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This study successfully prepared different loading levels of cyano-functionalized RCC3 molecular cage silica gel stationary phase (RCC3-CN@SiO 2 ) through aldehyde-amine condensation reaction and subsequent modification strategies. Fourier transform infrared spectroscopy, thermogravimetric analysis, nitrogen adsorption-desorption, and scanning electron microscopy confirmed the successful synthesis of RCC3-CN@SiO 2 chromatographic stationary phase. The research demonstrates that due to hydrophobic/hydrophilic interactions, π-π interactions, hydrogen bonding, and size-selective porous structure, the stationary phase effectively separates moderately polar and weakly polar compounds in reversed-phase liquid chromatography (RPLC) mode, exhibiting hydrophobic selectivity comparable to the commercial DaisoC18-RP columns. Additionally, the tertiary amine and cyanogen groups on the molecular cage surface enhance the interaction with polar compounds, successfully separating nucleosides, sulfonamides, amino acids, and sugars in hydrophilic interaction chromatography (HILIC) mode. Further applications in the separation analysis of acidic drugs, alkaline drugs, cinnamic acid natural products, and chiral compounds demonstrate the multifunctional chromatographic capabilities for diverse compound types. Compared to Unitary Diol commercial columns, the prepared stationary phase showed significant advantages in wide polarity range separation performance. Moreover, through nucleoside compound separation mode switching analysis, RCC3-CN@SiO 2 stationary phase further validates its favorable performance in both RPLC and HILIC modes, demonstrating extensive potential applications in the field of analytical chemistry. Importantly, the stationary phase exhibits efficient separation of nucleoside compounds in pure water systems, aligning with the principles of green analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The separation of enantiomers using chiral membranes has garnered much research interest. In this study, the enantioseparation of amino acids using chiral membranes, namely graphene oxide-ethylenediamine-maltodextrin (GO-EDA-MD) and GO-EDA-hydroxypropyl-MD (GO-EDA-HP-MD), was evaluated. HP-MD and MD were investigated as chiral selectors due to their inherent chirality. Various characterization techniques, including atomic force microscopy, Fourier transform infrared spectrometry, field emission scanning electron microscopy, water contact angle analysis, tensile properties, and thermal gravimetric analysis were employed to analyze the membrane structures. The evaluation of enantioseparation performance was conducted by employing tryptophan, phenylalanine, and tyrosine enantiomers. Optimal conditions for enantiomer separation were achived using a GO-EDA-HP-MD chiral composite (1.75 wt%), a feed concentration of 10 mg/L for each enantiomer, a separation time of 15 min, and a membrane effective surface area of 1.0 cm 2 . Also, the bovine serum albumin rejection was 90.0 %, and the water flux reached 37.1 L m -2 h -1 . The highest enantiomeric excess (ee.%) values were 46.33 %, 76.97 %, and 73.04 % for tryptophan, phenylalanine, and tyrosine, respectively. The impact of voltage on ee.% and substance flux was also explored. This membrane was able to separate enantiomers successfully., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study employed a diverse approach to extract antioxidant peptides from red seaweed Palmaria palmata , recognized for its comparatively high protein content. Initially, an aqueous extraction of the entire seaweed was performed, followed by enzymatic hydrolysis of the solid residues prepared from the first step. The effects of three different pH levels (3, 6, and 9) during the aqueous extraction were also examined. Results indicated that the solid fraction from the sequential extraction process contained significantly higher levels of proteins and amino acids than other fractions ( p < 0.05). Furthermore, the solid fractions (IC 50 ranging from 2.29 to 8.15 mg.mL -1 ) demonstrated significantly greater free radical scavengers than the liquid fractions (IC 50 ranging from 9.03 to 10.41 mg.mL -1 or not obtained at the highest concentration tested) at both stages of extraction ( p < 0.05). Among the solid fractions, those produced fractions under alkaline conditions were less effective in radical scavenging than the produced fractions under acidic or neutral conditions. The fractions with most effective metal ion chelating activity were the solid fractions from the enzymatic stage, particularly at pH 3 (IC 50 = 0.63 ± 0.04 mg.mL -1 ) and pH 6 (IC 50 = 0.89 ± 0.07 mg.mL -1 ), which were significantly more effective than those from the initial extraction stage ( p < 0.05). Despite no significant difference in the total phenolic content between these solid fractions and their corresponding liquid fractions (3.79 ± 0.05 vs. 3.48 ± 0.02 mg.mL -1 at pH 3 and 2.43 ± 0.22 vs. 2.51 ± 0.00 mg.mL -1 at pH 6) ( p > 0.05), the observed antioxidant properties may be attributed to bioactive amino acids such as histidine, glutamic acid, aspartic acid, tyrosine, and methionine, either as free amino acids or within proteins and peptides.

TLC is used globally, yet less attention has been paid to TLC (in enantioseparation) despite its advantages. The present paper describes/reviews successfully practiced direct approaches of 'chiral additive in achiral stationary phase' (as an application of in-home thought out, prepared, tested, and modified chiral stationary phase), 'pre-mixing of chiral reagent with the enantiomeric mixture' (an approach using both achiral phases during chromatographic separation) and 'chiral additive in mobile phase', and chiral ligand exchange for enantioseparation of DL-amino acids, their derivatives, and some active pharmaceutical ingredients. It provided efficient enantioseparation, quantitative determination, and isolation of native forms via in-situ formation of non-covalent diastereomeric pair. The mechanism of enantioseparation in these approaches has been discussed along with the isolation and establishment of the structure of diastereomers. This may help chemists gain useful insights into fields outside their specialization and the experts get brief accounts of recent key developments, providing solutions for sustainable development of less expensive methods for control of enantiomeric purity and isolation of native enantiomers., (© 2024 John Wiley & Sons Ltd.)

