A comparison of nicotine content methods to produce a UPC2-MS2 method for the analysis of nicotine and minor alkaloids in SPECTRUM nicotine research cigarettes.

Nicotine is the principal alkaloid in tobacco and has been the primary subject of scientific investigation for its pharmacological effects contributing to tobacco use, dependence, withdrawal, and physical harm. Related minor alkaloids, accounting for less than 6% of alkaloid content in tobacco leaves, may also mirror some of the same pharmacological effects. To detect such low concentrations of the minor alkaloids, tobacco product methods produced by the Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) using gas chromatography and flame ionization detection (GC-FID) have been adapted for use with gas chromatography-mass spectrometry (GC-MS). Nicotine and minor alkaloid content in SPECTRUM Nicotine Research Cigarettes (NRC) have previously been determined using GC-FID; however, the minor alkaloids were unable to be detected or quantitated. This study employed UltraPerformance Convergence Chromatography (UPC2) system coupled with tandem mass spectrometry (MS2) to determine the nicotine and minor alkaloid content in NRC tobacco products. CORESTA Recommended Methods (CRMs) were adapted for their sample preparative procedures for optimal extraction followed by detection with UPC2-MS2. These results were compared to two separate CRMs that used GC-FID and GC-MS2 as well as an alternative method with GC-MS2 detection. The GC-FID and GC-MS2 CRM preparations along with the alternative GC-MS2 were unable to detect the analytes in every NRC formulation, whereas the UPC2-MS2 extraction and detection method was able to quantify every analyte in every NRC formulation. This increased sensitivity demonstrates the utility of the UPC2-MS2 analytical method in accurately detecting and quantifying nicotine and minor alkaloids in tobacco filler.

Thermal Degradants Identified from the Vaping of Vitamin E Acetate.

Studies have suggested that vitamin E acetate (VEA), when used in an electronic vaping device, undergoes thermal degradation and is considered one of the main contributors in e-cigarette or vaping product use-associated lung injury (EVALI). Using a Borgwaldt 5.1 linear smoker, a SVS250 Electronic Vaporizer and two types of tank systems, VEA was analyzed for degradation products produced via the Cooperation Centre for Scientific Research Relative to Tobacco method 81 when the filter containing vaporized VEA was extracted using acetonitrile. Two of the major products identified were 2,3,5,6-tetramethyl-1,4-benzoquinone and 2,6,10,14-tetramethyl-1-pentadecene, which were confirmed using analytical standards and gas chromatography-high-resolution mass spectrometry (GC-HRMS). Additional synthesis of 4-acetoxy-2,3,5,6-tetramethyl-2,4-cyclohexadienone and subsequent characterization using nuclear magnetic resonance and GC-HRMS suggested that this is not one of the products produced. Identification of these degradants will allow future studies to quantify and examine the degradants in vivo and in vitro as biomarkers for exposure and toxicity assessment.

Fabrication of polyethylene terephthalate (PET) nanoparticles with fluorescent tracers for studies in mammalian cells.

Fluorescent nanoparticles (NPs) comprising polyethylene terephthalate (PET) with a hydrodynamic diameter of 158 ± 2 nm were synthesized in a bottom-up approach. Concentration-dependent uptake and cytotoxicity of PET NPs in macrophages are shown. The fabrication of well-characterized NPs, derived from high-commodity polymers, will support future studies to assess effects on biological systems.

Cannabinoid receptor subtype influence on neuritogenesis in human SH-SY5Y cells.

