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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.

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.

Purpose A tert-leucinate derivative synthetic cannabinoid, methyl (2S)-2-([1-(4-fluorobutyl)-1H-indazole-3-carbonyl]amino)-3,3-dimethylbutanoate (4F-MDMB-BINACA, 4F-MDMB-BUTINACA or 4F-ADB) is known to adversely impact health. This study aimed to evaluate the suitability of three different modes of monitoring metabolism: HepG2 liver cells, fungus Cunninghamella elegans (C. elegans) and pooled human liver microsomes (HLM) for comparison with human in-vivo metabolism in identifying suitable urinary marker(s) for 4F-MDMB-BINACA intake. Methods Tentative structure elucidation of in-vitro metabolites was performed on HepG2, C. elegans and HLM using liquid chromatography-tandem mass spectrometry and high-resolution mass spectrometry analysis. In-vivo metabolites obtained from twenty authentic human urine samples were analysed using liquid chromatography-Orbitrap mass spectrometry. Results Incubation with HepG2, C. elegans and HLM yielded nine, twenty-three and seventeen metabolites of 4F-MDMB-BINACA, respectively, formed via ester hydrolysis, hydroxylation, carboxylation, dehydrogenation, oxidative defluorination, carbonylation or reaction combinations. Phase II metabolites of glucosidation and sulfation were also exclusively identified using C. elegans model. Eight in-vivo metabolites tentatively identified were mainly products of ester hydrolysis with or without additional dehydrogenation, N-dealkylation, monohydroxylation and oxidative defluorination with further oxidation to butanoic acid. Metabolites with intact terminal methyl ester moiety, i.e., oxidative defluorination with further oxidation to butanoic acid, were also tentatively identified. Conclusions The in-vitro models presented proved useful in the exhaustive metabolism studies. Despite limitations, HepG2 identified the major 4F-MDMB-BINACA ester hydrolysis metabolite, and C. elegans demonstrated the capacity to produce a wide variety of metabolites. Both C. elegans and HLM produced all the in-vivo metabolit, Funding Agencies|Australian Government Research Training Program ScholarshipAustralian GovernmentDepartment of Industry, Innovation and Science; Australian Research CouncilAustralian Research Council

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 onsite 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 plasmaassisted 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 paperbased substrate using a HAuCl4 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.

Purpose A tert-leucinate derivative synthetic cannabinoid, methyl (2S)-2-([1-(4-fluorobutyl)-1H-indazole-3-carbonyl]amino)-3,3-dimethylbutanoate (4F-MDMB-BINACA, 4F-MDMB-BUTINACA or 4F-ADB) is known to adversely impact health. This study aimed to evaluate the suitability of three different modes of monitoring metabolism: HepG2 liver cells, fungus Cunninghamella elegans (C. elegans) and pooled human liver microsomes (HLM) for comparison with human in-vivo metabolism in identifying suitable urinary marker(s) for 4F-MDMB-BINACA intake. Methods Tentative structure elucidation of in-vitro metabolites was performed on HepG2, C. elegans and HLM using liquid chromatography-tandem mass spectrometry and high-resolution mass spectrometry analysis. In-vivo metabolites obtained from twenty authentic human urine samples were analysed using liquid chromatography-Orbitrap mass spectrometry. Results Incubation with HepG2, C. elegans and HLM yielded nine, twenty-three and seventeen metabolites of 4F-MDMB-BINACA, respectively, formed via ester hydrolysis, hydroxylation, carboxylation, dehydrogenation, oxidative defluorination, carbonylation or reaction combinations. Phase II metabolites of glucosidation and sulfation were also exclusively identified using C. elegans model. Eight in-vivo metabolites tentatively identified were mainly products of ester hydrolysis with or without additional dehydrogenation, N-dealkylation, monohydroxylation and oxidative defluorination with further oxidation to butanoic acid. Metabolites with intact terminal methyl ester moiety, i.e., oxidative defluorination with further oxidation to butanoic acid, were also tentatively identified. Conclusions The in-vitro models presented proved useful in the exhaustive metabolism studies. Despite limitations, HepG2 identified the major 4F-MDMB-BINACA ester hydrolysis metabolite, and C. elegans demonstrated the capacity to produce a wide variety of metabolites. Both C. elegans and HLM produced all the in-vivo metabolit, Funding Agencies|Australian Government Research Training Program ScholarshipAustralian GovernmentDepartment of Industry, Innovation and Science; Australian Research CouncilAustralian Research Council

