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The increasing occurrence of saxitoxins in freshwaters is becoming a concern for water treatment facilities owing to its structural properties which make it resistant to oxidation at pH, 8. Hence, it is crucial to be able to monitor these toxins in surface and drinking water to protect public health. This review aims to outline the current state of knowledge related to the occurrence of saxitoxins in freshwaters and its removal strategies and provide a critical assessment of the detection methods to provide a basis for further development. Temperature and nutrient content are some of the factors that influence the production of saxitoxins in surface waters. A high dose of sodium hypochlorite with sufficient contact time or activated carbon has been shown to efficiently remove extracellular saxitoxins to meet the drinking water guidelines. While HILIC-MS has proven to be a powerful technology for more sensitive and reliable detection of saxitoxin and variants after solid phase extraction, ELISA is cost-effective and easy to use and is used by Ohio EPA for surveillance with a limit of detection of 0.015 μg/L. However, there is a need for the development of cost-effective and sensitive techniques that can quantify the variants of saxitoxin. [ABSTRACT FROM AUTHOR]

Monitoring the levels of amino acids (AAs) in biological cell cultures provides key information to understand the regulation of cell growth and metabolism. Saccharomyces cerevisiae can naturally excrete AAs, making accurate detection and determination of amino acid levels within the cultivation medium pivotal for gaining insights into this still poorly known process. Given that most AAs lack ultraviolet (UV) chromophores or fluorophores necessary for UV and fluorescence detection, derivatization is commonly utilized to enhance amino acid detectability via UV absorption. Unfortunately, this can lead to drawbacks such as derivative instability, labor intensiveness, and poor reproducibility. Hence, this study aimed to develop an accurate and stable hydrophilic interaction liquid chromatography‐tandem mass spectrometry analytical method for the separation of all 20 AAs within a short 17‐min run time. The method provides satisfactory linearity and sensitivity for all analytes. The method has been validated for intra‐ and inter‐day precision, accuracy, recovery, matrix effect, and stability. It has been successfully applied to quantify 20 AAs in samples of yeast cultivation medium. This endeavor seeks to enhance our comprehension of amino acid profiles in the context of cell growth and metabolism within yeast cultivation media. [ABSTRACT FROM AUTHOR]

The high throughput in genome sequencing and metabolic model (MM) reconstruction has democratised bioinformatics approaches such as flux balance analysis. Fluxes' prediction accuracy greatly relates to the deepness of the MM curation for a specific organism starting from the cell composition. One component is the cell wall, which is a functional barrier (cell shape, exchanges) with the environment. The bacterial cell wall (BCW), including its thickness, structure, and composition, has been extensively studied in Escherichia coli but poorly described for other organisms. The peptidoglycan (PG) layer composing the BCW is usually thinner in Gram− bacteria than in Gram+ bacteria. In both bacteria groups, PG is a polymeric mesh-like structure of amino acids and sugars, including N-acetylglucosamine, N-acetylmuramic acid, and amino acids. In this study, we propose a high-throughput method to characterise and quantify PG in Gram-positive and Gram-negative bacteria using acidic hydrolysis and hydrophilic interaction liquid chromatography coupled with mass spectrometry (HILIC-MS). The method showed a relatively short time frame (11 min analytical run), low inter- and intraday variability (3.2% and 4%, respectively), and high sensitivity and selectivity (limits of quantification in the sub mg/L range). The method was successfully applied on two Gram-negative bacteria (Escherichia coli K12 MG1655, Bacteroides thetaiotaomicron DSM 2079) and one Gram-positive bacterium (Streptococcus salivarius ssp. thermophilus DSM20259). The PG concentration ranged from 1.6% w/w to 14% w/w of the dry cell weight. The results were in good correlation with previously published results. With further development, the PG concentration provided by this newly developed method could reinforce the curation of MM. [ABSTRACT FROM AUTHOR]

Mammalian hibernators undergo substantial changes in metabolic function throughout the seasonal hibernation cycle. We report here the polar metabolomic profile of white adipose tissue isolated from active and hibernating thirteen-lined ground squirrels (Ictidomys tridecemlineatus). Polar compounds in white adipose tissue were extracted from five groups representing different timepoints throughout the seasonal activity-torpor cycle and analyzed using hydrophilic interaction liquid chromatography-mass spectrometry in both the positive and negative ion modes. A total of 224 compounds out of 660 features detected after curation were annotated. Unsupervised clustering using principal component analysis revealed discrete clusters representing the different seasonal timepoints throughout hibernation. One-way analysis of variance and feature intensity heatmaps revealed metabolites that varied in abundance between active and torpid timepoints. Pathway analysis compared against the KEGG database demonstrated enrichment of amino acid metabolism, purine metabolism, glycerophospholipid metabolism, and coenzyme A biosynthetic pathways among our identified compounds. Numerous carnitine derivatives and a ketone that serves as an alternate fuel source, beta-hydroxybutyrate (BHB), were among molecules found to be elevated during torpor. Elevated levels of the BHB-carnitine conjugate during torpor suggests the synthesis of beta-hydroxybutyrate in white adipose mitochondria, which may contribute directly to elevated levels of circulating BHB during hibernation. [ABSTRACT FROM AUTHOR]

