Effect of erythrophagocytosis-induced ferroptosis during angiogenesis in atherosclerotic plaques.

Intraplaque (IP) angiogenesis is a key feature of advanced atherosclerotic plaques. Because IP vessels are fragile and leaky, erythrocytes are released and phagocytosed by macrophages (erythrophagocytosis), which leads to high intracellular iron content, lipid peroxidation and cell death. In vitro experiments showed that erythrophagocytosis by macrophages induced non-canonical ferroptosis, an emerging type of regulated necrosis that may contribute to plaque destabilization. Erythrophagocytosis-induced ferroptosis was accompanied by increased expression of heme-oxygenase 1 and ferritin, and could be blocked by co-treatment with third generation ferroptosis inhibitor UAMC-3203. Both heme-oxygenase 1 and ferritin were also expressed in erythrocyte-rich regions of carotid plaques from ApoE-/- Fbn1C1039G+/- mice, a model of advanced atherosclerosis with IP angiogenesis. The effect of UAMC-3203 (12.35 mg/kg/day) on atherosclerosis was evaluated in ApoE-/- Fbn1C1039G+/- mice fed a western-type diet (WD) for 12 weeks (n = 13 mice/group) or 20 weeks (n = 16-21 mice/group) to distinguish between plaques without and with established IP angiogenesis, respectively. A significant decrease in carotid plaque thickness was observed after 20 weeks WD (87 ± 19 µm vs. 166 ± 20 µm, p = 0.006), particularly in plaques with confirmed IP angiogenesis or hemorrhage (108 ± 35 µm vs. 322 ± 40 µm, p = 0.004). This effect was accompanied by decreased IP heme-oxygenase 1 and ferritin expression. UAMC-3203 did not affect carotid plaques after 12 weeks WD or plaques in the aorta, which typically do not develop IP angiogenesis. Altogether, erythrophagocytosis-induced ferroptosis during IP angiogenesis leads to larger atherosclerotic plaques, an effect that can be prevented by ferroptosis inhibitor UAMC-3203.

Investigating the Potential of Drift Tube Ion Mobility for the Analysis of Oxidized Lipids.

Epilipids, a subset of the lipidome that comprises oxidized, nitrated, and halogenated lipid species, show important biochemical activity in the regulation of redox lipid metabolism by influencing cell fate decisions, including death, health, and aging. Due to the large chemical diversity, reversed-phase liquid chromatography-high-resolution mass spectrometry (RPLC-HRMS) methods have only a limited ability to separate numerous isobaric and isomeric epilipids. Ion mobility spectrometry (IMS) is a gas-phase separation technique that can be combined with LC-HRMS to improve the overall peak capacity of the analytical platform. Here, we illustrate the advantages and discuss the current limitations of implementing IMS in LC-HRMS workflows for the analysis of oxylipins and oxidized complex lipids. Using isomeric mixtures of oxylipins, we demonstrated that while deprotonated ions of eicosanoids were poorly resolved by IMS, sodium acetate and metal adducts (e.g., Li, Na, Ag, Ba, K) of structural isomers often showed ΔCCS% above 1.4% and base peak separation with high-resolution demultiplexing (HRDm). The knowledge of the IM migration order was also used as a proof of concept to help in the annotation of oxidized complex lipids using HRDm and all-ion fragmentation spectra. Additionally, we used a mixture of deuterium-labeled lipids for a routine system suitability test with the purpose of improving harmonization and interoperability of IMS data sets in (epi)lipidomics.

Untargeted hair lipidomics: comprehensive evaluation of the hair-specific lipid signature and considerations for retrospective analysis.

Lipidomics investigates the composition and function of lipids, typically employing blood or tissue samples as the primary study matrices. Hair has recently emerged as a potential complementary sample type to identify biomarkers in early disease stages and retrospectively document an individual's metabolic status due to its long detection window of up to several months prior to the time of sampling. However, the limited coverage of lipid profiling presented in previous studies has hindered its exploitation. This study aimed to evaluate the lipid coverage of hair using an untargeted liquid chromatography-high-resolution mass spectrometry lipidomics platform. Two distinct three-step exhaustive extraction experiments were performed using a hair metabolomics one-phase extraction technique that has been recently optimized, and the two-phase Folch extraction method which is recognized as the gold standard for lipid extraction in biological matrices. The applied lipidomics workflow improved hair lipid coverage, as only 99 species could be annotated using the one-phase extraction method, while 297 lipid species across six categories were annotated with the Folch method. Several lipids in hair were reported for the first time, including N-acyl amino acids, diradylglycerols, and coenzyme Q10. The study suggests that hair lipids are not solely derived from de novo synthesis in hair, but are also incorporated from sebum and blood, making hair a valuable matrix for clinical, forensic, and dermatological research. The improved understanding of the lipid composition and analytical considerations for retrospective analysis offers valuable insights to contextualize untargeted hair lipidomic analysis and facilitate the use of hair in translational studies.

