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Detecting the use of bisphosphonates (BPs) in equine athletes is of interest to regulators and laboratories due to the threat to welfare issues for the potential to provide analgesic effects and manipulating bone structure. The detection of BPs in biological matrices is challenging due to erratic biological elimination and inconsistent analytical recoveries. Therefore, complementary approaches are needed to provide evidence of their misuse in racehorses. BPs have two sub-classes: nitrogenous and non-nitrogenous. This study investigated plasma elimination following administration of one example from each sub-class, together with changes in endogenous eicosanoid and corticosteroids. Zoledronic acid (ZA) and tiludronic acid (TA) were administered by IV infusion to 8 thoroughbred horses with an 11-month washout period between each administration. Sample preparation for quantification of BPs by liquid chromatography-tandem mass spectrometry (LC-MS/MS) utilised a two-step solid phase extraction (SPE) consisting of polymeric reversed-phase followed by weak anion exchange prior to derivatisation using trimethyl orthoacetate. Endogenous biomarkers were analysed after protein precipitation and SPE with polymeric reversed-phase prior to liquid chromatography-high resolution mass spectrometry (LC-HRMS) using data independent acquisition. The LC-MS/MS analysis showed ZA was undetectable after 8 h post-administration while TA was detected up to the final collection point of 28 days post-administration. The LC-HRMS analysis utilised targeted (i.e., prior inclusion list of compounds) approaches to monitor level changes of eicosanoid and corticosteroid biomarkers. Putative biomarkers were identified and now subject to validation for translation into routine sample analysis for improved retrospectivity to detecting BP misuse in equine plasma., (© 2024 The Author(s). Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Immunoglobulin E (IgE)-mediated immediate hypersensitivity reactions are the most concerning adverse events after penicillin antibiotics (PENs) administration because of their rapid progression and potential for fatal outcome. However, the diagnosis of allergic death is a forensic challenge because it mainly depends on nonspecific characteristic morphological changes, as well as exclusion and circumstantial evidence. In this study, an untargeted metabolomics approach based on liquid chromatography-mass spectrometry (LC-MS) was used to screen potential forensic biomarkers of fatal anaphylactic shock induced by four PENs (benzylpenicillin (BP), amoxicillin (AMX), oxacillin (OXA), and mezlocillin (MEZ)), and analyzed the metabolites, metabolic pathway and the mechanism which were closely related to the allergic reactions. The metabolomics results discovered that a total of 24 different metabolites in all four anaphylactic death (AD) groups, seven of which were common metabolites. A biomarker model consisting of six common metabolites (linoleic acid, prostaglandin D2, lysophosphatidylcholine (18:0), N-acetylhistamine, citric acid and indolelactic acid) AUC value of Receiver Operating Characteristic (ROC) curve was 0.978. Metabolism pathway analysis revealed that the pathogenesis of PENs-induced AD is closely related to linoleic acid metabolism. Our results revealed that the metabolomic profiling has potential in PENs-induced AD post-mortem diagnosis and metabolic mechanism investigations., (© 2024. The Author(s).)

Ketamine has recently become an anesthetic drug used in human and veterinary clinical medicine for illicit abuse worldwide, but the detection of illicit abuse and inference of time intervals following ketamine abuse are challenging issues in forensic toxicological investigations. Here, we developed methods to estimate time intervals since ketamine use is based on significant metabolite changes in rat serum over time after a single intraperitoneal injection of ketamine, and global metabolomics was quantified by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Thirty-five rats were treated with saline (control) or ketamine at 3 doses (30, 60, and 90 mg/kg), and the serum was collected at 21 time points (0 h to 29 d). Time-dependent rather than dose-dependent features were observed. Thirty-nine potential biomarkers were identified, including ketamine and its metabolites, lipids, serotonin and other molecules, which were used for building a random forest model to estimate time intervals up to 29 days after ketamine treatment. The accuracy of the model was 85.37% in the cross-validation set and 58.33% in the validation set. This study provides further understanding of the time-dependent changes in metabolites induced by ketamine abuse., (© 2024. The Author(s).)

Clozapine (CLZ) -related accidents or crimes are common in the world. Oral fluid drug detection is a convenient measure of dealing with things like that. There has not been any literature reported detailedly the representation rule of clozapine and its metabolites in oral fluid so far. The study aimed to describe the pharmacokinetics of CLZ and its metabolites N-desmethylclozapine and clozapine-N-oxide in human oral fluid after a single 12.5 mg oral dose of CLZ. Twenty-nine volunteers, including 20 males and 9 females, were recruited, and 2 mL oral fluid was collected from each participant at post-consumption time-points of prior (zero), 0.5, 1.5, 3, 5, 8, 12, 24, 36, 51, 82, and 130 h, respectively. Analytes of interest were extracted with solid-phase extraction and analyzed with liquid chromatography tandem mass spectrometry method. Pharmacokinetic parameters were calculated using the pharmacokinetic software DAS according to the non-compartment model. The maximum concentration, the time of maximum concentration, oral clearance, and the elimination half-life of clozapine were 16.57 ± 9.63 ng/mL, 4.53 ± 3.61 h, 57.65 ± 23.77 L/h and 53.58 ± 52.28 h, respectively. The maximum concentration, the time of maximum concentration, and the elimination half-life of the metabolite N-desmethylclozapine were 3.08 ± 1.19 ng/mL, 9.38 ± 9.33 h and 62.67 ± 82.57 h, respectively; of clozapine-N-oxide were 1.15 ± 0.36 ng/mL, 4.53 ± 2.19 h and 19.15 ± 23.11 h, respectively. It was the first study on the pharmacokinetics of CLZ and its metabolites in the oral fluid of Chinese healthy volunteers, and it provided a basis for the therapeutic drug monitoring and toxicological interpretation in clozapine-related cases., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: A rapid and reliable method was developed and validated for the simultaneous analysis of 52 antibiotics (cephalosporins, penicillins, carbapenems, lincosamides, quinolones, nitroimidazoles, macrolides, sulfonamides, tetracyclines, glycopeptide) in urine and whole blood by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS)., Method: Analytes were extracted by dilution or protein precipitation and analyzed on an Agilent 1260 HPLC system coupled to an Agilent 6470 Triple Quadrupole Mass Spectrometer., Results: The method attended method validation criteria. The limits of detection were equal or lower than 2.0 ng/mL, whereas the limits of quantification ranged from 0.1 to 10.0 ng/mL, from 0.1 to 5.0 ng/mL, in urine and whole blood, respectively. For all analytes, the bias and intra- and inter-day precision values were less than 14.7%. The ranges of recovery values of all antibiotics were 76.5-124.5% in whole blood and 76.3-121.8% in urine, values of the effects were lower than 25% in two matrices. No evidence of carryover was observed. The study of sample stability showed that almost all analytes were stable at 24 °C for 24 h, all analytes were stable at -20 °C for 14 days and at -80 °C for 30 days. Freeze-thaw cycles stability showed that antibiotics were stable except for imipenem. Autosampler stability study showed that all analytes were stable for 24 h, except for imipenem and amoxicillin. Applicability was proven by analyzing authentic whole blood (n = 86) and urine (n = 79) samples from patients under antibiotics treatment. Therefore, this method was applied to the analysis 3 forensic allergy cases, which were positive for at least one analyte., Conclusions: A simple, sensitive and high-throughput method for the simultaneous determination of different classes of antibiotics in urine and whole blood samples was developed and applied. This sensitive method was successfully applied to forensic cases., (© 2024. The Author(s), under exclusive licence to Japanese Association of Forensic Toxicology.)

