Simultaneous determination of carbonyl compounds related to thermal treatment and oxidative stability of infant formulas by gas-diffusion microextraction and high-performance liquid chromatography with ultraviolet detection.

Infant formulae are the only possible alternative to breastfeeding during the first year of life, so it is crucial to assure their innocuousness. Infant formula undergoes heat treatments to ensure safety and shelf life. However, such processes impact health as they lead to the formation of malondialdehyde, acrolein, and α-dicarbonyl compounds, related to Maillard reaction. Thus, there is a need for improved analytical methods to ensure the safety, quality, and nutritional value of infant formulae, and also exploring the potential of specific compounds as indicators for quality control and monitoring purposes. We developed and validated a novel, efficient, and cost-effective method using gas-diffusion microextraction for the simultaneous quantification of carbonyl compounds in infant formula. Malondialdehyde, acrolein, glyoxal, methylglyoxal, and diacetyl were detected as o-phenylenediamine derivatives using HPLC with UV detection. Parameters influencing extraction efficiency were studied using an asymmetric screening design. The validated method has shown excellent linearity, sensitivity, accuracy, and precision. It was applied to analyze 26 infant formula samples, including starter, follow-up, and special formulated powdered infant formula. Methylglyoxal was found in all samples (0.201-3.153 µg mL-1), while malondialdehyde was present only in certain starter formulas (1.033-1.802 µg mL-1). Acrolein (0.510-3.246 µg mL-1), glyoxal (0.109-1.253 µg mL-1), and diacetyl (0.119-2.001 µg mL-1) were detected in various sample types. Principal components and hierarchical cluster analyses have showcased distinct sample clustering based on analyte contents. This study presents a novel methodology for the analysis of markers of thermal treatment and oxidative stability in infant formula. It contributes to the characterization of the products' composition and quality control of infant formulae, thereby enhancing their safety and nutritional adequacy. This study also presents the first reported quantification of acrolein in infant formula and introduces the application of the acrolein-o-phenylenediamine derivative for food analysis.

Nutritional Composition and Untargeted Metabolomics Reveal the Potential of Tetradesmus obliquus, Chlorella vulgaris and Nannochloropsis oceanica as Valuable Nutrient Sources for Dogs.

The growing pet population is questioning the sustainability of the pet food system. Although microalgae may constitute a more sustainable food resource, the assessment of their potential for canine diets is almost non-existent. The present study aimed to evaluate the potential of three microalgae species (Tetradesmus obliquus, Chlorella vulgaris and Nannochloropsis oceanica) grown locally in industrial photobioreactors as alternative food resources for dogs. A detailed characterization of their nutritional composition and metabolomic profile was carried out and related to the nutritional requirements of dogs. Overall, the essential amino acid content exceeded the amounts required for dogs at all life stages, except methionine and cysteine. The three microalgae were deficient in linoleic acid, N. oceanica presented a linolenic acid content below requirements and T. obliquus and C. vulgaris were deficient in arachidonic and eicosapentaenoic acids. The fiber was mainly composed of insoluble dietary fiber. The mineral profile varied greatly with the microalgae species, demonstrating their different potential for dog feeding. Untargeted metabolomics highlighted glycolipids, glycerolipids and phospholipids as the most discriminating compounds between microalgae species. Overall, the results support the potential of T. obliquus, C. vulgaris and N. oceanica as valuable macro- and micro-nutrients sources for dog feeding.

A Novel Approach for Monitoring the Volatile Metabolome in Biological Samples from Ruminants through Miniaturized Liquid-Liquid Extraction and Multiclass Gas Chromatography Analysis.

A straightforward and versatile methodology for the extraction of volatile metabolites in biological samples from ruminants for gas chromatography analysis is proposed. The methodology was applied in the determination of multiclass metabolites (short-chain fatty acids, aldehydes, alcohols, ketones, esters, phenols, and sulfides) in different analytical matrices (rumen fluid, urine, and feces) collected from Holstein cows. The 24 multiclass volatile metabolites reported in the different biological samples and their respective concentrations were critically discussed in the context of digestive physiology. Most detected compounds are derived from the rumen and lower gut fermentation of carbohydrates, proteins, and lipids or their metabolism, being consistent with the prior state of the art. The proposed method also takes advantage of the already existing tools in animal nutrition laboratories, providing a novel methodological ground that can generate relevant bioanalytical information with a significant impact on ruminant's nutritional studies.

Unravelling the phytonutrients and antioxidant properties of European Vicia faba L. seeds.

