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A total of 324 compounds were systematically characterized by feature-based molecular networking. This work provides an efficient strategy for the rapid discovery and characterization of unknown compounds in natural products.

The availability of various high-purity unsaturated fatty acids has a wide range of needs due to their different activities. The nonlinear preparative chromatography behavior and principle for purification of palmitoleic acid with octadecyl bonded stationary phases were studied. The peak broadening and the concentration distribution of the target compounds were used to compare different C18 stationary phases. In preparative liquid chromatography, the C18 stationary phases with low, medium, and high bonding density showed different peak broadening and concentration distribution results. Medium bonding density C18 was suitable for the purification of ethyl palmitoleic acid. The forward broadening was much greater than the backward broadening on medium bonding density C18 column. And the highest concentration distribution of impurities and the main peak was not crossed in this column. Due to the low content of crude ethyl palmitoleic acid sample, a two-step purified method yields an oily product with purity of 96.57% in the GC method. This method would be universal and extensible for constructing purification method for other unsaturated fatty acids.

Plant-derived alkaloids are bioactive natural ingredients, but their contents are relatively low in plants. Therefore, efficient enrichment of alkaloids is a prerequisite for purification and further pharmacological research. In this study, an efficient and simple strategy for enrichment of steroidal alkaloid in Fritillaria was developed for the first time based on fluorinated reverse-phase stationary phase (FC8HL). Superior selectivity between alkaloids and non-alkaloids was achieved in a non-aqueous system, and a simple solvent system containing low-content additives was applied to elute alkaloids. Key parameters that affected the elution were investigated, including different types of buffer salts and optimized concentrations. The optimized elution system was then applied to selectively enrich alkaloids from five species of Fritillaria. Its practicability was further demonstrated by enrichment of alkaloids from Fritillaria cirrhosa D.Don at a preparative level. This developed method has great potential for other types of hydrophobic alkaloids. This article is protected by copyright. All rights reserved.

ATP citrate lyase (ACLY) is a key enzyme in glucolipid metabolism with therapeutic prospect for treating hyperlipidemia and various cancers. Much effort has been put into discovering ACLY inhibitors. However, current screening approaches have limitations in sensitivity, portability and high-throughput. To develop a general screening assay, we investigated series of conditions affecting the enzymatic reaction based on the ADP-Glo luminescence assay. Bovine serum albumin (0.001%) added triggered strong and stable fluorescence signal. The optimized assay was validated and applied to screen our natural product library. Two novel inhibitors were identified with IC

Historical marquisette curtains were composed of lightweight fabrics, woven in an open-mesh and leno-type weave, usually made of silk, and found in Qing imperial buildings. As panel curtains, they were exposed to light, and so underwent fading. This study investigated the manufacturing technology and fading mechanism of dyed marquisette fabric from the Studio of Cleansing Fragrance, the Palace Museum (Beijing). The technological aspects were identified. The types of weave, fiber, and adhesive used to fix the curtain to the wooden frame were identified through microscopic observation and infrared spectroscopy. A color change characterization was performed based on UV-visible diffuse reflectance spectra. The textile colorant was identified as malachite green (MG), and its degradation by light was subsequently studied by dynamic photolysis experiments in a kinetic solution for the rapid exploration of by-products. The main degradation pathways were thus identified and the factors responsible for the induced color changes were discussed. A comparison of the liquid chromatography-mass spectrometry (LC–MS) results of the products derived from the photolysis method as well as of the samples extracted from the object allowed for the identification of the presence of different degradation pathways in the faded and unfaded parts of the textile. A metabolomics analysis was applied to account for the differences in the degradation pathways.

Bottom-up proteomics has been increasingly applied in clinical research to study the disease pathophysiology and to discover disease biomarkers. However, glycoproteomic analysis always requires tedious experimental steps for intact glycopeptide enrichment, which has been the technique bottleneck for large-scale analysis of clinical samples. Herein, we developed an automated glycopeptide enrichment method for the analysis of serum site-specific N-glycoproteome. This automated method allowed for processing one sample within 20 min. It showed higher enrichment specificity, more intact glycopeptide identifications, and better quantitative reproducibility than the traditional manual method using microtip enrichment devices. We further applied this method to investigate the serum site-specific N-glycosylation changes between four patients with pancreatic cancer and seven healthy controls. The principal component analysis of intact N-glycopeptides showed good clustering across cancer and normal groups. Furthermore, we found that the site-specific glycoforms, monofucosylated and nonsialylated oligosaccharides, on IgG1 site 180 expressed a significant decrease in pancreatic cancer patients compared to healthy controls. Together, the automated method is a powerful tool for site-specific N-glycoproteomic analysis of complex biological samples, and it has great potential for clinical utilities.