Accurate estimation of the postmortem interval (PMI) is crucial in forensic medico-legal investigations to understand case circumstances (e.g. narrowing down list of missing persons or include/exclude suspects). Due to the complex decomposition chemistry, estimation of PMI remains challenging and currently often relies on the subjective visual assessment of gross morphological/taphonomic changes of a body during decomposition or entomological data. The aim of the current study was to investigate the human decomposition process up to 3 months after death and propose novel time-dependent biomarkers (peptide ratios) for the estimation of decomposition time. An untargeted liquid chromatography tandem mass spectrometry-based bottom-up proteomics workflow (ion mobility separated) was utilized to analyse skeletal muscle, collected repeatedly from nine body donors decomposing in an open eucalypt woodland environment in Australia. Additionally, general analytical considerations for large-scale proteomics studies for PMI determination are raised and discussed. Multiple peptide ratios (human origin) were successfully proposed (subgroups < 200 accumulated degree days (ADD), < 655 ADD and < 1535 ADD) as a first step towards generalised, objective biochemical estimation of decomposition time. Furthermore, peptide ratios for donor-specific intrinsic factors (sex and body mass) were found. Search of peptide data against a bacterial database did not yield any results most likely due to the low abundance of bacterial proteins within the collected human biopsy samples. For comprehensive time-dependent modelling, increased donor number would be necessary along with targeted confirmation of proposed peptides. Overall, the presented results provide valuable information that aid in the understanding and estimation of the human decomposition processes., (© 2023. The Author(s).)

Natural compounds in plants are often unevenly distributed, and determining the best sampling locations to obtain the most representative results is technically challenging. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can provide the basis for formulating sampling guideline. For a succulent plant sample, ensuring the authenticity and in situ nature of the spatial distribution analysis results during MSI analysis also needs to be thoroughly considered. In this study, we developed a well-established and reliable MALDI-MSI method based on preservation methods, slice conditions, auxiliary matrices, and MALDI parameters to detect and visualize the spatial distribution of mescaline in situ in Lophophora williamsii . The MALDI-MSI results were validated using liquid chromatography-tandem mass spectrometry. Low-temperature storage at -80°C and drying of "bookmarks" were the appropriate storage methods for succulent plant samples and their flower samples, and cutting into 40 μm thick sections at -20°C using gelatin as the embedding medium is the appropriate sectioning method. The use of DCTB (trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile) as an auxiliary matrix and a laser intensity of 45 are favourable MALDI parameter conditions for mescaline analysis. The region of interest semi-quantitative analysis revealed that mescaline is concentrated in the epidermal tissues of L. williamsii as well as in the meristematic tissues of the crown. The study findings not only help to provide a basis for determining the best sampling locations for mescaline in L. williamsii , but they also provide a reference for the optimization of storage and preparation conditions for raw plant organs before MALDI detection., Key Points: An accurate in situ MSI method for fresh water-rich succulent plants was obtained based on multi-parameter comparative experiments.Spatial imaging analysis of mescaline in Lophophora williamsii was performed using the above method.Based on the above results and previous results, a sampling proposal for forensic medicine practice is tentatively proposed., (© The Author(s) 2023. Published by OUP on behalf of the Academy of Forensic Science.)

HPLC-MS/MS analysis and postmortem distribution or postmortem redistribution of paraquat and its two metabolites in poisoning death cases were reported. Paraquat, monoquat, and paraquat monopyridone were extracted from the sample with acetonitrile or methanol, respectively, detected by ZORBAX HILIC Plus (4.6 × 100 mm, 3.5 μm) chromatographic column, with 0.1 % formic acid aqueous solution - 0.1 % formic acid acetonitrile solution (v/v) as mobile phase. Paraquat, monoquat, and paraquat monopyridone had a good linear relationship within the range of 10-1000, 1-400, and 1-1000 ng/mL (or g), the correlation coefficient (r) were all ≥ 0.9996. Their detection limits were lower than 1 ng/mL (or g). The detection accuracy was 91.25∼113.44 %. The intra-day and inter-day precision were 1.51-3.99 % and 1.92-4.93 %, respectively. This method was used to detect and analyze four rare paraquat poisoning cases. The distribution of paraquat, monoquat, and paraquat monopyridone is uneven, which is relatively high in the heart, blood, lung, and kidney. Heart blood/Peripheral blood ratio of paraquat, monoquat, paraquat monopyridone concentration in two poisoned cases were 1.4, 2.0, 1.5 and 1.9, 1.3, 1.2, which showed a location dependent postmortem redistribution. This is the first time that HPLC-MS/MS and the postmortem distribution or postmortem redistribution of paraquat metabolites in poisoned death cases have been reported. This research provides scientific basis for forensic identification of paraquat poisoning cases and extraction of biological specimen., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The use of catechol-O-methyltransferase inhibitors may mask doping agents, primarily levodopa, administered to racehorses and prolong the stimulating effects of dopaminergic compounds such as dopamine. It is known that 3-methoxytyramine is a metabolite of dopamine and 3-methoxytyrosine is a metabolite of levodopa thus these compounds are proposed to be potential biomarkers of interest. Previous research established a urinary threshold of 4,000 ng/mL for 3-methoxytyramine to monitor misuse of dopaminergic agents. However, there is no equivalent biomarker in plasma. To address this deficiency a rapid protein precipitation method was developed and validated to isolate target compounds from 100 µL equine plasma. A liquid chromatography-high resolution accurate mass (LC-HRAM) method using an IMTAKT Intrada amino acid column provided quantitative analysis of 3-methoxytyrosine (3-MTyr) with lower limit of quantification of 5 ng/mL. Reference population profiling (n = 1129) investigated the expected basal concentrations for raceday samples from equine athletes and showed a right-skewed distribution (skewness = 2.39, kurtosis = 10.65) which resulted from large variation (RSD = 71%) within the data. Logarithmic transformation of the data provided a normal distribution (skewness = 0.26, kurtosis = 3.23) resulting in the proposal of a conservative threshold for plasma 3-MTyr of 1,000 ng/mL at a 99.995% confidence level. A 12-horse administration study of Stalevo® (800 mg L-DOPA, 200 mg carbidopa, 1600 mg entacapone) revealed elevated 3-MTyr concentrations for 24-hours post-administration., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Combined diazepam-ethanol poisoning is common in forensic toxicology. Both diazepam and ethanol can inhibit the central nervous system via the γ-aminobutyric acid (GABA) ligand gated chloride ion channel, GABA A Receptor (GABA A R). As the common target of diazepam and ethanol, whether GABA A R is the key target of their combined action remains unclear. This study aimed to explore their interaction based on the synergistic mechanisms between diazepam and ethanol targeting the GABA A R. Four models were built in silico based on the crystal structure of GABA A R. Molecular dynamic processes of the four models were simulated by the GPU-accelerated pmemd.cuda program in the Amber18 package. Results showed that ethanol inclined to combine the adjacent GABA or diazepam sites, minimized fluctuations of the root-mean-square deviation (RMSD) in the molecular dynamic process of GABA or diazepam binding the GABA A R, and increased the release of binding energy of GABA or diazepam binding the GABA A R. Results also showed that diazepam had less effect on the RMSD fluctuation or the binding energy release of GABA binding GABA A R. The formation of complex of diazepam and GABA A R could minimize the RMSD fluctuation and increase binding energy release of ethanol binding GABA A R. Thus, ethanol, bridging GABA and diazepam, could strengthen the complex of GABA binding the GABA A R, as well as the complex of diazepam binding the GABA A R. However, whether diazepam binds GABA A R or not, it cannot affect GABA binding the GABA A R; and yet the complex of diazepam and GABA A R can stabilize the complex of ethanol and GABA A R., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The current approach to equine anti-doping is focused on the targeted detection of prohibited substances. However, as new substances are rapidly being developed, the need for complimentary methods for monitoring is crucial to ensure the integrity of the racing industry is upheld. Lipidomics is a growing field involved in the characterisation of lipids, their function and metabolism in a biological system. Different lipids have various biological effects throughout the equine system including platelet aggregation and inflammation. A certain class of lipids that are being reviewed are the eicosanoids (inflammatory markers). The use of eicosanoids as a complementary method for monitoring has become increasingly popular with various studies completed to highlight their potential. Studies including various corticosteroids, non-steroidal anti-inflammatories and cannabidiol have been reviewed to highlight the progress lipidomics has had in contributing to the equine anti-doping industry. This review has explored the techniques used to prepare and analyse samples for lipidomic investigations in addition to the statistical analysis and potential for lipidomics to be used for a longitudinal assessment in the equine anti-doping industry.