Introduction: Polar metabolites in Caenorhabditis elegans (C. elegans) have predominantly been analyzed using hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC-MS). Capillary electrophoresis coupled to mass spectrometry (CE-MS) represents another complementary analytical platform suitable for polar and charged analytes. Objective: We compared CE-MS and HILIC-MS for the analysis of a set of 60 reference standards relevant for C. elegans and specifically investigated the strengths of CE separation. Furthermore, we employed CE-MS as a complementary analytical approach to study polar metabolites in C. elegans samples, particularly in the context of longevity, in order to address a different part of its metabolome. Method: We analyzed 60 reference standards as well as metabolite extracts from C. elegans daf-2 loss-of-function mutants and wild-type (WT) samples using HILIC-MS and CE-MS employing a Q-ToF-MS instrument. Results: CE separations showed narrower peak widths and a better linearity of the estimated response function across different concentrations which is linked to less saturation of the MS signals. Additionally, CE exhibited a distinct selectivity in the separation of compounds compared to HILIC-MS, providing complementary information for the analysis of the target compounds. Analysis of C. elegans metabolites of daf-2 mutants and WT samples revealed significant alterations in shared metabolites identified through HILIC-MS, as well as the presence of distinct metabolites. Conclusion: CE-MS was successfully applied in C. elegans metabolomics, being able to recover known as well as identify novel putative biomarkers of longevity. [ABSTRACT FROM AUTHOR]

Mammalian hibernators undergo substantial changes in metabolic function throughout the seasonal hibernation cycle. We report here the polar metabolomic profile of white adipose tissue isolated from active and hibernating thirteen-lined ground squirrels (Ictidomys tridecemlineatus). Polar compounds in white adipose tissue were extracted from five groups representing different timepoints throughout the seasonal activity-torpor cycle and analyzed using hydrophilic interaction liquid chromatography-mass spectrometry in both the positive and negative ion modes. A total of 224 compounds out of 660 features detected after curation were annotated. Unsupervised clustering using principal component analysis revealed discrete clusters representing the different seasonal timepoints throughout hibernation. One-way analysis of variance and feature intensity heatmaps revealed metabolites that varied in abundance between active and torpid timepoints. Pathway analysis compared against the KEGG database demonstrated enrichment of amino acid metabolism, purine metabolism, glycerophospholipid metabolism, and coenzyme A biosynthetic pathways among our identified compounds. Numerous carnitine derivatives and a ketone that serves as an alternate fuel source, beta-hydroxybutyrate (BHB), were among molecules found to be elevated during torpor. Elevated levels of the BHB-carnitine conjugate during torpor suggests the synthesis of beta-hydroxybutyrate in white adipose mitochondria, which may contribute directly to elevated levels of circulating BHB during hibernation.

Tetrodotoxins (TTXs), the pufferfish venom traditionally associated with Indo-Pacific area, has been reported during last decades in ever wider range of marine organisms and ever more geographical areas, including shellfish in Europe. Wild mussels (Mytilus galloprovincialis) grown in the Marche Region (N Adriatic Sea, Italy) were shown to be prone to TTX contamination during the warm season, with a suspected role of Vibrio alginolyticus characterized by non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS)-encoding genes. This work aimed to deepen the knowledge about the toxin's origin and the way through which it accumulates in mussels. A two-year study (spring–summer 2020–2021) confirmed the recurrent presence of TTX (11–68 µg kg−1) in the official monitored natural mussel beds of the Conero Riviera. During 2021, a supplementary nonroutine monitoring of a natural mussel bed in the same area was carried out weekly from June until August for TTXs and/or the presence of V. alginolyticus. Biotic (mussels, mesozooplankton, worms and phytoplankton); abiotic (water and sediment) matrices and phytoplankton assemblage characterizations were studied. Mussels showed relevant TTX contamination levels (9–296 µg kg−1) with extremely rapid TTX accumulation/depletion rates. The toxin presence in phytoplankton and its distribution in the different mussel tissues supports its possible exogenous origin. The V. alginolyticus count trend overlaps that of TTX contamination in mussels, and similar trends were reported also for some phytoplankton species. The role of V. alginolyticus carrying NRPS or PKS genes as a possible TTX source and of phytoplankton as a "potential vector" should therefore be further investigated. [ABSTRACT FROM AUTHOR]

Glycation products produced by the non-enzymatic reaction between reducing carbohydrates and amino compounds have received increasing attention in both food- and health-related research. Although liquid chromatography mass spectrometry (LC-MS) methods for analyzing glycation products already exist, only a few common advanced glycation end products (AGEs) are usually covered by quantitative methods. Untargeted methods for comprehensively analyzing glycation products are still lacking. The aim of this study was to establish a method for simultaneously characterizing a wide range of free glycation products using the untargeted metabolomics approach. In this study, Maillard model systems consisting of a multitude of heterogeneous free glycation products were chosen for systematic method optimization, rather than using a limited number of standard compounds. Three types of hydrophilic interaction liquid chromatography (HILIC) columns (zwitterionic, bare silica, and amide) were tested due to their good retention for polar compounds. The zwitterionic columns showed better performance than the other two types of columns in terms of the detected feature numbers and detected free glycation products. Two zwitterionic columns were selected for further mobile phase optimization. For both columns, the neutral mobile phase provided better peak separation, whereas the acidic condition provided a higher quality of chromatographic peak shapes. The ZIC-cHILIC column operating under acidic conditions offered the best potential to discover glycation products in terms of providing good peak shapes and maintaining comparable compound coverage. Finally, the optimized HILIC-MS method can detect 70% of free glycation product features despite interference from the complex endogenous metabolites from biological matrices, which showed great application potential for glycation research and can help discover new biologically important glycation products. [ABSTRACT FROM AUTHOR]