The profiling of natural products is important in modern biological sciences and new drug development. However, the separation and characterization of complex herbal extracts are significantly challenging for researchers in the biochemical field. Herein, an offline two-dimensional mixed-mode liquid chromatography × reversed-phase liquid chromatography system is developed. Our system exhibits high orthogonality and is composed of a newly prepared stationary phase in the first dimension and a traditional C

Qingfei Paidu Decoction is a Chinese medicine formula that has been proved effective in the treatment of coronavirus disease 2019. However, the comprehensive separation and characterization of Qingfei Paidu Decoction are of a great challenge due to the diversity of chemical components in a wide range of polarity. In this study, a triplex off-line two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry is developed for the analysis of Qingfei Paidu Decoction. One reversed-phase liquid chromatography×hydrophilic interaction liquid chromatography system and two reversed-phase liquid chromatography×reversed phase liquid chromatography systems were constructed to separate polar components and weak-polar components in Qingfei Paidu Decoction, respectively. Benefiting from the good orthogonality of two-dimensional liquid chromatography and high sensitivity of quadrupole time-of-flight MS, chemical components with different polarities and content were discovered. A total of 749 peaks were detected in positive and negative ionization mode and presented as a four-dimensional data plot. Meanwhile, 498 compounds belonging to 14 categories were tentatively identified. These results provide good supplementary to elucidate the material basis of Qingfei Paidu Decoction. The triplex off-line two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry strategy can be a powerful and efficient tool for the separation and characterization of chemical substances in traditional Chinese medicine formulas.

Natural products, especially alkaloids, are one of the most valuable, potential drug leads in drug discovery. As an efficient tool for complex samples, two-dimensional liquid chromatography (2D-LC) has become a powerful means of analysis and separation of natural alkaloids. Method development of 2D-LC is of great importance because it helps to enhance the selectivity, resolution and peak capacity of a separation system. However, due to the diversity of the nature and subclasses of natural alkaloids, peak tailing occurs frequently, making alkaloid separation complicated and time-consuming. To conquer such difficulties, we proposed a guide for column selection and combination in 2D-LC so as to improve the alkaloid separation. Based on a comprehensive evaluation of applicability and orthogonality of several columns, this guide would provide a simple but clear starting point for column selection of 2D-LC method development. The evaluation included seven columns to involve most separation mechanisms reported in alkaloid separation, and 49 natural alkaloid standards of various subclasses and natures. Detailed studies of peak shapes of every column were carried out as well, providing useful references to better understand the peak tailing issues of some analytes on specific columns. Subsequently, a 2D-LC method was developed using our guide to isolate an alkaloid sample from U. rhynchophylla, generating symmetrical peaks and a high orthogonality of 80.3%. Further, this evaluation process would help to have a quick understanding when a new stationary phase is designed.

Silica-based positively-charged stationary phase bonding phenylaminopropyl (named PHN) was found to produce symmetrical peak shape and higher sample loading for basic compounds. In this work, firstly, surface charge property of the PHN was evaluated by ζ-potential and retention of NO3−. A considerable amount of pH-dependent positive charges was confirmed more than that on CSH Phenyl-Hexyl, a commercial positively-charged phenyl stationary phase. Then chromatographic evaluation of standard alkaloids revealed that PHN could offer better peak shape and higher column efficiency at lower pH, and it functioned well under a wide range of buffer ionic strength. The PHN also showed different selectivity for basic compounds compared to the CSH Phenyl-Hexyl. Furthermore, it provided superior peak shape for high sample mass, demonstrating potential applications of this stationary phase in a preparative scale. These results can be explained by the strong charge intensity of the PHN stationary phase. Finally, the PHN was applied to separate a fraction from rhizomes of Corydalis decumbens, and purify dehydrocorybulbine from Corydalis yanhusuo W.T. Wang. Our study indicated the advantages and potential applications of the phenylaminopropyl bonded PHN stationary phase for basic compound separation.

