweat-collecting patches (hereafter referred to as 'sweat patches') were applied to the test persons' skin. One strand of hair and one sweat patch were collected daily from each patient. Analyses were performed using GC-MS/MS (hair) and LC-MS/MS (serum, sweat patches). After 4 days of skin contact, methadone was detectable in the formerly drug-free hair strands in all 15 study participants. EDDP was detectable in 34 of 75 hair strands, with the maximum number of positive results (11 EDDP-positive hair strands) being detected after 5 days. These results show that transfer of methadone and EDDP to drug-free hair is possible through close skin contact with individuals taking part in methadone substitution programmes. A correlation between serum concentration, sweat concentration and substance concentration in hair strands could not be demonstrated, but a tendency towards higher concentrations due to longer contact time is clearly evident.

Not only smoking is deadly: fatal ingestion of e-juice-a case report.

A fatal case of nicotine intoxication by oral intake of a nicotine solution, sold via the Internet, is reported. The concentrated nicotine solution (72 mg/mL) is usually diluted with polypropylene, polyethylene glycol or glycerine, respectively, in order to allow the user to generate their own solution for vaporisation in electronic cigarettes (e-juice). A 34-year-old man was found lifeless by his parents, who reported that their son had been in good health and had shown no hints of suicidal behaviour. The medicolegal autopsy revealed unspecific findings. Toxicological analysis revealed nicotine concentrations of 5.5 mg/L in femoral venous blood, 136 mg/L in heart blood, 12.0 mg/kg in brain tissue, 42.6 mg/kg in kidney tissue, 89.5 mg/kg in lung tissue and a total amount of 3,950 mg in the gastric contents. Cotinine concentrations were 0.9 mg/L in femoral venous blood, 7.6 mg/L in heart blood, 0.4 mg/kg in brain tissue, 0.9 mg/kg in kidney tissue and 0.8 mg/kg in lung tissue. No cotinine was detected in the gastric contents. The nicotine level measured in the femoral blood was in good accordance with the levels reported in other fatal cases caused by oral or patch application of nicotine. Moreover, the high level of nicotine in lung and kidney tissue, compared to that within femoral blood, strikingly emphasises the strong effect of post-mortem redistribution, underlined by the comparably low concentration of nicotine in the brain. The extremely high level of nicotine in the heart blood is more likely due to the high concentration in the gastric contents, due to oral intake, and by accumulation of the basic substance in the acidic gastric contents. This further highlights the effect of post-mortem redistribution. The mother of the deceased later admitted that her son had been suffering from psychosis and that she found a package containing five nicotine solution vials of the brand "Titanium Ice" (of 50 mL each). Three of the vials were empty. The nicotine concentration in the e-juice Titanium Ice was confirmed by HPLC analysis.

Assisted suicide by fentanyl intoxication due to excessive transdermal application.

Herein, we report a case of an assisted suicide committed by application of 34 matrix-based fentanyl-containing transdermal therapeutic systems (TTS) with different release rates. The TTS were supplied by the husband but administered by the deceased herself. Besides routine systematic toxicological analysis (STA), the concentrations of fentanyl and norfentanyl were determined in the blood (femoral and heart), urine, stomach content, brain, lung tissue, musculus iliopsoas, liver, kidney, bile and in some of the used TTS by LC-MS/MS. Blood levels of fentanyl were 60.6 µg/L in femoral blood and 94.1 µg/L in heart blood. These concentrations are in good concordance with levels described in cases with accidental or lethal suicidal fentanyl patch application. The organ distribution indicates an influence of post-mortem redistribution. The levels of residual fentanyl in the TTS were also determined. STA furthermore revealed supratherapeutic levels of bromazepam. Thus, the cause of death was a combination of fentanyl and bromazepam intoxication. However, considering the determined levels of fentanyl and norfentanyl in the entire set of specimens and the high toxicity in comparison to bromazepam, fentanyl was the leading toxic noxa.

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.

In vitro phase I metabolism of the synthetic cannabimimetic JWH-018.

A potent synthetic cannabinoid receptor agonist, JHW-018, was recently detected as one of the most prominent active agents in abusively used incenses such as Spice and other herbal blends. The high pharmacological and addictive potency of JWH-018 highlights the importance of elucidating the metabolism of JWH-018, without which a meaningful insight into its pharmacokinetics and its toxicity would not be possible. In the present study, the cytochrome P450 phase I metabolites of JWH-018 were investigated, after in vitro incubation of the drug with human liver microsomes, followed by liquid chromatography-tandem mass spectrometry analysis. This revealed monohydroxylation of the naphthalene ring system, the indole moiety, and the alkyl side chain. In addition, observations were made of dihydroxylation of the naphthalene ring system, and the indole moiety, or as result of a combination of monohydroxylations of both the naphthalene ring system and the indole moiety or the alkyl side chain, or a combination of monohydroxylations of both the indole ring system and the alkyl side chain. There is also evidence of trihydroxylation at different locations of the hydroxyl groups in the molecule. Furthermore, dehydration of the alkyl side chain, in combination with both monohydroxylation and dihydroxylation as well as arene oxidation of the naphthalene ring system, combined with both monohydroxylation and dihydroxylation at different sites of oxidation were found. N-dealkylation also in combination with both monohydroxylation and dihydrodiol formation of the N-dealkylated metabolite was detected. Finally, a metabolite was found carboxylated at the alkyl side chain.