Nowadays, an increasing number of people worldwide use induction heating cookers to cook rice for consumption. This work reveals the influence of induction heating cooker heating modes on the quality attributes of cooked rice. Three heating modes, including bottom coil heating mode (mode 1), corner coil heating mode (mode 2), and side coil heating (mode 3), were used. Among the three modes, mode 2 allowed for an intermediate heating rate during rice cooking. For mode 2, the minimized temperature difference between the upper layer (including the central upper layer and peripheral upper layer) and the lower layer (including the central lower layer and peripheral lower layer) can reduce the effect of water absorption time difference on rice quality. Consequently, the rice cooked using mode 2 exhibited improved matrix uniformity, as indicated by the similar moisture content (59.92-61.89%), hardness (15.87-18.24 N), and water mobility (the relaxation time and peak area of the third relaxation peak) of rice samples at four different positions in the pot. The rice cooked by mode 2 showed better texture appearance and a more uniform porous microstructure. Consistently, the cooked rice samples by mode 2 at different positions did not show substantial differences in their starch digestion features., Competing Interests: Authors JK, JT, SF, and YW were employed by Zhuhai Gree Electric Appliances Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kong, Tao, Fu, Wen, Zhao and Zhang.)

Purpose: JWH-424, (8-bromo-1-naphthyl)(1-pentyl-1H-indol-3-yl)methanone, is a synthetic cannabinoid, which is a brominated analogue of JWH-018, one of the best-known synthetic cannabinoids. Despite the structural similarity to JWH-018, little is known about JWH-424 including its metabolism. The aim of the study was to compare human liver microsomes (HLM) and the fungus Cunninghamella elegans as the metabolism catalysts for JWH-424 to better understand the characteristic actions of the fungus in the synthetic cannabinoid metabolism., Methods: JWH-424 was incubated with HLM for 1 h and Cunninghamella elegans for up to 72 h. The HLM incubation mixtures were diluted with methanol and fungal incubation mixtures were extracted with dichloromethane and reconstituted in methanol before analyses by liquid chromatography-high-resolution mass spectrometry (LC-HRMS)., Results: HLM incubation resulted in production of ten metabolites through dihydrodiol formation, hydroxylation, and/or ipso substitution of the bromine with a hydroxy group. Fungal incubation led to production of 23 metabolites through carboxylation, dihydrodiol formation, hydroxylation, ketone formation, glucosidation and/or sulfation., Conclusions: Generally, HLM models give good predictions of human metabolites and structural analogues are metabolised in a similar fashion. However, major hydroxy metabolites produced by HLM were those hydroxylated at naphthalene instead of pentyl moiety, the major site of hydroxylation for JWH-018. Fungal metabolites, on the other hand, had undergone hydroxylation mainly at pentyl moiety. The metabolic disagreement suggests the necessity to verify the human metabolites in authentic urine samples, while H9 and H10 (hydroxynaphthalene), H8 (ipso substitution), F22 (hydroxypentyl), and F17 (dihydroxypentyl) are recommended for monitoring of JWH-424 in urinalysis., (© 2022. The Author(s), under exclusive licence to Japanese Association of Forensic Toxicology.)

The conventional detection of exogenous drugs in equine doping samples has been used for confirmation and subsequent prosecution of participants responsible. In recent years, alternative methods using indirect detection have been investigated due to the expanding number of pharmaceutical agents available with the potential of misuse. The monitoring of endogenous biomarkers such as hydrocortisone (HC) has been studied in equine urine with an international threshold of 1 μg/ml established; however, there is no current threshold for equine plasma. The aim of this research was to investigate plasma concentrations of HC and cortisone (C) in race day samples compared to an administration of Triamcinolone Acetonide (TACA). The reference population (n = 1150) provided HC (6 to 145 ng/ml) and C (0.7 to 13 ng/ml) levels to derive the HC to C ratio (HC/C). Population reference limits (PRLs) were proposed for HC/C values at 0.2 (lower) and 61 (upper). Administration of TACA resulted in down-regulation of HC/C values below the estimated PRLs for up to 96 h post-administration. This indirect detection period was longer than the detection of TACA for 72 h. The use of individual reference limits (IRLs) for HC/C values was investigated to support the Equine Biological Passport (EBP), an intelligence model developed by Racing NSW for longitudinal monitoring of biomarkers., (© 2022 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Equine urine analysis has evolved over time to detect thousands of urinary compounds for doping control in the horse racing industry. The longitudinal assessment of 3-methoxytyramine to tyramine ratio (3-MT/T) values in equine urine by GC-MS profiling was investigated to support the Racing NSW Equine Biological Passport (EBP) for detection of dopaminergic manipulation in racehorses. This involved comparison of routine urine samples to administration studies of Sinemet, a common Parkinson's disease medication containing levodopa. Using an endogenous reference compound (ERC) in a urinary ratio enabled greater confidence to provide intelligence of pharmaceutical manipulation as distinct from physiological variation. Population reference limits (PRLs) of 776 ng/ml for urinary 3-MT and 5.3 for 3-MT/T, together with the use of individual reference limits (IRLs), are proposed., (© 2022 John Wiley & Sons, Ltd.)

Metabolomics is a multidisciplinary field providing workflows for complementary approaches to conventional analytical determinations. It allows for the study of metabolically related groups of compounds or even the study of novel pathways within the biological system. The procedural stages of metabolomics; experimental design, sample preparation, analytical determinations, data processing and statistical analysis, compound identification and validation strategies are explored in this review. The selected approach will depend on the type of study being conducted. Experimental design influences the whole metabolomics workflow and thus needs to be properly assessed to ensure sufficient sample size, minimal introduced and biological variation and appropriate statistical power. Sample preparation needs to be simple, yet potentially global in order to detect as many compounds as possible. Analytical determinations need to be optimised either for the list of targeted compounds or a universal approach. Data processing and statistical analysis approaches vary widely and need to be better harmonised for review and interpretation. This includes validation strategies that are currently deficient in many presented workflows. Common compound identification approaches have been explored in this review. Metabolomics applications are discussed for clinical and forensic toxicology, human and equine sports anti-doping and veterinary residues., (© 2022 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

The proliferation of new psychoactive substances (NPS) has necessitated the development and improvement of current practices for the detection and identification of known NPS and newly emerging derivatives. High-resolution mass spectrometry (HRMS) is quickly becoming the industry standard for these analyses due to its ability to be operated in data-independent acquisition (DIA) modes, allowing for the collection of large amounts of data and enabling retrospective data interrogation as new information becomes available. The increasing popularity of HRMS has also prompted the exploration of new ways to screen for NPS, including broad-spectrum wastewater analysis to identify usage trends in the community and metabolomic-based approaches to examine the effects of drugs of abuse on endogenous compounds. In this paper, the novel applications of HRMS techniques to the analysis of NPS is reviewed. In particular, the development of innovative data analysis and interpretation approaches is discussed, including the application of machine learning and molecular networking to toxicological analyses., (© 2022. The Author(s).)

