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Background: Exhaled breath (EB) aerosol was in principle shown to be a suitable matrix for bioanalysis of volatile but also non-volatile compounds. This attracted particular interest in the field of drug analysis. However, a big gap still exists in the understanding how and which drugs and/or their metabolites are excreted into exhaled breath and could thus actually be detected. The current study aimed to develop an analytical workflow for the qualitative detection of non-volatile drugs in EB aerosol microparticles., Results: The analyte selection covered different drug classes such as antihypertensives, anticonvulsants or opioid analgesics to investigate and understand the excretion of drugs and their metabolites into EB aerosol. A device for collecting aerosol particles from the lung through impaction was used for the non-invasive sampling procedure. Three expiration cycles per participant and device were collected. The sample preparation consisted of a collector extraction with methanol. Qualitative method development and validation were performed using reversed-phase liquid chromatography (LC) coupled to orbitrap-based high-resolution mass spectrometry (HRMS). Qualitative method validation was done according to published recommendations and international guidelines. Parameters such as selectivity, carry-over, limits of detection and identification, recovery, matrix effects, and long-term stability were evaluated. The limits of detection ranged from 100 pg/collector to 10,000 pg/collector. The procedure was finally used to analyze a total of 31 patient EB samples and demonstrated that e.g., tilidine and its metabolite nortilidine as well as tramadol and its active metabolite O-desmethyltramadol can be detected in EB aerosol., Significance and Novelty: The work shows a comprehensive workflow for elucidating drug excretion into exhaled breath aerosol. This bioanalytical strategy and the corresponding novel data from this study are the foundation for further method development and to better understand, which drugs and their metabolites can be addressed by exhaled breath aerosol bioanalysis., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Felix Mahfoud reports financial support was provided by German Research Foundation. Felix Mahfoud reports financial support was provided by German Cardiac Society. Felix Mahfoud reports financial support was provided by German Heart Foundation. Felix Mahfoud reports a relationship with Ablative Solutions, Medtronic, ReCor Medical, Amgen, Astra-Zeneca, Bayer, Boehringer Ingelheim, Inari, Merck, Servier, and Terumo that includes: consulting or advisory and speaking and lecture fees. If there are other authors, they 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 © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Unsuitable sample preparation may result in loss of important analytes and consequently affect the outcome of untargeted metabolomics. Due to species differences, different sample preparations may be required within the same biological matrix. The study aimed to compare the in-house sample preparation method for urine with methods from literature and to investigate the transferability of sample preparation from human urine to rat urine. A total of 12 different conditions for protein precipitation were tested, combining four different extraction solvents and three different reconstitution solvents using an untargeted liquid-chromatography high resolution mass spectrometry (LC-HRMS) metabolomics analysis. Evaluation was done based on the impact on feature count, their detectability, as well as the reproducibility of selected compounds. Results showed that a combination of methanol as extraction and acetonitrile/water (75/25) as reconstitution solvent provided improved results at least regarding the total feature count. Additionally, it was found that a higher amount of methanol was most suitable for extraction of rat urine among the tested conditions. In comparison, human urine requires significantly less volume of extraction solvent. Overall, it is recommended to systematically optimize both, the extraction method, and the reconstitution solvent for the used biofluid and the individual analytical settings., 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 © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

New psychoactive substances (NPS) are an issue of global concern posing a serious threat to the healthcare systems. Consumption of some NPS has been associated with toxic effects on the liver amongst others. However, data concerning their (cyto-)toxicity are usually not available. For a straightforward assessment of their cytotoxic potential, a simplified strategy measuring six different cytotoxicity indicating parameters simultaneously by a high content screening assay (HCSA) was developed. Its applicability was further investigated using nine NPS from heterogeneous chemical classes. HepG2 cells were incubated with NPS for 48 h at a low and high concentration (7.81 and 125 µM), respectively. To study metabolism-mediated effects on their cytotoxicity, cells were additionally incubated with the unspecific cytochrome (CYP) P450 inhibitor 1-aminobenzotriazole. Four fluorescence dyes were used to monitor cell count, nuclear size, and nuclear intensity (all Hoechst33342), mitochondrial membrane potential (TMRM), cytoplasmic calcium levels (CAL-520), and plasma membrane integrity (TOTO-3). Amongst the investigated NPS, ephylone, CUMYL-CBMICA, and dibutylone showed a strong cytotoxic potential, affecting two parameters at 7.81 µM. 5-MeO-MiPT showed moderate effects by impairing one parameter at 7.81 and one at 125 µM. Furthermore, at the high concentration of 5-MeO-MiPT, an effect of metabolism on cytotoxicity was observed. The HCSA confirmed the cytotoxic potential of ephylone and 5-MeO-MiPT, as the investigated concentrations were in the range of their published blood concentrations which induced liver damages after intake. The mitochondrial membrane potential was the parameter with the highest sensitivity and thus considered as suitable "cytobiomarker". In turn, parameters showing a high variability or unexpected effects such as cytosolic calcium levels and plasma membrane integrity might be omitted in the future. Even though 5-MeO-MiPT showed metabolism-based effects, HepG2 are known to have limited metabolic activity compared to cell lines such as HepaRG. Therefore, in further experiments cell lines with higher CYP-expression needs to be included and findings compared. Nevertheless, the simplified HCSA-based strategy allowed to screen NPS from diverse chemical groups for a first assessment of the cytotoxic properties of the parent compound. This information is crucial for a thorough risk assessment of NPS not only for public health authorities., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Volumetric absorptive microsampling (VAMS), an emerging microsampling technique, is expected to overcome some disadvantages of dried blood spots. This study aimed to develop and evaluate a VAMS-based strategy for quantifying ten frequently prescribed antihypertensive drugs (AHD) (amlodipine, bisoprolol, candesartan, carvedilol, lercanidipine, losartan carboxylic acid, metoprolol, nebivolol, telmisartan, valsartan) in finger prick blood (FPB) within the scope of adherence monitoring. The straightforward workflow consisted of VAMS tip hydration and subsequent precipitation. Samples were analyzed by using reversed phase ultra-high performance liquid chromatography coupled to orbitrap mass spectrometry operating in parallel reaction monitoring mode. The analytical procedure was successfully validated based on international recommendations for most of the analytes. Selectivity and within/between-run accuracy and precision were in accordance with the recommendations. Internal standard normalized matrix factor met recommended criteria for all analytes at HT 20%, 40%, and 60% except for amlodipine were the CV exceeded 15% at HT 20% (CV 18%). Dilution integrity was given for all substances, covering the quantification in the upper part of the therapeutic range of selected AHD. Long-term stability in VAMS tips was tested and revealed degradation of lercanidipine after one week of storage at 24 °C. A proof of concept of the analytical applicability was done by quantification of selected AHD in VAMS tips and matched plasma samples. Results revealed that determined concentration in FPB by VAMS and plasma cannot be used interchangeably, and thus that specific reference ranges have to be established. However, a novel VAMS application was implemented in the context of adherence monitoring for at least the investigated AHD., 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 © 2021 Elsevier B.V. All rights reserved.)

Background: There is an unmet need for an accessible, less invasive, cost-effective method to facilitate clinical trial enrollment and aid in clinical Alzheimer's disease (AD) diagnosis. APOE genotype affects the clearance and deposition of amyloid-beta (Aβ) with APOE4 carriers having increased risk while APOE2 alleles appear to be protective. Lower plasma Aβ42/40 correlates with brain amyloidosis. In response, C 2 N has developed the PrecivityAD™ test; plasma LC-MS/MS assays for Aβ isoform quantitation and qualitative APOE isoform-specific proteotyping., Methods: In accord with CLIA standards, we developed and validated assay performance: precision, accuracy, linearity, limit of detection (LoD), interferences., Results: Within-day precision varied from 1.5-3.0% (Aβ40) and 2.5-8.4% (Aβ42). Total (within-lab) variability was 2.7-7.7% (Aβ40) and 3.1-9.5% (Aβ42). Aβ40 quantitation was linear from 10 to 1780 pg/mL; Aβ42 was linear from 2 to 254 pg/mL. LoD was 11 and 2 pg/mL for Aβ40 and Aβ42, respectively. APOE proteotypes were 100% concordant with genotype, while LoD (fM) was much lower than APOE concentrations observed in plasma (mM)., Conclusions: The PrecivityAD™ assays are precise, accurate, sensitive, and linear over a wide analytical range, free from significant interferences, and suitable for use in the clinical laboratory., (Copyright © 2021 C2N Diagnostics. Published by Elsevier B.V. All rights reserved.)

