First eco-toxicological evidence of ivabradine effect on the marine bacterium Vibrio fischeri: A chiral view.

Ivabradine (S-ivabradine) is a contemporary antihypertensive drug designed and commercialized for cardiovascular diseases treatment over the world. In this work the enantiomer-specific stability and acute toxicity of ivabradine to the marine bacterium Vibrio fischeri as well as the potential mechanism of action were investigated for the first time. With this aim, real concentrations of ivabradine enantiomers under abiotic and biotic conditions were determined by Capillary Electrophoresis (CE) with cyclodextrins (CDs) as chiral selectors. A moderate chiral stability without enantiomeric interconversion was observed for ivabradine. The bioluminescence inhibition method revealed an enantioselective toxicity of ivabradine to marine bacterium. The order of ecotoxicity was R-ivabradine < racemic ivabradine < S-ivabradine with EC50 (t = 5 min) values about 75.98, 11.11 and 7.93 mg/L, respectively. Confocal Live/Dead stained images showed that bacterial envelops cells were seriously damaged after exposure to S-ivabradine. S-ivabradine also disturbed the esterase activity and significantly increased the ROS level compared with the control. Thus, oxidative stress originating membrane cells damage and enzymatic activity changes was shown to be the primary mechanism of S-ivabradine toxicity to marine bacterium. Our results highlight the need for more eco-toxicological evaluations of the cardiovascular drug S-ivabradine on other aquatic organisms to establish the risk on the environment.

Stereoselective separation of dimethenamid by cyclodextrin electrokinetic chromatography using deep eutectic solvents.

An Electrokinetic Chromatography (EKC) method was developed in this work enabling for the first time the separation of the four stereoisomers of the acetamide herbicide dimethenamid. A screening of different anionic cyclodextrins (CDs) revealed that the use of a single CD system did not allow the separation of the four dimethenamid stereoisomers while dual systems improved the chiral separation. The combination of 15 mM (2-carboxyethyl)-ß-CD (CE-ß-CD) with 10 mM methyl-γ-CD (M-γ-CD) originated the partial separation of dimethenamid stereoisomers. To obtain the baseline separation between all consecutive peaks, the effect of the addition of ionic liquids and deep eutectic solvents to the CDs dual system was investigated. While ionic liquids did not improve the chiral separation obtained with the CDs dual system, the addition of deep eutectic solvents showed generally beneficial effects on the separation in terms of resolution. The influence of the nature of the deep eutectic solvent was studied and the effects of the ready-made deep eutectic solvent and its components on the separation were compared. Choline chloride-D-fructose (ChCl-D-fructose) when added to the CDs dual system under optimized conditions (15 mM CE-ß-CD, 10 mM M-γ-CD, 1.5 % ChCl-D-fructose (2:1) in a 100 mM borate buffer (pH 9.0), a separation voltage of 25 kV and a temperature of 20 ˚C) enabled separating the four stereoisomers of dimethenamid in 21 min with resolutions between consecutive peaks of 6.0, 2.1 and 1.5. The analytical characteristics of the developed method were evaluated and considered adequate to achieve the stereoselective analysis of dimethenamid-P in commercial agrochemical formulations. Results demonstrated the potential of the method to control the quality of these formulations and to determine the stereoisomeric purity of dimethenamid-P in these products.

Stereoselective separation of sulfoxaflor by electrokinetic chromatography and applications to stability and ecotoxicological studies.

An Electrokinetic Chromatography method was developed for the stereoselective analysis of sulfoxaflor, a novel sulfoximine agrochemical with two chiral centers. A screening with fourteen negatively charged CDs was performed and Succinyl-ß-CD (Succ-ß-CD) was selected. A 15 mM concentration of this CD in a 100 mM borate buffer (pH 9.0), using an applied voltage of 20 kV and a temperature of 15 °C made possible the baseline separation of the four stereoisomers of sulfoxaflor in 13.8 min. The evaluation of the linearity, accuracy, precision, LODs and LOQs of the method developed showed its performance to be applied to the analysis of commercial agrochemical formulations, the evaluation of the stability of sulfoxaflor stereoisomers under biotic and abiotic conditions, and to predict, for the first time, sulfoxaflor toxicity (using real concentrations instead of nominal concentrations), on two non-target aquatic organisms, the freshwater plant, Spirodela polyrhiza, and the marine bacterium, Vibrio fischeri.