Based on the specific interaction of Ag + and cytosine-cytosine (C-C) base mismatch and using berberine (Ber) as the fluorescent probe and Exonuclease I (Exo I) as the background fluorescence reducing tool, a label-free Exo I-assisted fluorescence aptamer sensing platform was established for the detection of silver ions with high sensitivity and selectivity. Exo I reduced the fluorescence background of the Ber/Ag + -aptamer complex to a level similar to that of Ber itself in the absence of Ag + . After introducing Ag + into the sensing system, it induces the aptamer rich in base C to form C-Ag + -C i-motif structure which are resistant to degradation mediated by Exo I. The concentration of Ber, Ag + -aptamer, Exo I and the temperature and reaction time for Exo I were all optimized. Under the optimal experimental conditions, the detection limit of Ag + was 4.4 nM and the linear range was from 0.0059 μM to 235.48 μM with a coefficient of determination (R 2 ) > 0.99. Moreover, the proposed strategy had been successfully applied to the detection of Ag + in tap water and human serum with a good recovery ranging from 88.4% to 106.9%., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest with any individual/organization for the present work., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The high morbidity and mortality of neuroinflammatory diseases drives significant interest in understanding the underlying mechanisms involved in the innate and adaptive immune response of the central nervous system (CNS). Diagnostic biomarkers are important to define treatable neuroinflammation. Metabolomics is a rapidly evolving research area offering novel insights into metabolic pathways, and elucidation of reliable metabolites as biomarkers for diseases. This review focuses on the emerging literature regarding the detection of neuroinflammation using cerebrospinal fluid (CSF) metabolomics in human cohort studies. Studies of classic neuroinflammatory disorders such as encephalitis, CNS infection and multiple sclerosis confirm the utility of CSF metabolomics. Additionally, studies in neurodegeneration and neuropsychiatry support the emerging potential of CSF metabolomics to detect neuroinflammation in common CNS diseases such as Alzheimer's disease and depression. We demonstrate metabolites in the tryptophan-kynurenine pathway, nitric oxide pathway, neopterin and major lipid species show moderately consistent ability to differentiate patients with neuroinflammation from controls. Integration of CSF metabolomics into clinical practice is warranted to improve recognition and treatment of neuroinflammation., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Organophosphorus pesticides (OP S ) are widely used in the world, and many poisoning cases were caused by them. Phorate intoxication is especially common in China. However, there are currently few methods for discriminating phorate poisoning death from phorate exposure after death and interpretation of false-positive results due to the lack of effective biomarkers. In this study, we investigated the metabonomics of rat plasma at different dose levels of acute phorate intoxication using ultra-performance liquid chromatography quadrupole-time of flight mass spectrometry (UPLC-Q-TOF-MS) analysis. A total of 11 endogenous metabolites were significantly changed in the groups exposed to phorate at LD 50 level and three times of LD50 (3LD 50 ) level compared with the control group, which could be potential biomarkers of acute phorate intoxication. Plasma metabonomics analysis showed that diethylthiophosphate (DETP) could be a useful biomarker of acute phorate intoxication. The levels of uric acid, acylcarnitine, succinate, gluconic acid, and phosphatidylcholine (PC) (36:2) were increased, while pyruvate level was decreased in all groups exposed to phorate. The levels of ceramides (Cer) (d 18:0/16:0), palmitic acid, and lysophosphatidylcholine (lysoPC) (18:1) were only changed after 3LD 50 dosage. The results of this study indicate that the dose-dependent relationship exists between metabolomic profile change and toxicities associated with apoptosis, fatty acid metabolism disorder, energy metabolism disorder especially tricarboxylic acid (TCA) cycle, as well as liver, kidney, and nervous system functions after acute exposure of phorate. This study shows that metabonomics is a useful tool in identifying biomarkers for the forensic toxicology study of phorate poisoning.

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., (© 2020 John Wiley & Sons, Ltd.)

The surge in the consumption of food products containing herbal aphrodisiacs has driven their widespread adulteration. A rapid screening strategy is, therefore, warranted to curb this problem. This study established an enzyme inhibition assay to screen phosphodiesterase 5 (PDE5) inhibitors as adulterants in selected food products. Fluorescein-labelled cyclic-3',5'-guanosine monophosphate was utilised as substrates for the PDE5A1 enzyme, aided by the presence of nanoparticle phosphate-binding beads on their fluorescence polarisation. The sample preparation was optimised to improve the enzyme inhibition efficiency and applied to calculate the threshold values of six blank food matrices. The assay was validated using sildenafil, producing an IC 50 of 4.2 nM. The applicability of the assay procedure was demonstrated by screening 55 distinct food samples. The results were subsequently verified using confirmatory liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis. Altogether, 49 samples inhibited the PDE5 enzyme above the threshold values (75.7%-105.5%) and were registered as potentially adulterated samples. The remaining six samples were marked as nonadulterated with percentage inhibition below the threshold values (-3.3%-18.2%). The LC-HRMS analysis agreed with the assay results for all food products except for the instant coffee premix (ICP) samples. False-positive results were obtained for the ICP samples at 32% (8/25), due to possible PDE5 inhibition by caffeine. Contrarily, all other food samples were found to produce 0% (0/30) false-positive or false-negative results. The broad-based assay, established via a simple mix-incubate-read format, exhibited promising potential for high-throughput screening of PDE5 inhibitors in various food products, except those with naturally occurring phosphodiesterase inhibitors such as caffeine., (© 2020 John Wiley & Sons, Ltd.)

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., (© 2020 John Wiley & Sons, Ltd.)

Aim: To explore the cerebrospinal fluid (CSF) metabolite features in acute neuroinflammatory diseases and identify potential biomarkers to diagnose and monitor neuroinflammation., Method: A cohort of 14 patients (five females, nine males; mean [median] age 7y 9mo [9y], range 6mo-13y) with acute encephalitis (acute disseminated encephalomyelitis n=6, unknown suspected viral encephalitis n=3, enteroviral encephalitis n=2, seronegative autoimmune encephalitis n=2, herpes simplex encephalitis n=1) and age-matched non-inflammatory neurological disease controls (n=14) were investigated using an untargeted metabolomics approach. CSF metabolites were analyzed with liquid chromatography coupled to high resolution mass spectrometry, followed by subsequent multivariate and univariate statistical methods., Results: A total of 35 metabolites could be discriminated statistically between the groups using supervised orthogonal partial least squares discriminant analysis and analysis of variance. The tryptophan-kynurenine pathway contributed nine key metabolites. There was a statistical increase of kynurenine, quinolinic acid, and anthranilic acid in patients with encephalitis, whereas tryptophan, 3-hydroxyanthrnailic acid, and kynurenic acid were decreased. The nitric oxide pathway contributed four metabolites, with elevated asymmetric dimethylarginine and argininosuccinic acid, and decreased arginine and citrulline in patients with encephalitis. An increase in the CSF kynurenine/tryptophan ratio (p<0.001), anthranilic acid/3-hydroxyanthranilic acid ratio (p<0.001), asymmetric dimethylarginine/arginine ratio (p<0.001), and neopterin (p<0.001) strongly predicted neuroinflammation., Interpretation: The combination of alterations in the tryptophan-kynurenine pathway, nitric oxide pathway, and neopterin represent a useful potential panel for neuroinflammation and holds potential for clinical translation practice., What This Paper Adds: The kynurenine/tryptophan and anthranilic acid/3-hydroxyanthranilic acid ratios hold great potential as biomarkers of neuroinflammation. Elevation of the asymmetric dimethylarginine/arginine ratio in acute brain inflammation shows dysregulation of the nitric oxide pathway., (© 2020 Mac Keith Press.)