Simultaneous quantification of THC-COOH, OH-THC, and further cannabinoids in human hair by gas chromatography-tandem mass spectrometry with electron ionization applying automated sample preparation.

The detection of Δ9 -tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in hair, for the purpose of identifying cannabis consumption, is conducted in many forensic laboratories. Since external contamination of hair with these cannabis components cannot be excluded, even after hair decontamination, only the detection of THC metabolites such as 11-nor-9-carboxy-Δ9 -tetrahydrocannabinol (THC-COOH) or 11-hydroxy-Δ9 -tetrahydrocannabinol (OH-THC), is considered to prove cannabis consumption. At present, testing for THC metabolites is not standard practice due to its analytical complexity. For these reasons, we developed a novel method for the detection of THC-COOH and OH-THC as well as THC, CBD, and CBN in one single analytical run using gas chromatography-tandem mass spectrometry (GC-MS/MS) with electron ionization. After manual hair washing and grinding, sample preparation was fully automated, by means of a robotic autosampler. The hair extraction took place by digestion with sodium hydroxide. A solid-phase extraction (SPE) was chosen for sample clean-up, using a mixed-mode anion exchange sorbent. Derivatization of all analytes was by silylation. The method has been fully validated according to guidelines of the Society of Toxicological and Forensic Chemistry (GTFCh), with a limit of detection (LOD) of 0.2 pg/mg for THC-COOH and OH-THC and 2 pg/mg for THC, CBD and CBN, respectively, thus fulfilling the Society of Hair Testing (SoHT) recommendations. The validated method has been successfully applied to our routine forensic case work and a summary of data from authentic hair samples is given, as well as data from proficiency tests.

Determination of 74 new psychoactive substances in serum using automated in-line solid-phase extraction-liquid chromatography-tandem mass spectrometry.

A detailed description is given of the development and validation of a fully automated in-line solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) method capable of detecting 90 central-stimulating new psychoactive substances (NPS) and 5 conventional amphetamine-type stimulants (amphetamine, 3,4-methylenedioxy-methamphetamine (MDMA), 3,4-methylenedioxy-amphetamine (MDA), 3,4-methylenedioxy-N-ethyl-amphetamine (MDEA), methamphetamine) in serum. The aim was to apply the validated method to forensic samples. The preparation of 150µL of serum was performed by an Instrument Top Sample Preparation (ITSP)-SPE with mixed mode cation exchanger cartridges. The extracts were directly injected into an LC-MS/MS system, using a biphenyl column and gradient elution with 2mM ammonium formate/0.1% formic acid and acetonitrile/0.1% formic acid as mobile phases. The chromatographic run time amounts to 9.3min (including re-equilibration). The total cycle time is 11min, due to the interlacing between sample preparation and analysis. The method was fully validated using 69 NPS and five conventional amphetamine-type stimulants, according to the guidelines of the Society of Toxicological and Forensic Chemistry (GTFCh). The guidelines were fully achieved for 62 analytes (with a limit of detection (LOD) between 0.2 and 4µg/L), whilst full validation was not feasible for the remaining 12 analytes. For the fully validated analytes, the method achieved linearity in the 5µg/L (lower limit of quantification, LLOQ) to 250µg/L range (coefficients of determination>0.99). Recoveries for 69 of these compounds were greater than 50%, with relative standard deviations≤15%. The validated method was then tested for its capability in detecting a further 21 NPS, thus totalling 95 tested substances. An LOD between 0.4 and 1.6µg/L was obtained for these 21 additional qualitatively-measured substances. The method was subsequently successfully applied to 28 specimens from routine forensic case work, of which 7 samples were determined to be positive for NPS consumption.

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.

Inhibition of 1,4-butanediol metabolism in human liver in vitro.

The conversion of 1,4-butanediol (1,4-BD) to gamma-hydroxybutyric acid (GHB), a drug of abuse, is most probably catalyzed by alcohol dehydrogenase, and potentially by aldehyde dehydrogenase. The purpose of this study was to investigate the degradation of 1,4-BD in cytosolic supernatant of human liver in vitro, and to verify involvement of the suggested enzymes by means of gas chromatography-mass spectrometry. The coingestion of 1,4-BD and ethanol (EtOH) might cause complex pharmacokinetic interactions in humans. Therefore, the effect of EtOH on 1,4-BD metabolism by human liver was examined in vitro. Additionally, the influence of acetaldehyde (AL), which might inhibit the second step of 1,4-BD degradation, was investigated. In case of a 1,4-BD intoxication, the alcohol dehydrogenase inhibitor fomepizole (4-methylpyrazole, FOM) has been discussed as an antidote preventing the formation of the central nervous system depressing GHB. Besides FOM, we tested pyrazole, disulfiram, and cimetidine as possible inhibitors of the formation of GHB from 1,4-BD catalyzed by human liver enzymes in vitro. The conversion of 1,4-BD to GHB was inhibited competitively by EtOH with an apparent K(i) of 0.56 mM. Therefore, the coingestion of 1,4-BD and EtOH might increase the concentrations and the effects of 1,4-BD itself. By contrast AL accelerated the formation of GHB. All antidotes showed the ability to inhibit the formation of GHB. In comparison FOM showed the highest inhibitory effectiveness. Furthermore, the results confirm strong involvement of ADH in 1,4-BD metabolism by human liver.