Headspace analysis of E-cigarette fluids using comprehensive two dimensional GC×GC-TOF-MS reveals the presence of volatile and toxic compounds.

The analysis of electronic cigarrete (E-cigarette) fluids by high performance liquid chromatography or gas chromatography (GC) coupled to mass spectrometry (MS), GC hyphenated to flame-ionisation detection, or nuclear magnetic resonance spectroscopy poses many challenges due to the complex matrix and extremely high number of compounds present. In order to overcome these challenges, this study focused on the detection of the multiple complex compounds classes produced by the pyrolysis of E-cigarette liquids using comprehensive two dimensional gas chromatography (GCxGC) coupled to time of flight (TOF)-MS. Gas samples were prepared by heating E-liquids inside aluminium tins for 5 min. The tins were placed in a sand bath, which was temperature controlled at 200 °C. The samples were collected using thermal desorption tubes connected to volatile organic compound (VOC) sampling pump attached and subsequently analysed using GCxGC-TOF-MS. The greater peak resolution obtained when using GCxGC-TOF-MS allowed to distinguish many toxic compounds and VOCs that could not be detected by the other methods mentioned above. As a result, a comprehensive list of volatile compounds emitted from E-cigarette fluids when heated was established, which might allow a better understanding of potential health effects of vaping. Heating E-liquids to moderate temperature results in the emission of over 1000 volatile compounds of which over 150 are toxic. These compounds are either present in the liquid or can be formed during storage or heating leading to a more complex volatile profile of E-cigarette liquids than previously assumed. The application of GCxGC-TOF-MS allows the elucidation of this profile and therefore a better understanding of possible health implications.

Finding the proverbial needle: Non-targeted screening of synthetic opioids in equine plasma.

Synthetic opioids are a class of compounds that are of particular concern due to their high potency and potential health impacts. With the relentless emergence of new synthetic opioid derivatives, non-targeted screening strategies are required that do not rely on the use of library spectra or reference materials. In this study, product ion searching, and Kendrick mass defect analysis were investigated for non-targeted screening of synthetic opioids. The estimated screening cut-offs for these techniques ranged between 0.05 and 0.1 ng/mL. These techniques were designed to not be reliant on a particular vendor's software, meaning that they can be applied to existing drug screening protocols, without requiring the development and validation of new analytical procedures. The efficacy of the developed techniques was tested through blind trials, with spiked samples inserted amongst authentic plasma samples, which demonstrated the usefulness of these methods for high-throughput screening. The use of a non-targeted screening workflow that contains complementary techniques can increase the likelihood of detecting compounds of interest within a sample, as well as the confidence in detections that are made.

Development and validation of a color spot test method for the presumptive detection of 25-NBOMe compounds.

The great increase of new psychoactive substances over the past decade has substantially transformed the illicit drug industry to an ever-changing dynamic market. 25-NBOMe compounds are just one of these new substance groups that pose a public health risk in many countries around the world. These highly potent, hallucinogenic phenethylamines have previously been sold as "legal highs" or "synthetic LSD" and the necessity to rapidly identify their presence is crucial. While there are many laboratory-based analytical methods capable of identifying these compounds, the lack of presumptive test methods indicates the need for a specific and timely test that could be used in the field. Herein we outline the developed chemical spot test that can selectively identify the presence of 25-NBOMe compounds and related analogs through the reaction with a substituted benzoquinone reagent under basic conditions. This test method has been comprehensively validated showing a high level of selectivity, specificity, and precision with only two other illicit substances producing similar positive results as 25-NBOMe and few false-negative results seen. The working limit of detection was determined to be 225 µg and there was no cross-reactivity from potential adulterants of significance. This test has also been shown to work directly with blotter papers containing 25-NBOMe compounds, indicating no interference from this common matrix and the ability to differentiate these compounds from LSD. This method shows a high potential to be translated to a field compatible test that is simple, rapid, and selective for 25-NBOMe compounds.

Towards compound identification of synthetic opioids in nontargeted screening using machine learning techniques.