The use of illicit drugs across the world causes issues for users, healthcare workers and the public. Therefore, rapid and reliable onsite testing methods to detect these drugs are required. In this study, seven commercial surface-enhanced Raman spectroscopy (SERS) substrates A-G were compared for the analysis of cocaine. These substrates were compared using scanning electron microscopy to study the surface structure and Raman spectroscopy and to determine if there was any enhancement of the cocaine bands. Substrate B provided the best enhancement of known cocaine vibrational bands, allowing the detection down to concentrations of 1 ng/mL in standards and 10 ng/mL extracted from the oral fluid. The results showed that SERS is an ideal method for future rapid onsite analysis of illicit drugs in oral fluid. Commercial SERS substrates were compared for the analysis of cocaine. Substrate B provided the best result and was further tested with lower concentrations and extracts from the oral fluid. The application to oral fluid testing could prove useful for future onsite analysis., (© 2020 John Wiley & Sons, Ltd.)

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., (© 2020 John Wiley & Sons Ltd.)

The presence of erectile dysfunction (ED) drugs in adulterated dietary supplements, mainly in pharmaceutical dosage forms, is frequently addressed in the literature. Little attention is given to food products despite their increasing adulteration trend. To address this knowledge gap targeted, suspected-target, and non-targeted strategies were utilised to analyse ED drugs and their analogues in powdered drink mix (PDM), honey, jelly, hard candy, and sugar-coated chewing gum using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). The method was optimised and validated using 23 target analytes, representing different ED drugs with structural similarities. The modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction exhibited insignificant matrix effect (ME) within - 9.2-8.8% and provided complete coverage of target analytes with acceptable extraction recovery (RE) within 75.5-123.9%, except for carbodenafil in the PDM matrix. Based on the ME and RE performance, the analytical method was validated to analyse 25 food samples that claimed to enhance male sexual performance. The method exhibited good specificity and linearity with a limit of detection within 10-70 ng/mL and limit of quantification of 80 ng/mL. Similarly, the accuracy and precision were satisfactory within 77.4-122.0% and< 16.7%RSD, respectively. The LC-HRMS targeted analysis, together with suspected-target and non-targeted screenings, identified and detected ten ED drugs from 24 food samples. The modified QuEChERS extraction with LC-HRMS-based method was demonstrated to be universally applicable to various food products, covering an extensive range of known and potentially novel ED drugs, which is valuable for routine casework., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this study., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The emergence of novel doping agents is a continuous issue for analysts who aim to maintain the integrity of horseracing together with the well-being and safety of the animals and riders involved. Untargeted mass spectrometric analysis presents a potential improvement for antidoping as it enables the detection of compounds being indirectly affected by an administered drug. In this study, liquid chromatography-high-resolution mass spectrometry was used to investigate a 12-horse administration study of the synthetic opioid, butorphanol. A mass spectrometric workflow capable of detecting metabolic differences for an extended period of time was successfully developed. This proof-of-concept study demonstrates the potential of untargeted workflows to provide a list of biomarkers of exposure and effect that are indicative of drug administration which may be implemented into routine testing for improved doping control., (© 2021 John Wiley & Sons, Ltd.)

The lucrative market of herbal remedies spurs rampant adulteration, particularly with pharmaceutical drugs and their unapproved analogues. A comprehensive screening strategy is, therefore, warranted to detect these adulterants and, accordingly, to safeguard public health. This study uses the data-dependent acquisition of liquid chromatography-quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS) to screen phosphodiesterase 5 (PDE5) inhibitors in herbal remedies using suspected-target and non-targeted strategies. For the suspected-target screening, we used a library comprising 95 PDE5 inhibitors. For the non-targeted screening, we adopted top-down and bottom-up approaches to flag novel PDE5 inhibitor analogues based on common fragmentation patterns. LC-QTOF-MS was optimised and validated for capsule and tablet dosage forms using 23 target analytes, selected to represent different groups of PDE5 inhibitors. The method exhibited excellent specificity and linearity with limit of detection and limit of quantification of <40 and 80 ng/mL, respectively. The accuracy ranged from 79.0% to 124.7% with a precision of <14.9% relative standard deviation. The modified, quick, easy, cheap, effective, rugged, and safe extraction provided insignificant matrix effect within -9.1%-8.0% and satisfactory extraction recovery of 71.5%-105.8%. These strategies were used to screen 52 herbal remedy samples that claimed to enhance male sexual performance. The suspected-target screening resulted in 33 positive samples, revealing 10 target analytes and 2 suspected analytes. Systematic MS and tandem MS interrogations using the non-targeted screening returned insignificant signals, indicating the absence of potentially novel analogues. The target analytes were quantified from 0.03 to 121.31 mg per dose of each sample. The proposed strategies ensure that all PDE5 inhibitors are comprehensively screened, providing a useful tool to curb the widespread adulteration of herbal remedies., (©2020 John Wiley & Sons, Ltd.)

Surface-enhanced Raman spectroscopy (SERS) technology is an attractive method for the prompt and accurate on-site screening of illicit drugs. As portable Raman systems are available for on-site screening, the readiness of SERS technology for sensing applications is predominantly dependent on the accuracy, stability and cost-effectiveness of the SERS strip. An atmospheric-pressure plasma-assisted chemical deposition process that can deposit an even distribution of nanogold particles in a one-step process has been developed. The process was used to print a nanogold film on a paper-based substrate using a HAuCl 4 solution precursor. X-ray photoelectron spectroscopy (XPS) analysis demonstrates that the gold has been fully reduced and that subsequent plasma post-treatment decreases the carbon content of the film. Results for cocaine detection using this substrate were compared with two commercial SERS substrates, one based on nanogold on paper and the currently available best commercial SERS substrate based on an Ag pillar structure. A larger number of bands associated with cocaine was detected using the plasma-printed substrate than the commercial substrates across a range of cocaine concentrations from 1 to 5000 ng/mL. A detection limit as low as 1 ng/mL cocaine with high spatial uniformity was demonstrated with the plasma-printed substrate. It is shown that the plasma-printed substrate can be produced at a much lower cost than the price of the commercial substrate.

