LogD-based modelling and ΔlogD as a proxy for pH-dependent action of ionizable chemicals reveal the relevance of both neutral and ionic species for fish embryotoxicity and possess great potential for practical application in the regulation of chemicals.

Depending on the ambient pH, ionizable substances are present in varying proportions in their neutral or charged form. The extent to which these two chemical species contribute to the pH-dependant toxicity of ionizable chemicals and whether intracellular ion trapping has a decisive influence in this context is controversially discussed. Against this background, we determined the acute toxicity of 24 ionizable substances at up to 4 different pH values on the embryonic development of the zebrafish, Danio rerio, and supplemented this dataset with additional data from the literature. The LC50 for some substances (diclofenac, propranolol, fluoxetine) differed by a factor of even >103 between pH5 and pH9. To simulate the toxicity of 12 acids and 12 bases, six models to calculate a pH-dependant logD value as a proxy for the uptake of potentially toxic molecules were created based on different premises for the trans-membrane passage and toxic action of neutral and ionic species, and their abilities to explain the real LC50 data set were assessed. Using this approach, we were able to show that both neutral and charged species are almost certainly taken up into cells according to their logD-based distribution, and that both species exert toxicity. Since two of the models that assume all intracellular molecules to be neutral overestimated the real toxicity, it must be concluded, that the toxic effect of a single charged intracellularly present molecule is, on the average, lower than that of a single neutral molecule. Furthermore, it was possible to attribute differences in toxicity at different pH values for these 24 ionizable substances to the respective deltas in logD at these pH levels with high accuracy, enabling particularly a full logD-based model on the basis of logPow as a membrane passage descriptor to be used for predicting potential toxicities in worst-case scenarios from existing experimental studies, as stipulated in the process of registration of chemicals and the definition of Environmental Quality Standards (EQS).

MALDI-TOF-MS integrated workflow for food authenticity investigations: An untargeted protein-based approach for rapid detection of PDO feta cheese adulteration.

Advances in Matrix-assisted Laser Desorption/Ionization -Time-Of-Flight Mass Spectrometry (MALDI-TOF-MS) have led to its supremacy for complex assessment of food authenticity studies, like dairy products fraud, holding promise for the discovery of potential authenticity (bio)markers. In this study, an integrated untargeted protein-based workflow in combination with advanced chemometrics is presented, to address authenticity challenges in PDO feta cheese which is legally manufactured by the mixture of sheep/goat milk. Potential markers attributed to specific animal origin were found from protein profiles acquired for authentic feta and white cheeses (prepared from cow milk), belonging to 4 kDa-18.5 kDa mass area. Rapid detection of feta cheese adulteration from cow milk was also achieved down to 1% adulteration level. The discriminative models showed high predictive ability for feta cheese authenticity (Q2 = 0.920, RMSEE = 0.053) and its adulteration (Q2 = 0.835, RMSEE = 0.121), introducing a reliable approach in routine analysis. The methodology was successfully applied in detection of cow milk in sheep yoghurt.

Heart rate as an early warning parameter and proxy for subsequent mortality in Danio rerio embryos exposed to ionisable substances.

Environmental risk assessments of organic chemicals usually do not consider pH as a key factor. Hence, most substances are tested at a single pH only, which may underestimate the toxicity of ionisable substances with a pKa in the range of 4-10. Thus, the ability to consider the pH-dependent toxicity would be crucial for a more realistic assessment. Moreover, there is a tendency in acute toxicity tests to focus on mortality only, while little attention is paid to sublethal endpoints. We used Danio rerio embryos exposed to ten ionisable substances (the acids diclofenac, ibuprofen, naproxen and triclosan and the bases citalopram, fluoxetine, metoprolol, propranolol, tramadol and tetracaine) at four external pH levels, investigating the endpoints mortality (LC50) and heart rate (EC20). Dose-response curves were fitted with an ensemble-model to determine the true uncertainty and variation around the mean endpoints. The ensemble considers eight (heart rate) or twelve (mortality) individual models for binominal and Poisson distributed data, respectively, selected based on the Akaike Information Criterion (AIC). In case of equally good models, the mean endpoint of all models in the ensemble was calculated, resulting in more robust ECx estimates with lower 'standard errors' as compared to randomly selected individual models. We detected a high correlation between mortality (LC50) at 96 hpf and reduced heart rate (EC20) at 48 hpf for all compounds and all external pH levels (r = 0.98). Moreover, the observed pH-dependent effects were strongly associated with log D and thus, likely driven by differences in uptake (toxicokinetic) rather than internal (toxicodynamic) processes. Prospectively, the a priori consideration of pH-dependent effects of ionisable substances might make testing at different pH levels redundant, while the endpoint of mortality might even be replaced by a reliable sublethal proxy that would reduce the exposure, accelerating the evaluation process.