The high throughput in genome sequencing and metabolic model (MM) reconstruction has democratised bioinformatics approaches such as flux balance analysis. Fluxes’ prediction accuracy greatly relates to the deepness of the MM curation for a specific organism starting from the cell composition. One component is the cell wall, which is a functional barrier (cell shape, exchanges) with the environment. The bacterial cell wall (BCW), including its thickness, structure, and composition, has been extensively studied in Escherichia coli but poorly described for other organisms. The peptidoglycan (PG) layer composing the BCW is usually thinner in Gram− bacteria than in Gram+ bacteria. In both bacteria groups, PG is a polymeric mesh-like structure of amino acids and sugars, including N-acetylglucosamine, N-acetylmuramic acid, and amino acids. In this study, we propose a high-throughput method to characterise and quantify PG in Gram-positive and Gram-negative bacteria using acidic hydrolysis and hydrophilic interaction liquid chromatography coupled with mass spectrometry (HILIC-MS). The method showed a relatively short time frame (11 min analytical run), low inter- and intraday variability (3.2% and 4%, respectively), and high sensitivity and selectivity (limits of quantification in the sub mg/L range). The method was successfully applied on two Gram-negative bacteria (Escherichia coli K12 MG1655, Bacteroides thetaiotaomicron DSM 2079) and one Gram-positive bacterium (Streptococcus salivarius ssp. thermophilus DSM20259). The PG concentration ranged from 1.6% w/w to 14% w/w of the dry cell weight. The results were in good correlation with previously published results. With further development, the PG concentration provided by this newly developed method could reinforce the curation of MM.

Advances in metabolomics have allowed the identification and characterization of saliva metabolites that can be used as biomarkers. However, discrepancies can be noted with the content of the same biomarker being increased or decreased for a given disease. Differences in the way saliva is collected, stored, and/or treated could cause these discrepancies. Indeed, there is no standardized method for saliva sampling and analysis. In this work, two chromatographic modes were used, i.e., RP-LC and HILIC both coupled to MS used in positive and negative ionization modes. The analytical conditions were optimized with a mixture of 90 compounds naturally present in saliva, representative of the wide range of molecular mass and polarity of salivary metabolites and being described as having a differential expression in various pathologies. These four methods were applied to the analysis of saliva samples collected by spitting, aspiration, or Salivette® with or without prior rinsing of the mouth. Rinsing had an effect on some metabolite concentrations. As it can induce an additional parameter of variability to the sampling, it seems therefore preferable to use methods without rinsing while effects of these parameters on the metabolites are investigated. Saliva obtained by spitting and aspiration gave statistically equivalent results for 84% of the metabolites studied. Conversely, Salivette® gave different results since the majority of the metabolites chosen for the study were not quantified in the samples. The Salivette® does not seem therefore to be a suitable sampling method for an untargeted analysis of the salivary metabolome, unlike aspiration and spitting. [ABSTRACT FROM AUTHOR]

Following in the footsteps of genomics and proteomics, metabolomics has revolutionised the way we investigate and understand biological systems. Rapid development in the last 25 years has been driven largely by technical innovations in mass spectrometry and nuclear magnetic resonance spectroscopy. However, despite the modest size of metabolomes relative to proteomes and genomes, methodological capabilities for robust, comprehensive metabolite analysis remain a major challenge. Therefore, development of new methods and techniques remains vital for progress in the field. Here, we review developments in LC-MS, GC–MS and NMR methods in the last few years that have enhanced quantitative and comprehensive metabolome coverage, highlighting the techniques involved, their technical capabilities, relative performance, and potential impact. [Display omitted] [ABSTRACT FROM AUTHOR]

Tetrodotoxins (TTXs), the pufferfish venom traditionally associated with Indo-Pacific area, has been reported during last decades in ever wider range of marine organisms and ever more geographical areas, including shellfish in Europe. Wild mussels (Mytilus galloprovincialis) grown in the Marche Region (N Adriatic Sea, Italy) were shown to be prone to TTX contamination during the warm season, with a suspected role of Vibrio alginolyticus characterized by non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS)-encoding genes. This work aimed to deepen the knowledge about the toxin’s origin and the way through which it accumulates in mussels. A two-year study (spring–summer 2020–2021) confirmed the recurrent presence of TTX (11–68 µg kg−1) in the official monitored natural mussel beds of the Conero Riviera. During 2021, a supplementary nonroutine monitoring of a natural mussel bed in the same area was carried out weekly from June until August for TTXs and/or the presence of V. alginolyticus. Biotic (mussels, mesozooplankton, worms and phytoplankton); abiotic (water and sediment) matrices and phytoplankton assemblage characterizations were studied. Mussels showed relevant TTX contamination levels (9–296 µg kg−1) with extremely rapid TTX accumulation/depletion rates. The toxin presence in phytoplankton and its distribution in the different mussel tissues supports its possible exogenous origin. The V. alginolyticus count trend overlaps that of TTX contamination in mussels, and similar trends were reported also for some phytoplankton species. The role of V. alginolyticus carrying NRPS or PKS genes as a possible TTX source and of phytoplankton as a “potential vector” should therefore be further investigated.

Glycation products produced by the non-enzymatic reaction between reducing carbohydrates and amino compounds have received increasing attention in both food- and health-related research. Although liquid chromatography mass spectrometry (LC-MS) methods for analyzing glycation products already exist, only a few common advanced glycation end products (AGEs) are usually covered by quantitative methods. Untargeted methods for comprehensively analyzing glycation products are still lacking. The aim of this study was to establish a method for simultaneously characterizing a wide range of free glycation products using the untargeted metabolomics approach. In this study, Maillard model systems consisting of a multitude of heterogeneous free glycation products were chosen for systematic method optimization, rather than using a limited number of standard compounds. Three types of hydrophilic interaction liquid chromatography (HILIC) columns (zwitterionic, bare silica, and amide) were tested due to their good retention for polar compounds. The zwitterionic columns showed better performance than the other two types of columns in terms of the detected feature numbers and detected free glycation products. Two zwitterionic columns were selected for further mobile phase optimization. For both columns, the neutral mobile phase provided better peak separation, whereas the acidic condition provided a higher quality of chromatographic peak shapes. The ZIC-cHILIC column operating under acidic conditions offered the best potential to discover glycation products in terms of providing good peak shapes and maintaining comparable compound coverage. Finally, the optimized HILIC-MS method can detect 70% of free glycation product features despite interference from the complex endogenous metabolites from biological matrices, which showed great application potential for glycation research and can help discover new biologically important glycation products.