Drug degradation as a consequence of putrefactive bacterial activity is a well-known factor that affects the identification and quantitation of certain substances of forensic interest. Current knowledge on putrefaction-mediated degradation of drugs is, however, significantly lacking. This study aimed to investigate the degradation of 4-methylmethcathinone (4-MMC or mephedrone) and to detect its degradation products in putrefied biological matrices containing 4-MMC. The bacteria species Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Proteus vulgaris were grown in brain-heart infusion broth, spiked with 4-MMC and incubated at 37°C for 24 h. Postmortem human blood and fresh porcine liver macerate were also left to putrefy in sample tubes at room temperature for 1 week. Structural elucidation was based on modern spectroscopic analyses including the use of high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. All four putrefactive bacteria were capable of degrading 4-MMC extensively under the experimental conditions explored. Of particular interest was the discovery of a novel degradation product common to all four bacterial species, which was assigned as 2-hydroxy-1-(4-methylphenyl)propan-1-one (HMP) based on the spectroscopic data. This degradation product was detectable in both postmortem human blood and porcine liver samples. The stability of the identified degradation products, especially HMP, should be further investigated to assess their validity of serving as marker analytes for monitoring 4-MMC in postmortem toxicology., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Association of Forensic Toxicologists, Inc. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The proliferation of adulterated health foods and beverages in the market demands a comprehensive analytical strategy to identify the adulterants, particularly those of isomeric phosphodiesterase 5 (PDE5) inhibitors. An instant coffee premix (ICP) purchased from an online retailer was flagged for suspected adulteration through PDE5 inhibition assay. The ICP was then analysed using suspected-target and non-targeted screenings of a liquid chromatography-quadrupole time-of-flight mass spectrometry. Based on these findings, a PDE5 inhibitor initially assigned as compound X was isolated from the ICP by employing a liquid chromatography-diode array detection before its structural elucidation with liquid chromatography-ultraviolet (LC-UV) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. The suspected-target screening matched the protonated molecule ([M + H] + ) precursor ion of compound X at m/z 499.2310 with two suspected analytes that are structural isomers of one another. The fragmentation patterns of compound X were comparable to those analogues in the dithiocarbodenafil group through the non-targeted screening. These findings, complemented by the LC-UV and NMR spectroscopy data, together with the chromatographic separation of related structural isomers, conclude the identity of compound X. To our best knowledge, this is the first study to report the presence of 3,5-dimethylpiperazinyl-dithiodesmethylcarbodenafil in an ICP sample.Key pointsThe herbal-based male sexual performance products' lucrative market has instigated their rampant adulteration, particularly with PDE5 inhibitors.The adulterated products may also contain analogues of the approved PDE5 inhibitors, which usually passed into the market undetected as they are not included in the routine targeted screening procedure.The present study detected, isolated, and identified an isomeric sildenafil analogue from an instant coffee premix sample using rapid qualitative assay and comprehensive analytical analysis.This paper highlighted the applicability of the established strategies for routine casework, particularly in a forensic drug testing laboratory., Competing Interests: No potential conflict of interest was reported by the authors., (© 2020 The Author(s). Published by Taylor & Francis Group on behalf of the Academy of Forensic Science.)

Thirdhand exposure to e-cigarette residue is likely to have harmful effects in children http://bit.ly/38a2umw., Competing Interests: Conflict of interest: H. Chen has nothing to disclose. Conflict of interest: G. Li has nothing to disclose. Conflict of interest: V.S.R.R. Allam has nothing to disclose. Conflict of interest: B. Wang has nothing to disclose. Conflict of interest: Y.L. Chan has nothing to disclose. Conflict of interest: C. Scarfo has nothing to disclose. Conflict of interest: M. Ueland has nothing to disclose. Conflict of interest: R. Shimmon has nothing to disclose. Conflict of interest: S. Fu has nothing to disclose. Conflict of interest: P. Foster has nothing to disclose. Conflict of interest: B.G. Oliver has nothing to disclose., (Copyright ©ERS 2020.)

Increasing reports of neurological and psychiatric complications due to psychostimulant synthetic cathinones (SCs) have recently raised public concern. However, the precise mechanism of SC toxicity is unclear. This paucity of understanding highlights the need to investigate the in-vitro toxicity and mechanistic pathways of three SCs: butylone, pentylone, and 3,4-Methylenedioxypyrovalerone (MDPV). Human neuronal cells of SH-SY5Y were cultured in supplemented DMEM/F12 media and differentiated to a neuronal phenotype using retinoic acid (10 μM) and 12- O -tetradecanoylphorbol-13-acetate (81 nM). Trypan blue and lactate dehydrogenase assays were utilized to assess the neurotoxicity potential and potency of these three SCs. To investigate the underlying neurotoxicity mechanisms, measurements included markers of oxidative stress, mitochondrial bioenergetics, and intracellular calcium (Ca 2+ ), and cell death pathways were evaluated at two doses (EC 15 and EC 40 ), for each drug tested. Following 24 h of treatment, all three SCs exhibited a dose-dependent neurotoxicity, characterized by a significant ( p < 0.0001 vs. control) production of reactive oxygen species, decreased mitochondrial bioenergetics, and increased intracellular Ca 2+ concentrations. The activation of caspases 3 and 7 implicated the orchestration of mitochondrial-mediated neurotoxicity mechanisms for these SCs. Identifying novel therapeutic agents to enhance an altered mitochondrial function may help in the treatment of acute-neurological complications arising from the illicit use of these SCs., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

As a widely consumed beverage, coffee tends to be a target for intentional adulteration. This study describes the application of modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) coupled to liquid chromatography-high-resolution mass spectrometry (LC-HRMS) for simultaneous screening, identification, and quantification of undeclared phosphodiesterase 5 (PDE5) inhibitors in instant coffee premixes (ICPs). The mass spectrometer was operated in auto MS/MS acquisition for simultaneous MS and MS/MS experiments. Qualitative establishments from the suspected-target screening and targeted identification processes led to an unambiguous analyte assignment from the protonated molecule ([M+H] + ) precursor ion which is subsequently used for quantification of 23 targeted PDE5 inhibitors. The analytical method validation covered specificity, linearity, range, accuracy, limit of detection (LOD), limit of quantification (LOQ), precisions, matrix effect (ME), and extraction recovery (RE). The specificity was established using the optimised chromatographic separation as well as the distinguishable [M+H] + precursor ion. The linearity of each target analyte was demonstrated with a coefficient of determination (r 2 ) of >0.9960 over the expected range of sample concentrations. The accuracy ranged from 88.1%-119.3% with LOD and LOQ of <70 ng/mL and 80 ng/mL, respectively. Excellent precisions were established within 0.4%-9.1% of the relative standard deviation. An insignificant ME within -5.2% to +8.7% was achieved using three different strategies of chromatography, sample extraction, and sample dilution. The RE was good for all target analytes within 84.7%-123.5% except for N-desethylacetildenafil at low (53.8%) and medium (65.1%) quality control levels. The method was successfully applied to 25 samples of ICPs where 17 of them were found to be adulterated with PDE5 inhibitors and their analogues. Further quantification revealed the total amount of these adulterants ranged from 2.77 to 121.64 mg per sachet., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Infiltration of leukocytes within the vessel at sites of inflammation and the subsequent generation of myeloperoxidase-derived oxidants, including hypochlorous acid, are key characteristics of atherosclerosis. Hypochlorous acid is a potent oxidant that reacts readily with most biological molecules, including DNA and RNA. This results in nucleic acid modification and the formation of different chlorinated products. These products have been used as biomarkers of inflammation, owing to their presence in elevated amounts in different inflammatory fluids and diseased tissue, including atherosclerotic lesions. However, it is not clear whether these materials are simply biomarkers, or could also play a role in the development of chronic inflammatory pathologies. In this study, we examined the reactivity of different chlorinated nucleosides with human coronary artery endothelial cells (HCAEC). Evidence was obtained for the incorporation of each chlorinated nucleoside into the cellular RNA or DNA. However, only 8-chloro-adenosine (8ClA) had a significant effect on the cell viability and metabolic activity. Exposure of HCAEC to 8ClA decreased glycolysis, and resulted in a reduction in ATP, with a corresponding increase in the chlorinated analogue, 8Cl-ATP in the nucleotide pool. 8ClA also induced sustained endoplasmic reticulum stress within the HCAEC, which resulted in activation of the unfolded protein response, the altered expression of antioxidant genes and culminated in the release of calcium into the cytosol and cell death by apoptosis. Taken together, these data provide new insight into pathways by which myeloperoxidase activity and resultant hypochlorous acid generation could promote endothelial cell damage during chronic inflammation, which could be relevant to the progression of atherosclerosis., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