Thrombotic complication has been an important symptom in critically ill patients with COVID-19. It has not been clear whether the virus spike (S) protein can directly induce blood coagulation in addition to inflammation. Heparan sulfate (HS)/heparin, a key factor in coagulation process, was found to bind SARS-CoV-2 S protein with high affinity. Herein, we found that the S protein can competitively inhibit the bindings of antithrombin and heparin cofactor II to heparin/HS, causing abnormal increase in thrombin activity. SARS-CoV-2 S protein at a similar concentration (~10 μg/mL) as the viral load in critically ill patients can cause directly blood coagulation and thrombosis in zebrafish model. Furthermore, exogenous heparin/HS can significantly reduce coagulation caused by S protein, pointing to a potential new direction to elucidate the etiology of the virus and provide fundamental support for anticoagulant therapy especially for the COVID-19 critically ill patients.

A new method based on solid-phase extraction-ultraperformance liquid chromatography (SPE-UPLC) was developed for the determination of five protopanaxadiol ginsenosides in ginseng. The ginsenosides were extracted from ground ginseng samples using water-saturated

The human natural killer-1 (HNK-1) epitope is a unique sulfated trisaccharide sequence presented on O- and N-glycans of various glycoproteins and on glycolipids. It is overexpressed in the nervous system and plays crucial roles in nerve regeneration, synaptic plasticity, and neuronal diseases. However, the investigation of functional roles of HNK-1 in a more complex glycan context at the molecular level remains a big challenge due to lack of access to related structurally well-defined complex glycans. Herein, we describe a highly efficient chemoenzymatic approach for the first collective synthesis of HNK-1-bearing O-mannose glycans with different branching patterns, and for their nonsulfated counterparts. The successful strategy relies on both chemical glycosylation of a trisaccharide lactone donor for the introduction of sulfated HNK-1 branch and substrate promiscuities of bacterial glycosyltransferases that can tolerate sulfated substrates for enzymatic diversification. Glycan microarray analysis with the resulting complex synthetic glycans demonstrated their recognition by two HNK-1-specific antibodies including anti-HNK-1/N-CAM (CD57) and Cat-315, which provided further evidence for the recognition epitopes of these antibodies and the essential roles of the sulfate group for HNK-1 glycan-antibody recognition.

Multilayer objects with different interfaces are quite typical for architectural heritage, and from them may be inferred the age, production process, and deterioration mechanism through analyzing characteristic compositions with advanced analytical techniques. The Meiwu ceiling in the Hall of Mental Cultivation of the Palace Museum was found to contain many paper-based layers during conservation. Once several surface strata were detached, a colorful layer of printed fabric textile was discovered integrally. Through microscopic observation and micro-attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) imaging, the overall structure consisted of 11 layers, namely, bast paper, cotton wiring, xuan paper, cotton printed fabric, two yellow board papers, bamboo paper, three wood pulp paper and surface coatings, and starch was considered as an organic adhesive. For identification of the printed fabric’s color palette, ultra-performance liquid chromatography (UPLC) combined with high-resolution quadrupole time-of-flight (QTOF) technology, non-invasive macro X-ray fluorescence (MA-XRF) and desorption electrospray ionization mass spectrometry imaging (DESI-MSI) were applied in situ. Seven industrial synthetic dyes, including auramine O, malachite green, and eosin Y with corresponding by-products, as well as chromium-based pigments considered as dark draft line, were confirmed. By X-ray diffraction (XRD), scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS), and micro Fourier transform infrared spectroscopy (micro FTIR, other results showed chalk soil and talc for the outermost coating. According to the synthetic time of industrial dyes and degradation degree of paper, there were at least four occurrences and their specific time periods were speculated.

Oligosaccharides are one of the most important components in mammalian milk. Milk oligosaccharides can promote colonization of gut microbiota and protect newborns from infections. The diversity and structures of MOs differ among mammalian species. MOs in human and farm animals have been well-documented. However, the knowledge on MOs in rat and mouse have been very limited even though they are the most-widely used models for studies of human physiology and disease. Herein, we use a high-sensitivity online solid-phase extraction and HILIC coupled with electrospray tandem mass spectrometry to analyze the acidic MOs in rat and mouse. Among the fifteen MOs identified, twelve were reported for the first time in rat and mouse together with two novel sulphated oligosaccharides. The complete list of acidic oligosaccharides present in rat and mouse milk is the baseline information of these animals and should contribute to biological/biomedical studies using rats and mice as models.