The epoxy propanol molecular cage bonded silica stationary phase, RCC3-GLD@silica, synthesized through the ring-opening reaction of secondary amine with epoxy propanol using RCC3-R as the scaffold unit, was successfully prepared as confirmed by infrared spectroscopy, thermogravimetric analysis, and nitrogen adsorption-desorption characterization. This stationary phase demonstrated excellent separation performance in both reversed-phase and hydrophilic chromatography modes, effectively separating a wide variety of compounds including alkylbenzenes, polycyclic aromatic hydrocarbons, phenols, anilines, sulfonamides, nucleosides, amino acids, sugars, and acids. The development of RCC3-GLD@silica benefits from the synergistic effects of its hydrophobic and hydrophilic actions, as evidenced by the U-shaped characteristic of the retention factor for nucleoside compounds with changes in the aqueous content of the mobile phase, further confirming the simultaneous presence of reversed-phase and hydrophilic chromatography mechanisms. Not only did this stationary phase successfully separate 33 compounds in reversed-phase chromatography mode, but it also separated 54 compounds in hydrophilic interaction chromatography mode, showcasing its broad separation capability from weakly polar to strongly polar compounds on a single chromatographic column. This indicates a wide application prospect in the field of chromatographic analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Enantioseparation of amino acids is considered as a challenging task due to the extreme structural similarity of their enantiomers. Herein, teicoplanin was modified with different chemical equivalents of azide groups and attached to silica particles by employing Click Chemistry for resolution of chiral amino acids for the first time. Interestingly, teicoplanin modified with 5-fold the chemical equivalent of azide groups (TK-2 CSP) exhibited superior amino acid separation ability compared to two other columns: one modified with only 1-fold the chemical equivalent of azide groups (TK-1 CSP), and the other modified with excess azide groups (TK-3 CSP). Additionally, the TK-2 CSP exhibited superior enantioselectivity when separating amino acids containing hydrophobic alkyl side chains in comparison to other teicoplanin-based CSPs. The TK-2 CSP column allows the baseline separation of 7 native amino acids. Molecular docking demonstrates that effective enantioseparation arises from distinct patterns of interaction between the host and guest molecules. Moreover, (p-methyl) phenylcarbaminoylated-teicoplanin CSP (TK-4, TK-5 CSP) were prepared by post-modification from TK-1 CSP and TK-2 CSP to isolate Fmoc-modified amino acids. This work explores the impact of various modification methods on the enantioseparation effects of host molecules and paves the way for expanding the potential applications of teicoplanin and macrocyclic glycopeptide molecules., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In this study, several amino acids deep eutectic solvents were prepared using L-valine and L-leucine as hydrogen bond acceptors, and L-lactic acid and glycerol as hydrogen bond donors. These amino acids' deep eutectic solvents were first used as buffer additives to construct several synergistic systems along with maltodextrin in capillary electrophoresis for the enantioseparations of four racemic drugs. Compared with single maltodextrin system, the separations of model drugs in the synergistic systems were significantly improved. Some key parameters affecting chiral separation such as maltodextrin concentration, deep eutectic solvent concentration, buffer pH, and applied voltage were optimized. In order to further understand the specific mechanism of the amino acids deep eutectic solvents in improving chiral separation, we first calculated the binding constants of maltodextrin with enantiomers using the capillary electrophoresis method in the two separation modes, respectively. We also used molecular simulation to calculate the binding free energy of maltodextrin with enantiomers. It is the first time that amino acids deep eutectic solvents were used for enantioseparation in capillary electrophoresis, which will greatly promote the development of deep eutectic solvents in the field of chiral separation., (© 2024 Wiley‐VCH GmbH.)

Marine rhodophyta are known to synthesize specific secondary metabolites, mycosporine-like amino acids (MAAs), to protect themselves from harmful UV-radiation. Shinorine and porphyra-334 are among the most abundant representatives of this compound class. In the present work, a novel approach for their isolation is described. As a first step, a fast centrifugal partition chromatography method, with an aqueous two-phase system comprising water, ethanol, ammonium sulfate and methanol in ascending mode, was developed to isolate the two MAAs from crude aqueous-methanolic extracts of three algal species within 90 min. The compounds could be isolated when just one of them was present in a sample or also both at the same time. By employing solid phase extraction as a second purification step, the individual MAAs were obtained in high purity and good quantity within a much shorter time frame than the established purification protocols, e.g., semi-preparative HPLC. For example, from 4 g Porphyra sp. (Nori) crude extract, 15.7 mg shinorine and 36.2 mg porphyra-334 were isolated. Both were highly pure, as confirmed by TLC, HPLC-MS and NMR analyses.

Through an appropriate computational protocol and environmental simulation, a satisfactory fit was observed for the theoretical electronic circular dichroism (ECD) spectra of 19 chiral amino acids (AAs), which correspondeds to the forms of the AAs in aqueous solution. Methods for enantioseparation of these chiral AAs by capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) were developed. Combining ECD with chromatographic separation methods, enantiomers were identified and quantified independent of a single enantiomer standard., Competing Interests: Declaration of Competing Interest There are no known conflicts to declare., (Copyright © 2021. Published by Elsevier B.V.)

Enantioseparation of nineteen ß 2 -amino acids has been performed by liquid chromatography on chiral stationary phases based on native teicoplanin and teicoplanin aglycone covalently bonded to 2.7 µm superficially porous silica particles. Separations were carried out in unbuffered (water/methanol), buffered [aqueous triethylammonium acetate (TEAA)/methanol] reversed-phase (RP) mode, and in polar-ionic (TEAA containing acetonitrile/methanol) mobile phases. Effects of pH in the RP mode, acid and salt additives, as well as counter-ion concentrations on chromatographic parameters have been studied. The structure of selectands (ß 2 -amino acids possessing aliphatic or aromatic side chains) and selectors (native teicoplanin or teicoplanin aglycone) was found to have a considerable influence on separation performance. Analysis of van Deemter plots and determination of thermodynamic parameters were performed to further explore details of the separation performance., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Reliable separation of peptides, amino acids and proteins as accurate as possible with the maximum conformation and biological activity is crucial and essential for drug discovery. Polysaccharide, as one of the most abundant natural biopolymers with optical activity on earth, is easy to be functionalized due to lots of hydroxyl groups on glucose units. Over the last few decades, polysaccharide derivatives are gradually employed as effective separation media. The highly-ordered helical structure contributes to complex, diverse molecular recognition ability, allowing polysaccharide derivatives to selectively interact with different analytes. This article reviews the development, application and prospects of polysaccharides as separation media in the separation of proteins, peptides and amino acids in recent years. The chiral molecules mechanism, advantages, limitations, development status and challenges faced by polysaccharides as separation media in molecular recognition are summarized. Meanwhile, the direction of its continued development and future prospects are also discussed., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We designed and synthesized three novel derivatization reagents bearing chiral 4-imidazolidinone, namely succinimidyl 2-(3-((benzyloxy)carbonyl)-1-methyl, ethyl, and -phenyl-5-oxoimidazolidin-4-yl)acetates (CIMs), for use in liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The CIMs were able to discriminate primary amines from other compounds such as secondary amines and phenols, based on their unique m/z reduction of precursor ion to form product ion in MS/MS. As amino acid derivatization reagents, the CIMs were compared in terms of enantioseparation of amino acid and detection sensitivity. CIM a -OSu with 1-methyl-5-oxoimidazolidinone moiety gave the best optical resolution and detection sensitivity among the CIM reagents. Next, we applied (R)-CIM a -OSu to determine amino acids in miso by LC-triple-quadrupole MS. The proposed method achieved simultaneous determination of 20 l-amino acids and two d-amino acids (d-alanine and d-serine) in the sample with a high sensitivity (limits of detection 5-238 fmol, signal-to-noise ratio 3.3). After derivatization with CIM a -OSu, it was possible to determine whether each peak in the chromatogram was a component of primary amine or not, by using a high-resolution orbitrap MS instrument., Competing Interests: Declaration of Competing Interest There are no conflicts of interest to declare., (Copyright © 2021. Published by Elsevier B.V.)