Background and Aims: Sex differences in cardiovascular prevention have been reported, yet the role of sex with regard to different modifiable risk factors such as low-density lipoprotein cholesterol (LDL-C), systolic blood pressure (BP), and glycated hemoglobin (HbA1c) in primary care settings is unclear. Therefore, we studied sex differences in assessment and measured values of LDL-C, BP, and HbA1c in primary and secondary cardiovascular prevention delivered by general practitioners., Methods: This cross-sectional study was based on electronic medical records of 59,092 primary care patients (51.9% women) aged 40-79 years in Switzerland. Multilevel regression was used to model associations of sex with assessment and measured values of LDL-C, BP, and HbA1c in 2018., Results: In both primary and secondary prevention, women had lower LDL-C assessment rates (age-adjusted odds ratio (aOR) 0.71 [95% confidence interval (CI) 0.67 to 0.75] and 0.70 [CI 0.51 to 0.95]), and higher measured LDL-C values than men (age-adjusted difference 0.30 mmol/L [CI 0.25 to 0.35] and 0.28 mmol/L [CI 0.07 to 0.48]). Compared with men, women in primary prevention displayed lower BP and HbA1c assessment frequencies (aOR 0.77 [CI 0.73 to 0.81] and 0.76 [CI 0.71 to 0.80]) and measured values (age-adjusted difference -2.49 mmHg [CI -2.99 to -1.79] and -0.19% [CI -0.24 to -0.14]), while there was no sex difference in secondary prevention. Age-dependent increases in measured values of LDL-C, BP, and HbA1c were greater in women than men., Conclusions: Control of LDL-C in women in primary care should be improved to reduce sex-based inequalities in prevention of cardiovascular disease., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Despite benefits of physical activity (PA), most Americans are not regularly active, with notable disparities for residents of low-income communities. PA is positively correlated with resource availability and quality, which can be measured and quantified by PA resource assessment tools. However, community members' perceptions are often not considered. This study incorporates community perceptions with systematic environmental observations to identify community PA resource priorities. The PA Space Methodology for Assessment and Prioritization (PASMAP) includes three phases. Phase 1: Promotora-researchers completed 57 PA Resource Assessments (PARAs) in colonias along the Texas-Mexico border assessing quantity and quality of features, amenities, and incivilities. Characteristics were ranked using average PARA scores from all PA spaces. Phase 2: community advisory board (CAB) members (n = 36 from 3 CABs) ranked the importance of each feature, amenity, and incivility respectively; rankings were averaged and ordered. Phase 3: differences between phases 1 and 2 were calculated. Large differences indicated high discordance between systematic observations and perceived importance. Phase 1: highest ranked PARA characteristics were sidewalks, non-street lighting, and noise. Phase 2: CAB members ranked trails/paths, sidewalks, play equipment, bathrooms, drinking fountains, substance abuse evidence, and litter most important. Phase 3: multiple characteristics had high discordance: trails/paths, fenced-in open fields (features), drinking fountains (amenity), and litter (incivility); low quantity/poor quality yet perceived as highly important. Discordant characteristics identified through PASMAP provide evidence-based, community-valued recommendations on PA resource priorities for planning and advocacy. Future work should incorporate perceptions from additional community members and apply PASMAP methods to other environmental assessments., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Being highly potent, New Synthetic Opioids (NSO) have become a public health concern. Little is known though about the metabolism and toxicokinetics (TK) of many of the non fentanyl NSO such as U-47700. Obtaining such data in humans is challenging and so we investigated if pigs were a suitable model species as TK model for U-47700. The metabolic fate of U-47700 was elucidated after intravenous administration to one pig in vivo and results were compared to metabolic patterns formed by different other in vitro systems (human and pig liver microsomes, human liver S9 fraction) and compared to rat and human in vivo data. Furthermore, monooxygenase isozymes responsible for the major metabolic steps were elucidated. In total, 12 phase I and 8 phase II metabolites of U-47700 could be identified. The predominant reactions were N-demethylation, hydroxylation, and combination of them followed by glucuronidation or sulfation. The most predominant monooxygenase catalyzed conversions were N-demethylation, and hydroxylation by CYP3A4 and 2B6, and FMO3 catalyzed N-oxidation. Similar main phase I metabolites were found in vitro as compared to in vivo (pig/human). The metabolic pattern elucidated in the pig was comparable to human in vivo data. Thus, pigs seem to be a suitable animal model for metabolism and further TK of U-47700., 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 © 2020 Elsevier B.V. All rights reserved.)

The analytical proof of a toxic mushroom and/or plant ingestion at an early stage of a suspected intoxication can be crucial for fast therapeutic decision making. Therefore, comprehensive analytical procedures need to be available. This study aimed to develop a strategy for the qualitative analysis of α- and β-amanitin, psilocin, bufotenine, muscarine, muscimol, ibotenic acid, and ricinine in human urine by means of hydrophilic interaction liquid chromatography-high resolution MS/MS (HILIC-HRMS/MS). Urine samples were prepared by hydrophilic-phase liquid-liquid extraction using dichloromethane and subsequent solid-phase extraction and precipitation, performed in parallel. Separation and identification of the biomarkers were achieved by HILIC using acetonitrile and methanol as main eluents and Orbitrap-based mass spectrometry, respectively. The method was validated as recommended for qualitative procedures and tests for selectivity, carryover, and extraction recoveries were included to also estimate the robustness and reproducibility of the sample preparation. Limits of identification were 1 ng/mL for α- and β-amanitin, 5 ng/mL for psilocin, bufotenine, muscarine, and ricinine, and 1500 ng/mL and 2000 ng/mL for ibotenic acid and muscimol, respectively. Using γ-amanitin, l-tryptophan-d5, and psilocin-d10 as internal standards, compensation for variations of matrix effects was shown to be acceptable for most of the toxins. In eight urine samples obtained from intoxicated individuals, α- and β-amanitin, psilocin, psilocin-O-glucuronide, muscimol, ibotenic acid, and muscarine could be identified. Moreover, psilocin-O-glucuronide and bufotenine-O-glucuronide were found to be suitable additional targets. The analytical strategy developed was thus well suited for analyzing several biomarkers of toxic mushrooms and plants in human urine to support therapeutic decision making in a clinical toxicology setting. To our knowledge, the presented method is by far the most comprehensive approach for identification of the included biomarkers in a human matrix., Competing Interests: Declaration of competing interest Authors have no competing interests to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Chronic non-communicable diseases share the pathomechanism of increased reactive oxygen species (ROS) production by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, known as Nox. The recent discovery that expression of Nox1, a Nox isoform that has been implicated in the pathogenesis of cardiovascular and kidney disease and cancer is regulated by the expression and activity of G protein-coupled estrogen receptor (GPER) led to the identification of orally active small-molecule GPER blockers as selective Nox1 downregulators (NDRs). Preclinical studies using NDRs have demonstrated beneficial effects in vascular disease, hypertension, and glomerular renal injury. These findings suggest the therapeutic potential of NDRs, which reduce Nox1 protein levels, not only for cardiovascular disease conditions including arterial hypertension, pulmonary hypertension, heart failure with preserved ejection fraction (HFpEF), and chronic renal disease, but also for other non-communicable diseases, such as cerebrovascular disease and vascular dementia, Alzheimer's disease, autoimmune diseases and cancer, in which elevated Nox1-derived ROS production plays a causal role., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Paper spray mass spectrometry (PSMS) is currently used in different analytical fields, but less effort has been made so far to use PSMS for highly polar compounds. Such analytes usually show poor performance in PSMS due to their high affinity for common paper substrates in addition to high matrix effects. In this study, strategies for hydrophobic modifications of commercially available paper substrates using ten different organosilanes were developed. The modified substrates were generated, characterized, and applied for PSMS analysis of polar toxins. By using the modified paper, PSMS performance of some of the toxins could be considerably increased, especially for orellanine, showing a more than 80-fold signal enhancement when substrates modified with chlorotrimethylsilane were used. For other toxins like ricinine, only small beneficial effects could be shown on PSMS performance when using modified substrates. Statistical equivalence tests showed sufficient ruggedness of the developed procedures also compared to conventional substrates. Thus, further systematic development of paper substrates modified with organosilane derivatives based on the presented study for application in PSMS should be encouraged., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Nonadherence to antihypertensive drugs therapy is known to be a serious issue in hypertension treatment. Liquid chromatography (LC) coupled to mass spectrometry (MS) was shown to allow the assessment of such nonadherence in blood and urine sample. However, their sampling may represent a logistical challenge and are often not favored by the patients. We questioned whether oral fluid (OF) might be an easier accessible alternative matrix for adherence monitoring of cardiovascular drugs (CD). A qualitative method for adherence monitoring of 78 commonly prescribed cardiovascular drugs in OF using LC high-resolution MS (LC-HRMS/MS) was therefore developed, validated, and used to study the presence of antihypertensive medication in OF. Selectivity, ion suppression and enhancement due coeluting analytes, carry over, limits of detection (LOD), limits of identification (LOI), recovery (RE), matrix effects (ME), and process efficiency (PE) were investigated. For demonstrating applicability, over 50 OF samples were investigated and data were compared to findings in blood and urine. Selectivity in OF was given for all compounds via their MS 2 spectra and no total suppression of signals could be observed. Determined LOI in OF for ten analytes was higher than the given therapeutic plasma concentration. Furthermore, RE, ME, and PE were in acceptable ranges for more than 65% of the compounds. In total, 208 prescriptions of CD to 57 patients were analyzed and demonstrated the suitability of for adherence monitoring in principle. OF was comparable to plasma regarding the drug categories and the frequencies of hits, except for acidic compounds but more hits could be found in urine samples. A analytical method using OF as analytical matrix was successfully developed. Application showed that it might be a suitable alternative for adherence monitoring of selected drugs in the future, particularly those having no acidic function., (Copyright © 2019 Elsevier B.V. All rights reserved.)

