Online Hydrophilic Interaction Chromatography Research Articles

Online LC-UV-ESI-MS/MS Comparative Analysis of Changes in Goat Colostrum N/O-Glycopatterns at Different Parities.

Goat milk oligosaccharides are complex carbohydrates with a variety of biological functions. Free oligosaccharides from goat milk show more similarity to human milk than cow milk. At present, changes in goat milk glycoconjugates at different parities remain poorly studied. Herein, we qualitatively and quantitatively compared the goat milk glycoprotein N/O-glycome at different parities using a stable isotope labeling followed by electrospray ionization mass spectrometry and online hydrophilic interaction chromatography. N-Glycans were mainly fucosylated and nonfucosylated nonsialylated, and both fucosylation and sialylation gradually increased with parity, amounting (at the third parity) to 1.25 times and 3.3 times those of the first parity, respectively. O-Glycans were mostly nonfucosylated and nonsialylated, and sialylation increased with increasing parity, and Neu5Ac-sialylated was up to 9 times higher in the third parity than in the first parity, whereas Neu5Gc-sialylated was 5.5 times higher. This study provides a reference for exploring an alternative milk source closest to human milk and for the development of humanized formula milk.

Bottom-up analysis using liquid chromatography-Fourier transform mass spectrometry to characterize fucosylated chondroitin sulfates from sea cucumbers.

Fucosylated chondroitin sulfates (FCSs) from sea cucumbers have repetitive structures that exhibit minor structural differences based on the organism from which they are recovered. A detailed characterization of FCSs and their derivatives is important to establish their structure–activity relationship in the development of new anticoagulant drugs. In the current study, online hydrophilic interaction chromatography–Fourier transform mass spectrometry (FTMS) was applied to analyze the FCS oligosaccharides generated by selective degradation from four species of sea cucumbers, Isostichopus badionotus, Pearsonothuria graeffei, Holothuria mexicana and Acaudina molpadioides. These depolymerized FCS fragments were quantified and compared using the glycomics software package, GlycReSoft. The quantified fragments mainly had trisaccharide-repeating compositions and showed significant differences in fucosylation (including its sulfation) among different species of sea cucumbers. Detailed analysis of FTMS ion peaks and top-down nuclear magnetic resonance spectroscopy of native FCS polysaccharides verified the accuracy of this method. Thus, a new structural model for FCS chains from these different sea cucumbers was defined. This bottom-up approach provides rich detailed structural analysis and provides quantitative information with high accuracy and reproducibility and should be suitable for the quality control in FCSs as well as their oligosaccharides.

Release, Separation, and Recovery of Monomeric Reducing N-Glycans with Pronase E Combined with 9-Chloromethyl Chloroformate and Glycosylasparaginase.

The glycan moiety of glycoproteins plays key roles in various biological processes. However, there are few versatile methods for releasing, separating, and recovering monomeric reducing N-glycans for further functional analysis. In this study, we developed a new method to achieve the release, separation, and recovery of monomeric reducing N-glycans using enzyme E (Pronase E) combined with 9-chloromethyl chloroformate (Fmoc-Cl) and glycosylasparaginase (GA). Ovalbumin, ribonuclease B, ginkgo, and pine nut glycoproteins were used as materials and sequentially enzymatically hydrolyzed with Pronase E, derivatized with Fmoc-Cl, and enzymatically hydrolyzed with GA. The products produced by this method were then detected by electrospray ionization mass spectrometry, high-performance liquid chromatography (HPLC), and online hydrophilic interaction chromatography (HILIC-MS) separation. The results showed that all N-glycans with essentially one amino acid obtained with Pronase E were labeled with Fmoc-Cl and could be efficiently separated and detected via HPLC and HILIC-MS. Finally, the isolated Asn-glycan derivatives were digested with GA, enabling the recovery of all monomeric reducing N-glycans modified by core α-1,3 fucose. This method was simple, inexpensive, and broadly applicable and could therefore be quite important for analysis of the structure-function relationships of glycans.