Purpose A tert-leucinate derivative synthetic cannabinoid, methyl (2S)-2-([1-(4-fluorobutyl)-1H-indazole-3-carbonyl]amino)-3,3-dimethylbutanoate (4F-MDMB-BINACA, 4F-MDMB-BUTINACA or 4F-ADB) is known to adversely impact health. This study aimed to evaluate the suitability of three different modes of monitoring metabolism: HepG2 liver cells, fungus Cunninghamella elegans (C. elegans) and pooled human liver microsomes (HLM) for comparison with human in-vivo metabolism in identifying suitable urinary marker(s) for 4F-MDMB-BINACA intake. Methods Tentative structure elucidation of in-vitro metabolites was performed on HepG2, C. elegans and HLM using liquid chromatography-tandem mass spectrometry and high-resolution mass spectrometry analysis. In-vivo metabolites obtained from twenty authentic human urine samples were analysed using liquid chromatography-Orbitrap mass spectrometry. Results Incubation with HepG2, C. elegans and HLM yielded nine, twenty-three and seventeen metabolites of 4F-MDMB-BINACA, respectively, formed via ester hydrolysis, hydroxylation, carboxylation, dehydrogenation, oxidative defluorination, carbonylation or reaction combinations. Phase II metabolites of glucosidation and sulfation were also exclusively identified using C. elegans model. Eight in-vivo metabolites tentatively identified were mainly products of ester hydrolysis with or without additional dehydrogenation, N-dealkylation, monohydroxylation and oxidative defluorination with further oxidation to butanoic acid. Metabolites with intact terminal methyl ester moiety, i.e., oxidative defluorination with further oxidation to butanoic acid, were also tentatively identified. Conclusions The in-vitro models presented proved useful in the exhaustive metabolism studies. Despite limitations, HepG2 identified the major 4F-MDMB-BINACA ester hydrolysis metabolite, and C. elegans demonstrated the capacity to produce a wide variety of metabolites. Both C. elegans and HLM produced all the in-vivo metabolit, Funding Agencies|Australian Government Research Training Program ScholarshipAustralian GovernmentDepartment of Industry, Innovation and Science; Australian Research CouncilAustralian Research Council

Purpose A tert-leucinate derivative synthetic cannabinoid, methyl (2S)-2-([1-(4-fluorobutyl)-1H-indazole-3-carbonyl]amino)-3,3-dimethylbutanoate (4F-MDMB-BINACA, 4F-MDMB-BUTINACA or 4F-ADB) is known to adversely impact health. This study aimed to evaluate the suitability of three different modes of monitoring metabolism: HepG2 liver cells, fungus Cunninghamella elegans (C. elegans) and pooled human liver microsomes (HLM) for comparison with human in-vivo metabolism in identifying suitable urinary marker(s) for 4F-MDMB-BINACA intake. Methods Tentative structure elucidation of in-vitro metabolites was performed on HepG2, C. elegans and HLM using liquid chromatography-tandem mass spectrometry and high-resolution mass spectrometry analysis. In-vivo metabolites obtained from twenty authentic human urine samples were analysed using liquid chromatography-Orbitrap mass spectrometry. Results Incubation with HepG2, C. elegans and HLM yielded nine, twenty-three and seventeen metabolites of 4F-MDMB-BINACA, respectively, formed via ester hydrolysis, hydroxylation, carboxylation, dehydrogenation, oxidative defluorination, carbonylation or reaction combinations. Phase II metabolites of glucosidation and sulfation were also exclusively identified using C. elegans model. Eight in-vivo metabolites tentatively identified were mainly products of ester hydrolysis with or without additional dehydrogenation, N-dealkylation, monohydroxylation and oxidative defluorination with further oxidation to butanoic acid. Metabolites with intact terminal methyl ester moiety, i.e., oxidative defluorination with further oxidation to butanoic acid, were also tentatively identified. Conclusions The in-vitro models presented proved useful in the exhaustive metabolism studies. Despite limitations, HepG2 identified the major 4F-MDMB-BINACA ester hydrolysis metabolite, and C. elegans demonstrated the capacity to produce a wide variety of metabolites. Both C. elegans and HLM produced all the in-vivo metabolit, Funding Agencies|Australian Government Research Training Program ScholarshipAustralian GovernmentDepartment of Industry, Innovation and Science; Australian Research CouncilAustralian Research Council