New psychoactive substances (NPS) are an emerging topic amongst abused compounds. New varieties appear constantly on the market, without any knowledge about their toxicodynamic and/or -kinetic properties and knowledge of their metabolism is crucial for the development of analytical methods employed for their detection. Controlled human studies would of course be best suited but due to ethical reasons and lack of preclinical safety data, they are usually not available. Often, in vitro models are used to evaluate similarities to human in vivo hepatic phase I and II metabolism and systems explored include primary human hepatocytes, pooled human S9 fraction, and HepaRG, a human hepatic cell line. All these in vitro models have considerable limitations and drug distribution, reabsorption, enterohepatic circulation, and renal elimination cannot be studied. In the recent years, zebrafish (Danio rerio) larvae (embryos) were discussed as a potential in vivo model to overcome these limitations. To date, no studies demonstrating its suitability for studying NPS metabolism in the context of analytical toxicology are available. The aim of this study was to elucidate whether zebrafish larvae can serve as a surrogate for human hepatic metabolism of NPS to develop toxicological screening procedures. Here, we used methyl 2-(1-(5-fluoropentyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamido)-3,3-dimethylbutanoate (7'N-5F-ADB), a new synthetic cannabinoid, whose human metabolism was recently described in the literature, as a model compound to evaluate zebrafish larvae as a new tool for metabolism studies. Different conditions for zebrafish larvae and HepaRG protocols were tested. As zebrafish larvae and HepaRG cell incubations provided the highest number of metabolites and the most authentic spectrum of human metabolites. The most suitable larvae protocol was the incubation via medium and the analysis of the extracted zebrafish larvae. The zebrafish larvae model might be a promising preclinical surrogate for human hepatic metabolism of NPS., (Copyright © 2019 Elsevier B.V. All rights reserved.)

New psychoactive substances (NPS) are still an emerging issue in clinical and forensic toxicology. Information about their cytotoxic potential is limited or even unavailable before distribution and thus their intake can be of high risk for consumers. The aim of the presented study was to develop a strategy to identify cytotoxic potential of NPS based on a high content screening assay (HCSA) using HepG2 cell line and four fluorescent dyes, namely Hoechst33342, TMRM, CAL-520, and TOTO-3. The HCSA was optimized to work without an automated analyzer by using the model compounds fluvastatin, paracetamol, propranolol, and simvastatin. The following parameters were monitored: stained nuclei as a measure for cell count as well as nuclear size and nuclear intensity (all Hoechst33342), mitochondrial membrane potential (TMRM), cytosolic calcium level (CAL-520), and plasma membrane integrity (TOTO-3). The present study showed strong cytotoxic potential for the NPS 5F-PB-22 and MDAI, moderate effects for MDMA, MDPV, methylone, cathinone, 4-MEC, and mephedrone, and no toxic effects for methamphetamine. To assess the metabolic suitability of HepG2 cells under the chosen conditions, cell culture supernatants were analyzed by liquid chromatography-high resolution-tandem mass spectrometry. Metabolites were merely detected for lipophilic drugs such as 5F-PB-22 and MDPV and in addition with a much lower abundance in comparison to the parent compound but the study only allowed a qualitative look for metabolites and the used liver cell line might not ideal when considering metabolism., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Among the emerging new psychoactive substances (NPS), compounds carrying an N-ortho-methoxybenzyl substituent, the so-called NBOMes, represented a highly potent group of new hallucinogens. Recently, 3,4-dimethoxyamphetamine (3,4-DMA)-NBOMe and 4-methylmethamphetamine (4-MMA)-NBOMe occurred, but no data on their pharmacokinetics were available. According to other NBOMes, they are expected to be extensively metabolized. For detection and identification of their phase I and II metabolites, nano liquid chromatography coupled to high resolution tandem mass spectrometry (nanoLC-HRMS/MS) was used. Rat urine was prepared by simple dilution and incubation mixtures with pooled human liver S9 fraction by precipitation. Furthermore, the results concerning detectability using the new nanoLC approach were compared to those obtained by conventional ultra-high performance LC (UHPLC). In addition, the detectability of the compounds by standard urine screening approaches (SUSAs) routinely used by the authors with UHPLC-HRMS/MS, LC-MS n , and GC-MS was tested. Both NBOMes were extensively metabolized mainly by O-demethylation and conjugation with glucuronic acid (3,4-DMA-NBOMe) or oxidation of the tolyl group to the corresponding carboxylic acid (4-MMA-NBOMe). The developed nanoLC-HRMS/MS approach was successfully applied for identification of 38 3,4-DMA-NBOMe metabolites and 33 4-MMA-NBOMe metabolites confirming its detection power. Furthermore, the solvent saving nanoLC system showed comparable results to the UHPLC-HRMS/MS approach. In addition, an intake of an estimated low common user's dose of the compounds was detectable by all SUSAs only via their metabolites. Suggested targets for urine screening procedures were O-demethyl- and O,O-bis-demethyl-3,4-DMA-NBOMe and their glucuronides and carboxy-4-MMA-NBOMe and its glucuronide and N-demethyl-carboxy-4-MMA-NBOMe., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A huge number of new chemical derivatives of known drugs of abuse, so-called new psychoactive substances (NPS), are sold and consumed without prior preclinical and clinical testing. For assessing the elimination behaviors, determination of the kinetic constants K m and V max of the cytochrome P450 (CYP) isoforms involved in the hepatic metabolism of NPS could help to predict their contributions to hepatic clearance, drug-drug interactions and polymorphisms. Therefore, the aims of the present study were to determine the K m and V max values for CYP isoforms using the substrate depletion approach for the six N-2-methoxybenzyl (NBOMe)-derived NPS 25B-NBOMe, 25C-NBOMe, 25I-NBOMe, 3,4-DMA-NBOMe, 4-EA-NBOMe, and 4-MMA-NBOMe. Furthermore, the contributions of each CYP isozyme to the hepatic net clearance were elucidated using the relative activity factor approach. Several CYPs including CYP1A2, CYP2B6, CYP2C19, CYP2D6, and CYP3A4 were identified to be involved in the metabolism of the investigated compounds. The determined K m values ranged from 0.010 μM (CYP2D6, 4-MMA-NBOMe) to 13 μM (CYP2B6, 4-EA-NBOMe). All NBOMes were good substrates of CYP2C19 and CYP2D6 resulting in very low K m values in the nanomolar range. The main contributors to hepatic net clearance were CYP2D6 for 25B-NBOMe (69%), 25C-NBOMe (83%), 25I-NBOMe (61%), 3,4-DMA-NBOMe (89%) as well as for 4-EA-NBOMe (62%) and CYP2C19 for 4-MMA-NBOMe (64%). As more than one isoform was involved in the particular steps, the risk of harm associated with drug-drug interactions might be considered low. However, in cases where substances with high contributions from polymorphically expressed CYP2C19 and CYP2D6 are encountered, inter-individual variations in metabolism and excretion cannot be excluded., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The WHO annually reports an increasing abuse of new psychoactive substances (NPS), which are a heterogeneous group of synthetic drugs and are consumed as substitute for controlled drugs of abuse. In this work, we focused on highly potent derivatives such those of phenethylamine (2C), N-2-methoxybenzyl phenethylamine (NBOMes), lysergic acid diethylamide (LSD), and fentanyl. Severe to fatal intoxications were described due to their high potency. Therefore, they have to be taken at very low doses resulting in low blood concentration in the low ng/mL range, which is a challenge for reliable analytical detection and quantification. The aim of this work was therefore to design a simple, robust, and fast method for simultaneous detection and quantification of multiple substances of the different classes in human blood plasma using liquid chromatography (LC) high resolution (HR) mass spectrometry (MS) with alternating HR full-scan (HRFS) MS and "All-ions fragmentation" (AIF) MS. The paper contains results of the method validation according to the EMA guideline, including intra-/interday accuracy and precision, matrix effects, storage and benchtop analyte stability as well as selectivity and carryover. All validation criteria were fulfilled for most tested compounds except for the NBOMe derivatives, one out of ten 2C-derivatives and butyryl fentanyl, which failed at accuracy and/or precision or at the acceptance criteria for matrix effect. Reasons for this are discussed and solutions presented. Despite some limitations, the HRFS + AIFMS analysis allowed detection of most of the analytes down to 0.1ng/mL, seamless integration of new or unexpected analytes, identification and quantification with no limitations on the number of monitored compounds, and reevaluation of the acquired data also concerning metabolism studies using group-indicating fragment ions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Interactions with the human breast cancer resistance protein (hBCRP) significantly influence the pharmacokinetic properties of a drug and can even lead to drug-drug interactions. As efflux pump from the ABC superfamily, hBCRP utilized energy gained by adenosine 5'-triphosphate (ATP) hydrolysis for the transmembrane movement of its substrates, while adenosine 5'-diphosphate (ADP) and inorganic phosphate were released. The ADP liberation can be used to detect interactions with the hBCRP ATPase. An ADP quantification method based on hydrophilic interaction liquid chromatography (HILIC) coupled to high resolution tandem mass spectrometry (HR-MS/MS) was developed and successfully validated in accordance to the criteria of the guideline on bioanalytical method validation by the European Medicines Agency. ATP and adenosine 5'-monophosphate were qualitatively included to prevent interferences. Furthermore, a setup consisting of six sample sets was evolved that allowed detection of hBCRP substrate or inhibitor properties of the test compound. The hBCRP substrate sulfasalazine and the hBCRP inhibitor orthovanadate were used as controls. To prove the applicability of the procedure, the effect of amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir on the hBCRP ATPase activity was tested. Nelfinavir, ritonavir, and saquinavir were identified as hBCRP ATPase inhibitors and none of the five HIV protease inhibitors turned out to be an hBCRP substrate. These findings were in line with a pervious publication., (Copyright © 2017 Elsevier B.V. All rights reserved.)