Metabolic signature of HepaRG cells exposed to ethanol and tumor necrosis factor alpha to study alcoholic steatohepatitis by LC-MS-based untargeted metabolomics.

Despite the high prevalence of alcoholic liver disease, its identification and characterization remain poor, especially in early stages such as alcoholic fatty liver disease and alcoholic steatohepatitis. This latter implies diagnostic difficulties, few therapeutic options and unclear mechanisms of action. To elucidate the metabolic alterations and pinpoint affected biochemical pathways, alcoholic steatohepatitis was simulated in vitro by exposing HepaRG cells to ethanol (IC10, 368 mM) and tumor necrosis factor alpha (TNF-α, 50 ng/mL) for 24 h. This combined exposure was compared to solely ethanol-exposed as well as -nonexposed cells. Four different metabolomics platforms were used combining liquid chromatography, high-resolution mass spectrometry and drift tube ion mobility to elucidate both intracellular and extracellular metabolic alterations. Some of the key findings include the influence of TNF-α in the upregulation of hepatic triglycerides and the downregulation of hepatic phosphatidylethanolamines and phosphatidylcholines. S-Adenosylmethionine showed to play a central role in the progression of alcoholic steatohepatitis. In addition, fatty acyl esters of hydroxy fatty acid (FAHFA)-containing triglycerides were detected for the first time in human hepatocytes and their alterations showed a potentially important role during the progression of alcoholic steatohepatitis. Ethoxylated phosphorylcholine was identified as a potential new biomarker of ethanol exposure.

Metabolic Signature of Ethanol-Induced Hepatotoxicity in HepaRG Cells by Liquid Chromatography-Mass Spectrometry-Based Untargeted Metabolomics.

Alcoholic liver disease is highly prevalent but poorly identified and characterized, leading to knowledge gaps, which impairs early diagnosis. Excessive alcohol consumption is known to alter lipid metabolism, followed by progressive intracellular lipid accumulation, resulting in alcoholic fatty liver disease. In this study, HepaRG cells were exposed to ethanol at IC10 and 1/10 IC10 for 24 and 48 h. Metabolic alterations were investigated intra- and extracellularly with liquid chromatography-high-resolution mass spectrometry. Ion mobility was added as an extra separation dimension for untargeted lipidomics to improve annotation confidence. Distinctive patterns between exposed and control cells were consistently observed, with intracellular upregulation of di- and triglycerides, downregulation of phosphatidylcholines and phosphatidylethanolamines, sphingomyelins, and S-adenosylmethionine, among others. Several intracellular metabolic patterns could be related to changes in the extracellular environment, such as increased intracellular hydrolysis of sphingomyelins, leading to increased phosphorylcholine secretion. Carnitines showed alterations depending on the size of their carbon chain, which highlights the interplay between ß-oxidation in mitochondria and peroxisomes. Potential new biomarkers of ethanol-induced hepatotoxicity have been observed, such as ceramides with a sphingadienine backbone, octanoylcarnitine, creatine, acetylcholine, and ethoxylated phosphorylcholine. The combination of the metabolic fingerprint and footprint enabled a comprehensive investigation of the pathophysiology behind ethanol-induced hepatotoxicity.

Guidelines and considerations for building multidimensional libraries for untargeted MS-based metabolomics.

INTRODUCTION: Feature annotation is crucial in untargeted metabolomics but remains a major challenge. The large pool of metabolites collected under various instrumental conditions is underrepresented in publicly available databases. Retention time (RT) and collision cross section (CCS) measurements from liquid chromatography ion mobility high-resolution mass spectrometers can be employed in addition to MS/MS spectra to improve the confidence of metabolite annotation. Recent advancements in machine learning focus on improving the accuracy of predictions for CCS and RT values. Therefore, high-quality experimental data are crucial to be used either as training datasets or as a reference for high-confidence matching. METHODS: This manuscript provides an easy-to-use workflow for the creation of an in-house metabolite library, offers an overview of alternative solutions, and discusses the challenges and advantages of using open-source software. A total of 100 metabolite standards from various classes were analyzed and subjected to the described workflow for library generation. RESULTS AND DISCUSSION: The outcome was an open-access available NIST format metabolite library (.msp) with multidimensional information. The library was used to evaluate CCS prediction tools, MS/MS spectra heterogeneities (e.g., multiple adducts, in-source fragmentation, radical fragment ions using collision-induced dissociation), and the reporting of RT.