Human milk oligosaccharides (HMOs) exhibit various biological activities for infants, such as serving as prebiotics, blocking pathogens, and aiding in brain development. HMOs are a complex mixture of hetero-oligosaccharides that are generally highly branched, containing multiple structural isomers and no intrinsic chromophores, presenting a challenge to both their resolution and quantitative detection. While liquid chromatography-mass spectrometry (LC-MS) has become the primary strategy for analysis of various compounds, the very polar and chromophore-free properties of native glycans hinder their separation in LC and ionization in MS. Various labeling approaches have been developed to achieve separation of glycans with higher resolution and greater sensitivity of detection. Here, we compared five commonly used labeling techniques [by 2-aminobenzamide, 2-aminopyridine, 2-aminobenzoic acid (2-AA), 2,6-diaminopyridine, and 1-phenyl-3-methyl-5-pyrazolone] for analyzing HMOs specifically under hydrophilic-interaction chromatography-mass spectrometry (HILIC-MS) conditions. The 2-AA labeling showed the most consistent deprotonated molecular ions, the enhanced sensitivity with the least structural selectivity, and the sequencing-informative tandem MS fragmentation spectra for the widest range of HMOs; therefore, this labeling technique was selected for further optimization under the porous graphitized carbon chromatography-mass spectrometry (PGC-MS) conditions. The combination strategy of 2-AA labeling and PGC-MS techniques provided online decontamination (removal of excess 2-AA, salts, and lactose) and resolute detection of many HMOs, enabling us to characterize the profiles of complicated HMO mixtures comprehensively in a simple protocol.

The identification and accurate quantitation of the various glycoforms contained in therapeutic monoclonal antibodies (mAbs) is one of the main analytical needs in the biopharmaceutical industry, and glycosylation represents a crucial critical quality attribute (CQA) that needs to be addressed. Currently, the reference method for performing such identification/quantitation consists of the release of the N-glycan moieties from the mAb, their labelling with a specific dye (e.g., 2-AB or RFMS) and their analysis by HILIC-FLD or HILIC-MS. In this contribution, the potential of a new cost- and time-effective analytical approach performed at the protein subunit level (middle-up) was investigated for quantitative purposes and compared with the reference methods. The robustness of the approach was first demonstrated by performing the relative quantification of the glycoforms related to a well characterized mAb, namely adalimumab. Then, the workflow was applied to various glyco-engineered mAb products (i.e., obinutuzumab, benralizumab and atezolizumab). Finally, the glycosylation pattern of infliximab (Remicade®) was assessed and compared to two of its commercially available biosimilars (Remsima® and Inflectra®). The middle-up analysis proved to provide accurate quantitation results and has the added potential to be used as multi-attribute monitoring method.

• Extended quantitative study of the main LMWC in a large set of 56 commercial green teas. • Changes observed in the indicators evaluated depend on processing conditions. • PCA was employed to evaluate differences between samples based on processing. • HCA was employed to explore similarities between samples based on origin. A comprehensive characterization of commercial green tea (CGT) with the employment of independent variables such as low molecular weight carbohydrates (LMWC), soluble solids, color and antioxidant capacity has been performed in this manuscript. Within the manuscript, a convenient HILIC-MS method, suitable to perform a simultaneous identification and quantification of all mono-, di-, oligosaccharides and cyclitols observed in green tea was introduced. The method covers all key analytes in a single chromatographic analysis. Fifty-six samples from different origins (n = 10) were evaluated to explore differences based on origin. In addition, commercial samples processed by pan-firing and steaming were used for comparative purposes, allowing the identification of putative processing markers. The results obtained contribute to gain a better knowledge of the variations, according to origin and processing, in composition and quality of CGT, commodity widely appreciated by the consumers. [ABSTRACT FROM AUTHOR]

• HILIC-MS analysis of oligonucleotides without the use of ion-pair reagents. • Applicable to oligodeoxy(ribo)nucleotides and phosphorothioates (PS). • Improved LC–MS sensitivity over ion-pair reverse phase methods. • Additional chromatographic developments required to match UV-based PS methods. Improving our understanding of nucleic acids, both in biological and synthetic applications, remains a bustling area of research for both academic and industrial laboratories. As nucleic acids research evolves, so must the analytical techniques used to characterize nucleic acids. One powerful analytical technique has been coupled liquid chromatography – tandem mass spectrometry (LC–MS/MS). To date, the most successful chromatographic mode has been ion-pairing reversed-phase liquid chromatography. Hydrophilic interaction liquid chromatography (HILIC), in the absence of ion-pair reagents, has been investigated here as an alternative chromatographic approach to the analysis of oligonucleotides. By combining a mobile phase system using commonly employed in liquid chromatography-mass spectrometry (LC–MS) - i.e., water, acetonitrile, and ammonium acetate - and a new, commercially available diol-based HILIC column, high chromatographic and mass spectrometric performance for a wide range of oligonucleotides is demonstrated. Particular applications of HILIC-MS for the analysis of deoxynucleic acid (DNA) oligomers, modified and unmodified oligoribonucleotides, and phosphorothioate DNA oligonucleotides are presented. Based on the LC–MS performance, this HILIC-based approach provides an attractive, sensitive and robust alternative to prior ion-pairing dependent methods with potential utility for both qualitative and quantitative analyses of oligonucleotides without compromising chromatographic or mass spectrometric performance. [ABSTRACT FROM AUTHOR]