In this study, a solid-phase extraction with liquid chromatography and tandem mass spectrometry method was developed to determine the degree of glycosylation of glycosylation sites and the ratio of free carrier protein to total carrier protein for glycoconjugate vaccines. To remove and enrich the glycosylated peptides, a solid-phase extraction method was developed, optimized, and hyphenated to liquid chromatography-tandem mass spectrometry. The developed solid-phase extraction with liquid chromatography-tandem mass spectrometry method was shown to possess a wide linear dynamic range (0.03-100 μg/mL), a high sensitivity (0.03 μg/mL for CRM197), good interday and intra-day precision (relative standard deviation of peak area < 3.3%), and good recoveries from vaccine matrix (90-105%). Finally, the method was utilized to determine the degree of glycosylation and free carrier protein to total carrier protein ratio for pneumococcal conjugate vaccines and meningococcal vaccines. For quality evaluation of glycoconjugate vaccines, the method could provide more information than the traditional size exclusion chromatography method. Fourteen and twelve reported glycosylation sites for CRM197- and tetanus toxin-based vaccines can be detected, respectively.

A sensitive, simple and rapid method for the simultaneous determination of eleven arsenic species has been developed. A high performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) technique was used for the analysis of eleven arsenic species in less than 17 min. Different extraction solutions were explored and the recovery results using water and aqueous acidic solvents, aqueous organic solvents and enzymes showed that 40 mg protease with 0.75 mL 0.5% hydrochloric acid (v/v) as the extraction agent gave the best experimental results. Species separation with ammonium carbonate solution as the mobile phase was conducted on an anion-exchange chromatographic column using gradient elution. The column temperature was 20 °C and kinetic energy discrimination (KED) was employed to eliminate spectral interference. The use of KED mode effectively removed interference from 75ArCl. The detection limit (LD) of the method was in the range of 0.11–0.59 μg kg−1. Repeatability values obtained for spiked real fish samples were in the range of 1.1%–7.6%. Accuracy was calculated based on the analysis of spiked real fish samples at five concentration levels. Obtained recoveries were 91%–106%. The validated method was used in a pilot study to analyze real samples of fish, the organic arsenic especially AsB was the major arsenic specie present in the analyzed samples, only trace amount of inorganic arsenic were detected. The analytical method should improve the assessment of human exposure associated with arsenic intake from fish.

A quick, simple, and reliable method was developed for the simultaneous determination of free and total choline and l-carnitine in infant formula employing a novel hydrophilic interaction liquid chromatography with tandem mass spectrometry method. Microwave-assisted hydrolysis was used to shorten the hydrolysis time to only 15 min. A novel Click XIon zwitterionic stationary phase was chosen because it gave better retention, perfect resolution, and sharper symmetrical peaks compared to traditional columns. The matrix effect under different experimental conditions was evaluated by using the matrix effect factor, which employs stable isotopically labeled internal standards and is more appropriate for evaluating the matrix effect related to endogenous analytes. The accuracy and precision of the method were validated with certified reference materials. The fortified recovery values for choline and l-carnitine were between 85.0 and 104% with relevant standard deviations

The conventional UV/Vis spectroscopy methods recommended by the European Pharmacopoeia (EP) for determining hexosamine, hexonic acid and methylpentose in pneumococcal polysaccharide vaccine (PPSV) hydrolates are time-consuming due to derivatization process (typically, an analysis cycle is more than 4 h) and improvements of selectivity and precision of the methods are in demand. In this study, a new approach based on hydrophilic interaction liquid chromatography and triple quadrupole mass spectrometry (HILIC-MS/MS) was optimized to overcome the drawbacks of the EP methods for simultaneous determination of methylpentose, hexose, hexosamine and hexonic acid in PPSV hydrolysates. The chromatographic, MS and sample hydrolysis conditions were systematically investigated. A zwitterionic column, Click Cys, using a gradient elution with a mobile phase of 10 mM ammonium formate (pH 4.3) in acetonitrile from 72% to 21% in 6 min was applied for separating the targets, which exhibited low column bleeding, easy equilibration and long-term stability. The HILIC-MS/MS method showed a high sensitivity (LOD = 0.98 μg L−1 for hexonic acid), a good repeatability (RSD of peak area less than 1.669%), accuracy (92.9%–104.2%), recovery (97.6%–99.3%) and a wide linear range. The RSD of retention time obtained from more than 3000 injections in three months was less than 1.64%. The new method was compared with the EP method for determining hexosamine in 23 serotypes of PPSV hydrolysates. The results indicated that the new HILIC-MS/MS method was highly selective, accurate, stable and extremely fast due to without need of derivatization, as compared to the conventional EP methods.

