Development of an easy-to-set-up multiple heart-cutting achiral-chiral LC-LC method for the analysis of branched-chain amino acids in commercial tablets.

In this paper, the development and application of a multiple heart-cutting achiral-chiral LC-LC method (mLC-LC) for the analysis of dansylated (Dns) branched-chain amino acids in commercial tablets are described. In the first dimension, a Waters Xbridge RP C18 achiral column was used under gradient conditions with buffered aqueous solution and acetonitrile. The elution order Dns-valine (Dns-Val) < Dns-isoleucine (Dns-Ile) < Dns-leucine (Dns-Leu) turned out with full resolution between adjacent peaks: 7.25 and 1.50 for the Val/Ile and the Ile/Leu pairs, respectively. A "research" validation study was performed, revealing high accuracy (Recovery%) and precision (RSD%) using two external set solutions, respectively, in the range 93.7%-104.1% and 0.4%-3.2%. The C18 column was connected via a two-position six-port switching valve to the quinidine-based Chiralpak quinidine-anion-exchange chiral column. A water/acetonitrile, 30/70 (v/v) with 50 mM ammonium acetate (apparent pH of 5.5) eluent allowed getting the three enantiomers' pairs resolved: RS equal to 4.3 for Dns-Val and Dns-Ile, and 1.7 for Dns-Leu. The application of the mLC-LC method confirmed that the content of Val, Ile, and Leu in the tablets was compliant with that labeled by the producer. Only l-enantiomers were found in the food supplement, as confirmed by LC-MS/MS analysis.

Plasma lipidomics analysis reveals altered profile of triglycerides and phospholipids in children with Medium-Chain Acyl-CoA dehydrogenase deficiency.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most prevalent mitochondrial fatty acid ß-oxidation disorder. In this study, we assessed the variability of the lipid profile in MCADD by analysing plasma samples obtained from 25 children with metabolically controlled MCADD (following a normal diet with frequent feeding and under l-carnitine supplementation) and 21 paediatric control subjects (CT). Gas chromatography-mass spectrometry was employed for the analysis of esterified fatty acids, while high-resolution C18-liquid chromatography-mass spectrometry was used to analyse lipid species. We identified a total of 251 lipid species belonging to 15 distinct lipid classes. Principal component analysis revealed a clear distinction between the MCADD and CT groups. Univariate analysis demonstrated that 126 lipid species exhibited significant differences between the two groups. The lipid species that displayed the most pronounced variations included triacylglycerols and phosphatidylcholines containing saturated and monounsaturated fatty acids, specifically C14:0 and C16:0, which were found to be more abundant in MCADD. The observed changes in the plasma lipidome of children with non-decompensated MCADD suggest an underlying alteration in lipid metabolism. Therefore, longitudinal monitoring and further in-depth investigations are warranted to better understand whether such alterations are specific to MCADD children and their potential long-term impacts.

Nutritional and lipidomics biomarkers of docosahexaenoic acid-based multivitamin therapy in pediatric NASH.

Two recent randomized controlled trials demonstrated improved radiographic, histological and hepatometabolic cues of non-alcoholic steatohepatitis (NASH) in pediatric patients treated with the ω-3 fatty acid docosahexaenoic acid (DHA) in combination with vitamin D (VD) or with choline (CHO) and vitamin E (VE), the DHA-VD and DHA-CHO-VE trials, respectively). In the present study we verified the nutritional compliance to these DHA-based multivitamin treatments; lipidomics biomarkers of the reported outcome on NASH indicators were also investigated. Samples were obtained from 30 biopsy-proven pediatric NASH patients of the DHA-CHO-VE trial randomized in multivitamin treatment group and placebo group (n = 15 each), and from 12 patients of the treatment group of the DHA-VD trial. All patients underwent 6-month therapy plus 6 months of follow-up. Plasma samples and clinical data were obtained at baseline and at the end of the study (12 months). Selected biomarkers included the free form of DHA and other ω-3 fatty acid arachidonic acid (AA), indices of the vitamin E status, and some hepatic metabolites of these lipids. Radiographic and histological improvements of treated patients were associated with increased concentrations of DHA, α-linolenic acid and α-tocopherol (i.e. VE), and with decreased AA that was also investigated in complex lipids by untargetd lipidomics. As a result a significantly lowered AA/DHA ratio was observed to represent the main indicator of the response to the DHA-based therapy. Furthermore, baseline levels of AA/DHA showed strong association with NAS and US improvement. A stable correction of DHA AA metabolism interaction is associated with the curative effect of this therapy and may represent a key nutritional endpoint in the clinical management of pediatric NASH.

Delving into the Polar Lipidome by Optimized Chromatographic Separation, High-Resolution Mass Spectrometry, and Comprehensive Identification with Lipostar: Microalgae as Case Study.

The work describes the chromatographic separation optimization of polar lipids on Kinetex-EVO, particularly focusing on sulfolipids in spirulina microalgae ( Arthrospira platensis). Gradient shape and mobile-phase modifiers (pH and buffer) were tested on lipid standards. Different conditions were evaluated, and resolution, peak capacity, and peak shape were calculated both in negative mode, for sulfolipids and phospholipids, and in positive mode, for glycolipids. A high-confidence lipid identification strategy was also applied. In collaboration with software creators and developers, Lipostar was implemented to improve the identification of phosphoglycerolipids and to allow the identification of glycosylmonoradyl- and glycosyldiradyl-glycerols classes, the last being the main focus of this work. By this approach, an untargeted screening also for searching lipids not yet reported in the literature could be accomplished. The optimized chromatographic conditions and database search were tested for lipid identification first on the standard mixture, then on the polar lipid extract of spirulina microalgae, for which 205 lipids were identified.

Passive Intestinal Absorption of Representative Plant Secondary Metabolites: A Physicochemical Study.

Natural products are generally ingested as part of traditional herbal decoctions or in the current diet. However, in natural product research, the bioavailability of secondary metabolites is often poorly investigated. In this work, a systematic study was carried out in order to highlight the physicochemical parameters that mainly influence the passive intestinal absorption of natural products. For this, a representative set of natural products including alkaloids, coumarins, flavonoid aglycones and glycosides, and carboxylic acids was selected and their physicochemical properties were predicted using relevant Volsurf+ descriptors. The chemical space obtained with this unbiased method was then correlated with experimental passive intestinal permeability data, which highlighted the main influence of lipophilicity, global hydrophilicity, size, and the ionisation state on passive intestinal absorption of natural products. Since the pH range encountered in the intestine is wide, the influence of the ionisation was investigated deeper experimentally. The ionisation state of weakly ionisable natural products, such as flavonoid aglycones, alkaloids, and carboxylic acids, was found to prevent the passive intestinal absorption of such natural products completely. In addition, the impact of solubility issues on passive permeability results was evaluated in cases of poorly water-soluble natural products, such as flavonoid aglycones and coumarins. The biomimetic fasted state simulated fluid-version 2 was found to improve the apparent solubility of such poorly soluble natural products without influencing their permeability behaviours. The use of such a solubilising buffer was found to be well adapted to the hexadecane membrane-parallel artificial membrane permeability assay and can circumvent the solubility issues encountered with poorly soluble natural products in such an assay.