• A comparison of HILIC-MS and CE-MS for metabolomics in blood serum is performed. • Five different terms (specific to metabolomics) are selected for comparison. • Concise outlook on the current promises and pitfalls of both techniques is discussed. • MS-DIAL is used for the first time to effectively align CE-MS peaks across samples. Cationic, anionic, zwitterionic and, partially polar metabolites are very important constituents of blood serum. Several of these metabolites underpin the core metabolism of cells (e.g., Krebs cycle, urea cycle, proteins synthesis, etc.), while others might be considered ancillary but still important to grasp the status of any organism through blood serum analysis. Due to its wide chemical diversity, modern metabolomics analysis of serum is still struggling to provide a complete and comprehensive picture of the polar metabolome, due to the limitations of each specific analytical method. In this study, two metabolomics-based analytical methods using the most successful techniques for polar compounds separation in human serum samples, namely hydrophilic interaction liquid chromatography (HILIC) and capillary electrophoresis (CE), are evaluated, both coupled to a high-resolution time-of-flight mass spectrometer via electrospray ionization (ESI-Q-TOF-MS). The performance of the two methods have been compared using five terms of comparison, three of which are specific to metabolomics, such as (1) compounds' detectability (2) Pezzatti score (Pezzatti et al. 2018), (3) intra-day precision (repeatability), (4) ease of automatic analysis of the data (through a common deconvolution alignment and extrapolation software, MS-DIAL, and (5) time & cost analysis. From this study, HILIC-MS proved to be a better tool for polar metabolome analysis, while CE-MS helped identify some interesting variables that gave it interest in completing metabolome coverage in metabolomics studies. Finally, in this framework, MS-DIAL demonstrates for the first time its ability to process CE data for metabolomics, although it is not designed for it. [ABSTRACT FROM AUTHOR]

Traditional SDS-PAGE method and its modern equivalent CE-SDS method are both widely applied to assess the purity of therapeutic monoclonal antibody (mAb) drug products. However, structural identification of low molecular weight (LMW) impurities using those methods has been challenging and largely based on empirical knowledges. In this paper, we present that hydrophilic interaction chromatography (HILIC) coupled with mass spectrometry analysis is a novel and orthogonal method to characterize such LMW impurities present within a purified mAb drug product sample. We show here that after removal of N-linked glycans, the HILIC method separates mAb-related LMW impurities with a size-based elution order. The subsequent mass measurement from a high-resolution accurate mass spectrometer provides direct and unambiguous identification of a variety of low-abundance LMW impurities within a single LC–MS analysis. Free light chain, half antibody, H2L species (antibody possessing a single light chain) and protein backbone-truncated species can all be confidently identified and elucidated in great detail, including the truncation sites and associated post-translational modifications. It is worth noting that this study provides the first example where the H2L species can be directly detected in a mAb drug product sample by intact mass analysis without prior enrichment. [ABSTRACT FROM AUTHOR]

Phosphorylated carbohydrates are indispensable cogs in several key metabolic wheels for all forms of life. Here, a straightforward liquid chromatography method coupled to mass spectrometry detection was developed for phosphorylated sugars. For separation of the targeted compounds, hydrophilic interaction chromatography (HILIC) was used with a bridged-ethylene hybrid amide column under alkaline conditions using triethylamine as a mobile phase modifier. Methylphosphonic acid was added to the aqueous mobile phase to reduce the tailing of compounds containing phosphate groups, which are known to interact with stainless steel components of the separation system. Under alkaline conditions and addition of methylphosphonic acid, the retention behavior can be attributed to both conventional HILIC mechanisms as well as ion-pairing interactions in the mobile phase. This hypothesis is supported by comparing the retention behavior of phosphorylated sugars and unmodified sugars. The HILIC method resolved eight biologically important phosphorylated sugars and thereby enables simultaneous detection and quantification of these compounds: fructose-1,6-bisphosphate, glucose-1-phosphate, glucose-6-phosphate, lactose-1-phosphate, mannose-6-phosphate, ribose-5-phosphate, sucrose-6-phosphate, and threhalose-6-phosphate. Fructose-1-phosphate and fructose-6-phosphate were not resolved but quantification of total fructose-phosphate is possible. [ABSTRACT FROM AUTHOR]

Selective determination of dithiocarbamate (DTC) fungicides is mainly performed by hydrophilic interaction liquid chromatography (HILIC). According to Crnogorac and Schwack, DTC analyses by HILIC only lead to meaningful results with a zwitterionic polymer-based hydrophilic interaction liquid chromatography (ZIC-pHILIC) column. Considering the limited availability of this special type of column and the importance of DTC residue analysis, several new HILIC columns were evaluated as alternatives to the ZIC-pHILIC column. Detection was carried out by ultraviolet light and by mass spectrometry (MS) on a single quadrupole mass spectrometer coupled to an electrospray ionization interface operating in negative mode. On nearly all tested columns, separation of dimethyldithiocarbamates, ethylenebis(dithiocarbamates), and propylenebis(dithiocarbamates) was achieved with ammonium acetate eluents (pH 6.8). However, due to ion suppression by the buffer and the limited alkaline pH stability, the tested silica-based columns were not suitable for the sensitive analysis of DTCs. The polymer-based iHILIC-Fusion was the only alternative that offered high MS sensitivity, when a buffer containing 15 mM aqueous ammonium hydroxide and 7.5 mM ammonium hydrogen carbonate (pH 9.8) was used, but the separation of the three DTC subclasses was poor. Thus, considering both selectivity and sensitivity, the originally proposed polymer-based ZIC-pHILIC column still outperformed all the tested newly available alternative HILIC columns. [ABSTRACT FROM AUTHOR]