New psychoactive substances (NPS) are an increasing problem in clinical and forensic toxicology. The knowledge of their metabolism is important for toxicological risk assessment and for developing toxicological urine screenings. Considering the huge numbers of NPS annually appearing on the market, metabolism studies should be realized in a fast, simple, cost efficient, and reliable way. Primary human hepatocytes (PHH) were recommended to be the gold standard for in vitro metabolism studies as they are expected to contain natural enzyme clusters, co-substrates, and drug transporters. In addition, they were already successfully used for metabolism studies of NPS. However, they also have disadvantages such as high costs and limited applicability without special equipment. The aims of the present study were therefore first to investigate exemplarily the phase I and phase II metabolism of six NPS (XLR-11, AB-PINACA, FUB-PB-22, 4-methoxy-α-PVP, 25-I-NBOMe, and meclonazepam) from different drug classes using pooled human S9 fraction (pS9) or pooled human liver microsomes combined with cytosol (pHLM/pHLC) after addition of the co-substrates for the main metabolic phase I and II reactions. Second to compare results to published data generated using primary human hepatocytes and human urine samples. Results of the incubations with pS9 or pHLM/pHLC were comparable in number and abundance of metabolites. Formation of metabolites, particularly after multi-step reactions needed a longer incubation time. However, incubations using human liver preparations resulted in a lower number of total detected metabolites compared to PHH, but they were still able to allow the identification of the main human urinary excretion products. Human liver preparations and particularly the pooled S9 fraction could be shown to be a sufficient and more cost-efficient alternative in context of metabolism studies also for developing toxicological urine screenings. It might be recommended to use the slightly cheaper pS9 fraction instead of a pHLM/pHLC combination. As formation of some metabolites needed a long incubation time, two sampling points at 60 and 360min should be recommended., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Dried matrix spot (DMS) technique as alternative sampling strategy, especially dried urine spots (DUS), might be an alternative for drug screening. So far only particular drugs or drug classes were covered in DMS screenings. Therefore, workup of DUS for a broad comprehensive library-based LC-MS n screening was developed. It consisted of enzymatic on-spot deconjugation followed by liquid extraction and LC-MS n analysis. This workup was compared to established urine precipitation (UP) and validated according to international guidelines for qualitative approaches, using exemplary compounds of several drug classes (antidepressants, benzodiazepines, cardiovascular drugs, neuroleptics, opioids, stimulants, etc.) with a broad range of (physico-)chemical properties and chromatographic behaviors. On-spot conjugate cleavage and liquid extraction was sufficient for most compounds and the validation results comparable to those obtained after simple UP. For demonstrating the applicability, 103 authentic urine samples, six rat urine samples after low dose substance administrations, and two proficiency tests for systematic toxicological urinalysis were worked up with the new DUS approach or by UP without or with conjugate cleavage. In the authentic urine samples, 112 different drugs out of 43 categories plus metabolites were identified via the used LC-MS n library. With the new DUS approach, 5% less positive hits could be found compared to the UP approach and 15% less than the latter after conjugate cleavage. The differences should be caused mainly by smaller urine volumes used for DUS. In the two proficiency tests, all 15 drugs could be detected. Unfortunately, all three approaches were not able to detect very low-dosed substances in rat urine samples. However, they could be detected using a more sensitive LC-high resolution-MS/MS approach showing that the DUS workup was also suitable for those. In conclusion, DUS might be an alternative sampling technique for comprehensive drug testing or adherence monitoring., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Orbitrap technology was successfully applied for broad metabolite-based LC-high resolution (HR)-MS screening of drugs in clinical and forensic toxicology. This paper aims to elucidate whether this technology can also be used for a corresponding blood plasma screening after simple precipitation without or with consecutive on-line extraction using turbulent flow chromatography (TurboFlow). The analytes were separated within 10 min and detected by a Q-Exactive mass spectrometer in full scan mode after positive/negative switching. In one single run, a target screening for about 700 relevant compounds was developed in parallel with data-dependent acquisition for unknowns. A compound was positively identified when the corresponding accurate mass precursor ion and the five most intense fragment ions were detected and the MS/MS spectrum fits well with the corresponding full HR-MS/MS reference library currently containing over 2000 parent drugs and 2500 metabolites. All over all run times were 17 min for precipitation and 21 min for TurboFlow after precipitation. Method validation was successfully performed for representative drugs and metabolites concerning recovery, matrix effect, process efficiency, and limits of detection and identification. Process efficiency data ranged for most analytes from 3 to 138% with coefficients of variation (CV) ≤ 20% for precipitation and from 1 to 156% with CV ≤ 20% for TurboFlow. Applicability studies showed that the developed method provided fast, simple, and robust screening and identification of a broad range of drugs within therapeutic ranges., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Tryptamines have emerged as new psychoactive substances (NPS), which are distributed and consumed recreationally without preclinical studies or safety tests. Within the alpha-methylated tryptamines, some of the psychoactive effects of the prototypical alpha-methyltryptamine (AMT) have been described decades ago and a contributing factor of its acute toxicity appears to involve the inhibition of monoamine oxidase (MAO). However, detailed information about analogs is scarce. Therefore, thirteen AMT analogs were investigated for their potential to inhibit MAO. An in vitro assay analyzed using hydrophilic interaction liquid chromatography-high resolution-tandem mass spectrometry was developed and validated. The AMT analogs were incubated with recombinant human MAO-A or B and kynuramine, a non-selective MAO substrate to determine the IC 50 values. The known MAO-A inhibitors 5-(2-aminopropyl)indole (5-IT), harmine, harmaline, yohimbine, and the MAO-B inhibitor selegiline were tested for comparison. AMT and all analogs showed MAO-A inhibition properties with IC 50 values between 0.049 and 166μM, whereas four analogs inhibited also MAO-B with IC 50 values between 82 and 376μM. 7-Me-AMT provided the lowest IC 50 value against MAO-A comparable to harmine and harmaline and was identified as a competitive MAO-A inhibitor. Furthermore, AMT, 7-Me-AMT, and nine further analogs inhibited MAO activity in human hepatic S9 fraction used as model for the human liver which expresses both isoforms. The obtained results suggested that MAO inhibition induced by alpha-methylated tryptamines might be clinically relevant concerning possible serotonergic and adrenergic effects and interactions with drugs (of abuse) particularly acting as monoamine reuptake inhibitors. However, as in vitro assays have only limited conclusiveness, further studies are needed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Comprehensive urine screening for drugs and metabolites by LC-HR-MS/MS using Orbitrap technology has been described with precipitation as simple workup. In order to fasten, automate, and/or simplify the workup, on-line extraction by turbulent flow chromatography and a dilute-and-shoot approach were developed and compared. After chromatographic separation within 10min, the Q-Exactive mass spectrometer was run in full scan mode with positive/negative switching and subsequent data dependent acquisition mode. The workup approaches were validated concerning selectivity, recovery, matrix effects, process efficiency, and limits of identification and detection for typical drug representatives