The Role of Kynurenines in Cognitive Dysfunction in Bipolar Disorder.

INTRODUCTION: Chronic low-grade inflammation is suggested to play a pathophysiological role in bipolar disorder (BD) and its related cognitive dysfunctions. Although kynurenine (KYN) pathway metabolites are key inflammatory mediators, studies investigating the association between KYN metabolism and cognition in BD are scarce. We aimed to explore the relationship between KYN metabolism and cognitive functioning across different mood states in BD. METHODS: Sixty-seven patients with BD (35 depressed and 32 [hypo] manic) and 29 healthy controls were included. Cognitive functioning was assessed at 3 time intervals (baseline, 4, and 8 months) assessing processing speed, sustained attention, verbal memory, working memory, and response inhibition. Plasma samples for quantification of 3-hydroxykynurenine, quinolinic acid, and kynurenic acid (KYNA) were concurrently provided. Linear mixed models were used for statistical analysis. RESULTS: The manic group showed deficits in all assessed cognitive domains with the exception of verbal memory at all test moments. The bipolar depression group showed deficits in the processing speed at all test moments. Throughout the whole follow-up period, KYNA was significantly lower in both patient groups than in controls. Only in the bipolar depression group, low KYNA was associated with worse global cognitive functioning (B = 0.114, p = 0.02) and slower processing speed in particular (B = 0.139, p = 0.03). CONCLUSION: Only in the bipolar depression group, lower KYNA was associated with worse cognitive functioning. Future large-scale longitudinal studies are warranted to confirm the role of KYN metabolites in cognitive impairment in patients with BD and the possible therapeutic implications of this relationship.

Comprehensive suspect screening for the identification of contaminants of emerging concern in urine of Flemish adolescents by liquid chromatography high-resolution mass spectrometry.

The increasing human exposure to contaminants of emerging concern (CECs) cannot be fully assessed by targeted biomonitoring methods alone as these are limited to a subset of known analytes. On the contrary, suspect screening approaches based on liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) allow the simultaneous detection of a high number of CECs and/or their (predicted) metabolites leading to a more comprehensive assessment of possible human exposure to these compounds. Within this study, 83 urine samples of Flemish adolescents (47 males, 36 females) collected in the frame of the 4th cycle of the Flemish Environment and Health Study (FLEHS IV) were selected with the aim of including a high and a low exposure group based on the overall exposure of 45 known contaminants. Samples were analyzed using a previously developed method involving a suspect screening approach to annotate CECs and their metabolites. The applied suspect list contained a total of >12,500 CECs and their known and predicted metabolites resulting from metabolization reactions, such as hydroxylation, glucuronidation and methylation. In total, 63 compounds were annotated at a confidence level of 3 or better, with most of the detected compounds not included in current biomonitoring programs. 5 out of the 63 compounds could be assigned with confidence level 2. Five compounds could unequivocally be identified (confidence level 1) through the comparison with reference standards. Personal care products were the main detected compound class (42% of detected compounds). Additionally, a detailed literature search indicated potential toxic effects for several of the detected CECs. Lastly, in the urine samples, a significantly higher number (p < 0.05) of compounds was detected in the high exposure group as opposed to the low exposure group. This difference could only be observed between high and low exposure load samples of female participants (p < 0.01).

Lipidomics profiling of zebrafish liver through untargeted liquid chromatography-high resolution mass spectrometry.