Purpose A tert-leucinate derivative synthetic cannabinoid, methyl (2S)-2-([1-(4-fluorobutyl)-1H-indazole-3-carbonyl]amino)-3,3-dimethylbutanoate (4F-MDMB-BINACA, 4F-MDMB-BUTINACA or 4F-ADB) is known to adversely impact health. This study aimed to evaluate the suitability of three different modes of monitoring metabolism: HepG2 liver cells, fungus Cunninghamella elegans (C. elegans) and pooled human liver microsomes (HLM) for comparison with human in-vivo metabolism in identifying suitable urinary marker(s) for 4F-MDMB-BINACA intake. Methods Tentative structure elucidation of in-vitro metabolites was performed on HepG2, C. elegans and HLM using liquid chromatography-tandem mass spectrometry and high-resolution mass spectrometry analysis. In-vivo metabolites obtained from twenty authentic human urine samples were analysed using liquid chromatography-Orbitrap mass spectrometry. Results Incubation with HepG2, C. elegans and HLM yielded nine, twenty-three and seventeen metabolites of 4F-MDMB-BINACA, respectively, formed via ester hydrolysis, hydroxylation, carboxylation, dehydrogenation, oxidative defluorination, carbonylation or reaction combinations. Phase II metabolites of glucosidation and sulfation were also exclusively identified using C. elegans model. Eight in-vivo metabolites tentatively identified were mainly products of ester hydrolysis with or without additional dehydrogenation, N-dealkylation, monohydroxylation and oxidative defluorination with further oxidation to butanoic acid. Metabolites with intact terminal methyl ester moiety, i.e., oxidative defluorination with further oxidation to butanoic acid, were also tentatively identified. Conclusions The in-vitro models presented proved useful in the exhaustive metabolism studies. Despite limitations, HepG2 identified the major 4F-MDMB-BINACA ester hydrolysis metabolite, and C. elegans demonstrated the capacity to produce a wide variety of metabolites. Both C. elegans and HLM produced all the in-vivo metabolit, Funding Agencies|Australian Government Research Training Program ScholarshipAustralian GovernmentDepartment of Industry, Innovation and Science; Australian Research CouncilAustralian Research Council

Purpose A tert-leucinate derivative synthetic cannabinoid, methyl (2S)-2-([1-(4-fluorobutyl)-1H-indazole-3-carbonyl]amino)-3,3-dimethylbutanoate (4F-MDMB-BINACA, 4F-MDMB-BUTINACA or 4F-ADB) is known to adversely impact health. This study aimed to evaluate the suitability of three different modes of monitoring metabolism: HepG2 liver cells, fungus Cunninghamella elegans (C. elegans) and pooled human liver microsomes (HLM) for comparison with human in-vivo metabolism in identifying suitable urinary marker(s) for 4F-MDMB-BINACA intake. Methods Tentative structure elucidation of in-vitro metabolites was performed on HepG2, C. elegans and HLM using liquid chromatography-tandem mass spectrometry and high-resolution mass spectrometry analysis. In-vivo metabolites obtained from twenty authentic human urine samples were analysed using liquid chromatography-Orbitrap mass spectrometry. Results Incubation with HepG2, C. elegans and HLM yielded nine, twenty-three and seventeen metabolites of 4F-MDMB-BINACA, respectively, formed via ester hydrolysis, hydroxylation, carboxylation, dehydrogenation, oxidative defluorination, carbonylation or reaction combinations. Phase II metabolites of glucosidation and sulfation were also exclusively identified using C. elegans model. Eight in-vivo metabolites tentatively identified were mainly products of ester hydrolysis with or without additional dehydrogenation, N-dealkylation, monohydroxylation and oxidative defluorination with further oxidation to butanoic acid. Metabolites with intact terminal methyl ester moiety, i.e., oxidative defluorination with further oxidation to butanoic acid, were also tentatively identified. Conclusions The in-vitro models presented proved useful in the exhaustive metabolism studies. Despite limitations, HepG2 identified the major 4F-MDMB-BINACA ester hydrolysis metabolite, and C. elegans demonstrated the capacity to produce a wide variety of metabolites. Both C. elegans and HLM produced all the in-vivo metabolit, Funding Agencies|Australian Government Research Training Program ScholarshipAustralian GovernmentDepartment of Industry, Innovation and Science; Australian Research CouncilAustralian Research Council

Infiltration of leukocytes within the vessel at sites of inflammation and the subsequent generation of myelo-peroxidase-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.