Application of Kinetically Optimised Online HILIC × RP-LC Methods Hyphenated to High Resolution MS for the Analysis of Natural Phenolics

Phenolics are a large group of secondary plant metabolites that are of interest because of their proposed health benefits. The analysis of plant phenolics is challenging due to their extreme structural diversity. Comprehensive two-dimensional liquid chromatography (LC × LC) coupled to high-resolution mass spectrometry (HR-MS) offers a powerful analytical tool for the analysis of such complex mixtures. Especially, the combination of hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RP-LC) is attractive for phenolic analysis due to the orthogonal group-type separations attainable. However, online hyphenation of HILIC and RP-LC is complicated by the relative elution strengths of the mobile phases used in both dimensions. Coupled to the inherent complexity of method development in LC × LC, this hampers the more widespread application of HILIC × RP-LC. In this study, a generic HILIC × RP-LC‒DAD-MS methodology for phenolic analysis utilising dilution of the first dimension flow and large volume injection in the second dimension is derived by kinetic optimisation of experimental parameters to provide maximum performance. The scope of the experimental configuration is demonstrated by its application to the analysis of rooibos tea, wine and grape samples containing a range of different flavonoid and non-flavonoid phenolic classes. Using this approach, excellent chromatographic performance was obtained, and a total of 149 phenolic compounds were tentatively identified in the investigated samples based on retention data in two dimensions, UV–Vis spectral as well as high- and low collision energy HR-MS data (72 in grape seeds, 32 in rooibos tea and 45 in wine and grapes) with minimal method development time. The results confirm the applicability of the proposed methodology for the detailed screening of phenolic constituents in natural products.

Predictive kinetic optimisation of hydrophilic interaction chromatography × reversed phase liquid chromatography separations: Experimental verification and application to phenolic analysis

Method development and optimisation for comprehensive two-dimensional liquid chromatography (LC × LC) is complex, since this involves the intricate relationships between a large number of experimental parameters with the aim of achieving three conflicting goals: maximising separation performance (peak capacity), minimising analysis time and minimising dilution. This is especially true for the on-line combination of hydrophilic interaction chromatography (HILIC) and reversed phase LC (RP-LC) due to the relative elution strengths of the mobile phases used in these modes, which has severe implications for the modulation process and dilution.In this study we report a predictive kinetic optimisation tool for on-line HILIC × RP-LC which is based on theoretical relationships between the optimisation goals, the target analyte properties and chromatographic parameters (column dimensions, flow rates, mobile phases, injection volumes, etc.), allowing all chromatographic parameters to vary simultaneously within defined ranges. Experimental restrictions, such as pressure limits, flow rates, etc., are enforced to ensure all results are practically achievable on a given instrumental configuration. A Pareto-optimality approach is then used to obtain optimal sets of experimental conditions, from which the one(s) best satisfying the requirements in terms of time, dilution and/or peak capacity can be chosen. To overcome the challenges associated with mobile phase incompatibility in the coupling of HILIC and RP-LC, splitting of the first dimension HILIC flow, dilution with an aqueous make-up, or a combination of both, were investigated to establish the best approach to minimise total dilution and maximise performance. The validity of the methodology is demonstrated by deriving optimal conditions for the HILIC × RP-LC separation of procyanidins on selected columns and subsequent experimental verification of the performance for the separation of a cocoa extract.

Comprehensive Two-Dimensional Hydrophilic Interaction Chromatography (HILIC) × Reversed-Phase Liquid Chromatography Coupled to High-Resolution Mass Spectrometry (RP-LC-UV-MS) Analysis of Anthocyanins and Derived Pigments in Red Wine.