Trapped Ion Mobility Incorporated in LC-HRMS Workflows as an Integral Analytical Platform of High Sensitivity: Targeted and Untargeted 4D-Metabolomics in Extra Virgin Olive Oil.

Trapped ion mobility spectrometry (TIMS) is a promising technique for the separation of isomers based on their mobility. In the present work, TIMS coupled to liquid chromatography (LC) and high-resolution mass spectrometry (HRMS) was applied as a comprehensive analytical platform to address authenticity challenges, focusing on extra virgin olive oil (EVOO). Isomers detected in EVOO's phenolic fraction, classified into secoiridoids group, were successfully separated. Thanks to parallel accumulation serial fragmentation (PASEF) acquisition mode, high-quality spectra were obtained, facilitating identification. Moreover, a four-dimensional (4D) untargeted metabolomics approach was implemented to evaluate EVOO's global profile in cases of both variety and geographical origin discrimination. Potential authenticity markers, attributed to isomers, were successfully identified through the proposed workflow that incorporates ion mobility information along with LC-HRMS analytical evidence (i.e., mass accuracy, retention time, isotopic pattern, MS/MS fragmentation). Our study establishes LC-TIMS-HRMS in food authenticity and highlights mobility-enhanced metabolomics in four dimensions.

Assessment of Environmental Pollution and Human Exposure to Pesticides by Wastewater Analysis in a Seven-Year Study in Athens, Greece.

Pesticides have been used in large amounts around the world for decades and are responsible for environmental pollution and various adverse effects on human health. Analysis of untreated wastewater can deliver useful information on pesticides' use in a particular area and allow the assessment of human exposure to certain substances. A wide-scope screening method, based on liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry, was applied, using both target and suspect screening methodologies. Daily composite influent wastewater samples were collected for seven or eight consecutive days in Athens between 2014 and 2020 and analyzed for 756 pesticides, their environmental transformation products and their human metabolites. Forty pesticides were quantified at mean concentrations up to 4.9 µg/L (tralkoxydim). The most abundant class was fungicides followed by herbicides, insect repellents, insecticides and plant growth regulators. In addition, pesticide transformation products and/or metabolites were detected with high frequency, indicating that research should be focused on them. Human exposure was evaluated using the wastewater-based epidemiology (WBE) approach and 3-ethyl-carbamoyl benzoic acid and cis-1,2,3,6-tetrahydrophthalimide were proposed as potential WBE biomarkers. Wastewater analysis revealed the presence of unapproved pesticides and indicated that there is an urgent need to include more transformation products in target databases.

Antihypertensive activity and molecular interactions of irbesartan in complex with 2-hydroxypropyl-ß-cyclodextrin.

Irbesartan (IRB) exerts beneficial effects either alone or in combination with other drugs on numerous diseases, such as cancer, diabetes, and hypertension. However, due to its high lipophilicity, IRB does not possess the optimum pharmacological efficiency. To circumvent this problem, a drug delivery system with 2-hydroxypropyl-ß-cyclodextrin (2-HP-ß-CD) was explored. The 1:1 complex between IRB and 2-HP-ß-CD was identified through ESI QTF HRMS. Dissolution studies showed a higher dissolution rate of the lyophilized IRB-2-HP-ß-CD complex than the tablet containing IRB at pH = 1.2. DSC results revealed the differences of the thermal properties between the complex and various mixtures consisting of the two components, namely IRB and 2-HP-ß-CD. Interestingly, depending on the way the mixture preparation was conducted, different association between the two components was observed. Molecular dynamics (MD) simulations predicted the favorable formation of the above complex and identified the dominant interactions between IRB and 2-HP-ß-CD. In vitro pharmacological results verified that the inclusion complex not only preserves the binding affinity of IRB for AT1R receptor, but also it slightly increases it. As the complex formulation lacks the problems of the tablet, our approach is a promising new way to improve the efficiency of IRB.

Wide-scope target screening of >2000 emerging contaminants in wastewater samples with UPLC-Q-ToF-HRMS/MS and smart evaluation of its performance through the validation of 195 selected representative analytes.