The constant evolution of the illicit drug market makes the identification of unknown compounds problematic. Obtaining certified reference materials for a broad array of new analogues can be difficult and cost prohibitive. Machine learning provides a promising avenue to putatively identify a compound before confirmation against a standard. In this study, machine learning approaches were used to develop class prediction and retention time prediction models. The developed class prediction model used a naïve Bayes architecture to classify opioids as belonging to either the fentanyl analogues, AH series or U series, with an accuracy of 89.5%. The model was most accurate for the fentanyl analogues, most likely due to their greater number in the training data. This classification model can provide guidance to an analyst when determining a suspected structure. A retention time prediction model was also trained for a wide array of synthetic opioids. This model utilised Gaussian process regression to predict the retention time of analytes based on multiple generated molecular features with 79.7% of the samples predicted within ±0.1 min of their experimental retention time. Once the suspected structure of an unknown compound is determined, molecular features can be generated and input for the prediction model to compare with experimental retention time. The incorporation of machine learning prediction models into a compound identification workflow can assist putative identifications with greater confidence and ultimately save time and money in the purchase and/or production of superfluous certified reference materials.

Evidence from a mouse model on the dangers of thirdhand electronic cigarette exposure during early life.

Thirdhand exposure to e-cigarette residue is likely to have harmful effects in children http://bit.ly/38a2umw.

Substances injected at the Sydney supervised injecting facility: A chemical analysis of used injecting equipment and comparison with self-reported drug type.

Providing information about substances injected can reduce the negative impact of illicit drug consumption and support people who inject drugs to make informed decisions. In Australia, information about drugs injected relies largely on periodic self-report surveys. For the first time, the analysis of the residual content of used injecting equipment was conducted in a supervised injecting facility (SIF) located in Sydney, Australia. The aim was to gain a better understanding of the substances injected by clients through: (1) chemical analyses of the content of used syringes; (2) comparison of these results with clients' self-reported drug use; and (3) assessing the usefulness of analysing other injecting equipment to detect substances used. During one week in February 2019, syringes and other injecting equipment were collected at the Sydney SIF. Their residual content was analysed by gas-chromatography/mass-spectrometry. Heroin was the most commonly detected substance (present in 51% of syringes), followed by methamphetamine (22%) and oxycodone (10%). In addition to the main psychoactive substance, cutting agents reported in the literature were also detected in used syringes. The main psychoactive substance identified by laboratory analysis reliably corresponded with users' self-reported drug type. Analytical confirmation of substances injected allows for the provision of better targeted harm reduction messaging based on timely and objective data. The approach used is amenable to clients and feasible in the Australian SIF context. Upscaling and wider implementation could be done through Needle and Syringe Programs, and would support the early detection of harmful substances entering drug markets and better inform harm reduction strategies.

Organic impurity profiling of 3,4-methylenedioxymethamphetamine (MDMA) synthesised from catechol and eugenol via 4-allylcatechol.

This work examines organic impurity profiles of 3,4-methylenedioxymethamphetamine (MDMA) that has been synthesised from the "pre-precursors" catechol (1,2-dihydroxybenzene) and eugenol, via a safrole intermediate. MDMA was synthesised from the catechol- and eugenol-derived safrole intermediate via two routes, which resulted in the synthesis of MDMA from catechol via two routes (Route 1A and 1B) and from eugenol via two routes (Route 2A and 2B). Twelve organic impurities were identified in MDMA synthesised via Routes 1A and 1B, and eleven organic impurities were identified in MDMA synthesised via Routes 2A and 2B. Route specific organic impurities were identified in MDMA that indicated the "pre-precursors" catechol and eugenol were used in the respective synthetic routes. Route specific organic impurities were also identified in MDMA that indicated the route used to synthesise safrole from the "pre-precursor" and the route used to synthesise MDMA from safrole. Thus, the use of the "pre-precursors" catechol and eugenol and the synthetic route utilised could be ascertained by the organic impurity profiling of MDMA under the conditions used here.

Collision-Induced Dissociation Studies of Synthetic Opioids for Non-targeted Analysis.