A quantitative method using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) was developed for the determination of 60 compounds, belonging to a variety of veterinary drug (VD) classes, in meat. The included analytes belong to the following VD classes: β-agonists, sulfanilamides, quinolones, macrolides, tetracyclines, β-lactams, nitroimidazoles, glucocorticoids, sex hormones, chloromycetins, sedatives, and olaquindox metabolite. The effective PRiME pass-through cleanup procedure was used to ensure high extraction efficiency and good sample cleanup after a simple liquid extraction of the meat samples with acetonitrile/water. The developed method was validated successfully. Mean recoveries for all analytes ranged from 80 to 116%, with the relative standard deviations (RSDs) ≤ 7.8%. Limits of quantification were in the range 0.05–3.0 μg kg−1 and limits of detection were in the range 0.1–10 μg kg−1. The matrix effect was evaluated for the different meat matrices and was found to be markedly different in different matrices. The validated method was used in a pilot study to analyze real samples of pork, beef, mutton, chicken, and pork liver, lambs’ liver, and chicken liver. Trace amounts of β-agonists, oxytetracycline, quinolones, chloromycetin sulfadimidine, and 3-methyl-quinoxaline-2-carboxylicacid were detected in these samples. In conclusion, this workflow can provide a simpler and more cost-effective alternative to conventional analytical methods and is compatible with processing large sample numbers in a short time period.

A mixed-mode reversed phase/positively charged repulsion stationary phase C8PN composed of octyl and amino group has been developed for separation of intact protein. Before the separation of proteins, a set of probe compounds were employed to evaluate the chromatographic properties of C8PN, demonstrating typical reversed phase/positively charged repulsion interaction on this stationary phase as estimated. Then the new C8PN stationary phase was used to separate a standard protein mixture on the reversed phase mode. Compared with a commercial C4 stationary phase, it showed different selectivity for some proteins. In order to better understand the properties of C8PN, the effect of acetonitrile content was investigated based on retention equation. Higher values of the equation parameters on C8PN demonstrated that the protein retentions were more sensitive to the change of acetonitrile content. Besides, the influences of buffer salt additives on the protein retentions were also studied. The retention factors of the proteins got larger with the increase of buffer salt concentration, which confirmed the positively charged repulsion interaction on the column. Finally, the C8PN was further applied to separate oxidized- and reduced- forms of Recombinant Human Growth Hormone. Our study indicated the advantages and application potential of mixed-mode reversed phase/positively charged repulsion stationary phase for intact protein separation.

Sialylation typically occurs at the terminal of glycans, and its aberration often correlates with diseases including neurological diseases and cancer. However, the analysis of glycoprotein sialylation in complex biological samples is still challenging due to their low abundance. Herein, a histidine-bonded silica (HBS) material with a hydrophilic interaction and switchable surface charge was fabricated to enrich sialylated glycopeptides (SGPs) from the digest of proteomics samples. High selectivity toward SGPs was obtained by combining the superior hydrophilicity and switchable-charge characteristics. During the enrichment of sialylated glycopeptides from bovine fetuin digest, seven glycopeptides were detected even at the ratio of 1:5000 with the nonsialylated glycopeptides, demonstrating the high specificity of SGP enrichment by using HBS material. Then, HBS material was further utilized to selectively enrich SGPs from the protein digest of human serum, and 487 glycosites were identified from only 2 μL of human serum; 92.0% of the glycopeptides contained at least one sialic acid, indicating good performance for SGP enrichment by using HBS material. Furthermore, the prepared HBS material also has great potential applications in the analysis of glycoprotein sialylation from other complex biological samples.