In this study, we examined aqueous extracts of the edible mushrooms Pleurotus ostreatus (oyster mushroom) and Lentinula edodes (shiitake mushroom). Proteome analysis was conducted using LC-Triple TOF-MS and showed the expression of 753 proteins by Pleurotus ostreatus , and 432 proteins by Lentinula edodes . Bioactive peptides: Rab GDP dissociation inhibitor, superoxide dismutase, thioredoxin reductase, serine proteinase and lectin, were identified in both mushrooms. The extracts also included promising bioactive compounds including phenolics, flavonoids, vitamins and amino acids. The extracts showed promising antiviral activities, with a selectivity index (SI) of 4.5 for Pleurotus ostreatus against adenovirus (Ad7), and a slight activity for Lentinula edodes against herpes simplex-II (HSV-2). The extracts were not cytotoxic to normal human peripheral blood mononuclear cells (PBMCs). On the contrary, they showed moderate cytotoxicity against various cancer cell lines. Additionally, antioxidant activity was assessed using DPPH radical scavenging, ABTS radical cation scavenging and ORAC assays. The two extracts showed potential antioxidant activities, with the maximum activity seen for Pleurotus ostreatus (IC50 µg/mL) = 39.46 ± 1.27 for DPPH; 11.22 ± 1.81 for ABTS; and 21.40 ± 2.20 for ORAC assays. This study encourages the use of these mushrooms in medicine in the light of their low cytotoxicity on normal PBMCs vis à vis their antiviral, antitumor and antioxidant capabilities.

Although covalent organic frameworks (COFs) have earned significant interest in separation applications, the use of COFs in biomolecule separation remains unexplored. We examined the ionic COF Py-BPy 2+ -COF as an ion exchange material for biomolecule separation. After characterizing the properties of the synthesized COF with a variety of techniques, binding experiments with both large and small biomolecules were performed. High adsorption capacities of amino acids with different hydrophobicity and charge, as well as proteins of different isoelectric points and molecular weights, were determined in batch equilibrium experiments. Desorption experiments with mixtures of model proteins demonstrated an ability to successfully separate one protein from another with the selectivity hypothesized to be a combination of the isoelectric point, hydrophobicity, and ability to penetrate the crystalline material. Overall, the results demonstrated that Py-BPy 2+ -COF can be exploited as a robust crystalline anion exchange biomolecule separation material.

Protein and amino acids in pollen are important nutritional components for larval development in several insect species, especially in Apoidea. The Bradford assay is a widely used method to measure relative protein content of pollen, which can shed light on pollen quality and consequences to fitness. Prior to using the Bradford assay, protein must be released from pollen grains, often using a mixture of chemical and mechanical fracturation methods. In this study, we tested the efficacy of protein extraction without using mechanical fracturation. We used pollen collected by the solitary bee Osmia lignaria Say to compare two known buffers associated with pollen protein analysis: phosphate-buffered saline and sodium hydroxide, and deionized water, and with different pollen weights from which we quantified protein using the Bradford assay. While all buffers and deionized water were useful in releasing protein from pollen grains collected by O. lignaria, the use of sodium hydroxide resulted in significantly higher protein quantification across all pollen weights. This methodological study can inform future studies of pollen nutrition in pollen-foraging species., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America.)

A modified C 18 column (Silpr-2MI-C18) was prepared using 2-methylindole and C 18 reagent. The extent of C 18 hydrocarbon chain, conjugative rings and anion exchange site provided multiple retention mechanisms, including reversed-phase liquid chromatography (RPLC), π-π interaction, hydrophilic interaction liquid chromatography (HILIC) and anion exchange chromatography (AEC). The separation of protected amino acids was investigated on the commercial C 18 and Silpr-2MI-C18 columns, while the chromatographic conditions, including methanol content and pH of the mobile phase, were studied. The separation arrangement of the hydrophilic amino acids was different on the Silpr-2MI-C18 column compared to the commercial C 18 column under RPLC mode. Furthermore, these amino acids were separated on the Silpr-2MI-C18 column under HILIC mode. The modified C 18 column was employed to separate amino acids, alkylbenzenes and polycyclic aromatic hydrocarbons under RPLC mode and inorganic anion under AEC mode. The results confirm that this new stationary phase of RPLC/HILIC/AEC has multiple interactions with different analytes. Effective retention of biological samples was found on the Silpr-2MI-C18 column by comparing the results obtained from the commercial C 18 column., (© 2021 John Wiley & Sons, Ltd.)

This study presents the validation of a high-performance liquid chromatography diode array detector (HPLC-DAD) method for the determination of different mycosporine-like amino acids (MAAs) in the red alga Bostrychia scorpioides . The investigated MAAs, named bostrychines, have only been found in this specific species so far. The developed HPLC-DAD method was successfully applied for the quantification of the major MAAs in Bostrychia scorpioides extracts, collected from four different countries in Europe showing only minor differences between the investigated samples. In the past, several Bostrychia spp. have been reported to include cryptic species, and in some cases such as B. calliptera , B. simpliciuscula , and B. moritziana , the polyphyly was supported by differences in their MAA composition. The uniformity in the MAA composition of the investigated B. scorpioides samples is in agreement with the reported monophyly of this Bostrychia sp.

Microalgae are known to be rich in protein. In this study, we aim to investigate methods of producing and purifying proteins of 98 microalgae including Chlorella vulgaris, Arthrospira platensis , Nostoc sp., Dunaliella salina , and Pleurochrysis carterae (Baltic Sea). Therefore, we studied their amino acid composition and developed a two-stage protein concentrate purification method from the microalgae biomass. After an additional stage of purification, the mass fraction of protein substances with a molecular weight greater than 50 kDa in the protein concentrate isolated from the biomass of the microalga Dunaliella salina increased by 2.58 times as compared with the mass fraction before filtration. In the protein concentrate isolated from the biomass of the microalga Pleurochrysis cartera , the relative content of the fraction with a molecular weight greater than 50.0 kDa reached 82.4%, which was 2.43 times higher than the relative content of the same fractions in the protein concentrate isolated from this culture before the two-stage purification. The possibilities of large-scale industrial production of microalgae biomass and an expanded range of uses determine the need to search for highly productive protein strains of microalgae and to optimize the conditions for isolating amino acids from them.

The average amino acid identity (AAI) is an index of pairwise genomic relatedness, and multiple studies have proposed its application in prokaryotic taxonomy and related disciplines. AAI demonstrates better resolution in elucidating taxonomic structure beyond the species rank when compared with average nucleotide identity (ANI), which is a standard criterion in species delineation. However, an efficient and easy-to-use computational tool for AAI calculation in large-scale taxonomic studies is not yet available. Here, we introduce a bioinformatic pipeline, named EzAAI, which allows for rapid and accurate AAI calculation in prokaryote sequences. The EzAAI tool is based on the MMSeqs2 program and computes AAI values almost identical to those generated by the standard BLAST algorithm with significant improvements in the speed of these evaluations. Our pipeline also provides a function for hierarchical clustering to create dendrograms, which is an essential part of any taxonomic study. EzAAI is available for download as a standalone JAVA program at http://leb.snu.ac.kr/ezaai .