Changes in anthocyanin chemistry represent some of the most important transformations involved in red wine aging. However, accurate analysis of the derived pigments, as required to study the evolution of anthocyanins and tannins during aging, is hampered by their extreme structural diversity, low levels, and the fact that many of these compounds have identical mass spectral characteristics. In this context, chromatographic separation is critical. In this contribution, the application of online hydrophilic interaction chromatography (HILIC) × reversed-phase liquid chromatography (RP-LC) separation coupled to high-resolution mass spectrometry (MS) is described for the detailed characterization of anthocyanins and their derived pigments in aged red wine. A systematic approach was followed for the optimization of HILIC × RP-LC separation parameters using a capillary liquid chromatography (LC) system in the first dimension and an ultrahigh-pressure LC system in the second dimension to ensure maximum sensitivity and performance. Ninety four (94) anthocyanin-derived pigments were tentatively identified in one- and six-year-old Pinotage wines using accurate mass and fragmentation information obtained using quadrupole-time-of-flight mass spectrometry (Q-TOF-MS). Online HILIC × RP-LC-MS was found to offer high-resolution separation, because of the combination of two different separation modes, while the structured elution order observed improved the certainty in compound identification. Therefore, this approach shows promise for the detailed elucidation of the chemical alteration of anthocyanins during wine aging.

Bottom-up low molecular weight heparin analysis using liquid chromatography-Fourier transform mass spectrometry for extensive characterization.

Low molecular weight heparins (LMWHs) are heterogeneous, polydisperse, and highly negatively charged mixtures of glycosaminoglycan chains prescribed as anticoagulants. The detailed characterization of LMWH is important for the drug quality assurance and for new drug research and development. In this study, online hydrophilic interaction chromatography (HILIC) Fourier transform mass spectrometry (FTMS) was applied to analyze the oligosaccharide fragments of LMWHs generated by heparin lyase II digestion. More than 40 oligosaccharide fragments of LMWH were quantified and used to compare LMWHs prepared by three different manufacturers. The quantified fragment structures included unsaturated disaccharides/oligosaccharides arising from the prominent repeating units of these LMWHs, 3-O-sulfo containing tetrasaccharides arising from their antithrombin III binding sites, 1,6-anhydro ring-containing oligosaccharides formed during their manufacture, saturated uronic acid oligosaccharides coming from some chain nonreducing ends, and oxidized linkage region oligosaccharides coming from some chain reducing ends. This bottom-up approach provides rich detailed structural analysis and quantitative information with high accuracy and reproducibility. When combined with the top-down approach, HILIC LC-FTMS based analysis should be suitable for the advanced quality control and quality assurance in LMWH production.

Comprehensive two-dimensional liquid chromatography coupled to the ABTS radical scavenging assay: a powerful method for the analysis of phenolic antioxidants

The on-line combination of comprehensive two-dimensional liquid chromatography (LC × LC) with the 2,2'-azino-bis(3-ethylbenzothiazoline)-6 sulphonic acid (ABTS) radical scavenging assay was investigated as a powerful method to determine the free radical scavenging activities of individual phenolics in natural products. The combination of hydrophilic interaction chromatography (HILIC) separation according to polarity and reversed-phase liquid chromatography (RP-LC) separation according to hydrophobicity is shown to provide much higher resolving power than one-dimensional separations, which, combined with on-line ABTS detection, allows the detailed characterisation of antioxidants in complex samples. Careful optimisation of the ABTS reaction conditions was required to maintain the chromatographic separation in the antioxidant detection process. Both on-line and off-line HILIC × RP-LC-ABTS methods were developed, with the former offering higher throughput and the latter higher resolution. Even for the fast analyses used in the second dimension of on-line HILIC × RP-LC, good performance for the ABTS assay was obtained. The combination of LC × LC separation with an on-line radical scavenging assay increases the likelihood of identifying individual radical scavenging species compared to conventional LC-ABTS assays. The applicability of the approach was demonstrated for cocoa, red grape seed and green tea phenolics.

Toward Unraveling Grape Tannin Composition: Application of Online Hydrophilic Interaction Chromatography × Reversed-Phase Liquid Chromatography–Time-of-Flight Mass Spectrometry for Grape Seed Analysis

Despite the significant importance of tannins in viticulture and enology, relatively little is known about the detailed chemical composition of these molecules. This is due to challenges associated with the accurate analytical determination of the highly structurally diverse proanthocyanidins which comprise tannins. In this contribution, we address this limitation by demonstrating how online comprehensive two-dimensional liquid chromatography (LC × LC) coupled to high resolution mass spectrometry (HR-MS) can be exploited as a powerful analytical approach for the detailed characterization of grape seed tannins. Hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RP-LC) were employed in the two dimensions to provide complementary information in terms of separation according to hydrophilicity and hydrophobicity, respectively. Online coupling of HILIC × RP-LC with fluorescence detection and electrospray ionization MS delivered high resolution analysis in a practical analysis time, while allowing selective detection and facilitating compound identification. Time-of-flight (TOF) MS provided high acquisition rates and sensitivity coupled to accurate mass information, which allowed detection of procyanidins up to a degree of polymerization (DP) of 16 and a degree of galloylation up to 8 in a red grape seed extract. This analytical methodology promises to shed new light on these important grape constituents and potentially on their evolution during wine production.