For the first time, seven European varieties of Vicia faba L. seeds were investigated for (1) their phytonutrients profile by HPLC-DAD-MS/MS, (2) total phenolic content (TPC), and (3) antioxidant capacity (DPPH and FRAP assays). A wide range of compounds were identified, namely alkaloids, organic acids, terpenoids, jasmonates, and phenolics; these two latter being the most abundant. TPC ranged between 2.62 and 4.3 mg (gallic acid equivalent) g-1 dry weight, for V. faba major variety Belshi and V. faba minor variety Bauska, respectively. The DPPH radical scavenging capacity showed poor correlation (r = 0.550, P = .041) with TPC, suggesting the presence of other antioxidant sources than phenolics. Still, FRAP was positively correlated with TPC (r = 0.709, P < .01) and DPPH (r = 0.819, P < .01). These results elucidated the phytonutrients and antioxidant properties of V. faba L. seeds as functional food sources.

Profiling of phenolic compounds and antioxidant properties of European varieties and cultivars of Vicia faba L. pods.

Vicia faba L. pods are a by-product generated from the industrial processing of beans for human and animal consumption. As phenolic compounds may play important roles in health, the present work envisaged the phenolic characterization of seven European varieties and cultivars of V. faba (major and minor) pods and the assessment of their antioxidant activity. The V. faba methanolic extracts were characterized by HPLC-DAD-MS/MS for identification of polyphenolic compounds. The total phenolic content and antioxidant capacity of the extracts were evaluated by colorimetric methods (Folin-Ciocalteu, DPPH scavenging capacity assay, and FRAP assay). Main compounds identified by HPLC-DAD-MS/MS were derivatives of caffeic acid, coumaric acid and kaempferol. The broad bean Jögeva variety presented the highest content of free and esterified phenolics (26.3 and 26.7 mg 100 g-1 dry weight, respectively), followed by the horse bean varieties Bauska and Lielplatones. These results were corroborated by the analysis of total phenolic content, DPPH scavenging capacity and FRAP. This study confirmed the rich phenolic content of V. faba pods suggesting to be an interesting novel source for animal nutrition, promoting product quality and consumers' health.

Determination of ammonia nitrogen in solid and liquid high-complex matrices using one-step gas-diffusion microextraction and fluorimetric detection.

This paper introduces a new method for a one-step determination of ammonia nitrogen (NH3) in high complex solid and liquid samples from the agricultural and livestock sectors. To this end, we developed a simultaneous extraction and fluorimetric labeling of NH3, using gas diffusion microextraction (GDME), followed by the fluorescence measurement under 96-well microplate format. The GDME ensured a selective diffusion of NH3 through a commercial hydrophobic membrane, and confined the acceptor solution, which included the fluorimetric labeling reagent o-phthalaldehyde (OPA). The OPA-NH3 labeling reaction was optimized resorting to a full factorial experimental design, which showed that the reducing agent (Na2SO3) concentration was critical to achieve the highest sensitivity. A similar optimization approach for GDME showed that time and temperature significantly influenced the sensitivity of the assay, and also that the modifications in these two factors could be used to adjust the sensitivity according to the concentrations present in the samples. In our final conditions, it was possible to quantify NH3 in the range between 0.38 and 6.27mgL-1 using a 10min extraction at 25°C in different samples (e.g., corn and grass silages, feces, urine). The developed method showed a high repeatability and reproducibility (intraday and interday relative standard deviations values of 4.5% and 9.5%, respectively) and an adequate limit of detection (0.22mgL-1). This new methodology also highlighted the simplicity and versatility of GDME for the determination of volatile components of high-complex matrices, which will certainly drive future developments in the analysis of environmental and biological samples.

Gas-diffusion microextraction coupled with spectrophotometry for the determination of formaldehyde in cork agglomerates.

In this work, a simple methodology was developed for the extraction and determination of free formaldehyde content in cork agglomerate samples. For the first time, gas-diffusion microextraction was used for the extraction of volatile formaldehyde directly from samples, with simultaneous derivatization with acetylacetone (Hantzsch reaction). The absorbance of the coloured solution was read in a spectrophotometer at 412 nm. Different extraction parameters were studied and optimized (extraction temperature, sample mass, volume of acceptor solution, extraction time and concentration of derivatization reagent) by means of an asymmetric screening. The developed methodology proved to be a reliable tool for the determination of formaldehyde in cork agglomerates with the following suitable method features: low LOD (0.14 mg kg-1) and LOQ (0.47 mg kg-1), r 2 = 0.9994, and intraday and interday precision of 3.5 and 4.9%, respectively. The developed methodology was applied to the determination of formaldehyde in different cork agglomerate samples, and contents between 1.9 and 9.4 mg kg-1 were found. Furthermore, formaldehyde was also determined by the standard method EN 717-3 for comparison purposes; no significant differences between the results of both methods were observed. Graphical abstract Representation of the GDME system and its main components.