Infiltration of leukocytes within the vessel at sites of inflammation and the subsequent generation of myelo-peroxidase-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.

Infiltration of leukocytes within the vessel at sites of inflammation and the subsequent generation of myelo-peroxidase-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.

Chronic cannabis use has been associated with impaired cognition and elevated psychological symptoms, particularly psychotic-like experiences.

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 (r2> 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.

Myeloperoxidase produces strong oxidants during the immune response to destroy invading pathogens. However, these oxidants can also cause tissue damage, which contributes to the development of numerous inflammatory diseases. Selenium containing compounds, including selenomethionine (SeMet) and 1,4-anhydro-5-seleno-D-talitol (SeTal), react rapidly with different MPO-derived oxidants to form the respective selenoxides (SeMetO and SeTalO). This study investigates the susceptibility of these selenoxides to undergo reduction back to the parent compounds by intracellular reducing systems, including glutathione (GSH) and the glutathione reductase and thioredoxin reductase systems. GSH is shown to reduce SeMetO and SeTalO, with consequent formation of GSSG with apparent second order rate constants, k2, in the range 10(3)-10(4)M(-1)s(-1). Glutathione reductase reduces both SeMetO and SeTalO at the expense of NADPH via formation of GSSG, whereas thioredoxin reductase acts only on SeMetO. The presence of SeMet and SeTal also increased the rate at which NADPH was consumed by the glutathione reductase system in the presence of N-chloramines. In contrast, the presence of SeMet and SeTal reduced the rate of NADPH consumption by the thioredoxin reductase system after addition of N-chloramines, consistent with the rapid formation of selenoxides, but only slow reduction by thioredoxin reductase. These results support a potential role of seleno compounds to act as catalytic scavengers of MPO-derived oxidants, particularly in the presence of glutathione reductase and NADPH, assuming that sufficient plasma levels of the parent selenoether can be achieved in vivo following supplementation.

Myeloperoxidase produces strong oxidants during the immune response to destroy invading pathogens. However, these oxidants can also cause tissue damage, which contributes to the development of numerous inflammatory diseases. Selenium containing compounds, including selenomethionine (SeMet) and 1,4-anhydro-5-seleno-D-talitol (SeTal), react rapidly with different MPO-derived oxidants to form the respective selenoxides (SeMetO and SeTalO). This study investigates the susceptibility of these selenoxides to undergo reduction back to the parent compounds by intracellular reducing systems, including glutathione (GSH) and the glutathione reductase and thioredoxin reductase systems. GSH is shown to reduce SeMetO and SeTalO, with consequent formation of GSSG with apparent second order rate constants, k2, in the range 10(3)-10(4)M(-1)s(-1). Glutathione reductase reduces both SeMetO and SeTalO at the expense of NADPH via formation of GSSG, whereas thioredoxin reductase acts only on SeMetO. The presence of SeMet and SeTal also increased the rate at which NADPH was consumed by the glutathione reductase system in the presence of N-chloramines. In contrast, the presence of SeMet and SeTal reduced the rate of NADPH consumption by the thioredoxin reductase system after addition of N-chloramines, consistent with the rapid formation of selenoxides, but only slow reduction by thioredoxin reductase. These results support a potential role of seleno compounds to act as catalytic scavengers of MPO-derived oxidants, particularly in the presence of glutathione reductase and NADPH, assuming that sufficient plasma levels of the parent selenoether can be achieved in vivo following supplementation.