Lipidomics analysis of zebrafish tissues has shown promising results to understand disease-related outcomes of exposure to toxic substances at a molecular level. However, knowledge about their lipidome is limited, as most untargeted studies only identify the lipids that are statistically significant in their setup. In this work, liquid chromatography-high resolution mass spectrometry was used to study different aspects of the analytical workflow, that is, extraction solvents (methanol/chloroform/water (3/2/2, v/v/v), methanol/dichloromethane/water (2/3/2, v/v/v) and methanol/methyl-tert-butyl ether/water (3/10/2.5, v/v/v), instrumental response, and strategies used for lipid annotation. The number of high-quality features (relative standard deviation of the intensity values ≤ 10% in the range 103 -107 counts) was affected by the dilution of lipid extracts, indicating that it is an important parameter for developing untargeted methods. The workflows used allowed the selection of a dilution factor to annotate 712 lipid species (507 bulk lipids) in zebrafish liver using four software (LipidMatch, LipidHunter, MS-DIAL, and Lipostar). Retention time mapping was a valuable tool to filter lipid annotations obtained from automatic software annotations. The lipid profiling of zebrafish livers will help in a better understanding of the true constitution of their lipidome at the species level, as well as in the use of zebrafish in toxicological studies.

Incorporating 'reason for use' into the prescribing process of medication: a survey on the opinion of patients in Flanders, Belgium.

BACKGROUND: A longstanding debate exists about including a 'reason for use' on prescriptions for medication. Little is known, however, about patients' opinions on this subject. METHODS: An internet-based questionnaire, consisting mainly of Likert scale questions, was distributed online to the general public in Belgium. Results from 1034 responses were analyzed using descriptive statistics. RESULTS: Opinions from patients toward including a 'reason for use' on medication prescriptions were generally positive. A clear majority of 62% increased to 74% after providing information about the possible link between indication and medication dose. A majority of the participants expressed a positive attitude regardless of the pathology involved, although sexually transmitted diseases were of greatest concern. Other important aspects differentiating the opinion positively was the transmission of this information in an electronic-only form and limiting it to the regular pharmacist excluding further use by third parties such as other pharmacies or insurance companies. Patients using multiple medicines and those frequenting the same pharmacy also had a more favorable opinion about including the reason for use. In addition, analysis of physician and pharmacist questionnaire responses, explicitly excluded from the main analysis, confirmed the known contrasting opinions in these subgroups. CONCLUSIONS: Patients have strong support for transferring information on the 'reason for use' of their prescriptions to their regular pharmacy if this is done in a secure and privacy-conscious way enabling increased patient safety and improved pharmaceutical care.

Ion Mobility-High-Resolution Mass Spectrometry (IM-HRMS) for the Analysis of Contaminants of Emerging Concern (CECs): Database Compilation and Application to Urine Samples.

Ion mobility mass spectrometry (IM-MS)-derived collision cross section (CCS) values can serve as a valuable additional identification parameter within the analysis of compounds of emerging concern (CEC) in human matrices. This study introduces the first comprehensive database of DTCCSN2 values of 148 CECs and their metabolites including bisphenols, alternative plasticizers (AP), organophosphate flame retardants (OP), perfluoroalkyl chemicals (PFAS), and others. A total of 311 ions were included in the database, whereby the DTCCSN2 values for 113 compounds are reported for the first time. For 105 compounds, more than one ion is reported. Moreover, the DTCCSN2 values of several isomeric CECs and their metabolites are reported to allow a distinction between isomers. Comprehensive quality assurance guidelines were implemented in the workflow of acquiring DTCCSN2 values to ensure reproducible experimental conditions. The reliability and reproducibility of the complied database were investigated by analyzing pooled human urine spiked with 30 AP and OP metabolites at two concentration levels. For all investigated metabolites, the DTCCSN2 values measured in urine showed a percent error of <1% in comparison to database values. DTCCSN2 values of OP metabolites showed an average percent error of 0.12% (50 ng/mL in urine) and 0.15% (20 ng/mL in urine). For AP metabolites, these values were 0.10 and 0.09%, respectively. These results show that the provided database can be of great value for enhanced identification of CECs in environmental and human matrices, which can advance future suspect screening studies on CECs.

Electrochemistry of Intact Versus Degraded Cephalosporin Antibiotics Facilitated by LC-MS Analysis.