and metabolites. The total workup time for on-line extraction was 6min, for the dilution approach 3min. For comparison, the established urine precipitation and evaporation lasted 10min. The validation results were acceptable. The limits for on-line extraction were comparable with those described for precipitation, but lower than for dilution. Thanks to the high sensitivity of the LC-HR-MS/MS system, all three workup approaches were sufficient for comprehensive urine screening and allowed fast, reliable, and reproducible detection of cardiovascular drugs, drugs of abuse, and other CNS acting drugs after common doses., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Insufficient physical activity and unhealthy eating behaviors are major contributors to the obesity epidemic in the United States. Identifying health behaviors and disparities in underserved communities is needed to guide the development of targeted interventions. The Community Assessment for Public Health Emergency Response (CASPER) is a set of tools designed for public health emergencies, but the utility of CASPER in non-emergency settings has not been explored. The purpose of this study was to use CASPER to obtain information on household-based behaviors of and barriers to fruit/vegetable consumption and physical activity, and explore the utility of these methods for future health assessments. Cross-sectional survey data included households (n=100) in a low-income neighborhood. Half of adults did not meet recommendations for fruit/vegetable consumption and 20% reported no physical activity during the previous week. Cost was significantly associated with healthy eating and physical activity in our community. Four primary advantages of using CASPER methodology included a user-friendly CDC toolkit, yield of a representative community sample with a relatively low sample size, low-cost/low-tech requirements for implementation, and the strengthening of an academic-practice-community partnership. Our work demonstrates the utility of CASPER for assessing healthy living in a geographically-defined community where household health behaviors and barriers are unknown., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Little is known about the role of flavin-containing monooxygenases (FMOs) in the metabolism of xenobiotics. FMO3 is the isoform in adult human liver with the highest impact on drug metabolism. The aim of the presented study was to elucidate the contribution of human FMO3 to the N-oxygenation of selected therapeutic drugs and drugs of abuse (DOAs). Its contribution to the in vivo hepatic net clearance of the N-oxygenation products was calculated by application of an extended relative activity factor (RAF) approach to differentiate from contribution of cytochrome P450 (CYP) isoforms. FMO3 and CYP substrates were identified using pooled human liver microsomes after heat inactivation and chemical inhibition, or single enzyme incubations. Kinetic parameters were subsequently determined using recombinant human enzymes and mass spectrometric analysis via authentic reference standards or simple peak areas of the products divided by those of the internal standard. FMO3 was identified as enzyme mainly responsible for the formation of N,N-diallyltryptamine N-oxide and methamphetamine hydroxylamine (>80% contribution for both). A contribution of 50 and 30% was calculated for the formation of N,N-dimethyltryptamine N-oxide and methoxypiperamide N-oxide, respectively. However, FMO3 contributed with less than 5% to the formation of 3-bromomethcathinone hydroxylamine, amitriptyline N-oxide, and clozapine N-oxide. There was no significant difference in the contributions when using calibrations with reference metabolite standards or peak area ratio calculations. The successful application of a modified RAF approach including FMO3 proved the importance of FMO3 in the N-oxygenation of DOAs in human metabolism., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Aims: Cardiac aging is associated with progressive structural changes and functional impairment, such as left ventricular hypertrophy, fibrosis and diastolic dysfunction. Aging also increases myocardial activity of endothelin-1 (ET-1), a multifunctional peptide with growth-promoting and pro-fibrotic activity. Because the G protein-coupled estrogen receptor (GPER) regulates vascular responsiveness to ET-1, we investigated whether GPER also plays a role in the regulation of the myocardial endothelin system with aging., Main Methods: Young (4month-old) and aged (24month-old) wild-type and Gper-deficient (Gper(-/-)) mice were studied. Gene expression levels of prepro-ET-1, endothelin converting enzymes ECE-1 and ECE-2, and endothelin ETA and ETB receptors were determined by qPCR in left ventricular myocardium., Key Findings: Aging markedly increased steady-state mRNA expression levels of ECE-1, ECE-2, ETA and ETB receptors (each p<0.001 vs. young mice). Deletion of Gper inhibited the age-dependent increase in ECE-2 and ETB receptor mRNA levels (57% and 40% reduction, respectively, each p<0.01 vs. wild-type mice), whereas gene expression of prepro-ET-1, ECE-1, and the ETA receptor was unaffected in Gper(-/-) mice., Significance: We identified a novel regulatory mechanism through which the endogenous Gper facilitates the age-dependent increase in myocardial expression of ECE-2 and the ETB receptor, which is compatible with an activating role of GPER for the local endothelin system with aging. Targeting GPER signaling by selective antagonists may therefore be considered a new therapeutic approach to reduce age-dependent increased ET-1 activity and the associated development of left ventricular hypertrophy, fibrosis and heart failure., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The field of new psychoactive substances (NPS) is highly dynamic and the situation changes from year to year. Therefore, the current review provides a timely update about the latest developments to help analysts keep the pace with NPS distribution. It covers PubMed-listed studies published between January 2014 and January 2016 dealing with the application of liquid chromatography (LC) coupled low- and high-resolution mass spectrometry (MS) for broad screenings for NPS in clinical (CT) and forensic (FT) toxicology. Latest developments and applications are highlighted and selected papers critically discussed. Comprehensive tables summarizing all discussed articles complete the overview. Finally, an outlook on the future of LC coupled MS in CT and FT is provided and readers will learn why low-resolution mass spectrometry might remain the standard for the next couple of years at least for easy-to-use quantitative screening procedures., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Aging is considered the most important nonmodifiable risk factor for cardiovascular disease and death after age 28 years. Because of demographic changes the world population is expected to increase to 9 billion by the year 2050 and up to 12 billion by 2100, with several-fold increases among those 65 years of age and older. Healthy aging and prevention of aging-related diseases and associated health costs have become part of political agendas of governments around the world. Atherosclerotic vascular burden increases with age; accordingly, patients with progeria (premature aging) syndromes die from myocardial infarctions or stroke as teenagers or young adults. The incidence and prevalence of arterial hypertension also increases with age. Arterial hypertension-like diabetes and chronic renal failure-shares numerous pathologies and underlying mechanisms with the vascular aging process. In this article, we review how arterial hypertension resembles premature vascular aging, including the mechanisms by which arterial hypertension (as well as other risk factors such as diabetes mellitus, dyslipidemia, or chronic renal failure) accelerates the vascular aging process. We will also address the importance of cardiovascular risk factor control-including antihypertensive therapy-as a powerful intervention to interfere with premature vascular aging to reduce the age-associated prevalence of diseases such as myocardial infarction, heart failure, hypertensive nephropathy, and vascular dementia due to cerebrovascular disease. Finally, we will discuss the implementation of endothelial therapy, which aims at active patient participation to improve primary and secondary prevention of cardiovascular disease., (Copyright © 2016 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.)