The diversity of O-linked glycan structures has drawn increasing attention due to its vital biological roles. However, intact O-glycopeptides with different glycans are typically not well elucidated using the current methods. In this work, an integrated strategy was developed for comprehensive analysis of O-GalNAc glycosylation by combining hydrophilic interaction chromatography (HILIC) tip enrichment, beam-type collision induced decomposition (beam-CID) detection, and in silico deglycosylation method for spectra interpretation. In this strategy, the intact O-GalNAc glycopeptides were selectively enriched and the original spectra obtained by time-of-flight (TOF)-CID were preprocessed using an in silico deglycosylation method, enabling direct searching without setting multiple glycosylation modifications, which could significantly decrease the search space. This strategy was applied to analyze the O-GalNAc glycoproteome of human serum, leading to identification of 407 intact O-GalNAc glycopeptides from 93 glycoproteins. About 81% of the glycopeptides contained at least one sialic acid, which could reveal the microheterogeneity of O-GalNAc glycosylation. Up until now, this is the largest data set of intact O-GalNAc glycoforms from complex biological samples at the proteome level. Furthermore, this method is readily applicable to study O-glycoform heterogeneity in other complex biological systems.

A rapid and robust separation method based on the positively charged reversed-phase (PGRP) stationary phase was developed for selective separation of saponins and xanthones from the rhizomes of Anemarrhena asphodeloides (A. asphodeloides). In this work, the chromatographic performances of three PGRP stationary phases with different surface positive charge densities were systematically evaluated by studying hydrophobicity, effects of pH and buffer concentration of the mobile phase, ion-exchange capacity, etc. The PGRP stationary phase exhibited reversed-phase/anion-exchange mixed-mode properties. Then the retention behaviors of xanthones were investigated. A good retention of xanthones was obtained at high pH and xanthones were easily eluted at low pH. The pH is like an on–off switch on the PGRP stationary phase that controls the retention of xanthones. Finally, this PGRP material was successfully applied to the selective separation of saponins and xanthones from A. asphodeloides. The result demonstrated that neutral and ionic compounds, such as xanthones and saponins from the rhizomes of A. asphodeloides, were separated selectively by modulating both the density of surface charges in the PGRP stationary phase and the pH of the mobile phase.

The human natural killer-1 (HNK-1) epitope is a unique sulfated trisaccharide sequence presented on

Click chemistry has attracted increasing attention for the synthesis of novel stationary phases. Considering the advantage of click chemistry, a strategy based on thiol-Michael addition was developed for the preparation of a new stationary phase herein, and a phenyl vinyl sulfone stationary phase (M-PVS) was prepared. The resulting M-PVS bonded silica was characterized by elemental analysis, solid-state 13C cross-polarization/magic-angle spinning NMR and infrared spectroscopy, confirming the successful immobilization of phenyl vinyl sulfone on the silica support. The retention properties of M-PVS were investigated and exhibited unambiguous reversed phase retention characteristics. Moreover, shape selectivity and silanol activity were studied to reveal the diverse interactions of M-PVS, including hydrophobic, π–π, hydrogen bonding, and ion-exchange interactions. In addition, de-wetting tolerance and hydrophilic properties were evaluated and a pronounced “U” retention curves were obtained, indicating enhanced retention for polar analytes and transitions of different interaction modes. Selectivity differences between M-PVS column, phenyl column and conventional C18 column were examined using series natural standards. The diverse interactions of M-PVS demonstrated its improved selectivity for the compounds with similar hydrophobic skeleton but different polar substituents.

Aberrant sialylation of glycoproteins is closely related to many malignant diseases, and analysis of sialylation has great potential to reveal the status of these diseases. However, in-depth analysis of sialylation is still challenging because of the high microheterogeneity of protein glycosylation, as well as the low abundance of sialylated glycopeptides (SGPs). Herein, an integrated strategy was fabricated for the detailed characterization of glycoprotein sialylation on the levels of glycosites and site-specific glycoforms by employing the SGP enrichment method. This strategy enabled the identification of up to 380 glycosites, as well as 414 intact glycopeptides corresponding to 383 site-specific glycoforms from only initial 6 μL serum samples, indicating the high sensitivity of the method for the detailed analysis of glycoprotein sialylation. This strategy was further employed to the differential analysis of glycoprotein sialylation between hepatocellular carcinoma patients and control samples, leading to the quantification of 344 glycosites and 405 site-specific glycoforms, simultaneously. Among these, 43 glycosites and 55 site-specific glycoforms were found to have significant change on the glycosite and site-specific glycoform levels, respectively. Interestingly, several glycoforms attached onto the same glycosite were found with different change tendencies. This strategy was demonstrated to be a powerful tool to reveal subtle differences of the macro- and microheterogeneity of glycoprotein sialylation.