The electrochemical detection of cephalosporins is a promising approach for the monitoring of cephalosporin levels in process waters. However, this class of antibiotics, like penicillins, is composed of chemically active molecules and susceptible to hydrolysis and aminolysis of the four membered ß-lactam ring present. In order to develop a smart monitoring strategy for cephalosporins, the influence of degradation (hydrolysis and aminolysis) on the electrochemical fingerprint has to be taken into account. Therefore, an investigation was carried out to understand the changes of the voltammetric fingerprints upon acidic and alkaline degradation. Changes in fingerprints were correlated to the degradation pathways through the combination of square wave voltammetry and liquid chromatography quadrupole time-of-flight analysis. The characteristic electrochemical signals of the ß-lactam ring disappeared upon hydrolysis. Additional oxidation signals that appeared after degradation were elucidated and linked to different degradation products, and therefore, enrich the voltammetric fingerprints with information of the state of the cephalosporins. The applicability of the electrochemical monitoring system was explored by the analysis of the intact and degraded industrial process waters containing the key intermediate 7-aminodeacetoxycephalosporanic acid (7-ADCA). Clearly, the intact process samples exhibited the expected core signals of 7-ADCA and could be quantified, while the degraded samples only showed the newly formed degradation products.

Diagnostic Accuracy of Biomarkers of Alcohol Use in Patients With Liver Disease: A Systematic Review.

BACKGROUND AND AIMS: Alcohol-related liver disease is the most frequent cause of cirrhosis and a major indication for liver transplantation. Several alcohol use biomarkers have been developed in recent years and are already in use in several centers. However, in patients with liver disease their diagnostic performance might be influenced by altered biomarker formation by hepatic damage, altered excretion by kidney dysfunction and diuretics use, and altered deposition in hair and nails. We systematically reviewed studies on the diagnostic accuracy of biomarkers of alcohol use in patients with liver disease and performed a detailed study quality assessment. METHODS: A structured search in PubMed/Medline/Embase databases was performed for relevant studies, published until April 28, 2019. The risk of bias and applicability concerns was assessed according to the adapted quality assessment of diagnostic accuracy studies-2 (QUADAS-2) checklist. RESULTS: Twelve out of 6,449 studies met inclusion criteria. Urinary ethyl glucuronide and urinary ethyl sulfate showed high sensitivity (70 to 89 and 73 to 82%, respectively) and specificity (93 to 99 and 86 to 89%, respectively) for assessing any amount of alcohol use in the past days. Serum carbohydrate-deficient transferrin showed low sensitivity but higher specificity (40 to 79 and 57 to 99%, respectively) to detect excessive alcohol use in the past weeks. Whole blood phosphatidylethanol showed high sensitivity and specificity (73 to 100 and 90 to 96%, respectively) to detect any amount of alcohol use in the previous weeks. Scalp hair ethyl glucuronide showed high sensitivity (85 to 100%) and specificity (97 to 100%) for detecting chronic excessive alcohol use in the past 3 to 6 months. Main limitations of the current evidence are the lack of an absolute gold standard to assess alcohol use, heterogeneous study populations, and the paucity of studies. CONCLUSIONS: Urinary and scalp hair ethyl glucuronide are currently the most validated alcohol use biomarkers in patients with liver disease with good diagnostic accuracies. Phosphatidylethanol is a highly promising alcohol use biomarker, but so far less validated in liver patients. Alcohol use biomarkers can complement each other regarding diagnostic time window. More validation studies on alcohol use biomarkers in patients with liver disease are needed.

Suicide by vaping the synthetic cannabinoid 4F-MDMB-BINACA: cannabinoid receptors and fluoride at the crossroads of toxicity?

A 22-year-old man was hospitalized after stating he would 'commit suicide in a non-detectable way'. He was admitted with a severe necrotizing pancreatitis and acute kidney injury, evolving to multiple organ failure. His condition rapidly deteriorated, and he died 11 days after hospital admission. Postmortem histopathology confirmed fulminant necrotizing pancreatitis, acute tubular necrosis, cerebral edema, pericentral/midzonal hepatocellular necrosis and acute respiratory distress syndrome. Metabolites of 4F-MDMB-BINACA, a synthetic cannabinoid, were detected in urine and serum collected at hospital admission. The same drug was found in a vapor fluid found in the man's apartment. As cannabis use has been etiologically linked to acute pancreatitis, we hypothesize that the more afferent and potent 4F-MDMB-BINACA could induce acute pancreatitis via stimulation of cannabinoid (CB)1-receptors. Alternatively, terminal fluorination could have induced a dose-dependent toxic effect on a wide range of cellular processes, leading to cell dysfunction and death. This is the first clinicopathological description of a lethal intoxication with 4F-MDMB-BINACA, following extensive vaping. Toxic effects could either relate to CB-receptor binding or to direct fluoride toxicity.

Identifying Electrochemical Fingerprints of Ketamine with Voltammetry and Liquid Chromatography-Mass Spectrometry for Its Detection in Seized Samples.