Hypochlorous acid (HOCl) and N-chloramines are produced by myeloperoxidase (MPO) as part of the immune response to destroy invading pathogens. However, MPO also plays a detrimental role in inflammatory pathologies, including atherosclerosis, as inappropriate production of oxidants, including HOCl and N-chloramines, causes damage to host tissue. Low molecular mass thiol compounds, including glutathione (GSH) and methionine (Met), have demonstrated efficacy in scavenging MPO-derived oxidants, which prevents oxidative damage in vitro and ex vivo. Selenium species typically have greater reactivity toward oxidants compared to the analogous sulfur compounds, and are known to be efficient scavengers of HOCl and other hypohalous acids produced by MPO. In this study, we examined the efficacy of a number of sulfur and selenium compounds to scavenge a range of biologically relevant N-chloramines and oxidants produced by both isolated MPO and activated neutrophils and characterized the resulting selenium-derived oxidation products in each case. A dose-dependent decrease in the concentration of each N-chloramine was observed on addition of the sulfur compounds (cysteine, methionine) and selenium compounds (selenomethionine, methylselenocysteine, 1,4-anhydro-4-seleno-L-talitol, 1,5-anhydro-5-selenogulitol) studied. In general, selenomethionine was the most reactive with N-chloramines (k2 0.8-3.4×10(3)M(-1) s(-1)) with 1,5-anhydro-5-selenogulitol and 1,4-anhydro-4-seleno-L-talitol (k2 1.1-6.8×10(2)M(-1) s(-1)) showing lower reactivity. This resulted in the formation of the respective selenoxides as the primary oxidation products. The selenium compounds demonstrated greater ability to remove protein N-chloramines compared to the analogous sulfur compounds. These reactions may have implications for preventing cellular damage in vivo, particularly under chronic inflammatory conditions.

Aim: Currently, procedures that identify the drugs 'destroyed' in adulterated urine specimens are very limited. This study aimed to determine the effect of pyridinium chlorochromate (PCC) on routine opiate assays and identify reaction products formed. Results/methodology: Opiate-positive urines adulterated with PCC (20 and 100 mM) were analyzed using CEDIA® immunoassay and GC-MS. Urine and water samples spiked with 6-monoacetylmorphine, morphine and its glucuronides (10 µg/ml) and PCC (0.02-100 mM) were monitored with LC-MS, and the products characterized. Conclusion: PCC significantly decreased the abundance of morphine, codeine and IS. Adulterated water and urine samples containing 6-monoacetylmorphine, morphine and morphine-3-glucuronide yielded morphinone-3-glucuronide, 7,14-dihydroxy-6-monoacetylmorphine, 7,8-diketo-6-monoacetylmorphine and 7,8-diketo-morphine (tentative assignment). Reaction pathways may be different in the two matrices.

Aim: Currently, procedures that identify the drugs 'destroyed' in adulterated urine specimens are very limited. This study aimed to determine the effect of pyridinium chlorochromate (PCC) on routine opiate assays and identify reaction products formed. Results/methodology: Opiate-positive urines adulterated with PCC (20 and 100 mM) were analyzed using CEDIA® immunoassay and GC-MS. Urine and water samples spiked with 6-monoacetylmorphine, morphine and its glucuronides (10 µg/ml) and PCC (0.02-100 mM) were monitored with LC-MS, and the products characterized. Conclusion: PCC significantly decreased the abundance of morphine, codeine and IS. Adulterated water and urine samples containing 6-monoacetylmorphine, morphine and morphine-3-glucuronide yielded morphinone-3-glucuronide, 7,14-dihydroxy-6-monoacetylmorphine, 7,8-diketo-6-monoacetylmorphine and 7,8-diketo-morphine (tentative assignment). Reaction pathways may be different in the two matrices.

The powerful oxidant HOCl (hypochlorous acid and its corresponding anion, −OCl) generated by the myeloperoxidase (MPO)–H2O2–Cl− system of activated leukocytes is strongly associated with multiple human inflammatory diseases; consequently there is considerable interest in inhibition of this enzyme. Nitroxides are established antioxidants of low toxicity that can attenuate oxidation in animal models, with this ascribed to superoxide dismutase or radical-scavenging activities. We have shown (M.D. Rees et al., Biochem. J. 421, 79–86, 2009) that nitroxides, including 4-amino-TEMPO (4-amino-2,2,6,6-tetramethylpiperidin-1-yloxyl radical), are potent inhibitors of HOCl formation by isolated MPO and activated neutrophils, with IC50 values of ~1 and ~6 µM respectively. The utility of tetramethyl-substituted nitroxides is, however, limited by their rapid reduction by biological reductants. The corresponding tetraethyl-substituted nitroxides have, however, been reported to be less susceptible to reduction. In this study we show that the tetraethyl species were reduced less rapidly than the tetramethyl species by both human plasma (89–99% decreased rate of reduction) and activated human neutrophils (62–75% decreased rate). The tetraethyl-substituted nitroxides retained their ability to inhibit HOCl production by MPO and activated neutrophils with IC50 values in the low-micromolar range; in some cases inhibition was enhanced compared to tetramethyl substitution. Nitroxides with rigid structures (fused oxaspiro rings) were, however, inactive. Overall, these data indicate that tetraethyl-substituted nitroxides are potent inhibitors of oxidant formation by MPO, with longer plasma and cellular half-lives compared to the tetramethyl species, potentially allowing lower doses to be employed.