Citrus pectins have demonstrated health benefits through direct interaction with Toll-like receptor 2. Methyl-ester distribution patterns over the homogalacturonan were found to contribute to such immunomodulatory activity, therefore molecular interactions with TLR2 were studied. Molecular-docking analysis was performed using four GalA-heptamers, GalA 7 Me 0 , GalA 7 Me 1,6 , GalA 7 Me 1,7 and GalA 7 Me 2,5 . The molecular relations were measured in various possible conformations. Furthermore, commercial citrus pectins were characterized by enzymatic fingerprinting using polygalacturonase and pectin-lyase to determine their methyl-ester distribution patterns. The response of 12 structurally different pectic polymers on TLR2 binding and the molecular docking with four pectic oligomers clearly demonstrated interactions with human-TLR2 in a structure-dependent way, where blocks of (non)methyl-esterified GalA were shown to inhibit TLR2/1 dimerization. Our results may be used to understand the immunomodulatory effects of certain pectins via TLR2. Knowledge of how pectins with certain methyl-ester distribution patterns bind to TLRs may lead to tailored pectins to prevent inflammation., 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 © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Meat colour is one of the cues available to the consumer to gauge overall meat quality and wholesomeness. Colour stability of meat is determined by several factors both inherent to the animal and post-slaughter conditions, including ageing, storage/packaging and display times. A hydrophilic interaction liquid chromatography–mass spectrometry (HILIC–MS) based metabolomics study was undertaken to identify and compare polar metabolites between ovine meat samples that were exposed to different durations of ageing, storage conditions, and display times. Primary metabolites comprising amino acids, sugars, nucleotides, nucleosides, organic acids and their breakdown products were mainly identified as discriminating factors. For the first time, boron complexes of sugar and malic acid were also tentatively identified. As expected, most compounds identified were related to myoglobin chemistry, and compounds with antioxidant properties were found in higher levels in colour stable samples. Supplementary studies identifying semi-polar, non-polar and volatile compounds will provide a holistic understanding of the chemical basis of colour stability in ovine meat. [ABSTRACT FROM AUTHOR]

Amino acids, neurotransmitters, purines, and pyrimidines are bioactive molecules that play fundamental roles in maintaining various physiological functions. Their metabolism is closely related to the health, growth, development, reproduction, and homeostasis of organisms. Most recently, comprehensive measurements of these metabolites have shown their potential as innovative approaches in disease surveillance or drug intervention. However, simultaneous measurement of these metabolites presents great difficulties. Here, we report a novel quantitative method that uses hydrophilic interaction ultra-high-performance liquid chromatography-tandem mass spectrometry (HILIC-UPLC-MS/MS), which is highly selective, high throughput, and exhibits better chromatographic behavior than existing methods. The developed method enabled the rapid quantification of 37 metabolites, spanning amino acids, neurotransmitters, purines, and pyrimidines pathways, within 6.5 min. The compounds were separated on an ACQUITY UPLC® BEH Amide column. Serum and brain homogenate were extracted by protein precipitation. The intra- and interday precision of all of the analytes was less than 11.34 %, and the accuracy was between −11.74 and 11.51 % for all quality control (QC) levels. The extraction recoveries of serum ranged from 84.58 % to 116.43 % and those of brain samples from 80.80 % to 119.39 %, while the RSD was 14.61 % or less for all recoveries. This method was used to successfully characterize alterations in the rat brain and, in particular, their dynamics in serum. The following study was performed to simultaneously test global changes of these metabolites in a serotonin antagonist p-chlorophenylalanine (PCPA)-induced anxiety and insomnia rat model to understand the effect and mechanism of PCPA. Taken together, these results show that the method is able to simultaneously monitor a large panel of metabolites and that this protocol may represent a metabolomic method to diagnose toxicological and pathophysiological states. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Quantification of energy and redox cofactors is of great value to synthetic biologists to infer the balance of energy metabolism in engineered microbial strains and assess each strain's potential for further improvement. Most currently used methods for intracellular cofactor measurement suffer from incomplete coverage, low reproducibility, suboptimal sensitivity or specificity. In this study, we described an SPE–HILIC/MS approach for simultaneous determination of six cofactor targets (ATP, ADP, NAD, NADH, NADP, NADPH) in Escherichia coli cells. Sufficient linearity, precision and metabolite recoveries of this new approach justified its reliability in targeted cofactor quantification. Our approach was then compared with conventional enzymatic assays to demonstrate its superior performance. We applied the SPE–HILIC/MS approach to profile shift of cofactor balances in several engineered E. coli strains with varying isobutanol production. Our cofactor analysis clearly revealed that optimal energy fitness was achieved in the highest-yield strain through combined modulation of a transhydrogenase and a NAD + kinase. Apart from the targeted cofactors, the SPE enrichment procedure also allowed for confident identification of 39 groups of polar metabolites mainly involved in central carbon metabolism in E. coli cells. [ABSTRACT FROM AUTHOR]

[Display omitted] • BRCs content was determined in 71 bee pollen samples by HILIC-MS. • Betonicine was the predominant compound in all samples. • It was possible to discriminate the apiaries and harvest periods by means of CDA. • Low classification rates (52%-75%) were observed regarding apiary of origin. • Satisfactory classification rates (76%-97%) were reported regarding harvest period. In the last years, an increase has been observed in the adulteration of bee pollen. Consequently, different tools are required to authenticate the origin of this product, such as a study of the profile and composition of a specific family of compounds. The present study investigates the potential of betaines and related compounds as markers of the apiary of origin and harvest period of 71 bee pollen samples. These were collected from four apiaries (Pistacho, Tío Natalio, Monte and Fuentelahiguera), located in the same geographical area (Guadalajara, Spain) and sampled during three consecutive harvest periods in the same year (April-May, June, July-August). They were analyzed by means of a previously developed methodology, which involved solvent extraction, hydrophilic interaction liquid chromatography coupled to mass spectrometry, and a statistical analysis of the data (canonical discriminant analysis). Variable amounts of betaines and related compounds were found in the samples, with four of these being identified in all of them (betonicine, betaine, trigonelline and choline); betonicine was the predominant compound in a concentration range of 264 to 52384 mg/kg. It was possible to statistically assign over 50 % of the samples to the corresponding apiary of origin, the best results being obtained for the Tío Natalio apiary (75 %); this classification was even better in the case of the harvest period, as more than 75 % of the samples were correctly assigned, and in two periods (April-May and June) a 90 % rate was obtained. [ABSTRACT FROM AUTHOR]