Enantiomeric separation of prothioconazole and prothioconazole-desthio by Capillary Electrophoresis. Degradation studies in environmental samples.

In this work, two analytical methodologies by Capillary Electrophoresis were developed. The first one enabled the rapid and cost-effective enantioseparation of prothioconazole and was applied to the analysis of prothioconazole-based commercial agrochemical formulations. The second methodology enabled the simultaneous enantioseparation of prothioconazole and its metabolite prothioconazole-desthio and was applied to degradation studies of both compounds in soil and sand samples. The influence of several experimental variables was investigated to develop both methodologies. The separation of prothioconazole enantiomers was achieved in 4.5 min with a resolution of 2.8 employing a neutral cyclodextrin (heptakis(2,3,6-tri-O-methyl)-ß-cyclodextrin). Given the nature of prothioconazole-desthio, a neutral cyclodextrin cannot be used for its chiral separation. For this reason, the simultaneous enantioseparation of prothioconazole and prothioconazole-desthio was achieved in 5.5 min with resolution values of 1.9 and 8.2, respectively, using a negatively charged cyclodextrin (sulfated-γ-cyclodextrin). The analytical characteristics of the developed methodologies were evaluated and both methods showed good performance to be applied to the quantitation of the enantiomers of prothioconazole in commercial agrochemical formulations (LOD 0.7 mg L-1) and to carry out degradation studies for both compounds in environmental matrices (LODs lower than 0.9 and 1.3 mg L-1 for prothioconazole and prothioconazole-desthio enantiomers, respectively). The recovery values obtained were in the range between 94-104 % for the agrochemical formulations, between 96-99 % for the sand samples and between 97-100 % for the soil samples.

Enantiomeric separation of panthenol by Capillary Electrophoresis. Analysis of commercial formulations and toxicity evaluation on non-target organisms.

The first CE methodology enabling the enantiomeric separation of panthenol was developed in this work. Electrokinetic chromatography with cyclodextrins (CD-EKC) was the CE mode employed for this purpose. The effect of different experimental variables such as the nature and concentration of the cyclodextrin, the temperature and the separation voltage was investigated. The best enantiomeric separation was obtained with 25 mM (2-carboxyethyl)-ß-CD (CE-ß-CD) in 100 mM borate buffer (pH 9.0), with a separation voltage of 30 kV and a temperature of 30 °C. Under these conditions, an enantiomeric resolution of 2.0 in an analysis time of 4.2 min was obtained, being the biologically active enantiomer d-panthenol (dexpanthenol) the second-migrating enantiomer. The analytical characteristics of the method were evaluated in terms of precision, accuracy, selectivity, linearity, LOD, and LOQ, showing a good performance for the quantitation of dexpanthenol in cosmetic and pharmaceutical formulations. The enantiomeric impurity (L-panthenol) could be detected at a 0.1% level with respect to the majority enantiomer, allowing to accomplish the requirements of the ICH guidelines. The method was also successfully applied to study the stability of panthenol under abiotic and biotic conditions and its toxicity on non-target organisms (the aquatic plant Spirodela polyrhiza).

Enantiomeric analysis of pyrethroids and organophosphorus insecticides.

The use of pesticides has increased sharply in the last decades, not only in agriculture, but also in industry, public health, and other areas. Pyrethroids and organophosphorus insecticides are among the most employed pesticides. These chemicals usually contain asymmetric chiral atoms; thus, they are characterized by stereoisomerism. Although most of these chiral pesticides are produced, used, and released as racemic mixtures, the different enantiomers of these compounds can present different insecticidal activity, different toxicity against vertebrates and invertebrates, and also different persistence in the environment. In fact, in some cases, only one enantiomer is active, while the other can be less active or even toxic to non-target organisms. Therefore, the development of enantioselective analytical methodologies enabling their determination presents a high interest. Different separation techniques, including high performance liquid chromatography, gas chromatography, supercritical fluid chromatography, and capillary electrophoresis, have been employed to achieve the chiral analysis of pyrethroids and organophosphorus insecticides. This review presents the characteristics of the stereoselective analytical methodologies developed with this aim from 2010 to April 2019 and their applications to the analysis of real samples as well as for toxicity and biodegradation studies.