The continual introduction of a large number of new psychoactive substances, along with the large turnover of these substances, necessitates the development of non-targeted detection strategies to keep pace with the ever-changing drug market. The production of certified reference materials often lags behind the introduction of new substances to the market, therefore these detection strategies need to be able to function without relying on reference materials or library spectra. Synthetic opioids have recently emerged as a drug class of particular concern due to the health issues caused by their incredibly high potency. A common method which has been used for non-targeted analysis in the past involves the identification of common product ions formed as a result of the fragmentation of the parent molecule. These common fragments can then potentially be used as markers to indicate the presence of a particular class of compounds within a sample. In this study, standards of a number of different synthetic opioids, including 14 fentanyl derivatives, 7 AH series opioids, 4 U series opioids, 4 W series opioids and MT-45, were subjected to collision-induced dissociation studies to determine how the compounds fragment. The spectra obtained from these studies included a number of diagnostic fragments common to the different opioid classes that, when used in combination, show potential for use as class predictors. By using simple data processing techniques, such as extracted ion chromatograms, these diagnostic product ions identified can be applied to a non-targeted screening workflow.

A Comparison of Two Ovine Lumbar Intervertebral Disc Injury Models for the Evaluation and Development of Novel Regenerative Therapies.

STUDY DESIGN: Large animal research. OBJECTIVE: Lumbar discectomy is the most commonly performed spinal surgical procedure. We investigated 2 large animal models of lumbar discectomy in order to study the regenerative capacity of mesenchymal stem cells following disc injury. METHODS: Twelve adult ewes underwent baseline 3-T magnetic resonance imaging (MRI) followed by lumbar intervertebral disc injury by either drill bit (n = 6) or annulotomy and partial nucleotomy (APN) (n = 6). Necropsies were performed 6 months later. Lumbar spines underwent 3-T and 9.4-T MRI prior to histological, morphological and biochemical analysis. RESULTS: Drill bit-injured (DBI) and APN-injured discs demonstrated increased Pfirrmann grades relative to uninjured controls (P < .005), with no difference between the 2 models. Disc height index loss was greater in the APN group compared with the DBI group (P < .005). Gross morphology injury scores were higher in APN than DBI discs (P < .05) and both were higher than controls (P < .005). Proteoglycan was reduced in the discs of both injury models relative to controls (P < .005), but lower in the APN group (P < .05). Total collagen of the APN group disc regions was higher than DBI and control discs (P < .05). Histology revealed more matrix degeneration, vascular infiltration, and granulation in the APN model. CONCLUSION: Although both models produced disc degeneration, the APN model better replicated the pathobiology of human discs postdiscectomy. We therefore concluded that the APN model was a more appropriate model for the investigation of the regenerative capacity of mesenchymal stem cells administered postdiscectomy.

Color Tests for the Preliminary Identification of New Psychoactive Substances.

Color tests are a key tool for the rapid and simple identification of seized illicit drugs. This chapter outlines a series of color tests that can be used for the preliminary identification of new psychoactive substances such as cathinones, piperazines, tryptamines, and amphetamine-type stimulants.

Color Spot Test As a Presumptive Tool for the Rapid Detection of Synthetic Cathinones.

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and other border protection agencies globally. Color testing is a presumptive identification technique indicating the presence or absence of a particular drug class using rapid and uncomplicated chemical methods. Owing to their relatively recent emergence, a color test for the specific identification of synthetic cathinones is not currently available. In this study, we introduce a protocol for the presumptive identification of synthetic cathinones, employing three aqueous reagent solutions: copper(II) nitrate, 2,9-dimethyl-1,10-phenanthroline (neocuproine) and sodium acetate. Small pin-head sized amounts (approximately 0.1-0.2 mg) of the suspected drugs are added to the wells of a porcelain spot plate, and each reagent is then added dropwise sequentially before heating on a hotplate. A color change from very light blue to yellow-orange after 10 min indicates the likely presence of synthetic cathinones. The highly stable and specific test reagent has the potential for use in the presumptive screening of unknown samples for synthetic cathinones in a forensic laboratory. However, the nuisance of an added heating step for the color change result limits the test to laboratory application and decreases the likelihood of an easy translation to field testing.