Herein, the fabrication of a fascinating multifunctional cyclodextrin (CD) chiral stationary phase and its chiral separation performance in capillary electrochromatography are proposed. A facile interfacial polymerization was used to anchor ethanediamine-β-cyclodextrin (EDA-β-CD) polymerized with trimesoyl chloride (TMC) and to form the chiral stationary phase (CSP) composite onto the surface wall of the capillary. The characters of prepared columns were confirmed by Fourier transform infrared spectroscopy (FT-IR), X-ray Photoelectron Spectrometer (XPS), scanning electron microscopy (SEM) and energy dispersive X-ray spectrometry (EDS). This novel CSP offers multi-typical interactions including hydrogen bonding, π-interaction, hydrophobic and electrostatic interaction as well as steric effects which contribute to prominent chiral recognition for Dansyl-DL-amino acids in CEC modes. The EDA-β-CD modified column showed eminent enantioseparation performance towards five Dansyl-DL-amino acids (the DL-forms of valine, threonine, leucine, phenylalanine, serine). Besides, the prepared columns were perfectly reproducible and stable. The relative standard deviations of the enantiomer retention times for intra-day (n = 5), inter-day (n = 3) runs and column-to-columns (n = 3) are below 0.54%, 1.35% and 4.89%, individually. This innovative chiral stationary phase shows a broader application view and scope in chiral recognition domain., Competing Interests: Declaration of Competing Interest Authors declare no conflict of interest., (Copyright © 2021. Published by Elsevier B.V.)

Evaluation of the chromatographic properties of covalently bonded hyperbranched stationary phase based on poly(styrene-divinylbenzene) (PS-DVB) and containing zwitterionic fragments in the structure of functional layer was conducted in suppressed ion chromatography (IC), reversed phase high performance liquid chromatography (RP HPLC), and hydrophilic interaction liquid chromatography (HILIC) modes. Besides the possibility of resolving 20 inorganic anions and organic acids using KOH eluent in suppressed IC, prepared resin provided the separation of alkylbenzenes in RP HPLC, water-soluble vitamins, amino acids, and sugars in HILIC mode. Trends in the retention of hydrophobic and polar analytes on the prepared stationary phase indicated the dominating effect of analyte nature on the retention mechanism and proved satisfactory hydrophilization of PS-DVB surface with hyperbranched functional layer for retaining polar compounds. The obtained results revealed good prospects of using hydrophobic PS-DVB substrate for preparing stationary phases for mixed-mode chromatography., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

The aim of this study was to establish a method for geographical origins identification of Panax notoginseng (P. notoginseng) based on abundant chromatographic spectral information. Characteristic fingerprints of P. notoginseng extracts samples were generated by Multi-wavelength Fusion Profiling (MWFP) method based on the HPLC fingerprints established at three wavelengths of 203 nm, 270 nm and 325 nm. The samples grouping results calculated with the averagely linear quantified fingerprint method (ALQFM) and the unsupervised statistical methods based on fusion fingerprints matches with the geographical origins. The Multi-wavelength Fusion Profiling (MWFP) method has been successfully applied to identification of geographical origins of P. notoginseng and shows the advantages compared with single-channel fingerprints. In addition, eight physiologically active components, including four saponins, two flavones and two amino acids, were identified from the most relevant ingredients of P. notoginseng geographical origins by fusion fingerprint-efficacy relationship analysis. Besides the recognized active saponins, other categories of active ingredients such as flavonoids and amino acids should be paid attention to in the producing areas identification or the quality judgment of P. notoginseng.

This study was designed to profile the metabolites of Isochrysis galbana , an indigenous and less explored microalgae species. 1 H Nuclear Magnetic Resonance (NMR) spectroscopy and Liquid Chromatography-Mass Spectrometry (LCMS) were used to establish the metabolite profiles of five different extracts of this microalga, which are hexane (Hex), ethyl acetate (EtOAc), absolute ethanol (EtOH), EtOH:water 1:1 (AqE), and 100% water (Aq). Partial least square discriminant analysis (PLS-DA) of the generated profiles revealed that EtOAc and Aq extracts contain a diverse range of metabolites as compared to the other extracts with a total of twenty-one metabolites, comprising carotenoids, polyunsaturated fatty acids, and amino acids, that were putatively identified from the NMR spectra. Meanwhile, thirty-two metabolites were successfully annotated from the LCMS/MS data, ten of which (palmitic acid, oleic acid, α-linolenic acid, arachidic acid, cholesterol, DHA, DPA, fucoxanthin, astaxanthin, and pheophytin) were similar to those present in the NMR profile. Another eleven glycerophospholipids were discovered using MS/MS-based molecular network (MN) platform. The results of this study, besides providing a better understanding of I. galbana 's chemical make-up, will be of importance in exploring this species potential as a feed ingredient in the aquaculture industry.

The recovery of amino acids and other important bioactive compounds from the comb penshell ( Atrina pectinata ) using subcritical water hydrolysis was performed. A wide range of extraction temperatures from 140 to 290 °C was used to evaluate the release of proteins and amino acids. The amount of crude protein was the highest (36.14 ± 1.39 mg bovine serum albumin/g) at 200 °C, whereas a further increase in temperature showed the degradation of the crude protein content. The highest amount of amino acids (74.80 mg/g) was at 230 °C, indicating that the temperature range of 170-230 °C is suitable for the extraction of protein-rich compounds using subcritical water hydrolysis. Molecular weights of the peptides obtained from comb penshell viscera decreased with the increasing temperature. SDS-PAGE revealed that the molecular weight of peptides present in the hydrolysates above the 200 °C extraction temperature was ≤ 1000 Da. Radical scavenging activities were analyzed to evaluate the antioxidant activities of the hydrolysates. A. pectinata hydrolysates also showed a particularly good antihypertensive activity, proving that this raw material can be an effective source of amino acids and marine bioactive peptides.

Spontaneous cereal fermentations involve diverse lactic acid bacteria (LAB) and yeasts which may include multifunctional and safe or unsafe strains. This study assessed acidification ability, safety, antifungal activity and free amino acids release ability of LAB and yeasts previously isolated from spontaneously fermented cereal doughs in Benin. Fourteen LAB and thirteen yeast strains were studied in liquid media and/or in a model cereal dough prepared in laboratory conditions. Antifungal activity was assessed against Candida glabrata in liquid medium. Amino acids were determined by pre-column derivatization and separation with reversed-phase HPLC. Antimicrobial susceptibility was analysed by minimum inhibitory concentration determination. The acidification ability was higher for LAB compared to yeast strains. All LAB strains retarded the growth of C. glabrata Cg1 with the highest inhibition recorded for Weissella confusa Wc1 and Wc2. The highest free amino acid content was found in the doughs fermented with Pichia kudriavzevii Pk2 and Pk3. All the LAB strains were susceptible to ampicillin, chloramphenicol, erythromycin, but displayed phenotypic resistance to kanamycin, streptomycin and tetracycline. Positive PCR amplicon of resistance genes were detected in the following cases: 2 LAB strains were positive for kanamycin (aph(3)III), 5 strains were positive for streptomycin (aadA and/or strA and/or strB) and 3 strains were positive for tetracycline (tet (L) and/or tet (M)). For yeasts, most of the P. kudriavzevii strains were resistant to amphotericin B, fluconazole and itraconazole opposite to K. marxianus and Saccharomyces cerevisiae strains which were susceptible. The results obtained are valuable for selecting safe and multifunctional strains for cereal fermentation in West Africa.

Supercritical fluid chromatography (SFC) has unique separative characteristics distinguished from those of HPLC and gas chromatography. At present, SFC is widely used and there are many applications in various biological, medical, and pharmaceutical fields. In this review, we focus on recently developed novel techniques related to SFC separation including: new column stationary phases, microfluidics, two-dimensional separation, and gas-liquid separation. In addition, we discuss the application of SFC using a water-containing modifier to biological molecules such as amino acids, peptides, and small proteins that had been challenging analytes.