In vitro urine adulteration is a well-documented practice adopted by individuals aiming to evade detection of drug use, when required to undergo mandatory sports and workplace drug testing. Potassium nitrite is an effective urine adulterant due to its oxidizing potential, and has been shown to mask the presence of many drugs of abuse. However, limited research has been conducted to understand its mechanism of action, and to explore the possibility of the drugs undergoing direct oxidation to form stable reaction products. In this study, opiates including morphine, codeine, morphine-3-glucuronide and morphine-6-glucuronide were exposed to potassium nitrite in water and urine to mimic the process of nitrite adulteration. It was found that two stable reaction products were detected by liquid chromatography-mass spectrometry (LC-MS) when morphine and morphine-6-glucuronide were exposed to nitrite. Isolation and elucidation using spectrometric and spectroscopic techniques revealed that they were 2-nitro-morphine and 2-nitro-morphine-6-glucuronide, respectively. These reaction products were also formed when an authentic morphine-positive urine specimen was fortified with nitrite. 2-Nitro-morphine was found to be stable enough to undergo the enzymatic hydrolysis procedure and also detectable by gas chromatography-mass spectrometry (GC-MS) after forming a trimethylsilyl derivative. On the contrary, morphine-3-glucuronide did not appear to be chemically manipulated when exposed to potassium nitrite in urine. These reaction products are not endogenously produced, are relatively stable and can be monitored with both LC-MS and GC-MS confirmatory techniques. As a result, these findings have revealed the possibility for the use of 2-nitro-morphine and 2-nitro-morphine-6-glucuronide as markers for the indirect monitoring of morphine and morphine-6-glucuronide in urine specimens adulterated with nitrite. 2013 John Wiley & Sons, Ltd.

RATIONALE Pyridinium chlorochromate (PCC) is the active ingredient of 'Urine Luck', a commercially available in vitro adulterating agent used to conceal the presence of drugs in a urine specimen. The exposure of codeine and its major glucuronide metabolite codeine-6-glucuronide (C6G) to PCC was investigated to determine whether PCC is an effective masking agent for these opiate compounds. METHODS Following the addition of PCC to both spiked and authentic codeine and C6G-positive urine specimens, the samples were monitored using liquid chromatography/mass spectrometry (LC/MS). Stable reaction products were identified and characterized using high-resolution MS analysis and, where possible, nuclear magnetic resonance (NMR) analysis. RESULTS It was determined that PCC effectively oxidizes codeine and C6G, thus altering the original codeine-to-C6G ratio in the urine specimen. Four reaction products were identified for codeine: codeinone, 14-hydroxycodeinone, 6-O-methylcodeine and 8-hydroxy-7,8-dihydrocodeinone. Similarly, three reaction products were identified for C6G: codeinone, codeine and a lactone of C6G (tentative assignment). CONCLUSIONS Besides addressing the complications added to interpretation, more investigation is warranted to further determine their potential for use as markers for monitoring the presence of codeine and C6G in urine specimens adulterated with PCC.

Drug testing programs are established to help achieve a drug-free work environment, promote fair competition in sport, facilitate harm minimization and rehabilitation programs, better manage patient care by clinicians and service law enforcement authorities. Urine remains the most popular and appropriate testing matrix for such purposes. However, urine is prone to adulteration, where chemicals, especially oxidizing chemicals, are purposely added to the collected urine specimens to produce a false-negative test result. This article will describe the effect of various popular oxidizing adulterants on urine drug test results, the countermeasures taken by laboratories in dealing with adulterated urine samples and the prospect of developing more robust and economical methods to combat urine adulteration in the future.