Mesenchymal progenitor cells primed with pentosan polysulfate promote lumbar intervertebral disc regeneration in an ovine model of microdiscectomy.

BACKGROUND CONTEXT: Neural compression associated with lumbar disc herniation is usually managed surgically by microdiscectomy. However, 10%-20% of patients re-present with debilitating back pain, and approximately 15% require further surgery. PURPOSE: Using an ovine model of microdiscectomy, the present study investigated the relative potential of pentosan polysulfate-primed mesenchymal progenitor cells (pMPCs) or MPC alone implanted into the lesion site to facilitate disc recovery. STUDY DESIGN: An ovine model of lumbar microdiscectomy was used to compare the relative outcomes of administering MPCs or pMPCs to the injury site postsurgery. METHODS: At baseline 3T magnetic resonance imaging (MRI) of 18 adult ewes was undertaken followed by annular microdiscectomy at two lumbar disc levels. Sheep were randomized into three groups (n=6). The injured controls received no further treatment. Defects of the treated groups were implanted with a collagen sponge and MPC (5×105 cells) or pMPC (5×105 cells). After 6 months, 3T MRI and standard radiography were performed. Spinal columns were dissected, individual lumbar discs were sectioned horizontally, and nucleus pulposus (NP) and annulus fibrosus (AF) regions were assessed morphologically and histologically. The NP and AF tissues were dissected into six regions and analyzed biochemically for their proteoglycans (PGs), collagen, and DNA content. RESULTS: Both the MPC- and pMPC-injected groups exhibited less reduction in disc height (p<.05) and lower Pfirrmann grades (p≤.001) compared with the untreated injury controls, but morphologic scores for the pMPC-injected discs were lower (p<.05). The PG content of the AF injury site region (AF1) of pMPC discs was higher than MPC and injury control AF1 (p<.05). At the AF1 and contralateral AF2 regions, the DNA content of pMPC discs was significantly lower than injured control discs and MPC-injected discs. Histologic and birefringent microscopy revealed increased structural organization and reduced degeneration in pMPC discs compared with MPC and the injured controls. CONCLUSIONS: In an ovine model 6 months after administration of pMPCs to the injury site disc PG content and matrix organization were improved relative to controls, suggesting pMPCs' potential as a postsurgical adjunct for limiting progression of disc degeneration after microdiscectomy.

Effect of drug precursors and chemicals relevant to clandestine laboratory investigation on plastic bags used for collection and storage.

In the area of clandestine laboratory investigations, plastic bags are used to collect and store evidence, such as solvents, precursors, and other compounds usually employed for the manufacturing of drugs (although liquids may be stored in glass containers within the bags first). In this study, three different types of plastic bags were provided by the NSW Police Force and investigated for their suitability for evidence collection: two different types of low-density polyethylene (LDPE) bags and one type of polyvinyl chloride (PVC) bag. Three different experiments were carried out: (1) storing relevant chemicals in the bags for up to three months; (2) exposing the bags including their content to accelerated conditions using a weatherometer, and (3) simulating an expected real case scenario. This study indicates that drugs and related chemicals stored in plastic bags may lead to a change in the composition of the chemical and an alteration or degradation of the plastic bag. All experiments led to the same conclusion: the polyvinyl chloride bags appeared to be the most affected. LDPE bags seem to be more appropriate for routine use, although it has been established they are not suitable for the collection of liquids (unless pre-packaged in, for instance, a glass container).

Synthesis and organic impurity profiling of 4-methoxymethamphetamine hydrochloride and its precursors.

4-Methoxymethamphetamine (PMMA) was synthesised from star anise and from 4-methoxytoluene and the organic impurity profiles examined. These two starting materials are unrestricted chemicals in many jurisdictions and contain the requisite functional groups and are thus well suited for clandestine manufacturers. trans-Anethole was extracted from star anise and oxidised to 4-methoxyphenyl-2-propanone (PMP2P). 4-Methoxytoluene was oxidised to anisaldehyde, converted to 4-methoxyphenyl-2-nitropropene, and then reduced to PMP2P. The PMP2P obtained by both methods was then converted to PMMA via the Leuckart reaction. 4-Methoxymethamphetamine hydrochloride (PMMA·HCl) was synthesised from PMMA using hydrogen chloride gas. Both of the examined synthetic methods were found to be feasible routes into PMMA·HCl. The products of each step were analysed by gas chromatography-mass spectrometry (GC-MS) and proton nuclear magnetic resonance spectroscopy (1H NMR). Impurities were examined in an attempt to identify route specific compounds, which may provide valuable information about the synthetic pathway and precursors.