Human SH-SY5Y neuroblastoma cells stably expressing exogenous CB1 (CB1XS) or CB2 (CB2XS) receptors were developed to investigate endocannabinoid signaling in the extension of neuronal projections. Expression of cannabinoid receptors did not alter proliferation rate, viability, or apoptosis relative to parental SH-SY5Y. Transcripts for endogenous cannabinoid system enzymes (diacylglycerol lipase, monoacylglycerol lipase, α/ß-hydrolase domain containing proteins 6 and 12, N-acyl phosphatidylethanolamine-phospholipase D, and fatty acid amide hydrolase) were not altered by CB1 or CB2 expression. Endocannabinoid ligands 2-arachidonoylglycerol (2-AG) and anandamide were quantitated in SH-SY5Y cells, and diacylglycerol lipase inhibitor tetrahydrolipstatin decreased 2-AG abundance by 90% but did not alter anandamide abundance. M3 muscarinic agonist oxotremorine M, and inhibitors of monoacylglycerol lipase and α/ß hydrolase domain containing proteins 6 &12 increased 2-AG abundance. CB1 receptor expression increased lengths of short (<30 µm) and long (>30 µm) projections, and this effect was significantly reduced by tetrahydrolipstatin, indicative of stimulation by endogenously produced 2-AG. Pertussis toxin, Gßγ inhibitor gallein, and ß-arrestin inhibitor barbadin did not significantly alter long projection length in CB1XS, but significantly reduced short projections, with gallein having the greatest inhibition. The rho kinase inhibitor Y27632 increased CB1 receptor-mediated long projection extension, indicative of actin cytoskeleton involvement. CB1 receptor expression increased GAP43 and ST8SIA2 mRNA and decreased ITGA1 mRNA, whereas CB2 receptor expression increased NCAM and SYT mRNA. We propose that basal endogenous production of 2-AG provides autocrine stimulation of CB1 receptor signaling through Gi/o, Gßγ, and ß-arrestin mechanisms to promote neuritogenesis, and rho kinase influences process extension.

Route of administration effects on nicotine discrimination in female and male mice.

BACKGROUND: Use of electronic cigarettes (e-cigarettes) has increased exponentially since their appearance on the U.S. market around 2007. To provide preclinical models of vaping that incorporate olfactory cues and chemosensory effects (including flavors) that play a role in human vaping behavior, the feasibility of using a modified e-cigarette device for delivery of aerosolized nicotine was examined in a nicotine discrimination procedure in mice. METHODS: Adult female and male C57BL/6 mice were trained to discriminate 0.75 mg/kg subcutaneous (s.c.) nicotine from saline. After determination of a s.c. nicotine dose-effect curve, aerosolized freebase nicotine and nicotine-containing tobacco products (i.e., non-flavored and Arctic Blast e-liquids) were evaluated. RESULTS: Nicotine (s.c.) dose-dependently substituted in mice of both sexes, although females showed less sensitivity and greater variability. By contrast, aerosolized nicotine, regardless of formulation, produced concentration-dependent increases up to maximum of 46-62% nicotine-associated responding. Brain nicotine concentrations for each sex were similar for s.c. 0.75 mg/kg nicotine and 30 mg/ml freebase nicotine. CONCLUSIONS: Mice of both sexes readily acquired s.c. nicotine discrimination, but females showed less sensitivity. Further, all three formulations of aerosolized nicotine produced increases in nicotine-like responding in mice of each sex. However, the maximum magnitude of these increases did not engender a similar degree of substitution as s.c. 0.75 mg/kg nicotine, despite similar brain concentrations of nicotine at 30 mg/ml aerosolized nicotine. Additional research is needed for determination of the reason(s); however, results here demonstrate initial feasibility for examination of the discriminative stimulus effects of vaped drugs such as nicotine.

Toxic by design? Formation of thermal degradants and cyanide from carboxamide-type synthetic cannabinoids CUMYL-PICA, 5F-CUMYL-PICA, AMB-FUBINACA, MDMB-FUBINACA, NNEI, and MN-18 during exposure to high temperatures.

PURPOSE: The use of novel synthetic cannabinoids as intoxicants continues in spite of associated health risks. These compounds are typically smoked or vaporized, but many synthetic cannabinoids contain thermally labile chemical moieties. This study investigated the thermal stability six carboxamide-type synthetic cannabinoids (CUMYL-PICA, 5F-CUMYL-PICA, AMB-FUBINACA, MDMB-FUBINACA, NNEI, and MN-18) in order to characterise potential user exposure to thermolysis products. METHODS: Compounds were heated sequentially to 200, 400, 600 and 800 °C using a thermolysis probe, and the resultant thermolysis products were analysed via GC-MS. A secondary analysis quantified thermolytically generated cyanide via LC-MS/MS. RESULTS: All six synthetic cannabinoids underwent thermal degradation when heated above 400 °C, and released a variety of potentially toxic products, including toluene, naphthalene, and 1-naphthalamine. Compound-specific degradants were tentatively identified together with a general degradative pathway for carboxamide-type synthetic cannabinoids, which proceeds via indole- or indazole-amide formation and subsequent dehydration to an indole- or indazole-carbonitrile. This degradative pathway culminated in the thermolytic liberation of cyanide, in amounts up to 27 µg per mg of starting material. CONCLUSIONS: People who smoke carboxamide-type synthetic cannabinoids are likely to be exposed to range of potentially toxic thermal degradants, including cyanide. These degradants could have significant health impacts in human users.