Drug testing programs are established to help achieve a drug-free work environment, promote fair competition in sport, facilitate harm minimization and rehabilitation programs, better manage patient care by clinicians and service law enforcement authorities. Urine remains the most popular and appropriate testing matrix for such purposes. However, urine is prone to adulteration, where chemicals, especially oxidizing chemicals, are purposely added to the collected urine specimens to produce a false-negative test result. This article will describe the effect of various popular oxidizing adulterants on urine drug test results, the countermeasures taken by laboratories in dealing with adulterated urine samples and the prospect of developing more robust and economical methods to combat urine adulteration in the future.

In vitro urine adulteration is a well-documented practice adopted by individuals aiming to evade detection of drug use, when required to undergo mandatory sports and workplace drug testing. Potassium nitrite is an effective urine adulterant due to its oxidizing potential, and has been shown to mask the presence of many drugs of abuse. However, limited research has been conducted to understand its mechanism of action, and to explore the possibility of the drugs undergoing direct oxidation to form stable reaction products. In this study, opiates including morphine, codeine, morphine-3-glucuronide and morphine-6-glucuronide were exposed to potassium nitrite in water and urine to mimic the process of nitrite adulteration. It was found that two stable reaction products were detected by liquid chromatography-mass spectrometry (LC-MS) when morphine and morphine-6-glucuronide were exposed to nitrite. Isolation and elucidation using spectrometric and spectroscopic techniques revealed that they were 2-nitro-morphine and 2-nitro-morphine-6-glucuronide, respectively. These reaction products were also formed when an authentic morphine-positive urine specimen was fortified with nitrite. 2-Nitro-morphine was found to be stable enough to undergo the enzymatic hydrolysis procedure and also detectable by gas chromatography-mass spectrometry (GC-MS) after forming a trimethylsilyl derivative. On the contrary, morphine-3-glucuronide did not appear to be chemically manipulated when exposed to potassium nitrite in urine. These reaction products are not endogenously produced, are relatively stable and can be monitored with both LC-MS and GC-MS confirmatory techniques. As a result, these findings have revealed the possibility for the use of 2-nitro-morphine and 2-nitro-morphine-6-glucuronide as markers for the indirect monitoring of morphine and morphine-6-glucuronide in urine specimens adulterated with nitrite. 2013 John Wiley & Sons, Ltd.

RATIONALE Pyridinium chlorochromate (PCC) is the active ingredient of 'Urine Luck', a commercially available in vitro adulterating agent used to conceal the presence of drugs in a urine specimen. The exposure of codeine and its major glucuronide metabolite codeine-6-glucuronide (C6G) to PCC was investigated to determine whether PCC is an effective masking agent for these opiate compounds. METHODS Following the addition of PCC to both spiked and authentic codeine and C6G-positive urine specimens, the samples were monitored using liquid chromatography/mass spectrometry (LC/MS). Stable reaction products were identified and characterized using high-resolution MS analysis and, where possible, nuclear magnetic resonance (NMR) analysis. RESULTS It was determined that PCC effectively oxidizes codeine and C6G, thus altering the original codeine-to-C6G ratio in the urine specimen. Four reaction products were identified for codeine: codeinone, 14-hydroxycodeinone, 6-O-methylcodeine and 8-hydroxy-7,8-dihydrocodeinone. Similarly, three reaction products were identified for C6G: codeinone, codeine and a lactone of C6G (tentative assignment). CONCLUSIONS Besides addressing the complications added to interpretation, more investigation is warranted to further determine their potential for use as markers for monitoring the presence of codeine and C6G in urine specimens adulterated with PCC.