Of the various methods available for high-performance liquid chromatography separation of enantiomers (of e.g. amino acids and amino group containing compounds) by the pre-column derivatization approach, use of Marfey's reagent has been most successful with continued application since its introduction in 1984. The reagent is prepared from difluoro dinitro benzene by nucleophilic substitution of one of its F atoms by l-alanine amide. There is flexibility to prepare several chiral variants (by substituting the F atom with different chiral auxiliaries) and to tailor the hydrophobicity and resolution, ultimately, of the diastereomeric derivatives. The present paper assesses and reviews applications of Marfey's reagent and its chiral variants (i.e. other FDNP reagents) for enantioseparation of certain amino group containing drugs/amino acids, and to provide some case studies on enantiomeric separations that are important for the pharmaceutical industry. Various explanations for separation mechanism and elution order using FDNP reagents are included and the question of the configuration of the corresponding enantiomer using an indirect approach has also been addressed., (© 2020 John Wiley & Sons, Ltd.)

Amino acids are essential compounds for living organisms, and their determination in biological fluids is crucial for the clinical analysis and diagnosis of many diseases. However, the detection of most amino acids is hindered by the lack of a strong chromophore/fluorophore or electrochemically active group in their chemical structures. The highly sensitive determination of amino acids often requires derivatization. Capillary electrophoresis is a separation technique with excellent characteristics for the analysis of amino acids in biological fluids. Moreover, it offers the possibility of precapillary, on-capillary, or postcapillary derivatization. Each derivatization approach has specific demands in terms of the chemistry involved in the derivatization, which is discussed in this review. The family of homocyclic o-dicarboxaldehyde compounds, namely o-phthalaldehyde, naphthalene-2,3-dicarboxaldehyde, and anthracene-2,3-dicarboxaldehyde, are powerful derivatization reagents for the determination of amino acids and related compounds. In the presence of suitable nucleophiles they react with the primary amino group to form both fluorescent and electroactive derivatives. Moreover, the reaction rate enables all of the derivatization approaches mentioned above. This review focuses on articles that deal with using these reagents for the derivatization of amino acids and related compounds for ultraviolet-visible spectrometry, fluorescence, or electrochemical detection. Applications in capillary and microchip electrophoresis are summarized and discussed., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A new capillary electrophoresis method was applied to chiral separation of three amino acids, including D,L-tryptophan, D,L-tyrosine and D,L-phenylalanine. The chiral resolution was attained in an untreated fused-sillica capillary using a dual chiral selector, which was made up of Cu(II)-L-histidine complex and β-cyclodextrin (CD). The cardinal factors influencing its separation efficiency, such as chiral selectors, buffer pH and applied voltage, were optimized. Best results were acquired by using a buffer consisting of 10 mmol/L Cu(II), 13 mmol/L L-histidine, 8 mmol/L β-CD, 5 mmol/L phosphate adjusted to pH 5.0 and 15 kV applied voltage. All enantiomers were entirely resolved within 20 min with high resolutions of 3.6~6.1. The analysis method was verified through the determination of D,L-tryptophan in terms of linearity, precision and accuracy. And the robustness of this method was proved. The Limit of Detection and Limit of Quantification for both enantiomers were 2.5 and 5 μg/mL, respectively. The method was perfectly applied to the determination of the enantiomeric purity of L-tryptophan. Furthermore, the interaction between Cu(II)-L-histidine complex and β-CD was also studied using Ultraviolet-visible and 1H NMR spectroscopy to explain the synergistic effect involved. The results illustrated that Cu(II)-L-histidine complex and β-CD played a synergistic role in the enantiomeric separation of chiral drugs, with good prospects for application., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Fast enantiomeric separation of amino acids was studied by liquid chromatography/mass spectrometry (LC/MS) on a chiral crown ether stationary phase. A chiral crown ether bonded silica column (3 mm internal diameter (i.d.), 5 cm long) packed with 3 μm particles was employed instead of a 15 cm column packed with 5 μm particles used in our previous study. In addition, the extra-column variance, becoming more serious for smaller columns, was reduced by replacing 0.127 mm i.d. post-column tubes with shorter, smaller-diameter (0.0635 mm i.d.) tubes. The results demonstrated the benefits of using shorter columns packed with smaller particles and the reduction of the extra-column band broadening for fast enantiomeric separation. Finally, the enantiomeric separation of 18 pairs of proteinogenic amino acids was achieved within 2 min with a resolution (Rs) > 1.5 for each pair using an isocratic mobile phase of acetonitrile/water/trifluoroacetic acid (ACN/W/TFA) = 96/4/0.5, and a flow rate 1.2 mL/min at 30°C. This is the highest throughput method for simultaneous chiral separation of all proteinogenic amino acids except proline to date., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Herein, D- and L-oligopeptides were explored for the first time as chiral ligands in a chiral ligand exchange capillary electrophoresis (CLE-CE) protocol with Zn(II) as central ion for separation of derived d,l-amino acid enantiomers (Dns-D,L-AAs). The effect of four D- and L-oligopeptides, including glycine-l-lysine (Gly-L-Lys), glycine-d-lysine (Gly-D-Lys), l-lysine-lysine-OH and l-lysine-lysine-lysine-lysine-OH on the CLE-CE separation efficiency were evaluated. Thermodynamic calculations and circular dichroism spectra properties showed that the ternary species [(Gly-L-Lys)Zn(II)(D-AAs)] and [(Gly-L-Lys)Zn(II)(L-AAs)] presented the best stereoselectivity, possibly due to entropic effects. Notably, the migration order of Dns-D-AAs and Dns-L-AAs could be tuned by using Gly-D-Lys as the ligand for the CLE-CE system. To obtain satisfactory CLE-CE performance, the concentration ratio of Zn(II) to Gly-L-Lys, the pH of the buffer solution, and the concentration of Zn(II) were investigated. Under the optimized CLE-CE conditions using 100.0 mM H 3 BO 3 , 10.0 mM NH 4 Ac, 3.0 mM Zn (II) and 12.0 mM Gly-L-Lys as the buffer solution at pH 8.30, nine pairs of Dns-D,L-AAs achieved baseline separation, with the partial separation of another five pairs. Furthermore, the proposed CLE-CE protocol, which presented a good linear relationship with the concentration of the test analytes in the range of 75.0-625.0 μM (r 2  ≥ 0.994) and the limit of detection of the method was 5.0 μM, was successfully applied in the kinetics study of l-asparaginase using l-asparagine as the substrate. Our strategy shows the great potential of L-dipeptides in the CLE-CE separation of D,L-AAs enantiomers and bio-applications., Competing Interests: Declaration of competing interest There is no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Miniaturized LC has evolved at an exponential rate over the last 50 years. In the past decade, it has received considerable attention in the field of bioanalytical separation science and technology due to the need to measure different classes of biomolecules present in a variety of matrixes on a global scale to gain a deeper understanding of complex biological processes. This field has become a dominant area underpinning the molecular omics research (e.g., proteomics, metabolomics, lipidomics, and foodomics), allowing key insights into the function and mechanism of small to very large biomolecules on a molecular level. This Feature highlights the recent advances in molecular omics focusing on miniaturized LC technology combined with mass spectrometry-based platforms, with a particular emphasis on the strategies adopted and applications using new and sensitive nanoscale analytical methodologies.