Delivery of nicotine aerosol to mice via a modified electronic cigarette device.

BACKGROUND: Although both men and women use e-cigarettes, most preclinical nicotine research has focused on its effects in male rodents following injection. The goals of the present study were to develop an effective e-cigarette nicotine delivery system, to compare results to those obtained after subcutaneous (s.c.) injection, and to examine sex differences in the model. METHODS: Hypothermia and locomotor suppression were assessed following aerosol exposure or s.c. injection with nicotine in female and male mice. Subsequently, plasma and brain concentrations of nicotine and cotinine were measured. RESULTS: Passive exposure to nicotine aerosol produced concentration-dependent and mecamylamine reversible hypothermic and locomotor suppressant effects in female and male mice, as did s.c. nicotine injection. In plasma and brain, nicotine and cotinine concentrations showed dose/concentration-dependent increases in both sexes following each route of administration. Sex differences in nicotine-induced hypothermia were dependent upon route of administration, with females showing greater hypothermia following aerosol exposure and males showing greater hypothermia following injection. In contrast, when they occurred, sex differences in nicotine and cotinine levels in brain and plasma consistently showed greater concentrations in females than males, regardless of route of administration. DISCUSSION: In summary, the e-cigarette exposure device described herein was used successfully to deliver pharmacologically active doses of nicotine to female and male mice. Further, plasma nicotine concentrations following exposure were similar to those after s.c. injection with nicotine and within the range observed in human smokers. Future research on vaped products can be strengthened by inclusion of translationally relevant routes of administration.

Thermolytic Degradation of Synthetic Cannabinoids: Chemical Exposures and Pharmacological Consequences.

Synthetic cannabinoids are manufactured clandestinely with little quality control and are distributed as herbal "spice" for smoking or as bulk compound for mixing with a solvent and inhalation via electronic vaporizers. Intoxication with synthetic cannabinoids has been associated with seizure, excited delirium, coma, kidney damage, and other disorders. The chemical alterations produced by heating these structurally novel compounds for consumption are largely unknown. Here, we show that heating synthetic cannabinoids containing tetramethylcyclopropyl-ring substituents produced thermal degradants with pharmacological activity that varied considerably from their parent compounds. Moreover, these degradants were formed under conditions simulating smoking. Some products of combustion retained high affinity at the cannabinoid 1 (CB1) and CB2 receptors, were more efficacious than (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55,940) in stimulating CB1 receptor-mediated guanosine 5'-O-(3-thiotriphosphate) (GTPγS) binding, and were potent in producing Δ9-tetrahydrocannabinol-like effects in laboratory animals, whereas other compounds had low affinity and efficacy and were devoid of cannabimimetic activity. Degradants that retained affinity and efficacy also substituted in drug discrimination tests for the prototypical synthetic cannabinoid 1-pentyl-3-(1-naphthoyl)indole (JWH-018), and are likely to produce psychotropic effects in humans. Hence, it is important to take into consideration the actual chemical exposures that occur during use of synthetic cannabinoid formulations to better comprehend the relationships between dose and effect.

Utilizing artificial neural networks in MATLAB to achieve parts-per-billion mass measurement accuracy with a fourier transform ion cyclotron resonance mass spectrometer.

Fourier transform ion cyclotron resonance mass spectrometry has the ability to realize exceptional mass measurement accuracy (MMA); MMA is one of the most significant attributes of mass spectrometric measurements as it affords extraordinary molecular specificity. However, due to space-charge effects, the achievable MMA significantly depends on the total number of ions trapped in the ICR cell for a particular measurement, as well as relative ion abundance of a given species. Artificial neural network calibration in conjunction with automatic gain control (AGC) is utilized in these experiments to formally account for the differences in total ion population in the ICR cell between the external calibration spectra and experimental spectra. In addition, artificial neural network calibration is used to account for both differences in total ion population in the ICR cell as well as relative ion abundance of a given species, which also affords mean MMA values at the parts-per-billion level.