Development and validation of an LC-MS/MS method for determination of B vitamins and some its derivatives in whole blood.

Obligate symbiotic bacteria associated with the insects feeding exclusively on vertebrate blood are supposed to complement B vitamins presumably lacking in their diet. Recent genomic analyses revealed considerable differences in biosynthetic capacities across different symbionts, suggesting that levels of B vitamins may vary across different vertebrate hosts. However, a rigorous determination of B vitamins content in blood of various vertebrates has not yet been approached. A reliable analytical method focused on B vitamin complex in blood can provide valuable informative background and understanding of general principles of insect symbiosis. In this work, a chromatographic separation of eight B vitamins (thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, biotin, folic acid, and cyanocobalamine), four B vitamin derivatives (niacinamide, pyridoxal-5-phosphate, 4-pyridoxic acid, and tetrahydrofolic acid), and 3 stable isotope labelled internal standards was developed. Detection was carried out using dual-pressure linear ion trap mass spectrometer in FullScan MS/MS and SIM mode. Except for vitamin B9 (tetrahydrofolic acid), the instrument quantitation limits of all analytes were ranging from 0.42 to 5.0 µg/L, correlation coefficients from 0.9997 to 1.0000, and QC coefficients from 0.53 to 3.2%. Optimization of whole blood sample preparation step was focused especially on evaluation of two types of protein-precipitation agents: trichloroacetic acid and zinc sulphate in methanol. The best results were obtained for zinc sulphate in methanol, but only nine analytes were successfully validated. Accuracy of the procedure using this protein-precipitating agent was ranging from 89 to 120%, precision from 0.5 to 13%, and process efficiency from 65 to 108%. The content of B vitamins in whole blood samples from human and various vertebrates is presented as an application example of this newly developed method.

Photodegradation of fluoroquinolones in aqueous solution under light conditions relevant to surface waters, toxicity assessment of photoproduct mixtures.

Photochemical degradation of fluoroquinolones ciprofloxacin, enrofloxacin and norfloxacin in aqueous solution under light conditions relevant to surface waters at neutral and alkaline pH was found to proceed readily with half-lives between 0.9 and 2.7 min. The products of photochemical degradation identified by HPLC-MS included defluorinated, hydroxylated, and decarboxylated structures as well as structures with opened cyclic structures. For all of the studied substances, the reaction pathways were influenced significantly by the pH of the reaction system, with more products formed at alkaline pH than at neutral pH: the ratios of products in neutral and alkaline pH were 16/26, 9/19, 15/23 for ciprofloxacin, enrofloxacin, and norfloxacin, respectively. The structures of photoproducts and pathways of photochemical degradation are proposed. The antibacterial activities of photoproduct mixtures tested on E. coli and S. epidermidis were significantly higher in comparison to parental antibiotics in the case of both ciprofloxacin and enrofloxacin with p-values less than 0.0001 in most cases. The effect of the photoproducts was shown to be dependent on the pH value of the original antibiotic solutions before photodegradation: for ciprofloxacin, antibacterial activity against E. coli was more notably pronounced with regard to neutral pH photoproducts, while a less significant, or in one case not significant, effect of pH was observed against S. epidermidis; for norfloxacin, antibacterial activity against both E. coli and S. epidermidis was especially high with regard to alkaline pH photoproducts.

Toxicity assessment of verapamil and its photodegradation products.

Pathways of photochemical degradation of a cardiovascular drug verapamil under conditions relevant to natural waters and the toxicity of the photoproducts to Daphnia magna were investigated. Photodegradation was shown to proceed via photocatalysed mechanism. Two main photodegradation pathways were recognised: the first leading to hydroxylation at the methylamino position followed by splitting of verapamil molecule into two fragments, and the second providing the main active metabolite of verapamil, norverapamil, and a series of norverapamil isomers, followed again by their splitting at the amino group position. Twenty-two products of photodegradation were identified. Toxicity assays in sublethal concentrations of the parental drug, of the photoproduct mixture, and of norverapamil revealed no direct negative response in Daphnia magna to verapamil. On the other hand, photochemical products significantly lowered the number of juveniles, number of clutches, and body size of Daphnia. The exposition of Daphnia to norverapamil showed the same but even more pronounced effects than its exposition to the mixture of photoproducts, which leads to the conclusion that norverapamil is mainly responsible for the toxicity of photoproduct mixture and represents a noteworthy threat to aquatic invertebrates.

Biosynthesis of poly-3-hydroxybutyrate from grass silage by a two-stage fermentation process based on an integrated biorefinery concept.

Grass silage as a renewable feedstock for an integrated biorefinery includes nutrients and carbon sources directly available in the press juice (PJ) and in lignocellulosic saccharides from the plant framework. Here, a novel two-stage fed-batch fermentation process for biosynthesis of poly-3-hydroxybutyrate (PHB) by Cupriavidus necator DSM 531 is presented. For bacterial growth, nutrient-rich PJ was employed as a fermentation medium, without any supplements. Saccharides derived from the mechano-enzymatic hydrolysis of the press cake (PC) were subjected to a lactic acid fermentation process, before the fermentation products were fed into the polymer accumulation phase. By combination of pH-stat feeding and cell recycling, the PHB content in 22 g L-1 total-dry cells reached 39% after 32 h of cultivation. Using mimicked hydrolyzate of diluted PJ artificially supplemented with glucose and xylose, the resulting cell dry weight of 21 g L-1 contained 42% PHB.