Methamphetamine (MA) is a highly addictive central nervous system stimulant. Drug addiction is not a static condition but rather a chronically relapsing disorder. Hair is a valuable and stable specimen for chronic toxicological monitoring as it retains toxicants and metabolites. The primary focus of this study was to discover the metabolic effects encompassing diverse pathological symptoms of MA addiction. Therefore, metabolic alterations were investigated in human hair following heavy MA abuse using both targeted and untargeted mass spectrometry and through integrated network analysis. The statistical analyses ( t -test, variable importance on projection score, and receiver-operator characteristic curve) demonstrated that 32 metabolites (in targeted metabolomics) as well as 417 and 224 ion features (in positive and negative ionization modes of untargeted metabolomics, respectively) were critically dysregulated. The network analysis showed that the biosynthesis or metabolism of lipids, such as glycosphingolipids, sphingolipids, glycerophospholipids, and ether lipids, as well as the metabolism of amino acids (glycine, serine and threonine; cysteine and methionine) is affected by heavy MA abuse. These findings reveal crucial metabolic effects caused by MA addiction, with emphasis on the value of human hair as a diagnostic specimen for determining drug addiction, and will aid in identifying robust diagnostic markers and therapeutic targets.

The young leaves of green tea become lighter in color than usual when protected from sunlight by a shading net for about two weeks while growing. These leaves are called "shaded white leaf tea" or SWLT. In the eluate of SWLT, the amount of amino acids (361 mg/L) was significantly higher than that in regular tea (53.5 mg/L). Since theanine and arginine, the first and second most abundant amino acids in SWLT, have significant antistress effects, we examined the antistress effect of SWLT on humans. SWLT or placebo green tea (3 g) was eluted with room-temperature water (500 mL). Participants consumed the tea for one week prior to pharmacy practice and continued for 10 days in the practice period. The state-trait anxiety inventory, an anxiety questionnaire, tended to be scored lower in the SWLT group than the placebo, but other stress markers showed no differences. The effect of the difference in SWLT components examined with mice showed that aspartic acid and asparagine, which are abundant in SWLT, counteracted the antistress effects of theanine and arginine. Large amounts of caffeine also interfered with SWLT's antistress effect. Thus, SWLT, which is high in caffeine and amino acids, suppressed depressant behavior in mice.

In this study, 31 racemates of Nα-FMOC (fluorenylmethoxycarbonyl) amino acids (AAs) with different chemico-physical characteristics (neutral nonpolar, neutral polar, acidic and basic) have been successfully resolved in fast enantioselective chromatography on recently-developed zwitterionic-teicoplanin chiral stationary phases (CSPs). The CSPs were prepared by covalently bonding the teicoplanin selector on fully-porous particles of narrow dispersion particle-size distribution (particle diameter 1.9 µm) and superficially-porous particles (2.0 µm). Both the zwitterionic-teicoplanin CSPs have proved to be ideal media for the separation of this important class of compounds. In particular, the zwitterionic CSP prepared on superficially-porous particles exhibited superior enantioselectivity and resolution, compared to that made of fully porous particles, in virtue of more favorable thermodynamics. The zwitterionic nature of these CSPs allowed avoiding the annoying effect of Donnan's exclusion of enantiomers from the stationary phase. This effect, on the opposite, was frequently observed on a commercial teicoplanin CSP (Teicoshell) employed for comparative purposes. Noticeably, on the zwitterionic-teicoplanin CSPs, by using either acetonitrile- or methanol-rich mobile phases (MPs), it was possible to favor speed over enantioresolution and vice versa. This work gives further replies to the request for rapid determination of enantiomeric excess of Nα-FMOC proteinogenic (and non-proteinogenic) AAs, typically used as preferred chiral synthons in the solid-phase synthesis of therapeutic peptides., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Nile perch wastewater was biodegraded using two Bacillus species to recover bioactive substances to enhance its reutilization value. The two Bacillus species successfully produced low-molecular-weight substances with a 47.8% degree of hydrolysis. The antioxidant activities of the Nile perch wastewater increased as the biodegradation proceeded, and the culture supernatant exhibited the highest DPPH (80.1%), ABTS (93.1%) and Fe 2+ chelating (88.5%) antioxidant activities at 60 h. The antioxidant potential of the biodegraded Nile perch wastewater was found to be higher than those of other fish hydrolysates. Moreover, the biodegraded Nile perch wastewater exhibited effective antimicrobial activity against Vibrio vulnificus, exhibiting a minimal inhibitory concentration of 585 μg mL -1 . Two-dimensional thin layer chromatography analysis revealed the specific amino acids responsible for the antioxidant activity, and molecular-weight cut-off ultrafiltration revealed that the <2-kDa fraction exhibited the highest antioxidant activity with the lowest IC 50 values (0.43 and 0.22 mg mL -1 for DPPH and ABTS antioxidant activities, respectively). This is the first report of the reutilization of Nile perch wastewater as a natural antioxidant and antimicrobial ingredient for nutraceuticals., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Measurements of the isotopic abundances in meteoritic amino acids have found enhancements of 2 H/H, 15 N/ 14 N, and 13 C/ 12 C in the amino acids in the meteorites studied. We show that they are consistent with the processing of the constituents of the meteorites by electron antineutrinos that would be expected from a core-collapse supernova or neutron-star merger. Using theoretical electron antineutrino cross-sections, we are able to predict these isotopic ratio variations depending on the time-integrated antineutrino flux at the site where the amino acids were processed.

A diethylammonio-propylsulfate amphoteric ionic resin was synthesized and employed as the stationary phase for high-performance liquid chromatography-mass spectrometry (LC-MS) separation and detection of amino acids. The influence of experimental conditions such as mobile phase composition, column length and temperature upon the amino acid separations was evaluated. However, temperature, addition of water-miscible organic solvent to the mobile phase and mobile phase gradients were not effective at improving the separations. In contrast, the use of an unbuffered pure water mobile phase proved successful for the separation and detection of amino acids. The observed order of elution seems to parallel the isoelectric points of the respective amino acids and suggests that this diethylammonio-propylsulfate stationary phase column functions as an amphoteric ion-exchanger. Under optimized chromatographic conditions, the detection limits for the amino acids were in range of 0.07-0.44 pmol (pico-mole). The method was also successfully applied for the analysis of an actual commercial sample., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

New zwitterionic (ZIC) stationary phases (SPs) are synthesized with the click and conventional bonding of tyrosine to silica gel. Infrared spectra and elemental analysis demonstrate the successful click and conventional bonding of this ZIC group on silica particles by the surface coverage including 2.36 and 0.75 µm m -2 , respectively. Given the above-mentioned explanation, the present study evaluated the retention mechanism and chromatographic manners of polar compounds on these new materials under hydrophilic interaction liquid chromatography (HILIC) conditions. Based on the results, the Click-Tyrosine Stationary Phase provided good HILIC characteristics when it was applied to separate phenolic compounds, amino acids, alkaloids, and nucleobases compared to bare silica gel SP and even conventional tyrosine SPs. Further, this new Click-Tyrosine-SP represented appropriate HILIC features and column efficiency (the theoretical plate number was up to 50,000 plates m -1 for thebaine). Furthermore, the study investigated the effect of solute polarity (the number of the hydroxyl group of phenolic compounds) and hydrophobicity (the number of the side chain of aliphatic amino acids) on retention behaviors. Finally, some important factors were studied as the potential variables for guiding the retention behavior of the polar compound in HILIC condition including solvent composition, salt concentration, and the buffer pH of the mobile phase., Competing Interests: Declaration of Competing Interest The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript, apart from those disclosed. No writing assistance was utilized in the production of this manuscript. The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