This paper presents the data on the optimisation and validation of a liquid chromatography-high-resolution mass spectrometry (LC-HRMS) to establish the presence of phosphodiesterase 5 (PDE5) inhibitors and their analogues as adulterants in instant coffee premixes. The method development data covered chromatographic optimisation for better analyte separation and isomeric resolution, mass spectrometry optimisation for high sensitivity and sample preparation optimisation for high extraction recovery (RE) and low matrix effect (ME). The validation data covered specificity, linearity, range, accuracy, limit of detection, limit of quantification, precisions, ME, and RE. The optimisation and validation data presented here is related to the article: "Determination of phosphodiesterase 5 (PDE5) inhibitors in instant coffee premixes using liquid chromatography-high-resolution mass spectrometry (LC-HRMS)" Mohd Yusop et al., 2019.

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.

Some food components can modulate the antigenicity of β-lactoglobulin (β-LG). This study investigated the role of oleic acid (OA) in reducing the antigenicity of β-LG. The results indicate the antigenicity of β-LG gradually decreased from 15 (sample with no OA) to 9.86, 7.51, and 6.01 µg/mL when interacting with OA during dynamic high-pressure microfluidization treatment at 0.1, 80, and 160 MPa. Although binding sites (n) of β-LG combined with OA at 0.1, 80, and 160 MPa decreased from 0.79 to 0.5 and 0.66, β-LG had a higher binding affinity (K a ) to OA than that of untreated β-LG. The values of K a for β-LG/OA at 0.1, 80, and 160 MPa were 5.51 × 10 6 , 17.43 × 10 6 , and 49.75 × 10 6 M -1 , respectively. The molecule dynamic simulation showed that the OA molecules located at both β-barrel (site 1) interacted with Lys60, Glu62, and Lys69 and outer surface site 2 consisting of Tyr20, Tyr42, Ser21, Glu157, and His161. Additionally, when binding with OA during the dynamic high-pressure microfluidization treatment, the conformation of β-LG changed, reflected by the decrease of fluorescence intensity and total sulfhydryl group content, the increase of surface sulfhydryl group content, and secondary structure changes of β-LG. These results deduce that some epitopes may be masked by OA or modified by the conformational changes, resulting in the decline of antigenicity of β-LG molecules., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Cocaine trafficking in the form of textile impregnation is routinely encountered as a concealment method. Raman spectroscopy has been a popular and successful testing method used for in situ screening of cocaine in textiles and other matrices. Quantitative analysis of cocaine in these matrices using Raman spectroscopy has not been reported to date. This study aimed to develop a simple Raman method for quantifying cocaine using atropine as the model analogue in various types of textiles. Textiles were impregnated with solutions of atropine in methanol. The impregnated atropine was extracted using less hazardous acidified water with the addition of potassium thiocyanate (KSCN) as an internal standard for Raman analysis. Despite the presence of background matrix signals arising from the textiles, the cocaine analogue could easily be identified by its characteristic Raman bands. The successful use of KSCN normalised the analyte signal response due to different textile matrix background interferences and thus removed the need for a matrix-matched calibration. The method was linear over a concentration range of 6.25-37.5 mg/cm 2 with a coefficient of determination (R 2 ) at 0.975 and acceptable precision and accuracy. A simple and accurate Raman spectroscopy method for the analysis and quantification of a cocaine analogue impregnated in textiles has been developed and validated for the first time. This proof-of-concept study has demonstrated that atropine can act as an ideal model compound to study the problem of cocaine impregnation in textile. The method has the potential to be further developed and implemented in real world forensic cases., (Copyright © 2017 John Wiley & Sons, Ltd.)

Traditional fingermark developing methods are unsuitable for developing and extracting latent blood fingermarks on dark surfaces at crime scene because of their ineffectiveness or tedious operation procedures. In the present work, an effective and simple method was developed to serve this purpose using a suspension of monodisperse silica nanoparticles (MSNs). A suspension of 0.1 g/mL of MSNs was prepared by dispersing MSNs ultrasonically into an ethanol solution containing 1.0% Tween 80 and then uniformly sprayed onto the latent blood fingermarks on black plastic bags. Approximately 20 s later, ethanol was sprayed to clean the superfluous developing liquid. After the ethanol had evaporated, the latent fingermarks became visible as a result of the photonic crystal effect produced by the MSNs that had adsorbed to the fingermark ridges. The developed fingermarks were then photographed using a digital camera under a white or monospectral light source, revealing fine ridges and clear fingermark details. This novel, simple and effective method uses the photonic crystal effect of MSNs to develop latent blood fingermarks without the need for surface functionalization and conjugation to dye or fluorescently label molecules. The method can detect latent blood fingermarks that have been retained on a black plastic bag surface for at least 30 d. Given the simplicity and effectiveness of the developed method, MSNs may be a useful alternative material for use in developing latent fingermarks. Further research on the topic is warranted., (© 2020 The Author(s). Published by Taylor & Francis Group on behalf of the Academy of Forensic Science.)

The number of new psychoactive substances keeps on rising despite the controlling efforts by law enforcement. Although metabolism of the newly emerging drugs is continuously studied to keep up with the new additions, the exact structures of the metabolites are often not identified due to the insufficient sample quantities for techniques such as nuclear magnetic resonance (NMR) spectroscopy. The aim of the study was to characterise several metabolites of the synthetic cannabinoid (1-pentyl-1H-indol-3-yl) (2,2,3,3-tetramethylcyclopropyl) methanone (UR-144) by NMR spectroscopy after the incubation with the fungus Cunninghamella elegans. UR-144 was incubated with C. elegans for 72 h, and the resulting metabolites were chromatographically separated. Six fractions were collected and analysed by NMR spectroscopy. UR-144 was also incubated with human liver microsomes (HLM), and the liquid chromatography-high resolution mass spectrometry analysis was performed on the HLM metabolites with the characterised fungal metabolites as reference standards. Ten metabolites were characterised by NMR analysis including dihydroxy metabolites, carboxy and hydroxy metabolites, a hydroxy and ketone metabolite, and a carboxy and ketone metabolite. Of these metabolites, dihydroxy metabolite, carboxy and hydroxy metabolites, and a hydroxy and ketone metabolite were identified in HLM incubation. The results indicate that the fungus is capable of producing human-relevant metabolites including the exact isomers. The capacity of the fungus C. elegans to allow for NMR structural characterisation by enabling production of large amounts of metabolites makes it an ideal model to complement metabolism studies.

