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Hexahydrocannabinol (HHC), 6,6,9-trimethyl-3-pentyl-6a,7,8,9,10,10a-hexahydrobenzo[c]chromen-1-ol, is a semi-synthetic cannabinoid that has presented challenges to analytical laboratories due to its emergence and spread in the drug market. The lack of information on human pharmacokinetics hinders the development and application of presumptive and confirmatory tests for reliably detecting HHC consumption. To address this knowledge gap, we report the analytical results obtained from systematic forensic toxicological analysis of body-fluid samples collected from three individuals suspected of drug-impaired driving after HHC consumption. Urine and plasma samples were analyzed using non-targeted liquid chromatography–high-resolution tandem mass spectrometry. The results provided evidence that HHC undergoes biotransformation reactions similar to other well-characterized cannabinoids, such as ∆9-tetrahydrocannabinol or cannabidiol. Notably, HHC itself was only detectable in plasma samples, not in urine samples. The observed Phase I reactions involved oxidation of C11 and the pentyl side chain, leading to corresponding hydroxylated and carboxylic acid species. Additionally, extensive glucuronidation of HHC and its Phase I metabolites was evident. [ABSTRACT FROM AUTHOR]

Systematic toxicological analysis (STA) is an important step in medicolegal investigations of death, poisoning, and drug use. The primary goal is the detection and confirmation of potentially toxic compounds in [...]

We present a rapid and informative mitoehondrial DNA profiling system, which has high forensic impact. The assay is based on the analysis of a 23-plex PCR by ion-pair reversed-phase high-performance liquid chromatography online hyphenated to electrospray ionization time-of-flight mass spectrometry (ICEMS). In a single 25-min run, an overall number of 627 nucleotide positions were screened. The vast majority of observed sequence variations were explainable by alterations of the allelic states of the 23 target SNPs, which were selected on their ability to increase forensic discrimination within West Eurasian populations. Within an Austrian population sample comprising 90 unrelated men, 14 different, nontarget SNP-related sequence variations-13 base substitutions and 1 deletion-were detected by ICEMS and confirmed by sequencing. All amplified sequences were located outside of the routinely sequenced hypervariable segments (HVS-I and HVS-II) of the noncoding control region. Accordingly, the genetic information obtained by the 23-plex PCRICEMS assay could be combined with HVS-I/HVS-II sequencing results to one highly discriminating mtDNA profile, which covered ~7.5% of the total mtDNA genome. With the 23-plex PCR-ICEMS assay, DNA mixtures were detected and the allelic ratios were accurately quantified. The observed robustness and sensitivity underlined the practical applicability of the assay in forensic science, which was proven by typing eight representative casework samples.

Buprenorphine is a semisynthetic opioid that is often used in opiate maintenance therapy. For this purpose, regular toxicological analyses of urine samples are mandatory. For fast analytical results, analyses are commonly performed by immunoassay, for example, Thermo Scientific™ CEDIA® Buprenorphine or Buprenorphine II assay. One drawback of immunoassay-based methods is the possible cross-reaction with other substances. Several drugs have already been checked for cross-reactivity to CEDIA® Buprenorphine II immunoassay. In contrast, cross-reactivities have not been checked for any food additives. In the present study, a cross-reaction of CEDIA® Buprenorphine II assay to steviol glucuronide was investigated. Steviol glucuronide is a phase II metabolite of the sugar substitute stevia. For our study, 32 urine samples of patients in rehabilitation centers were collected. These samples were tested positive with the CEDIA® Buprenorphine II immunoassay. These findings were suspicious, because it was highly unlikely that the patients in those institutions had access to buprenorphine. The absence or presence of buprenorphine in urine samples was evaluated by a validated gas chromatography–mass spectrometry method. In order to determine the concentration of steviol glucuronide in urine samples, a liquid chromatography–tandem mass spectrometry method has been developed and fully validated according to the respective guidelines of the German Society of Toxicological and Forensic Chemistry. The cross-reactivity of steviol glucuronide in the CEDIA® Buprenorphine II immunoassay was observed at concentrations above 15,000 µg/L. These findings demonstrate that food additives should also be considered as compounds that may reduce the selectivity of immunoassays and emphasize the importance of confirming implausible results by selective analytical methods. [ABSTRACT FROM AUTHOR]

Buprenorphine is a commonly used opioid in pain therapy as well as in opiate maintenance therapy. Immunoassays are quick and cost-effective methods for the necessary toxicological urine analysis of maintenance therapy patients. In this study a novel enzymatic immunoassay, the Thermo Fisher Scientific CEDIA Buprenorphine II assay (Bup2) was evaluated for the detection of buprenorphine, norbuprenorphine and their conjugated metabolites in human urine samples. The Bup2 assay has a cut-off of 10 ng/mL with ±25% controls, whereas the existing CEDIA Buprenorphine assay (Bup1) has a cut-off of 5 ng/mL and ±40% controls. Both assays were analyzed on a Thermo Scientific Indiko Plus benchtop analyzer. Seven-day precision studies of Bup2 assay demonstrated excellent precision of 7.2–10.6%. No crossover between control samples and the cut-off level were observed. Urine samples of 120 patients undergoing opiate maintenance therapy were collected. Immunoassay results of Bup1 and Bup2 were confirmed by gas chromatography mass spectrometry (GC/MS) for buprenorphine and norbuprenorphine as well as for their glucuronides. Comparison showed a specificity of 0.99 between the Bup2 assay and GC/MS, whereas the Bup1 assay had a specificity 0.70 due to 21 false positive samples. The reason is a known cross-reactivity of the Bup1 assay to opiate compounds. The Bup2 assay revealed one false positive result close to the cut-off value; no specific candidate possibly causing a cross-reaction was detected by GC/MS and liquid chromatography tandem mass-spectrometry (LC/MS/MS) methods. The data presented demonstrate an excellent correlation of the Bup2 assay to GC/MS, showing improved specificity and sensitivity when compared to the Bup1 assay. Thus, the Bup2 assay is highly suitable for urine testing, even for opiate maintenance patients receiving high doses of morphine. [ABSTRACT FROM AUTHOR]