Development and dissolution studies of bisphosphonate (clodronate)-containing hydroxyapatite-polylactic acid biocomposites for slow drug delivery.

An increase in clinical demand on the controlled release of bisphosphonates (BPs) due to complications associated with systemic administration, has been the current driving force on the development of BP drug-release systems. Bisphosphonates have the ability to bind to divalent metal ions, such as Ca2+ , in bone mineral and prevent bone resorption by influencing the apoptosis of osteoclasts. Localized delivery using biodegradable materials, such as polylactic acid (PLA) and hydroxyapatite (HAp), which are ideal in this approach, have been used in this study to investigate the dissolution of clodronate (non-nitrogen-containing bisphosphonate) in a new release system. The effects of coral structure-derived HAp and the release kinetics of the composites were evaluated. The release kinetics of clodronate from PLA-BP and PLA-HAp-BP systems seemed to follow the power law model described by Korsmeyer-Peppas. Drug release was quantified by 31 P-NMR with detection and quantification limits of 9.2 and 30.7 mM, respectively. The results suggest that these biocomposite systems could be tuned to release clodronate for both relatively short and prolonged period of time. In addition to drug delivery, the degradation of HAp supplies both Ca2+ and phosphate ions that can help in bone mineralization. Copyright © 2015 John Wiley & Sons, Ltd.

The mechanical properties of plastic evidence bags used for collection and storage of drug chemicals relevant to clandestine laboratory investigations.

The effectiveness of three types of plastic bags used by the New South Wales Police Force for the storage of clandestine drug evidence has been investigated through a comparison of mechanical properties. The tensile and tear properties of "as received" low-density polyethylene (LDPE) and poly(vinyl chloride) (PVC) bags do not show major differences such that one type would be favoured over the other. However, the mechanical properties of the bags once exposed to a range of chemicals routinely collected as drug evidence have been shown to be influenced as a result of different chemical interactions. Although an interaction of reagents/solvents with an additive within the LDPE bags is proposed to influence the mechanical properties of the bags, the change in properties has been shown to be less severe than that observed for the PVC bag, where softening and damage of the bags results due to absorption of reagents.

Organic impurity profiling of methylone and intermediate compounds synthesized from catechol.

This work examined the synthesis and organic impurity profile of methylone prepared from catechol. The primary aim of this work was to determine whether the synthetic pathway used to prepare 3,4-methylenedioxypropiophenone could be ascertained through analysis of the synthesized methylone. The secondary aim was the structural elucidation and origin determination of the organic impurities detected in methylone and the intermediate compounds. The organic impurities present in the reaction products were identified using GC-MS and NMR spectroscopy. Six organic impurities were detected in 1,3-benzodioxole and identified as the 1,3-benzodioxole dimer, 1,3-benzodioxole trimer, [1,3] dioxolo[4,5-b]oxanthrene, 4,4'-, 4,5'-, and 5,5'-methylenebis-1,3-benzodioxole. Six organic impurities were detected in 3,4-methylenedioxypropiophenone and identified as (2-hydroxyphenyl) propanoate, [2-(chloromethoxy) phenyl] propanoate, (2-propanoyloxyphenyl)propanoate, 5-[1-(1,3-benzodioxol-5-yl)prop-1-enyl]-1,3-benzodioxole, (5E)- and (5Z)-7-(1,3-benzodioxol-5-yl)-5-ethylidene-6-methyl-cyclopenta[f][1,3]benzodioxole). Exploratory synthetic experiments were also conducted to unambiguously identify the organic impurities detected in 3,4-methylenedioxypropiophenone. Two organic impurities were detected in 5-bromo-3,4-methylenedioxypropiophenone and identified as [2-(chloromethoxy)phenyl] propanoate and 3,4-methylenedioxypropiophenone. Five organic impurities were detected in methylone and identified as 3,4-methylenedioxypropiophenone, 1-(1,3-benzodioxol-5-yl)-N-methyl-propan-1-imine, 1-(1,3-benzodioxol-5-yl)-2-methylimino-propan-1-one, 1-(1,3-benzodioxol-5-yl)-N1,N2-dimethyl-propane-1,2-diimine and butylated hydroxytoluene. The origin of these organic impurities was also ascertained, providing valuable insight into the chemical profiles of methylone and the intermediate compounds. However, neither the catechol precursor nor the 1,3-benzodioxole intermediate could be identified based on the organic impurities detected in the synthesized methylone using standard techniques. This demonstrated that the organic impurity profiling of methylone had limitations in the determination of precursor chemical and synthetic pathways used. Copyright © 2017 John Wiley & Sons, Ltd.