Introduction: Chronic cannabis use has been associated with impaired cognition and elevated psychological symptoms, particularly psychotic-like experiences. While Δ 9 -tetrahydrocannabinol (THC) is thought to be primarily responsible for these deleterious effects, cannabidiol (CBD) is purported to have antipsychotic properties and to ameliorate cognitive, symptomatic, and brain harms in cannabis users. However, this has never been tested in a prolonged administration trial in otherwise healthy cannabis users. Here, we report the first study of prolonged CBD administration to a community sample of regular cannabis users in a pragmatic trial investigating potential restorative effects of CBD on psychological symptoms and cognition. Materials and Methods: Twenty frequent cannabis users (16 male, median age 25 years) underwent a 10-week open-label trial of 200 mg of daily oral CBD treatment, while continuing to use cannabis as usual. The majority of participants were daily cannabis users who had used cannabis for several years (median 5.5 years of regular use). Participants underwent psychological and cognitive assessments at baseline (BL) and post-treatment (PT) and were monitored weekly throughout the trial. Results: CBD was well tolerated with no reported side effects; however, participants retrospectively reported reduced euphoria when smoking cannabis. No impairments to cognition were found, nor were there deleterious effects on psychological function. Importantly, participants reported significantly fewer depressive and psychotic-like symptoms at PT relative to BL, and exhibited improvements in attentional switching, verbal learning, and memory. Increased plasma CBD concentrations were associated with improvements in attentional control and beneficial changes in psychological symptoms. Greater benefits were observed in dependent than in nondependent cannabis users. Conclusions: Prolonged CBD treatment appears to have promising therapeutic effects for improving psychological symptoms and cognition in regular cannabis users. Our findings require replication given the lack of a placebo control in this pragmatic trial, but suggest that CBD may be a useful adjunct treatment for cannabis dependence., Competing Interests: No competing financial interests exist.

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.

Chemical 'spot' tests are a presumptive illicit drug identification technique commonly used by law enforcement, border security personnel, and forensic laboratories. The simplicity, low cost, and rapid results afforded by these tests make them particularly attractive for presumptive identification globally. In this paper, we review the development of these long-established methods and discuss color test recommendations and guidelines. A search of the scientific literature revealed the chemical reactions occurring in many color tests are either not actively investigated or reported as unknown. Today, color tests face many challenges, from the appearance of new psychoactive substances to concerns regarding selectivity, sensitivity, and safety. Advances in technology have seen color test reagents used in digital image color analysis, solid sensors, and microfluidic devices for illicit drug detection. This summarizes current research and suggests the future of presumptive color testing., (Copyright © 2017 John Wiley & Sons, Ltd.)

Purpose: Identifying intake of synthetic cannabinoids generally requires the metabolism data of the drugs so that appropriate metabolite markers can be targeted in urine testing. However, the continuous appearance of new cannabinoids during the last decade has made it difficult to keep up with all the compounds including {1-[(1-methylpiperidin-2-yl)methyl]-1 H -indol-3-yl}(naphthalen-1-yl)methanone (AM1220). In this study, metabolism of AM1220 was investigated with human liver microsomes and the fungus Cunninghamella elegans ., Methods: Metabolic stability of AM1220 was analysed by liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode after 1 µM incubation in human liver microsomes for 30 min. Tentative structure elucidation of metabolites was performed on both human liver microsome and fungal incubation samples using liquid chromatography-high-resolution mass spectrometry., Results: Half-life of AM1220 was estimated to be 3.7 min, indicating a high clearance drug. Nine metabolites were detected after incubating human liver microsomes while seven were found after incubating Cunninghamella elegans , leading to 11 metabolites in total (five metabolites were common to both systems). Demethylation, dihydrodiol formation, combination of the two, hydroxylation and dihydroxylation were the observed biotransformations., Conclusions: Three most abundant metabolites in both human liver microsomes and Cunninghamella elegans were desmethyl, dihydrodiol and hydroxy metabolites, despite different isomers of dihydrodiol and hydroxy metabolites in each model. These abundant metabolites can potentially be useful markers in urinalysis for AM1220 intake., Competing Interests: Compliance with ethical standardsThe authors declare no conflicts of interest.This article does not contain any studies with human participants or animals performed by any of the authors.

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.

Neurotransmitters play crucial roles in physiological functions and their imbalances have demonstrated association in the pathology of several diseases. The measurement of neurotransmitters possesses a great potential as a significant clinical tool. This study presents the development and validation of an LC-MS/MS method for simultaneous quantification of multi-class neurotransmitters associated with dopamine, tryptophan and glutamate-γ-aminobutyric acid pathways. A total of ten neurotransmitters and their metabolites (dopamine, epinephrine, metanephrine, tryptophan, serotonin, kynurenic acid, kynurenine, anthranilic acid, GABA, glutamic acid) were determined based on a simple and rapid 'dilute and shoot' method using minimal urine volume. The chromatographic separation was achieved using a Poroshell 120 Bonus-RP LC Column in combination with a gradient elution within an 8.5-min time frame. The method exhibited good sensitivity as the limits of quantification ranged between 0.025 and 0.075 μg/mL with acceptable matrix effects (< ± 14.5%), no carryover and good linearity (r 2  > 0.98). The accuracy and precision for all analytes were within tolerances, at < ± 9.9% mean relative error (MRE) and < 8.6% relative standard deviation (RSD), respectively. The method was successfully applied in measuring the neurotransmitter concentrations in urine of healthy donors. Furthermore, the undertaken stability experiments indicated that acidified urine specimens allowed the analytes to be stable for prolonged durations in comparison to those untreated. The study also reveals the performance of the method is unaffected by the absence of expensive deuterated reference standards under the experimental conditions employed which further simplifies the analytical procedures and provides a significant cost saving for running the assay. Graphical abstract The quantification of multi-class neurotransitters associated with the dopamine, tryptophan and GABA-glutamate pathways using a simple 'dilute and shoot' LC-MS/MS method.

Hallucinogenic phenethylamines such as 2,5-dimethoxyphenethylamines (2C-X) and their N-(2-methoxybenzyl) derivatives (25X-NBOMe) have seen an increase in novel analogues in recent years. These rapidly changing analogues make it difficult for laboratories to rely on traditional targeted screening methods to detect unknown new psychoactive substances (NPS). In this study, twelve 2C-X, six 2,5-dimethoxyamphetamines (DOX), and fourteen 25X-NBOMe derivatives, including two deuterated derivatives (2C-B-d 6 and 25I-NBOMe-d 9 ), were analyzed using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Collision-induced dissociation (CID) experiments were performed using collision energies set at 10, 20, and 40 eV. For 2C-X and DOX derivatives, common losses were observed including neutral and radical losses such as NH 3 (17.0265 Da), •CH 6 N (32.0500 Da), C 2 H 7 N (45.0578 Da) and C 2 H 9 N (47.0735 Da). 2C-X derivatives displayed common product ions at m/z 164.0837 ([C 10 H 12 O 2 ] +• ), 149.0603 ([C 9 H 9 O 2 ] + ), and 134.0732 ([C 9 H 10 O] +• ) while DOX derivatives had common product ions at m/z 178.0994 ([C 11 H 14 O 2 ] +• ), 163.0754 ([C 10 H 11 O 2 ] + ), 147.0804 ([C 10 H 11 O] + ), and 135.0810 ([C 9 H 11 O] + ). 25X-NBOMe had characteristic product ions at m/z 121.0654 ([C 8 H 9 O] + ) and 91.0548 ([C 7 H 7 ] + ) with minor common losses corresponding to 2-methylanisole (C 8 H 10 O, 122.0732 Da), 2-methoxybenzylamine (C 8 H 11 NO, 137.0847 Da), and •C 9 H 14 NO (152.1074 Da). Novel analogues of the selected classes can be detected by applying neutral loss filters (NLFs) and extracting the common product ions. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)