Electrochemistry (EC) is a mature scientific discipline aimed to study the movement of electrons in an oxidation-reduction reaction. EC covers techniques that use a measurement of potential, charge, or current to determine the concentration or the chemical reactivity of analytes. The electrical signal is directly converted into chemical information. For in-depth characterization of complex electrochemical reactions involving the formation of diverse intermediates, products and byproducts, EC is usually combined with other analytical techniques, and particularly the hyphenation of EC with mass spectrometry (MS) has found broad applicability. The analysis of gases and volatile intermediates and products formed at electrode surfaces is enabled by differential electrochemical mass spectrometry (DEMS). In DEMS an electrochemical cell is sampled with a membrane interface for electron ionization (EI)-MS. The chemical space amenable to EC/MS (i.e., bioorganic molecules including proteins, peptides, nucleic acids, and drugs) was significantly increased by employing electrospray ionization (ESI)-MS. In the simplest setup, the EC of the ESI process is used to analytical advantage. A limitation of this approach is, however, its inability to precisely control the electrochemical potential at the emitter electrode. Thus, particularly for studying mechanistic aspects of electrochemical processes, the hyphenation of discrete electrochemical cells with ESI-MS was found to be more appropriate. The analytical power of EC/ESI-MS can further be increased by integrating liquid chromatography (LC) as an additional dimension of separation. Chromatographic separation was found to be particularly useful to reduce the complexity of the sample submitted either to the EC cell or to ESI-MS. Thus, both EC/LC/ESI-MS and LC/EC/ESI-MS are common. © 2013 The Authors. Mass Spectrometry Reviews published by Wiley Periodicals, Inc. Mass Spec Rev 34:64-92, 2015 [ABSTRACT FROM AUTHOR]

Ca 2+ -influx through L-type Ca 2+ -channels (LTCCs) is associated with activity-related stressful oscillations of Ca 2+ levels within dopaminergic (DA) neurons in the substantia nigra (SN), which may contribute to their selective degeneration in Parkinson's disease (PD). LTCC blockers were neuroprotective in mouse neurotoxin models of PD, and isradipine is currently undergoing testing in a phase III clinical trial in early PD. We report no evidence for neuroprotection by in vivo pretreatment with therapeutically relevant isradipine plasma levels, or Ca v 1.3 LTCC deficiency in 6-OHDA-treated male mice. To explain this finding, we investigated the pharmacological properties of human LTCCs during SN DA-like and arterial smooth muscle (aSM)-like activity patterns using whole-cell patch-clamp recordings in HEK293 cells (Ca v 1.2 α1-subunit, long and short Ca v 1.3 α1-subunit splice variants; β3/α2δ1). During SN DA-like pacemaking, only Ca v 1.3 variants conducted Ca 2+ current (I Ca ) at subthreshold potentials between action potentials. SN DA-like burst activity increased integrated I Ca during (Ca v 1.2 plus Ca v 1.3) and after (Ca v 1.3) the burst. Isradipine inhibition was splice variant and isoform dependent, with a 5- to 11-fold lower sensitivity to Ca v 1.3 variants during SN DA-like pacemaking compared with Ca v 1.2 during aSM-like activity. Supratherapeutic isradipine concentrations reduced the pacemaker precision of adult mouse SN DA neurons but did not affect their somatic Ca 2+ oscillations. Our data predict that Ca v 1.2 and Ca v 1.3 splice variants contribute differentially to Ca 2+ load in SN DA neurons, with prominent Ca v 1.3-mediated I Ca between action potentials and after bursts. The failure of therapeutically relevant isradipine levels to protect SN DA neurons can be explained by weaker state-dependent inhibition of SN DA LTCCs compared with aSM Ca v 1.2. SIGNIFICANCE STATEMENT The high vulnerability of dopamine (DA) neurons in the substantia nigra (SN) to neurodegenerative stressors causes Parkinson's disease (PD). Ca 2+ influx through voltage-gated L-type Ca 2+ channels (LTCCs), in particular Ca v 1.3, appears to contribute to this vulnerability, and the LTCC inhibitor isradipine is currently being tested as a neuroprotective agent for PD in a phase III clinical trial. However, in our study isradipine plasma concentrations approved for therapy were not neuroprotective in a PD mouse model. We provide an explanation for this observation by demonstrating that during SN DA-like neuronal activity LTCCs are less sensitive to isradipine than Ca v 1.2 LTCCs in resistance blood vessels (mediating dose-limiting vasodilating effects) and even at supratherapeutic concentrations isradipine fails to reduce somatic Ca 2+ oscillations of SN DA neurons., (Copyright © 2017 the authors 0270-6474/17/376762-17$15.00/0.)

We herein present the supramolecular construction of a completely fluorescent unquenched multichromophoric wheel consisting of boron dipyrromethene dyes arranged perpendicularly to the circular plane.