The first example of application of nanosized polystyrene-based cation exchanger (NSCE) with sulfo groups as a dynamic coating of capillary walls was demonstrated. The conditions of dynamic coating formation were optimized and ensured the long-term stability of the coating. Capillary-to-capillary and day-to-day repeatabilities were 4% and 3%, correspondingly. The NSCE coating stability at various pH and influence of pH on the EOF mobility were investigated. The developed NSCE-modified coated capillaries provided improved resolution (R s = 0.9-3.2 for catecholamines and R s = 1.7-2.8 for amino acids) and efficiencies (330-520 ×10 3 t.p./m) of basic analytes, which are 1.5 times higher compared to untreated capillary. The optimized conditions were as follows: 50 mM phosphate buffer solution at pH 2.2 with 5 μM NSCE. The effect of the NSCE concentration in BGE on the electrophoretic mobilities of the analytes was investigated. The various online concentration techniques were tested in order to decrease the LODs. The simultaneous application of NSCE capillaries and field-amplified sample stacking provided the lowest LODs of catecholamines and amino acids and allowed to determine these analytes in human urine., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An ethylenediamine dicarboxyethyl diacetamido-bridged bis(β-cyclodextrin) was firstly synthesized through the reaction of 6-deoxy-6-amino-β-cyclodextrin (NH 2 -CD) with ethylenediaminetetraacetic dianhydride. Then it was bonded onto the surface of silica gel SBA-15 to obtain an ethylenediamine dicarboxyethyl diacetamido-bridged bis(β-CD)-bonded chiral stationary phase (EBCDP). The structures of the bridged bis(β-CD) and EBCDP were characterized by infrared spectroscopy, mass spectrometry, elemental analysis and thermogravimetric analysis, accordingly. The chiral chromatographic performances of EBCDP were systematically evaluated by separating 28 racemic analytes in the reversed-phase or polar organic mode, including eight flavanones, eight bolckers, five dansyl-amino acids, three DL-amino acids and four other common drugs. As a result, the relatively high enantioselectivity of EBCDP was observed in comparison with a native β-CD-CSP (CDSP). All selected analytes were separated on EBCDP, of which 20 analytes had resolutions up to baseline, 2'-hydroxyflavanone and arotinolol had resolutions up to 4.35 and 2.05 in about 30 min, respectively, whereas CDSP only separated 11 analytes with low resolutions (0.55~1.69). Moreover, EBCDP was able to utilize the complexation of the bridging linker (ethylenediamine dicarboxyethyl diamide group, EDTA-based) to realize direct separations of DL-amino acids with a mobile phase containing copper ion (Cu 2+ ), which was similar to the chiral ligand exchange chromatography. Unlike the native cyclodextrin with small cavity (~242 Å 3 ), the bridged bis(β-CD) combined two β-CD units with a bridging linker, having a well-organized "pseudo-cavity" as an organic whole to encapsulate more analytes, which made EBCDP have broad-spectrum applications in chiral separations., Competing Interests: Declaration of Competing Interest The authors have declared no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

Flow injection chromatography (FIC) or sequential injection chromatography (SIC) is a low-pressure liquid chromatography technique that uses flow injection or sequential injection hardware. Due to the constraints of this hardware, the separation resolution is low; often no more than 3-5 components are resolved. We have recently demonstrated the excellent resolving power of narrow open tubular (OT) columns for various biomolecules, and only moderate elution pressures are needed to carry out these separations. In this paper, we incorporate a narrow OT column with FIC and construct an FIC system using a pressure chamber and two injection valves to implement gradient elution. The resultant system not only improves the resolution but also reduces the system cost. When we use the system to separate peptides from trypsin-digested cytochrome C, we can resolve dozens of peptides (with resolutions of 0.5 or greater) at a speed of 12 samples per hour. When we use this system to separate a mixture containing 3 amino acids, we can base-line resolve these compounds at a speed of 1800 sample per hour., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background Quantification of plasma amino acids is key to the diagnosis of inherited defects of amino acid synthesis, catabolism and transport, many of which present as clinical emergencies. The utility of this test is limited by the long analysis time and subsequent inability of laboratories to provide results in real-time. Traditionally, analysis has been performed by ion exchange chromatography (IEC) but recently there has been a move towards liquid chromatography tandem mass spectrometry (LC-MS/MS) which provides the potential for faster analysis. However, the necessity to derivatise the sample and/or utilise an ion-pair reagent, combined with lack of commercially available stable isotope internal standards (IS) has prevented laboratories fully exploiting the benefits of this methodology. We describe an underivatised LC-MS/MS method enabling patient results to be reported with an improved turnaround time (<1 h). Methods Methanolic IS was added to plasma (10 μL) to precipitate protein. Following centrifugation amino acids were analysed by LC-MS/MS using selected reaction monitoring (SRM) for each analyte and corresponding IS. Results Patient samples (n = 57) and external quality assessment (EQA) material (n = 11) were analysed and results compared with IEC. Comparable accuracy and precision were obtained with 15-min analysis time. Conclusions This method enables the analysis of a clinically comprehensive amino acid profile without the need for derivatisation/ion-pair reagents and benefitting from improved analytical quantitation through multipoint calibration and use of stable isotope IS. The analysis time is fast in comparison to IEC, improves efficiency of laboratory workflow and enables stat analysis of clinically urgent samples.

Most HPLC enantiomer separations are performed with columns packed with a chiral stationary phase (CSP) operated with an achiral mobile phase. The intrinsically limited chemical selectivity of most CSPs to the simultaneous resolution of several pairs of enantiomers means that complex mixtures of diverse pairs of enantiomers cannot be resolved in a single run due to peak overlapping. Moreover, some drawbacks remain when the analyte is present in very complex samples containing other achiral compounds which can co-elute with the enantiomer peaks. Multidimensional chromatography becomes an option to increase peak capacity and resolve these samples. The aim of this work was to study an online fully comprehensive 2D-LC mode utilizing a combination of a chiral column in the first dimension and an achiral column in the second dimension. The 2D-LC system was built with an active flow splitter pump in order to easily adjust the volume of sample transferred into the second dimension and to independently optimize the flow rate in the first dimension. The present LCxLC method was optimized for the separation of amino acids present in honey samples, taking into account key parameters that influence the bidimensional peak capacity (orthogonality, sampling frequency, etc.). The amino acids have been preconcentrated on a cation-exchange column followed by derivatization. Several amino acids present in different honey samples have been identified and the data generated has been analyzed by principal component analysis., (Copyright © 2019. Published by Elsevier B.V.)