Data Processing Algorithms for Analysis of High Resolution MSMS Spectra of Peptides with Complex Patterns of Posttranslational Modifications

The emergence of efficient fragmentation methods such as electron capture dissociation (ECD) and electron transfer dissociation (ETD) provides the opportunity for detailed structural characterization of heavily covalently modified large peptides and small proteins such as intact histones. Even with effective gas phase ion isolation so that a single molecular precursor ion is selected, the MSMS spectrum of a heavily modified peptide may reveal the presence of a mixture of peptides with the same amino acid sequence and the same total number of posttranslational modification (PTM) moieties (same PTM composition) but with different PTM configurations or site-specific occupancy isoforms. Currently available data analysis methods depend on a deisotoping procedure, which becomes less effective when spectra (fragmentation patterns) contain many overlapping isotopic distributions. Peptide database search engines can only identify the most abundant PTM configuration (PTM arrangement on different residues) in such mixtures. To identify all the PTM configurations present in these mixtures and to estimate their relative abundances, we extended our fragment assignment by visual assistance program to search for ions representing all possible configurations, subjected to the total PTM composition constraint. This resulted in the identification of PTM configurations supported by unique fragment ions, and their relative abundances were estimated by use of a non-negative least squares procedure.

Simultaneous separation of hydrophilic and hydrophobic compounds by using an online HILIC‐RPLC system with two detectors

In the present study, we developed a novel online hydrophilic interaction chromatography (HILIC)-RPLC separation system for simultaneous separation of both hydrophilic and hydrophobic solutes in a complex sample with one injection. A HILIC Si column and a C18 column were hyphenated with an interface including two electronic 2-position valves, a solvent pump, and a solute transfer column. By using column-switching technique, the nonretained hydrophobic solutes were eluted out of the HILIC column and transferred into the C18 column to perform further separation. The hydrophilic solutes were separated on the HILIC column at the same time. One detector was equipped for each column to record the individual chromatograms. By separating a standard mixture and a traditional Chinese medicine (TCM) extract, the developed HILIC-RPLC system demonstrates its potential for "entire-components" separation of complex samples with improved peak capacity and throughput compared with the common single-column LC.

Spermine isolated and identified as the major trypanocidal compound from the snake venom of Eristocophis macmahoni causes autophagy in Trypanosoma brucei

The snake venom from the leaf-nosed viper Eristocophis macmahoni was analyzed regarding its toxic effects on the bloodstream form of Trypanosoma brucei. A considerable trypanocidal effect was measured with an IC 50 value of 186 ng/ml in bloodstream form parasites. Following several high performance liquid chromatography (HPLC) separation steps, the major trypanocidal activity was assigned to a single fraction by in vitro toxicity assays. Analysis by off-line ESI-MS n revealed an m/z value of 202.2 for the precursor ion and fragment ions of m/z=129.1 (MS 2) and 112.1 (MS 3), respectively, clearly corresponding to the molecular mass and the fragmentation pattern of the polyamine spermine. Quantification of spermine within the viper venom using an on-line hydrophilic interaction chromatography (HILIC) ESI-MS method revealed that this compound constituted approximately 1% of the dry venom mass. The polyamine oxidase activity in the fetal calf serum used for cultivation was responsible for a trypanocidal effect of pure spermine in the low micromolar range, whereas the antitrypanosomal activity of crude snake venom was virtually independent from serum, suggesting the oxidation of spermine by intrinsic venom components. Using fetal calf serum, spermine was shown to induce autophagy in the parasites using transmission electron microscopy (TEM).