In this study including the field of metabolomics approach for food, the evaluation of untargeted compounds using HILIC–ESI/TOF/MS and multivariate statistical analysis method is proposed for the assessment of classification, contamination and degradation of infant formula. HILIC mode is used to monitor more detected numbers in infant formulas in the ESI-positive scan mode than the reversed phase. The repeatability of the non-targeted contents from 4 kinds of infant formulas based on PCA was less than the relative standard deviation of 15% in all groups. The PCA pattern showed that significant differences in the classification of types and origins, the contamination of melamine and the degradations for one week were evaluated using HILIC–ESI/TOF/MS. In the S-plot from the degradation test, we could identify two markers by comparison to standards as nicotinic acid and nicotinamide. With this strategy, the differences from the untargeted compounds could be utilized for quality and safety assessment of infant formula. [ABSTRACT FROM AUTHOR]

In the present work two different approaches, a semi-quantitative and a Derringer function approach, were developed to assist column selection for method development in targeted metabolomics. These approaches were applied in the performance assessment of three HILIC columns with different chemistries (an amide, a diol and a zwitterionic phase). This was the first step for the development of a HILIC UPLC–MS/MS method that should be capable to analyze a large number of polar metabolites. Two gradient elution profiles and two mobile phase pH values were tested for the analysis of multi-analyte mixtures. Acquired chromatographic data were firstly treated by a ratiometric, “semi-quantitative” approach which quantifies various overall analysis parameters (e.g. the percent of detected compounds, retentivity and resolved critical pairs). These parameters were used to assess chromatographic performance in a rather conventional/traditional and cumbersome/labor-intensive way. Secondly, a comprehensive and automated comparison of the three columns was performed by monitoring several well-known chromatographic parameters (peak width, resolution, tailing factor, etc.) using a lab-built programming script which calculates overall desirability utilizing Derringer functions. Derringer functions exhibit the advantage that column performance is ultimately expressed in an objective single and quantitative value which can be easily interpreted. In summary, results show that each column exhibits unique strengths in metabolic profiling of polar compounds. The applied methodology proved useful for the selection of the most effective chromatographic system during method development for LC–MS/MS targeted metabolomics, while it could further assist in the selection of chromatographic conditions for the development of multi-analyte methods. [ABSTRACT FROM AUTHOR]

Tetrodotoxins (TTXs), the pufferfish venom traditionally associated with Indo-Pacific area, has been reported during last decades in ever wider range of marine organisms and ever more geographical areas, including shellfish in Europe. Wild mussels ( Mytilus galloprovincialis ) grown in the Marche Region (N Adriatic Sea, Italy) were shown to be prone to TTX contamination during the warm season, with a suspected role of Vibrio alginolyticus characterized by non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS)-encoding genes. This work aimed to deepen the knowledge about the toxin's origin and the way through which it accumulates in mussels. A two-year study (spring-summer 2020-2021) confirmed the recurrent presence of TTX (11-68 µg kg -1 ) in the official monitored natural mussel beds of the Conero Riviera. During 2021, a supplementary nonroutine monitoring of a natural mussel bed in the same area was carried out weekly from June until August for TTXs and/or the presence of V. alginolyticus . Biotic (mussels, mesozooplankton, worms and phytoplankton); abiotic (water and sediment) matrices and phytoplankton assemblage characterizations were studied. Mussels showed relevant TTX contamination levels (9-296 µg kg -1 ) with extremely rapid TTX accumulation/depletion rates. The toxin presence in phytoplankton and its distribution in the different mussel tissues supports its possible exogenous origin. The V. alginolyticus count trend overlaps that of TTX contamination in mussels, and similar trends were reported also for some phytoplankton species. The role of V. alginolyticus carrying NRPS or PKS genes as a possible TTX source and of phytoplankton as a "potential vector" should therefore be further investigated., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

[Display omitted] Among all the –omics approaches, proteomics and metabolomics have received increased attention over the last decade. Both approaches have reached a certain level of maturity, showing their relevance in numerous clinical applications, including biomarkers discovery, improved diagnosis, staging, and prognosis of diseases, as well as a better knowledge on various (patho-)physiological processes. Analytically, reversed-phase liquid chromatography – mass spectrometry (RPLC-MS) is considered the golden standard in proteomics and metabolomics, due to its ease of use and reproducilibity. However, RPLC-MS alone is not sufficient to resolve the complexity of the proteome, while very polar metabolites are typically poorly retained. In this context, hydrophilic interaction chromatography (HILIC) represents an attractive complementary approach, due to its orthogonal separation mechanism. This review presents an overview of the literature reporting the application of HILIC-MS in metabolomics and proteomics. For metabolomics the focus is on the analysis of bioactive lipids, amino acids, organic acids, and nucleotides/nucleosides, whereas for proteomics the analysis of complex samples and protein post-translational modifications therein using bottom-up, middle up/down proteomics and intact protein analysis is discussed. The review handles the technological aspects related to the use of HILIC-MS in both proteomics and metabolomics, paying attention to stationary phases, mobile phase conditions, injection volume and column temperature. Recent trends and developments in the application of HILIC-MS in proteomics and metabolomics are also presented and discussed, highlighting the advantages the technique can provide in addition or complementary to RPLC-MS, as well as the current limitations and possible solutions. [ABSTRACT FROM AUTHOR]