Herein, a straightforward electrochemical approach for the determination of ketamine in street samples and seizures is presented by employing screen-printed electrodes (SPE). Square wave voltammetry (SWV) is used to study the electrochemical behavior of the illicit drug, thus profiling the different oxidation states of the substance at different pHs. Besides, the oxidation pathway of ketamine on SPE is investigated for the first time with liquid chromatography-high-resolution mass spectrometry. Under the optimized conditions, the calibration curve of ketamine at buffer solution (pH 12) exhibits a sensitivity of 8.2 µA µM-1, a linear relationship between 50 and 2500 µM with excellent reproducibility (RSD = 2.2%, at 500 µM, n = 7), and a limit of detection (LOD) of 11.7 µM. Subsequently, binary mixtures of ketamine with adulterants and illicit drugs are analyzed with SWV to investigate the electrochemical fingerprint. Moreover, the profile overlapping between different substances is addressed by the introduction of an electrode pretreatment and the integration of a tailor-made script for data treatment. Finally, the approach is tested on street samples from forensic seizures. Overall, this system allows for the on-site identification of ketamine by law enforcement agents in an easy-to-use and rapid manner on cargos and seizures, thereby disrupting the distribution channel and avoiding the illicit drug reaching the end-user.

A mood state-specific interaction between kynurenine metabolism and inflammation is present in bipolar disorder.

OBJECTIVES: Cytokines are thought to contribute to the pathogenesis of psychiatric symptoms by kynurenine pathway activation. Kynurenine metabolites affect neurotransmission and can cause neurotoxicity. We measured inflammatory markers in patients with bipolar disorder (BD) and studied their relation to kynurenine metabolites and mood. METHODS: Patients with BD suffering from an acute mood episode were assigned to the depressive (n = 35) or (hypo)manic (n = 32) subgroup. Plasma levels of inflammatory markers [cytokines, C-reactive protein] and kynurenine metabolites [tryptophan (TRP), kynurenine (KYN), 3-hydroxykynurenine (3-HK), quinolinic acid (QA), kynurenic acid (KYNA)] were measured on 6 time points during 8 months follow-up. Biological marker levels in patients were compared to controls (n = 35) and correlated to scores on mood scales. Spearman correlations and linear mixed models were used for statistical analysis. RESULTS: Twenty patients of the manic subgroup, 29 of the depressive subgroup, and 30 controls completed the study. The manic subgroup had a rapid remission of mood symptoms, but in the depressive subgroup subsyndromal symptoms persisted. No differences in inflammation were found between groups. A strong correlation between tumor necrosis factor-α and KYN, KYN/TRP, 3-HK and QA (ρ > 0.60) was specific for the manic group, but only at baseline (during mania). The depressive subgroup had a lower neuroprotective ratio (KYNA/3-HK, P = .0004) and a strong association between interferon-y and kynurenine pathway activation (P < .0001). KYNA was low in both patient groups versus controls throughout the whole follow-up (P = .0008). CONCLUSIONS: Mania and chronic depressive symptoms in BD are accompanied by a strong interaction between inflammation and a potentially neurotoxic kynurenine metabolism.

Cephalosporin Antibiotics: Electrochemical Fingerprints and Core Structure Reactions Investigated by LC-MS/MS.

Electrochemistry and exploiting electrochemical fingerprints is a potent approach to address newly emerging surveillance needs, for instance, for antibiotics. However, a comprehensive insight into the electrochemical oxidation behavior and mechanism is required for this sensing strategy. To address the lack of knowledge of the voltammetric behavior of the cephalosporin antibiotics, a selection of cephalosporin antibiotics and two main intermediates were subjected to an electrochemical study of their redox behavior by means of pulsed voltammetric techniques and small-scale electrolysis combined with HPLC-MS/MS analyses. Surprisingly, the detected oxidation products did not fit the earlier suggested oxidation of the sulfur group to the corresponding sulfoxide. The influence of different side chains, both at the three and seven position of the ß-lactam core structure on the electrochemical fingerprint, were investigated. Additional oxidation signals at lower potentials were elucidated and linked to different side chains. These signals were further exploited to allow simultaneous detection of different cephalosporins in one voltammetric sweep. These fundamental insights can become the building blocks for a new on-site screening method.

Unraveling the Mechanisms Behind the Complete Suppression of Cocaine Electrochemical Signals by Chlorpromazine, Promethazine, Procaine, and Dextromethorphan.