6-Monoacetylmorphine (6-MAM), being a unique metabolite of heroin, is routinely tested in urine samples to monitor heroin use. However, detection of 6-MAM-related opiates such as morphine is known to be affected by in vitro urine adulteration using oxidizing adulterants such as potassium nitrite. This study aimed to investigate the fate of 6-MAM after exposure to nitrite and to identify any formed oxidation products that may potentially be used for monitoring heroin abuse despite nitrite adulteration. Potassium nitrite (0.05 M and 0.6 M) was reacted with 6-MAM (5-10,000 ng/mL) in both water and blank urine with pH adjusted to range from 3 to 8. Following reaction at room temperature for varying periods, the reaction mixtures were monitored by both the CEDIA® Heroin Metabolite (6-AM) immunoassay and liquid chromatography-mass spectrometry (LC-MS) methods. Structural elucidation of the isolated oxidation products was based on mass spectrometry and nuclear magnetic resonance spectroscopic evidence. Nitrite, under acidic environment (pH < 7), was shown to be effective in masking the detection of 6-MAM by both the CEDIA® immunoassay and the LC-MS methods. 2-Nitro-6-monoacetylmorphine (2-nitro-MAM) was identified as the sole oxidation product, which remained detectable in urine for at least 11 days under the experimental conditions investigated. 2-Nitro-MAM was detectable in a urine sample of a heroin user after nitrite exposure. 2-Nitro-MAM has shown potential to serve as a marker for monitoring heroin abuse when urine is adulterated with nitrite. Certification of 2-nitro-MAM reference standard for further development of its quantitative testing methods is thus warranted.

6-Monoacetylmorphine (6-MAM), being a unique metabolite of heroin, is routinely tested in urine samples to monitor heroin use. However, detection of 6-MAM-related opiates such as morphine is known to be affected by in vitro urine adulteration using oxidizing adulterants such as potassium nitrite. This study aimed to investigate the fate of 6-MAM after exposure to nitrite and to identify any formed oxidation products that may potentially be used for monitoring heroin abuse despite nitrite adulteration. Potassium nitrite (0.05 M and 0.6 M) was reacted with 6-MAM (5-10,000 ng/mL) in both water and blank urine with pH adjusted to range from 3 to 8. Following reaction at room temperature for varying periods, the reaction mixtures were monitored by both the CEDIA® Heroin Metabolite (6-AM) immunoassay and liquid chromatography-mass spectrometry (LC-MS) methods. Structural elucidation of the isolated oxidation products was based on mass spectrometry and nuclear magnetic resonance spectroscopic evidence. Nitrite, under acidic environment (pH < 7), was shown to be effective in masking the detection of 6-MAM by both the CEDIA® immunoassay and the LC-MS methods. 2-Nitro-6-monoacetylmorphine (2-nitro-MAM) was identified as the sole oxidation product, which remained detectable in urine for at least 11 days under the experimental conditions investigated. 2-Nitro-MAM was detectable in a urine sample of a heroin user after nitrite exposure. 2-Nitro-MAM has shown potential to serve as a marker for monitoring heroin abuse when urine is adulterated with nitrite. Certification of 2-nitro-MAM reference standard for further development of its quantitative testing methods is thus warranted.

6-Monoacetylmorphine (6-MAM), being a unique metabolite of heroin, is routinely tested in urine samples to monitor heroin use. However, detection of 6-MAM-related opiates such as morphine is known to be affected by in vitro urine adulteration using oxidizing adulterants such as potassium nitrite. This study aimed to investigate the fate of 6-MAM after exposure to nitrite and to identify any formed oxidation products that may potentially be used for monitoring heroin abuse despite nitrite adulteration. Potassium nitrite (0.05 M and 0.6 M) was reacted with 6-MAM (5-10,000 ng/mL) in both water and blank urine with pH adjusted to range from 3 to 8. Following reaction at room temperature for varying periods, the reaction mixtures were monitored by both the CEDIA® Heroin Metabolite (6-AM) immunoassay and liquid chromatography-mass spectrometry (LC-MS) methods. Structural elucidation of the isolated oxidation products was based on mass spectrometry and nuclear magnetic resonance spectroscopic evidence. Nitrite, under acidic environment (pH < 7), was shown to be effective in masking the detection of 6-MAM by both the CEDIA® immunoassay and the LC-MS methods. 2-Nitro-6-monoacetylmorphine (2-nitro-MAM) was identified as the sole oxidation product, which remained detectable in urine for at least 11 days under the experimental conditions investigated. 2-Nitro-MAM was detectable in a urine sample of a heroin user after nitrite exposure. 2-Nitro-MAM has shown potential to serve as a marker for monitoring heroin abuse when urine is adulterated with nitrite. Certification of 2-nitro-MAM reference standard for further development of its quantitative testing methods is thus warranted.