New psychoactive substances (NPS) are not tested for their cytochrome P450 (CYP) inhibition potential before consumption. Therefore, this potential was explored for tryptamine-derived NPS (TDNPS) including alpha-methyl tryptamines (AMTs), dimethyl tryptamines (DMTs), diallyl tryptamines (DALTs), and diisopropyl tryptamines (DiPTs) using test substrates preferred by the Food and Drug Administration in a cocktail assay. All tested TDNPS with the exception of DMT inhibited CYP2D6 activity with IC50 values below 100μM. DALTs inhibited CYP2D6 activity similar to paroxetine and quinidine and CYP1A2 activity comparable to fluvoxamine. 5-Methoxy-N,N-diallyltryptamine reduced in vivo the caffeine metabolism in rats consistent with in vitro results. Five of the AMTs also inhibited CYP1A2 activity comparable to amiodarone. AMT and 6-F-AMT inhibited CYP2A6 activity in the range of the test inhibitor tranylcypromine. CYP2B6 activity was inhibited by 19 tryptamines, but weakly compared to efavirenz. CYP2C8 activity was inhibited by five of the tested TDNPS and three showed values comparable to trimethoprim and gemfibrozil. Six tryptamines inhibited CYP2C9 and seven CYP2C19 activities comparable to fluconazole and chloramphenicol, respectively. Nineteen compounds showed inhibition of CYP2E1 and 18 of CYP3A activity, respectively. These results showed that the CYP inhibition by TDNPS might be clinically relevant, but clinical studies are needed to explore this further., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Lefetamine was first marketed in the 1940s as an opioid analgesic. Since withdrawal symptoms were observed during treatment, it became a controlled substance. Its N-ethyl and N-iso-propyl derivatives appeared on the illicit drug market in 2008. Metabolism studies for lefetamine and these derivatives showed that N-dealkylation was the initial step for all three substances in rats. The involvement of the ten most important human cytochrome P450 (CYP) isozymes in this N-dealkylation should be studied now. Studies showed the involvement of CYP1A2, CYP2B6, CYP2C19, and CYP3A4 in N-dealkylation of all three compounds and additionally CYP2D6 for lefetamine and NEDPA. All kinetic profiles followed classic Michaelis-Menten kinetics. Using the relative activity factor approach, the following net clearances were calculated: for lefetamine 8% by CYP1A2, 72% by CYP2B6, 2% by CYP2C19, 1% by CYP2D6, and 17% by CYP3A4; for NEDPA 27% by CYP1A2, 30% by CYP2B6, 23% by CYP2C19, 4% by CYP2D6, and 17% by CYP3A4; for NPDPA 18% by CYP1A2, 24% by CYP2B6, 28% by CYP2C19, and 30% by CYP3A4. In addition, calculated data were compared to chemical inhibition studies in human liver microsomes. Due to the involvement of at least four enzymes in the initial metabolic steps, the risk of CYP-related drug-drug or drug-food interactions should be low., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: Wastewater analysis is a new approach developed to estimate drug (of abuse) consumption in large communities, such as cities or even whole countries., Aims: This paper presents data on the loads of amphetamine and methamphetamine measured in ten wastewater treatment plants in different parts of a German federal state. It provides an estimation of the intensity of the consumption and a comparison to other regions in Germany and Europe., Methods: Consumption of amphetamine and methamphetamine was estimated by analysis of drug residues in composite 24h samples of wastewater after mechanical treatment over one week by liquid chromatography-high resolution tandem mass spectrometry. Samples were collected from the inlet of ten wastewater treatment plants (WWTP) in the federal state of Saarland, representing bigger cities (>200,000 inhabitants), medium sized cities (>50,000 inhabitants), small cities (>25,000 inhabitants), and villages (<25,000 inhabitants). In each WWTP, samples were taken daily for seven consecutive days in July 2014., Results: We observed differences of amphetamine versus methamphetamine loads (expressed as mg/day/1000 inhabitants): Amphetamine loads were much higher in all tested WWTPs indicating a low prevalence of methamphetamine abuse in the federal state of Saarland at the tested period. These findings are in line with previous reports about the distribution of amphetamine and methamphetamine in Germany and Europe., Conclusions: The approach confirms that wastewater analysis can provide valuable data about the abuse pattern of drugs of abuse in cities and larger areas. It can be useful for planning interventions aimed at specific areas and substances., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

LC-high resolution (HR)-MS well established in proteomics has become more and more important in bioanalysis of small molecules over the last few years. Its high selectivity and specificity provide best prerequisites for its use in broad screening approaches. Therefore, Orbitrap technology was tested for developing a general metabolite-based LC-HR-MS/MS screening approach for urinalysis of drugs necessary in clinical and forensic toxicology. After simple urine precipitation, the drugs and their metabolites were separated within 10 min and detected by a Q-Exactive mass spectrometer in full scan with positive/negative switching, and subsequent data dependent acquisition (DDA) mode. Identification criteria were the presence of accurate precursor ions, isotopic patterns, five most intense fragment ions, and comparison with full HR-MS/MS library spectra. The current library contains over 1900 parent drugs and 1200 metabolites. The method was validated for typical drug representatives and metabolites concerning recovery, matrix effects, process efficiency, and limits showed acceptable results. The applicability was tested first for cardiovascular drugs, which should be screened for in poisoning cases and for medication adherence of hypertension patients. The novel LC-HR-MS/MS method allowed fast, simple, and robust urine screening, particularly for cardiovascular drugs showing the usefulness of Orbitrap technology for drug testing., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Human esterases such as the human carboxylesterases (hCES) are important for the catalytic ester hydrolysis of xenobiotics and they play an important role in the detoxification of drugs (e.g., cocaine) but also in the activation of prodrugs (e.g., ramipril). Therefore, the aim of the presented study was to characterize the enzyme-catalyzed ester hydrolysis of ten drugs (cocaine, dimethocaine, ethylphenidate, 4-fluoro-3α-tropacocaine, 4-fluoro-3β-tropacocaine, heroin, methylphenidate, mitragynine, ramipril, and thebacon) by different esterase-containing systems (recombinant hCES1b, hCES1c, and hCES2, pooled human liver microsomes, pooled human liver S9 fraction, and pooled human plasma). Michaelis-Menten kinetic studies were done using in vitro incubations with the aforementioned enzyme-containing systems and LC coupled to ion trap MS for analysis. Ramipril and heroin were used as known model substrates to ensure reliable incubation conditions. The hydrolysis reactions followed classic Michaelis-Menten kinetics with exception of cocaine and 4-fluoro-3α-tropacocaine, for which hydrolysis rate was too low for reliable modeling. The substrates were mainly metabolized by the following enzymes systems: cocaine, hCES1c; dimethocaine, human plasma esterases; ethylphenidate, hCES1c; 4-fluoro-3β-tropacocaine, human plasma esterases; heroin, hCES2; methylphenidate, hCES1c; mitragynine, hCES1c; ramipril, hCES1b; thebacon, hCES2. Compounds bearing a small alcohol part and a larger acyl part showed higher affinity to hCES1 while those with a large alcohol part showed higher affinity to hCES2. The collected data are important for prediction of drug-drug or drug-food interactions as well as for individual variations in metabolism of drugs of abuse due to enzyme polymorphisms., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Aims: Aging, a physiological process and main risk factor for cardiovascular and renal diseases, is associated with endothelial cell dysfunction partly resulting from NADPH oxidase-dependent oxidative stress. Because increased formation of endothelium-derived endothelin-1 (ET-1) may contribute to vascular aging, we studied the role of NADPH oxidase function in age-dependent contractions to ET-1., Main Methods: Renal arteries and abdominal aortas from young and old C57BL6 mice (4 and 24 months of age) were prepared for isometric force measurements. Contractions to ET-1 (0.1-100 nmol/L) were determined in the presence and absence of the NADPH oxidase-selective inhibitor gp91ds-tat (3 μmol/L). To exclude age-dependent differential effects of NO bioactivity between vascular beds, all experiments were conducted in the presence of the NO synthase inhibitor L-NAME (300 μmol/L)., Key Findings: In young animals, ET-1-induced contractions were 6-fold stronger in the renal artery than in the aorta (p<0.001); inhibition of NADPH oxidase by gp91ds-tat reduced the responses to ET-1 by 50% and 72% in the renal artery and aorta, respectively (p<0.05). Aging had no effect on NADPH oxidase-dependent and -independent contractions to ET-1 in the renal artery. In contrast, contractions to ET-1 were markedly reduced in the aged aorta (5-fold, p<0.01 vs. young) and no longer sensitive to gp91ds-tat., Significance: The results suggest an age-dependent heterogeneity of NADPH oxidase-mediated vascular contractions to ET-1, demonstrating an inherent resistance to functional changes in the renal artery but not in the aorta with aging. Thus, local activity of NADPH oxidase differentially modulates responses to ET-1 with aging in distinct vascular beds., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: Aging is a major risk factor for carotid artery disease and stroke. Endothelin-1 (ET-1) and angiotensin II (Ang II) are important modifiers of vascular disease, partly through increased activity of NADPH oxidase and vasoconstrictor prostanoids. Since the renin-angiotensin and endothelin systems become activated with age, we hypothesized that aging affects NADPH oxidase- and prostanoid-dependent contractions to ET-1 and Ang II., Main Methods: Carotid artery rings of young (4 month-old) and old (24 month-old) C57BL6 mice were pretreated with the NO synthase inhibitor L-NAME to exclude differential effects of NO. Contractions to ET-1 and Ang II were determined in the presence and absence of the NADPH oxidase-selective inhibitor gp91ds-tat or the thromboxane-prostanoid receptor antagonist SQ 29,548. Gene expression of endothelin and angiotensin receptors was measured by qPCR., Key Findings: Aging reduced ET-1-induced contractions and diminished ETA but increased ETB receptor gene expression levels. Gp91ds-tat inhibited contractions to ET-1 in young and to a greater extent in old animals, whereas SQ 29,548 had no effect. Ang II-induced contractions were weak compared to ET-1 and unaffected by aging, gp91ds-tat, and SQ 29,548. Aging had also no effect on AT1A and AT1B receptor gene expression levels., Significance: Aging in carotid arteries decreases ETA receptor gene expression and responsiveness to ET-1, which nevertheless becomes increasingly dependent upon NAPDH oxidase activity with age; responses to Ang II and gene expression of its receptors are however unaffected. These findings suggest that physiological aging differentially regulates functional responses to G protein-coupled receptor agonists and the signaling pathways associated with their activation., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Drugs of abuse are not tested for cytochrome P450 (CYP) inhibition potential before distribution. Therefore, a cocktail assay should be developed for testing the inhibition potential for all relevant CYPs. The following CYP test substrates and selective inhibitors were incubated in pooled human liver microsomes: phenacetin (alpha-naphthoflavone for CYP1A2), coumarin (tranylcypromine, CYP2A6), bupropion (sertraline, CYP2B6), amodiaquine (trimethoprim, CYP2C8), diclofenac (sulfaphenazole, CYP2C9), omeprazole (fluconazole, CYP2C19), dextromethorphan (quinidine, CYP2D6), chlorzoxazone (clomethiazole, CYP2E1), testosterone (verapamil, CYP3A). Samples were analyzed after protein precipitation using a Thermo Fisher Q-Exactive LC-high-resolution-MS/MS. The