Mammalian cells have been used in various research fields. More recently, cultured cells have been used as the cell source of "cultured meat." Cell cultivation requires media containing nutrients, of which glucose and amino acids are the essential ones. These nutrients are generally derived from grains or heterotrophic microorganisms, which also require various nutrients derived from grains. Grain culture, in turn, requires many chemical fertilizers and agrochemicals, which can cause greenhouse gas emission and environmental contamination. Furthermore, grain production is greatly influenced by environmental changes. In contrast, microalgae efficiently synthesize various nutrients using solar energy, water, and inorganic substances, which are widely used in the energy sector. In this study, we aimed to apply nutrients extracted from microalgae in the culture media for mammalian cell cultivation. Glucose was efficiently extracted from Chlorococcum littorale or Arthrospira platensis using sulfuric acid, whereas 18 of the 20 proteinogenic amino acids were efficiently extracted from Chlorella vulgaris using hydrochloric acid. We further investigated whether nutrients present in the algal extracts could be used in mammalian cell cultivation. Although almost all C2C12 mouse myoblasts died during cultivation in a glucose- and amino acid-free medium, the cell death was rescued by adding algal extract(s) into the nutrient-deficient media. This indicates that nutrients present in algal extracts can be used for mammalian cell cultivation. This study is the first step toward the establishment of a new cell culture system that can reduce environmental loads and remain unaffected by the impact of environmental changes., (© 2019 American Institute of Chemical Engineers.)

UV-absorbing compounds, such as mycosporine-like amino acids (MAAs), are a group of secondary metabolites present in many marine species, including red seaweeds. In these organisms, the content and proportion of the composition of MAAs vary, depending on the species and several environmental factors. Its high cosmetic interest calls for research on the content and composition of MAAs, as well as the dynamics of MAAs accumulation in seaweeds from different latitudes. Therefore, this study aimed to survey the content of UV-absorbing MAAs in three Subantarctic red seaweeds during a seasonal cycle. Using spectrophotometric and HPLC techniques, the content and composition of MAAs of intertidal Iridaea tuberculosa , Nothogenia fastigiate , and Corallina officinalis were assessed. Some samples were also analyzed using high-resolution mass spectrometry coupled with HPLC-ESI-MS in order to identify more precisely the MAA composition. I. tuberculosa exhibited the highest MAA values (above 1 mg g -1 of dried mass weight), while C. officinalis showed values not exceeding 0.4 mg g -1 . Porphyra-334 was the main component in N. fastigiata , whereas I. tuberculosa and C. officinalis exhibited a high content of palythine. Both content and composition of MAAs varied seasonally, with high concentration recorded in different seasons, depending on the species, i.e., winter ( I. tuberculosa ), spring ( N. fastigiata ), and summer ( C. officinalis ). HPLC-ESI-MS allowed us to identify seven different MAAs. Two were recorded for the first time in seaweeds from Subantarctic areas (mycosporine-glutamic acid and palythine-serine), and we also recorded an eighth UV-absorbing compound which remains unidentified., Competing Interests: The authors declare no conflict of interest.

Two new copolymer-grafted silica stationary phases were prepared and employed in hydrophilic interaction chromatography (HILIC). 2-(Dimethylamino)ethyl methacrylate (DMAEMA) are copolymerized with itaconic acid (IA) and acrylic acid (AA) respectively, via thiol-ene click reaction on silica surface with deep eutectic solvents (DES) as new solvents. The obtained poly(DMAEMA-co-itaconic acid)-grafted silica (Sil-PDM-PIA) and poly(DMAEMA-co-acrylic acid)-grafted silica (Sil-PDM-PAA) were characterized by Fourier transform infrared spectroscopy, elemental analysis and solid-state 13 C NMR spectra. Their hydrophilic interaction performances were evaluated by separating nucleosides, nucleobases, saccharides, and amino acids. Compared with previous reported poly(itaconic acid)-grafted silica (Sil-PIA) and poly(acrylic acid)-grafted silica (Sil-PAA) stationary phases, these two new copolymer-grafted silica performed higher selectivity and better separation for polar analytes in HILIC., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The crude drug ysypó hû ( Adenocalymma marginatum DC., Bignoniaceae) is used traditionally by the Guarani of Eastern Paraguayan as a male sexual enhancer. The aim of the present study was to identify the main constituents of the crude drug and to evaluate the in vitro inhibitory activity towards the enzyme phosphodiesterase-5 (PDE-5). The main compounds were isolated by counter-current chromatography (CCC). The metabolites were identified by spectroscopic and spectrometric means. The chemical profiling of the extracts was assessed by high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS/MS). The crude extract and main isolated compounds were tested for their PDE-5 inhibitory activity using commercial kits. The iridoid theviridoside and 4-hydroxy-1-methylproline were isolated as the main constituent of the crude drug. Four chlortheviridoside hexoside derivatives were detected for the first time as natural products. Chemical profiling by HPLC-MS/MS led to the tentative identification of nine iridoids, six phenolics, and five amino acids. The crude extracts and main compounds were inactive towards PDE-5 at concentrations up to 500 µg/mL. Iridoids and amino acid derivatives were the main compounds occurring in the Paraguayan crude drug. The potential of ysypó hû as a male sexual enhancer cannot be discarded, since other mechanisms may be involved.

Mycosporine-like amino acids (MAAs) have gained considerable attention as highly active photoprotective candidates for human sunscreens. However, more studies are necessary to evaluate the extraction efficiency of these metabolites in solvents compatible with cosmetics and their subsequent analysis by HPLC. In the present study, MAA extraction using distilled water and 20% methanol in four Rhodophyta was investigated. Different re-dissolution solvents and a C8 and C18 columns were tested for the HPLC analysis. Porphyra-334, shinorine, palythine, palythine-serine, asterina-330, and palythinol were identified by HPLC/ESI-MS.These MAAs were better isolated with the C8 column and using methanol as re-dissolution solvents. Regarding total MAAs concentrations, no differences between the two solvents were found. The highest values were observed injecting them directly in the HPLC. According to these results, distilled water could be an excellent extraction solvent in the production of MAAs extracts and quantification of their total concentrations for different uses in the industry. Nevertheless, the extraction of MAAs using 20% methanol and re-dissolution in pure methanol after dryness is the best option to characterize and identify the most common MAAs in these red algae. Our results entail important implications regarding the use of red macroalgae as promising candidates as environment-friendly sources of natural sunscreens.

Gut microbiota has been implicated in major diseases affecting the human population and has also been linked to triglycerides and high-density lipoprotein levels in the circulation. Recent development in metabolomics allows classifying the lipoprotein particles into more details. Here, we examine the impact of gut microbiota on circulating metabolites measured by Nuclear Magnetic Resonance technology in 2309 individuals from the Rotterdam Study and the LifeLines-DEEP cohort. We assess the relationship between gut microbiota and metabolites by linear regression analysis while adjusting for age, sex, body-mass index, technical covariates, medication use, and multiple testing. We report an association of 32 microbial families and genera with very-low-density and high-density subfractions, serum lipid measures, glycolysis-related metabolites, ketone bodies, amino acids, and acute-phase reaction markers. These observations provide insights into the role of microbiota in host metabolism and support the potential of gut microbiota as a target for therapeutic and preventive interventions.