The present work investigates the challenges accompanied by the electrochemical cocaine detection in physiological conditions (pH 7) in the presence of chlorpromazine, promethazine, procaine, and dextromethorphan, frequently used cutting agents in cocaine street samples. The problem translates into the absence of the cocaine oxidation signal (signal suppression) when in a mixture with one of these compounds, leading to false negative results. Although a solution to this problem was provided through earlier experiments of our group, the mechanisms behind the suppression are now fundamentally investigated via electrochemical and liquid chromatography quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) strategies. The latter was used to confirm the passivation of the electrodes due to their interaction with promethazine and chlorpromazine. Electron transfer mechanisms were further identified via linear sweep voltammetry. Next, adsorption experiments were performed on the graphite screen printed electrodes both with and without potential assistance in order to confirm if the suppression of the cocaine signals is due to passivation induced by the cutting agents or their oxidized products. The proposed strategies allowed us to identify the mechanisms of cocaine suppression for each cutting agent mentioned. Suppression due to procaine and dextromethorphan is caused by fouling of the electrode surface by their oxidized forms, while for chlorpromazine and promethazine the suppression of the cocaine signal is related to the strong adsorption of these (nonoxidized) cutting agents onto the graphite electrode surface. These findings provide fundamental insights in possible suppression and other interfering mechanisms using electrochemistry in general not only in the drug detection sector.

Mass spectrometric strategies for the investigation of biomarkers of illicit drug use in wastewater.

The analysis of illicit drugs in urban wastewater is the basis of wastewater-based epidemiology (WBE), and has received much scientific attention because the concentrations measured can be used as a new non-intrusive tool to provide evidence-based and real-time estimates of community-wide drug consumption. Moreover, WBE allows monitoring patterns and spatial and temporal trends of drug use. Although information and expertise from other disciplines is required to refine and effectively apply WBE, analytical chemistry is the fundamental driver in this field. The use of advanced analytical techniques, commonly based on combined chromatography-mass spectrometry, is mandatory because the very low analyte concentration and the complexity of samples (raw wastewater) make quantification and identification/confirmation of illicit drug biomarkers (IDBs) troublesome. We review the most-recent literature available (mostly from the last 5 years) on the determination of IDBs in wastewater with particular emphasis on the different analytical strategies applied. The predominance of liquid chromatography coupled to tandem mass spectrometry to quantify target IDBs and the essence to produce reliable and comparable results is illustrated. Accordingly, the importance to perform inter-laboratory exercises and the need to analyze appropriate quality controls in each sample sequence is highlighted. Other crucial steps in WBE, such as sample collection and sample pre-treatment, are briefly and carefully discussed. The article further focuses on the potential of high-resolution mass spectrometry. Different approaches for target and non-target analysis are discussed, and the interest to perform experiments under laboratory-controlled conditions, as a complementary tool to investigate related compounds (e.g., minor metabolites and/or transformation products in wastewater) is treated. The article ends up with the trends and future perspectives in this field from the authors' point of view. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 37:258-280, 2018.

Suspect and Nontargeted Strategies to Investigate in Vitro Human Biotransformation Products of Emerging Environmental Contaminants: The Benzotriazoles.

Benzotriazole derivatives (BTRs) are high production volume chemicals involved in a wide range of applications and consumer products resulting in their ubiquitous presence in environmental matrices. Yet, the human exposure assessment to these chemicals is limited since it is based only on the analysis of parent compounds in biological matrices. The objective of this study was to investigate the in vitro human biotransformation for three widely used BTRs and to stepwise examine the role of Phase I and II enzymes (cytochrome P450 (CYP), uridine glucuronic acid transferase (UGT), and sulfotransferase (SULT)) in their biotransformation. Extracts with generated biotransformation products (bioTPs) were analyzed using liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS), followed by their identification based on a workflow combining suspect and nontargeted strategies. Ten bioTPs were identified for 1H-benzotriazole, 14 for tolyltriazole, and 14 for 5-chloro-1H-benzotriazole. Most of the proposed bioTPs were identified and structurally elucidated for the first time. Based on these findings, possible bioTPs and metabolic transformation pathways were subsequently predicted for other structurally close BTR derivatives. Our findings provide new identified in vitro biotransformation products for future biomonitoring studies and emphasize that it is important to investigate the biotransformation pathway to assess overall exposure to xenobiotics.