IC50 values were calculated by plotting the concentration of the formed metabolite, relative to the control sample, over the logarithm of the inhibitor concentration. They were determined either for single substrate or the cocktail incubation. Unfortunately, the cocktail assay had to be split because of interferences during incubation caused by substrates or metabolites, but the mixture of both incubates could be analyzed in one analytical run. The IC50 values determined in the single substrate or both cocktail incubations were comparable among themselves and with published data. In conclusion, the new inhibition cocktail assay was reproducible and applicable for testing the inhibition potential of drugs of abuse as exemplified for 2,5-dimethoxy-4-iodo-amfetamine (DOI)., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Sewage profiling as a mean to estimate consumption of drugs of abuse is gaining increasing attention. However, only scarce data are available so far on the impact of microbial biotransformation on the presence and hence detectability of drugs of abuse and their metabolites in wastewater (WW) samples. The aim of this work was therefore to study the biotransformation pathways of the novel psychoactive substance 3,4-methylenedioxypyrovalerone (MPDV) in WW by incubating it, based on the OECD guideline 314 A. MDPV was incubated (100 μg/L) for 10d at 22 °C in WW from a local WW treatment plant. Furthermore, urine and feces collected from rats administered 20mg MDPV/kg BW were incubated correspondingly. Samples were worked-up either by centrifugation/filtration and solid-phase (HCX) extraction or QuEChERS. High resolution (HR) mass spectra (MS) were recorded using an Orbitrap mass spectrometer. All products were identified via their HR-MS(2) spectra and chromatographic properties. The observed biotransformations in WW were: demethylenation and subsequent O-methylation, hydroxylation at the phenyl part, hydroxylation at the pyrrolidine part with subsequent methylation or oxidation, N-demethylation, and hydroxylation at the alkyl part as well as combination of them. In total, 12 biotransformation products were identified after 10 days of incubation. Three of these biotransformation products were previously reported to be also rat and human metabolites. No additional MDPV biotransformation products could be found after incubating the rat urine and feces samples. Instead, the urinary phase II glucuronides were nearly completely cleaved after one day of WW incubation. The presented study indicates that demethylenyl-methyl MDPV, the most abundant metabolite in human urine, should be the best indicator in WW to estimate its use., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The 2,5-dimethoxyphenethylamine-derived designer drugs (so-called "2Cs") recently became of great importance on the illicit drug market as stimulating hallucinogens. They are distributed and consumed as "novel psychoactive substances" (NPS) without any safety testing at the forefront. As previous studies have shown, the 2Cs are mainly metabolized by O-demethylation, N-acetylation, or deamination. Therefore, the aim of this study was to elucidate the role of the recombinant human N-acetyltransferase (NAT) isoforms 1 and 2 in the phase II metabolism of 2Cs. For these studies, cDNA-expressed recombinant human NATs were used and formation of metabolites after incubation was measured using GC-MS. NAT2 could be shown to be the only isoform catalyzing the reaction in vitro, hence it should be the only relevant enzyme for in vivo acetylation. In general, all metabolite formation reactions followed classic Michaelis-Menten kinetics and the affinity to human NAT2 was increasing with the volume of the 4-substituent. In consequence, a slow acetylator phenotype or inhibition of NAT2 could lead to decreased N-acetylation and might lead to an increased risk of side effects caused by these novel psychoactive substances., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Methylenedioxy designer drugs of abuse such as 3,4-methylenedioxymethamphetamine (MDMA) can be selectively toxic to serotonergic neurons and glutathione (GSH) adducts have been implicated in its neurotoxicity. The catecholic demethylenyl metabolites of MDMA, 3,4-dihydroxymethamphetamine and 3,4-dihydroxyamphetamine, are metabolically oxidized to the corresponding ortho-quinones, which are highly reactive intermediates. These intermediates can then be conjugated with GSH preventing cellular damage. Furthermore, glutathionyl transferase (GST) activity was described to be irreversibly inhibited by the catechols dopamine, α-methyldopa and their GSH conjugates. Therefore, the aims of the present work were the detection and characterization of GSH conjugates of ten methylenedioxy drugs of abuse and their phase I metabolites as well as to assess their inhibition potency on GST activity. The substrates were incubated using human placental GST with or without preincubation by cytochrome P450 enzymes preparations. GST inhibition was tested using chlorodinitrobenzene GSH conjugation as marker reaction. GSH conjugates were analyzed and characterized using LC-high-resolution-MS/MS. For confirmation of postulated fragmentation patterns, formation of GSH conjugates of selected deuterated analogs (deuterated analogue approach, DAA) of the investigated drugs was explored. For the methylenedioxy amphetamines the following steps could be identified: conjugation of the parent compounds at position 2, 5, 6, of the demethylenyl metabolites at position 2 and 5, and of the further deaminated demethylenyl metabolites at position 2. For the β-keto-phenylalkylamine and pyrrolidinophenone, conjugation of the demethylenyl metabolites and of the deaminated demethylenyl metabolites at position 2 could be identified. The DAA allowed the differentiation of the 2 and 5/6 isomers by confirmation of the postulated mass spectral fragments. Finally, the tested drugs and phase I metabolites showed no inhibition potency on GST activity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Dimethocaine (DMC), a synthetic derivative of cocaine, is distributed and consumed as "new psychoactive substance" (NPS) without any safety testing at the forefront. It is mainly metabolized by N-acetylation, N-deethylation or hydroxylation. Therefore, the aim of the presented study was to determine the human NAT and P450 isozymes involved in this major metabolic steps, to measure the kinetics of the reactions, and to estimate the contribution on in vivo hepatic clearance. For these studies, cDNA-expressed NATs and P450s were used and formation of metabolites after incubation was measured using LC-MS or LC-MS(n). For N-acetylation, NAT2 could be shown to be the only isoform catalyzing the reaction in vitro hence assuming to be the only relevant enzyme for in vivo acetylation. Kinetic profiles of all P450 catalyzed metabolite formations followed classic Michaelis-Menten behavior with enzyme affinities (Km values) between 3.6 and 220 μM. Using the relative activity factor approach, the net clearances for deethylation of DMC were calculated to be 3% for P450 1A2, 1% for 2C19, <1% for 2D6, and 96% for 3A4. The net clearances for hydroxylation of DMC were calculated to be 32% for P450 1A2, 5% for 2C19, 51% for 2D6, and 12% for 3A4. Furthermore, these data were confirmed by chemical inhibition tests in human liver microsomes. As DMC is metabolized via two main steps and different P450 isoforms were involved in the hepatic clearance of DMC, a clinically relevant interaction with single P450 inhibitors should not be expected. However, a slow acetylation phenotype or inhibition of NAT2 could lead to decreased N-acetylation and hence leading to an increased risk of side effects caused by this arylamine., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Poisonings with Amanita phalloides toxins require fast diagnosis in order to avoid expensive and unnecessary therapies. Initial clinical assessment in combination with urinary amanitin analysis is necessary for a definite diagnosis. Therefore, a simple, fast, and robust method was developed for reliable detection of α- and β-amanitin as well as for fully validated quantification of α-amanitin in human urine. After simple dilution and centrifugation of the urine sample, a fast on-line extraction using a Transcend TLX-II system based on turbulent flow chromatography (TurboFlow) was established. A new TurboFlow mode was introduced, the pseudo quick elute mode (PQEM), which had more options for method optimization than the generic quick elute mode (QEM). It allowed running several modes in one valve arrangement. The PQEM showed better practicability in routine and emergency analysis than the previously used methods. After extraction, the fast 15min LC-high resolution (HR)-MS/MS analysis allowed reliable identification of α- and β-amanitin based on fragments identified using so-called HR pseudo MS(3) experiments. According to international recommendations, the requirements for full validation including the parameters selectivity, calibration, accuracy, precision, recovery, matrix effects, and stability were fulfilled for α-amanitin. The method was successfully applied to the analysis of authentic urine samples containing amatoxins. In conclusion, this method allowed the determination of amatoxins using the novel PQEM in a faster, robust, and more reliable way than existing methods, making it suitable for daily routine and especially emergency toxicological analysis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Arabidopsis CRINKLY4 (ACR4), a receptor-like kinase required for plant growth and development, possesses an extracellular ligand binding domain, a transmembrane helix, and an intracellular domain (ICD). The ICD contains the juxtamembrane (JMD) and the C-terminal (CTD) subdomains, which flank the core kinase domain (KD), with at least 16 autophosphorylation sites. Phosphorylation sites are often docking sites for the modification-dependent recruitment of interacting proteins that orchestrate many downstream signaling events. In this context, we have specifically probed the role of the two phosphorylation sites Ser(475) and Thr(478) in the JMD using mutagenesis and phage-peptide screening techniques. Thus, naïve and phosphorylated 15-mer peptides derived from the JMD were panned against a 21-amino acid random phage peptide library. The phosphorylated peptide preferentially recognized the consensus sequence LxSLL. This sequence harbors the LxxLL motif, a known protein-protein interaction motif that is also present in the N-terminal lobe of the KD. We demonstrate the binding of JMD peptides to the KD and also show through kinetic analyses of mutants that phosphorylation of Ser(475) and Thr(478) in the JMD is necessary for optimal substrate phosphorylation in vitro. Our experiments suggest that an intramolecular interaction can occur between the JM and the N-terminal lobe of the KD., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Acute myeloid leukemia (AML) is characterized by dysregulated gene expression and abnormal patterns of DNA methylation; the relationship between these events is unclear. Many AML patients are now being treated with hypomethylating agents, such as decitabine (DAC), although the mechanisms by which it induces remissions remain unknown. The goal of this study was to use a novel stromal coculture assay that can expand primary AML cells to identify the immediate changes induced by DAC with a dose (100nM) that decreases total 5-methylcytosine content and reactivates imprinted genes (without causing myeloid differentiation, which would confound downstream genomic analyses). Using array-based technologies, we found that DAC treatment caused global hypomethylation in all samples (with a preference for regions with higher levels of baseline methylation), yet there was limited correlation between changes in methylation and gene expression. Moreover, the patterns of methylation and gene expression across the samples were primarily determined by the intrinsic properties of the primary cells, rather than DAC treatment. Although DAC induces hypomethylation, we could not identify canonical target genes that are altered by DAC in primary AML cells, suggesting that the mechanism of action of DAC is more complex than previously recognized.