An Insight on Salting-out Assisted Liquid-Liquid Extraction for Phytoanalysis.

INTRODUCTION: Salting-out assisted liquid-liquid extraction (SALLE) is a technique that, although simple and not requiring any complex equipment, is very powerful and versatile. It has obtained growing interest in bioanalysis particularly when combined with chromatographic techniques. OBJECTIVES: Herein, fennel seeds (Foeniculum vulgare Mill.) were used as a case-study to show the application of SALLE in phytochemical analysis. MATERIAL AND METHODS: SALLE combined with HPLC-UV-MS/MS and GC-MS. RESULTS: By HPLC-UV-MS/MS analysis of the organic extract it was possible to identify various phenolic compounds, including quercetin derivatives, caffeic acid, p-coumaric acid and chlorogenic acid. The main compounds identified by GC-MS were estragole, fenchone, anisaldehyde, anethole, benzaldehyde, camphor and apiole. CONCLUSION: HPLC and GC analysis of the extracts showed that it is possible to isolate, in only one step, a wide range of compounds with distinct properties, allowing a detailed phytochemical analysis. Copyright © 2017 John Wiley & Sons, Ltd.

Isolation of Cells Specialized in Anticancer Alkaloid Metabolism by Fluorescence-Activated Cell Sorting.

Plant specialized metabolism often presents a complex cell-specific compartmentation essential to accomplish the biosynthesis of valuable plant natural products. Hence, the disclosure and potential manipulation of such pathways may depend on the capacity to isolate and characterize specific cell types. Catharanthus roseus is the source of several medicinal terpenoid indole alkaloids, including the low-level anticancer vinblastine and vincristine, for which the late biosynthetic steps occur in specialized mesophyll cells called idioblasts. Here, the optical, fluorescence, and alkaloid-accumulating properties of C. roseus leaf idioblasts are characterized, and a methodology for the isolation of idioblast protoplasts by fluorescence-activated cell sorting is established, taking advantage of the distinctive autofluorescence of these cells. This achievement represents a crucial step for the development of differential omic strategies leading to the identification of candidate genes putatively involved in the biosynthesis, pathway regulation, and transmembrane transport leading to the anticancer alkaloids from C. roseus.

Chromatographic analysis of methylglyoxal and other α-dicarbonyls using gas-diffusion microextraction.

Many α-dicarbonyl compounds such as methylglyoxal, diacetyl and pentane-2,3-dione are important quality markers of processed foods. They are produced by enzymatic and chemical processes, the Maillard reaction is the most known chemical route for α-dicarbonyl formation. In the case of methylglyoxal, there are obstacles to be overcome when analysing this compound due to its high reactivity, low volatility and low concentration. The use of extraction techniques based on the volatilization of methylglyoxal (like solid-phase microextraction) showed to be ineffective for the methylglyoxal extraction from aqueous solutions. Therefore, derivatization is typically applied to increase analyte's volatility. In this work a new methodology for the extraction and analysis of methylglyoxal and also diacetyl and pentane-2,3-dione from selected food matrices is presented. It is based on a gas-diffusion microextraction step followed by high performance liquid chromatographic analysis. It was successfully applied to port wines, black tea and soy sauce. Methylglyoxal, diacetyl and pentane-2,3-dione were quantified in the following concentration ranges: 0.24-1.74 mg L(-1), 0.1-1.85 mg L(-1) and 0.023-0.15 mg L(-1), respectively. The main advantages over existing methodologies are its simplicity in terms of sample handling, not requiring any chemical modification of the α-dicarbonyls prior to the extraction, low reagent consumption and short time of analysis.

Application of gas-diffusion microextraction to the analysis of free and bound acetaldehyde in wines by HPLC-UV and characterization of the extracted compounds by MS/MS detection.

In wines, the presence of high levels of acetaldehyde (AA) not only is responsible for undesirable characteristic odours but can also cause health adverse effects. Such sensorial activity of AA can be overcome by adding sulphites during winemaking, due to the formation of adducts between AA and sulphites, which lower the sensorial impact of AA. Nevertheless, bound AA can be released during wine storage; therefore, the knowledge of its total amount can be important to estimate the long-term wine quality. The proposed methodology is based on the extraction of AA from wines using gas-diffusion microextraction and determination by liquid chromatography. Free and bound forms of AA could be differentiated and determined using an alkaline hydrolysis step to dissociate the sulphites-AA adducts. This methodology was successfully applied to different wine types, with free AA values ranging between 5 and 26 mg L(-1) and total form between 154 and 906 mg L(-1). Bound AA was above 90% of the total content determined for all samples analysed, and higher amounts were obtained for white wines (around 98%). Other carbonyl compounds were also identified in the extracts using mass spectrometry.