A new optimization strategy for purification of alkaloids from Rhizoma Corydalis using preparative liquid chromatography was developed, featuring a selective separation of different types of alkaloids into different parts by a reversed-phase/weak cation-exchange mixed-mode column (named C18WCX) at first. The total alkaloids of Rhizoma Corydalis were divided into four fractions with fraction III and IV corresponding to the tertiary type medium bases and the quaternary type strong bases, respectively. For fraction III, a conventional C18 column was used to isolate tertiary alkaloids using acetonitrile and 0.1% phosphoric acid (adjusted with triethylamine to pH 6.0) as mobile phases. High selectivity and symmetrical peak shapes of tertiary alkaloids were obtained, resulting in six main tertiary alkaloids isolated in a single run. As strong bases, quaternary alkaloids often suffer from serious peak tailing problem on conventional C18 columns. Therefore, a silica-based strong cation-exchange (SCX) column was used for purification of fraction IV. On the SCX column, good peak shapes in high sample loading were achieved. Five main quaternary alkaloids were isolated and identified from the fraction in one-step. The procedures presented effective for the preparative isolation and purification of alkaloids from Rhizoma Corydalis.

Human milk oligosaccharides (HMOs) are one of the most abundant ingredients in breast milk, and they play a beneficial role for newborns and are important for infant health. The peripheral fucosylated sequences of HMOs, such as the histo-blood group ABH(O) and Lewis a, b, x, and y antigens, are determined by the expression of the secretor (Se) and Lewis (Le) genes in the mammary gland, and are often the recognition motifs and serve as decoy receptors for microbes. In this work, we developed a method for determination of secretor status and Lewis blood phenotype and assignment of Lewis blood-group epitopes. The method was based on electrostatic repulsion/hydrophilic interaction chromatography coupled with tandem mass spectrometry (ERLIC-MS/MS). A specifically designed stationary phase, aspartic acid-bonded silica (ABS), was used to separate the acidic and neutral HMOs by electrostatic repulsion followed by HILIC. Negative-ion electrospray MS/MS was then used for analysis of secretor status and Lewis blood phenotypes and assignment of important epitopes of HMOs from the lactating mothers by selecting a specific set of unique fragment ions.

Gangliosides, widely distributed in tissues and body fluid, have been connected to the therapy of cancer and brain related diseases. The complexity of the gangliosides structures with different polar moieties coexisting, a carbohydrate moiety and a ceramide chain, make it a great challenge in separation and analysis science. This study aimed to develop a strategy on the basis of high-accuracy data collected by an offline two-dimensional (2D) supercritical fluid chromatography (SFC) × reversed phase liquid chromatography (RPLC)/quadrupole time-of-flight (Q-ToF) system, and to integrate an in-house library with self-developed software for fast screening and identification of gangliosides from a complex sample (swine brain extract). Subsequent positive-mode MS/MS was used to validate the identified gangliosides. Finally, 153 gangliosides were separated and 79 of them were identified by the in-house library and self-developed software, 4-fold more than those by manual identification (18 gangliosides). Among the identified ones, 20 were detected in swine brain for the first time. This study established an offline 2D SFC × RPLC system and provided a new method for fast screening and automatic identification of gangliosides in complex mixtures. It will be conducive to further study of biological functions of gangliosides.

A practical method was established for the definition of chromatographic parameters in preparative liquid chromatography. The parameters contained both the peak broadening level under different amounts of sample loading and the concentration distribution of the target compound in the elution. The parameters of the peak broadening level were defined and expressed as a matrix, which consisted of sample loading, the forward broadening and the backward broadening levels. The concentration distribution of the target compound was described by the heat map of the elution profile. The most suitable stationary phase should exhibit the narrower peak broadening and it was best to broaden to both sides to compare to the peak under analytical conditions. Besides, the concentration distribution of the target compounds should be focused on the middle of the elution. The guiding principles were validated by purification of amitriptyline from the mixture of desipramine and amitriptyline. On the selected column, when the content of the impurity desipramine was lower than 0.1%, the recovery of target compound was much higher than the other columns even when the sample loading was as high as 8.03 mg/cm(3). The parameters and methods could be used for the evaluation and selection of stationary phases in preparative chromatography.