The pharmacokinetics of various important drugs are known to be significantly influenced by the human ABC transporter P-glycoprotein (P-gp), which may lead to clinically relevant drug-drug interactions. In contrast to therapeutic drugs, emerging drugs of abuse (DOA) are sold and consumed without any safety pharmacology testing. Only some studies on their metabolism were published, but none about their affinity to the transporter systems. Therefore, 47 DOAs from various classes were tested for their P-gp affinity using human P-gp (hP-gp) to predict possible drug-drug interactions. DOAs were initially screened for general hP-gp affinity and further characterized by modeling classic Michaelis-Menten kinetics and assessing their K(m) and V(max) values. Among the tested drugs, 12 showed a stimulation of ATPase activity. The most intensive stimulating DOAs were further investigated and compared with the known P-gp model substrates sertraline and verapamil. ATPase stimulation kinetics could be modeled for the entactogen 3,4-methylenedioxy-α-ethylphenethylamine (3,4-BDB), the hallucinogen 2,5-dimethoxy-4-iodoamphetamine (DOI), the abused alkaloid glaucine, the opioid-like drugs N-iso-propyl-1,2-diphenylethylamine (NPDPA), and N-(1-phenylcyclohexyl)-3-ethoxypropanamine (PCEPA), with K(m) and V(max) values within the same range as for verapamil or sertraline. As a consequence interactions with other drugs being P-gp substrates might be considered to be very likely and further studies should be encouraged., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

This paper reviews analytical approaches published in 2002-2012 for chiral drug analysis and their relevance in research and practice in the field of clinical and forensic toxicology. Separation systems such as gas chromatography, high performance liquid chromatography, capillary electromigration, and supercritical fluid chromatography, all coupled to mass spectrometry, are discussed. Typical applications are reviewed for relevant chiral analytes such as amphetamines and amphetamine-derived designer drugs, methadone, tramadol, psychotropic and other CNS acting drugs, anticoagulants, cardiovascular drugs, and some other drugs. Usefulness of chiral drug analysis in the interpretation of analytical results in clinical and forensic toxicology is discussed as well., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Aims: An increase in intracellular vascular smooth muscle cell calcium concentration (VSMC [Ca(2+)](i)) is essential for endothelin-1 (ET-1)-induced vasoconstriction. Based on previous findings that activation of the G protein-coupled estrogen receptor (GPER) inhibits vasoconstriction in response to ET-1 and regulates [Ca(2+)](i) in cultured VSMC, we investigated whether endogenous GPER regulates ET-1-induced changes in VSMC [Ca(2+)](i) and constriction of intact arteries., Main Methods: Pressurized carotid arteries of GPER-deficient (GPER(0)) and wildtype (WT) mice were loaded with the calcium indicator fura 2-AM. Arteries were stimulated with the GPER-selective agonist G-1 or solvent followed by exposure to ET-1. Changes in arterial diameter and VSMC [Ca(2+)](i) were recorded simultaneously. Vascular gene expression levels of ET(A) and ET(B) receptors were determined by qPCR., Key Findings: ET-1-dependent vasoconstriction was increased in arteries from GPER(0) compared to arteries from WT mice. Despite the more potent vasoconstriction to ET-1, GPER deficiency was associated with a marked reduction in the ET-1-stimulated VSMC [Ca(2+)](i) increase, suggesting an increase in myofilament force sensitivity to [Ca(2+)](i). Activation of GPER by G-1 had no effect on vasoconstriction or VSMC [Ca(2+)](i) responses to ET-1, and expression levels of ET(A) or ET(B) receptor were unaffected by GPER deficiency., Significance: These results demonstrate that endogenous GPER inhibits ET-1-induced vasoconstriction, an effect that may be associated with reduced VSMC Ca(2+) sensitivity. This represents a potential mechanism through which GPER could contribute to protective effects of endogenous estrogen in the cardiovascular system., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Different elimination was reported for the two enantiomers of the designer drug 3,4-methylenedioxyethylamphetamine (MDEA) in vivo. In the present work, the enantioselectivity of glucuronidation and sulfation of the MDEA phase I metabolites 3,4-dihydroxyethylamphetamine (DHEA) and 4-hydroxy-3-methoxyethylamphetamine (HMEA) was investigated. First, glucuronide standards were synthesized using rat liver microsomes. Incubations were performed with recombinant human UDP-glucuronyltransferases (UGT) and pooled human liver microsomes (pHLM) for glucuronidation and using recombinant human sulfotransferases (SULT) and pooled human liver cytosol (pHLC) for sulfation. Product formation experiments were performed by quantification of the phase II metabolites using liquid chromatography-high-resolution mass spectrometry. Additionally, substrate depletion experiments were conducted by gas chromatography-mass spectrometry after chiral derivatization for sulfation. UGT2B7, 2B15, and 2B17 were involved in glucuronidation of HMEA and SULT1A1 and SULT1A3 and SULT1A3 and SULT1E1 in the sulfation of DHEA and HMEA, respectively. SULTs provided much higher affinity, whereas UGTs showed higher capacities. Marked stereoselectivity could be observed for UGT2B15, UGT2B17, and pHLM toward S-HMEA, for SULT1A3 and pHLC toward S-DHEA and for SULT1A3 and pHLC toward R-HMEA. In conclusion, the phase II metabolism might also contribute to the observed different pharmacokinetic properties of MDEA., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy) is excreted in human urine mainly as conjugates of its metabolites 3,4-dihydroxymethamphetamine (DHMA) and 4-hydroxy-3-methoxymethamphetamine (HMMA). The glucuronidation kinetics of HMMA showed high capacities, but also high K(m) values, unlikely to be reached after recreational user's doses. Therefore, the aim of the present work was to investigate the sulfation of DHMA and HMMA by human sulfotransferases (SULTs) in pooled human liver cytosol (pHLC). The kinetic data showed deviation from typical Michaelis-Menten kinetics. The overall efficiency for HMMA sulfation was calculated to be 2-10 times higher than for glucuronidation. As the sulfation of both MDMA metabolites showed substrate inhibition effects, their inhibitory potential towards typical sulfation reactions in pHLC was tested. The following substrates for typical sulfation reactions were used: nitrophenol, dopamine, estradiol, and dehydroepi androsten dione. Inhibition was observed towards dopamine sulfation by DHMA and HMMA, but not by MDMA. The 1/V vs. 1/S plots indicated a mixed-type or competitive inhibition model for DHMA and HMMA, respectively. In conclusion, the presented data indicated that sulfation of HMMA should be the major conjugation reaction observed in humans. Furthermore, both, DHMA and HMMA, were identified as inhibitors of dopamine sulfation., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)