Inhibitory Kinetics of Azachalcones and their Oximes on Mushroom Tyrosinase: A Facile Solid-state Synthesis.

A solid-state-based mechanochemical process was used to synthesize novel azachalcones and their oximes as tyrosinase inhibitors. Their inhibitory activities on mushroom tyrosinase using l-3,4-dihydroxyphenylalanine as a substrate were investigated. Two of the novel oxime derivatives synthesized were seen to be more potent than the positive control, kojic acid. Both the compounds 1b and 2b inhibited the diphenolase activity of tyrosinase in a dose-dependent manner with their IC50 values of 15.3 and 12.7 µm, respectively. The kinetic analysis showed that their inhibition mechanism was reversible. Both the novel oxime compounds displayed competitive inhibition with their Ki values of 5.1 and 2.5 µm, respectively. This method minimizes waste disposal problems and provides a simple, efficient, and benign method for the synthesis of novel tyrosinase inhibitors for use as skin-whitening agents or as anti-browning food additives.

Dimethylsulfoniopropionate, superoxide dismutase and glutathione as stress response indicators in three corals under short-term hyposalinity stress.

Corals are among the most active producers of dimethylsulfoniopropionate (DMSP), a key molecule in marine sulfur cycling, yet the specific physiological role of DMSP in corals remains elusive. Here, we examine the oxidative stress response of three coral species (Acropora millepora, Stylophora pistillata and Pocillopora damicornis) and explore the antioxidant role of DMSP and its breakdown products under short-term hyposalinity stress. Symbiont photosynthetic activity declined with hyposalinity exposure in all three reef-building corals. This corresponded with the upregulation of superoxide dismutase and glutathione in the animal host of all three species. For the symbiont component, there were differences in antioxidant regulation, demonstrating differential responses to oxidative stress between the Symbiodinium subclades. Of the three coral species investigated, only A. millepora provided any evidence of the role of DMSP in the oxidative stress response. Our study reveals variability in antioxidant regulation in corals and highlights the influence life-history traits, and the subcladal differences can have on coral physiology. Our data expand on the emerging understanding of the role of DMSP in coral stress regulation and emphasizes the importance of exploring both the host and symbiont responses for defining the threshold of the coral holobiont to hyposalinity stress.

Evaluation of Novel Chalcone Oximes as Inhibitors of Tyrosinase and Melanin Formation in B16 Cells.

A series of hydroxy-substituted chalcone oxime derivatives were synthesized. These compounds were then evaluated for their inhibitory activities on tyrosinase and melanogenesis in murine B16F10 melanoma cells. The structures of the synthesized compounds were confirmed by (1) H NMR, (13) C NMR, FTIR, and HRMS. Two of the compounds exhibited much higher tyrosinase inhibitory activities (IC50 values of 4.77 and 7.89 µM, respectively) than the positive control, kojic acid (IC50 : 22.25 µM). Kinetic studies revealed them to act as competitive tyrosinase inhibitors with their Ki values of 5.25 and 8.33 µM, respectively. Both the compounds inhibited melanin production and tyrosinase activity in B16 cells. Docking results confirmed that the active inhibitors strongly interacted with the mushroom tyrosinase residues.