This study presents the development and validation of a comprehensive quantitative target methodology for the analysis of 2316 emerging pollutants in water based on Ultra-Performance Liquid Chromatography Quadrupole-Time-Of-Flight Mass Spectrometry (UPLC-Q-ToF-HRMS/MS). Target compounds include pesticides, pharmaceuticals, drugs of abuse, industrial chemicals, doping compounds, surfactants and transformation products, among others. The method was validated for 195 analytes, chosen to be representative of the chemical space of the target list, enabling the assessment of the performance of the method. The method involves a generic sample preparation based on mixed mode solid phase extraction, a UPLC-QTOF-MS/MS screening method using Data Independent Acquisition (DIA) mode, which provides MS and MS/MS spectra simultaneously and an elaborate strong post-acquisition evaluation of the data. The processing method was optimized to provide a successful identification rate >95 % and to minimize the number of false positive results (< 5 %). Decision limit (CCα) and detection capability (CCß) were also introduced in the validation scheme to provide more realistic metrics on the performance of a HRMS-based wide-scope screening method. A new system of identification points (IPs) based on the one described in the Commission Decision 2002/657/EC was applied to communicate the confidence level in the identification of the analytes. This system considers retention time, mass accuracy, isotopic fit and fragmentation; taking full advantage of the capacities of the HRMS instruments. Finally, 398 contaminants were detected and quantified in real wastewater.

Host-Guest Interactions between Candesartan and Its Prodrug Candesartan Cilexetil in Complex with 2-Hydroxypropyl-ß-cyclodextrin: On the Biological Potency for Angiotensin II Antagonism.

Renin-angiotensin aldosterone system inhibitors are for a long time extensively used for the treatment of cardiovascular and renal diseases. AT1 receptor blockers (ARBs or sartans) act as antihypertensive drugs by blocking the octapeptide hormone Angiotensin II to stimulate AT1 receptors. The antihypertensive drug candesartan (CAN) is the active metabolite of candesartan cilexetil (Atacand, CC). Complexes of candesartan and candesartan cilexetil with 2-hydroxylpropyl-ß-cyclodextrin (2-HP-ß-CD) were characterized using high-resolution electrospray ionization mass spectrometry and solid state 13C cross-polarization/magic angle spinning nuclear magnetic resonance (CP/MAS NMR) spectroscopy. The 13C CP/MAS results showed broad peaks especially in the aromatic region, thus confirming the strong interactions between cyclodextrin and drugs. This experimental evidence was in accordance with molecular dynamics simulations and quantum mechanical calculations. The synthesized and characterized complexes were evaluated biologically in vitro. It was shown that as a result of CAN's complexation, CAN exerts higher antagonistic activity than CC. Therefore, a formulation of CC with 2-HP-ß-CD is not indicated, while the formulation with CAN is promising and needs further investigation. This intriguing result is justified by the binding free energy calculations, which predicted efficient CC binding to 2-HP-ß-CD, and thus, the molecule's availability for release and action on the target is diminished. In contrast, CAN binding was not favored, and this may allow easy release for the drug to exert its bioactivity.

Assessment of the Acute Toxicity, Uptake and Biotransformation Potential of Benzotriazoles in Zebrafish ( Danio rerio) Larvae Combining HILIC- with RPLC-HRMS for High-Throughput Identification.

The current study reports on the toxicity, uptake, and biotransformation potential of zebrafish (embryos and larvae) exposed to benzotriazoles (BTs). Acute toxicity assays were conducted. Cardiac function abnormalities (pericardial edema and poor blood circulation) were observed from the phenotypic analysis of early life zebrafish embryos after BTs exposure. For the uptake and biotransformation experiment, extracts of whole body larvae were analyzed using liquid chromatography-high-resolution tandem mass spectrometry (UPLC-Q-TOF-HRMS/MS). The utility of hydrophilic interaction liquid chromatography (HILIC) as complementary technique to reversed phase liquid chromatography (RPLC) in the identification process was investigated. Through HILIC analyses, additional biotransformation products (bio-TPs) were detected, because of the enhanced sensitivity and better separation efficiency of isomers. Therefore, reduction of false negative results was accomplished. Both oxidative (hydroxylation) and conjugative (glucuronidation, sulfation) metabolic reactions were observed, while direct sulfation proved the dominant biotransformation pathway. Overall, 26 bio-TPs were identified through suspect and nontarget screening workflows, 22 of them reported for the first time. 4-Methyl-1- H-benzotriazole (4-MeBT) demonstrated the highest toxicity potential and was more extensively biotransformed, compared to 1- H-benzotriazole (BT) and 5-methyl-1- H-benzotriazole (5-MeBT). The extent of biotransformation proved particularly informative in the current study, to explain and better understand the different toxicity potentials of BTs.