Bispecific antibodies (bsAbs) are considered as an important class of biopharmaceutical drugs, with about 160 products in clinical trials. From an analytical point of view, the correct chain-association is one of the most critical challenge to monitor during bsAbs development and production. In the present study, a full analytical workflow has been developed based on the use of various chromatographic modes: size exclusion chromatography (SEC), ion exchange chromatography (IEX), reversed phase liquid chromatography (RPLC), and hydrophilic interaction chromatography (HILIC), all combined with high resolution mass spectrometry (MS). This analytical strategy was applied to Hemlibra® (emicizumab), which is certainly the most successful commercial bsAb to date. Using this strategy, it was possible to monitor the presence of mispaired bsAb species and detect and identify additional post-translational modifications (PTMs). [Display omitted] • Full analytical characterization of the bispecific antibody emicizumab was performed. • Multiple LC-MS strategies in a multi-level approach were used. • Intact level SEC-MS and CEX-MS allowed unveiling size and charge variants. • Middle-up level RPLC-MS and HILIC-MS allowed characterizing PTMs and glycan profile. • Overall LC-MS approaches ensured the monitoring of the correct chains association. [ABSTRACT FROM AUTHOR]

The identification and accurate quantitation of the various glycoforms contained in therapeutic monoclonal antibodies (mAbs) is one of the main analytical needs in the biopharmaceutical industry, and glycosylation represents a crucial critical quality attribute (CQA) that needs to be addressed. Currently, the reference method for performing such identification/quantitation consists of the release of the N-glycan moieties from the mAb, their labelling with a specific dye (e.g., 2-AB or RFMS) and their analysis by HILIC-FLD or HILIC-MS. In this contribution, the potential of a new cost- and time-effective analytical approach performed at the protein subunit level (middle-up) was investigated for quantitative purposes and compared with the reference methods. The robustness of the approach was first demonstrated by performing the relative quantification of the glycoforms related to a well characterized mAb, namely adalimumab. Then, the workflow was applied to various glyco-engineered mAb products (i.e., obinutuzumab, benralizumab and atezolizumab). Finally, the glycosylation pattern of infliximab (Remicade®) was assessed and compared to two of its commercially available biosimilars (Remsima® and Inflectra®). The middle-up analysis proved to provide accurate quantitation results and has the added potential to be used as multi-attribute monitoring method. [ABSTRACT FROM AUTHOR]

Citrus pectins were studied by enzymatic fingerprinting using a simultaneous enzyme treatment with endo-polygalacturonase (endo-PG) from Kluyveromyces fragilis and pectin lyase (PL) from Aspergillus niger to reveal the methyl-ester distribution patterns over the pectin backbone. Using HILIC-MS combined with HPAEC enabled the separation and identification of the diagnostic oligomers released. Structural information on the pectins was provided by using novel descriptive parameters such as degree of blockiness of methyl-esterified oligomers by PG (DB PGme ) and degree of blockiness of methyl-esterified oligomers by PL (DB PLme ). This approach enabled us to clearly differentiate citrus pectins with various methyl-esterification patterns. The simultaneous use of PG and PL showed additional information, which is not revealed in digests using PG or PL alone. This approach can be valuable to differentiate pectins having the same DM and to get specific structural information on pectins and therefore to be able to better predict their physical and biochemical functionalities., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

[Display omitted] • Graphene/fibrous silica nanohybrids were synthesized and characterized. • A G/KCC-1 based SPE method was developed for enriching phospholipids. • Lipidomics phenotype of clam was performed by HILIC-MS analysis. • The proposed method was verified to be accurate and efficient. Polyunsaturated phospholipids are abundant in clam (Corbicula fluminea) but difficult to be fully extracted. Herein, graphene/fibrous silica (G/KCC-1) nanohybrids were synthesized, characterized, and applied for solid-phase extraction (SPE) of phospholipids in clam. The effectiveness of G/KCC-1 SPE was verified by hydrophilic interaction chromatography mass spectrometry (HILIC-MS) based lipidomics and statistical analysis. The ions of PE 16:0/18:1 (m / z 716.4), PC 16:0/20:5 (m / z 824.6) and etc. were regarded as the main difference among the crude lipids, acetone washed extract, and eluate of G/KCC-1 SPE. Finally, this method was validated in terms of linearity (R2 0.9965 to 0.9981), sensitivity (LOD 0.19–0.51 μg·mL−1 and LOQ 0.48 – 1.47 μg·mL−1), and precision (RSD intra-day ≤ 7.16% and RSD inter-day ≤ 7.30%). In conclusion, the G/KCC-1 SPE and HILIC-MS method was shown to be accurate and efficient in selective extracting and phenotyping phospholipids in C. fluminea. [ABSTRACT FROM AUTHOR]

• Cellulose and pectin were the major constituents of mountain tea. • Mountain tea pectin was composed mostly of HG segments. • Acid-extracted pectin consisted of high Mw populations (60−100 kDa). • The presence of pectin blocks with a different Me-esterification pattern was demonstrated. The present study was conducted to investigate the structural characteristics of an acid-extracted polysaccharide fraction from mountain tea. The monosaccharide composition revealed that uronic acids (72.4 mol%) considerably predominated in the fraction, followed by smaller amounts of galactose (14.5 mol%) and glucose (6.2 mol%). The fraction contained mostly a highly methyl-esterified homogalacturonan (HG) – 71 mol%. The pectin had a high molecular weight population (∼60−100 kDa). Enzymatic fingerprinting was employed with a combination of HG degrading enzymes and LC-HILIC-MS, HPAEC, HPSEC to examine the structure in greater detail. Unsaturated oligomers released indicated the presence of large blocks of highly methyl-esterified GalA residues. Furthermore, the presence of blocks of non-esterified GalA residues and partly methyl-esterified and acetylated GalA residues in HG domain was demonstrated. The research findings provide a basis for further investigations regarding biological activity and commercial exploitation of mountain tea. [ABSTRACT FROM AUTHOR]