A method was developed for the determination of glyphosate residues in plant-derived food using a two-step solid phase extraction (SPE) combined with mixed-mode liquid chromatography-tandem mass spectrometry (LC-MS/MS). The samples were extracted with water. Then, the extracting solution was pretreated by a C18 cartridge to remove protein and weak-polar interferences and further directly extracted using a strong anion exchange (SAX) cartridge to remove neutral and basic substances. The obtained glyphosate residues from the SPE were separated on a hydrophilic interaction/weak anion-exchange (HILIC/WAX) column and detected by mass spectrometry with negative electrospray ionization (ESI-) in multiple reaction monitoring (MRM) mode. This approach was evaluated by five different kinds of plant-derived food (soybean, corn, carrot, apple, and spicy cabbage) matrices in terms of matrix effect and recovery. Results showed that two-step SPE and mixed-mode chromatography separation provided the method with a very low matrix effect, and the spiked recoveries of glyphosate were satisfied in the range of 83.1 to 100.8 % at three spiked levels. The limit of quantification (LOQ) and detection (LOD) of the method in different matrices were 0.016–0.026 and 0.005–0.008 mg kg−1, respectively. The procedure was validated and showed good accuracy and precision over a large linear range of 0.02–10 mg kg−1.

The separation of basic macrolide antibiotics suffers from peak tailing and poor efficiency on traditional silica-based reversed-phase liquid chromatography columns. In this work, a C18HCE column with positively charged surface was applied to the separation of macrolides. Compared with an Acquity BEH C18 column, the C18HCE column exhibited superior performance in the aspect of peak shape and separation efficiency. The screening of mobile phase additives including formic acid, acetic acid and ammonium formate indicated that formic acid was preferable for providing symmetrical peak shapes. Moreover, the influence of formic acid content was investigated. Analysis speed and mass spectrometry compatibility were also taken into account when optimizing the separation conditions for liquid chromatography coupled with tandem mass spectrometry. The developed method was successfully utilized for the determination of macrolide residues in a honey sample. Azithromycin was chosen as the internal standard for the quantitation of spiramycin and tilmicosin, while roxithromycin was used for erythromycin, tylosin, clarithromycin, josamycin and acetylisovaleryltylosin. Good correlation coefficients (r(2) > 0.9938) for all macrolides were obtained. The intra-day and inter-day recoveries were 73.7-134.7% and 80.7-119.7% with relative standard deviations of 2.5-8.0% and 3.9-16.1%, respectively. Outstanding sensitivity with limits of quantitation (S/N ≥ 10) of 0.02-1 μg/kg and limits of detection (S/N ≥ 3) of 0.01-0.5 μg/kg were achieved.

A mixed-mode chromatographic method based on surface electrostatic exclusion and reversed-phase chromatography was established for the determination of alkaloids present in

Human milk glycans provide a broad range of carbon sources for gut microbes in infants. Levels of protein glycosylation in human milk vary during lactation and may also be affected by the stages of gestation and lactation and by the secretor status of the mother. This was the first study to evaluate systematically dynamic changes in human milk oligosaccharides and fucosylated N-glycans in the milk of Chinese mothers with different secretor statuses during 6 months of lactation. Given the unique single nucleotide polymorphism site (rs1047781, A385T) on the fucosyltransferase 2 gene among Chinese populations, our report provides a specific insight into the milk glycobiome of Chinese mothers, which may exert effects on the gut microbiota of infants that differ from findings from other study cohorts., The milk glycobiome has a significant impact on the gut microbiota of infants, which plays a pivotal role in health and development. Fucosylated human milk oligosaccharides (HMOs) and N-glycans on milk proteins are beneficial for the development of healthy gut microbiota, and the fucosylation levels of these glycans can be affected by the maternal fucosyltransferase 2 gene (FUT2). Here, we present results of longitudinal research on paired milk and stool samples from 56 Chinese mothers (CMs) and their breast-fed children. Changes of HMOs and fucosylated N-glycans in milk of CMs at different lactation stages were detected, which allowed characterization of the major differences in milk glycans and consequential effects on the gut microbiome of infants according to maternal FUT2 status. Significant differences in the abundance of total and fucosylated HMOs between secretor and nonsecretor CMs were noted, especially during early lactation. Despite a tendency toward decreasing milk protein concentrations, the fucosylation levels of milk N-glycans increased during late lactation. The changes in the levels of fucosylated HMOs and milk N-glycans were highly correlated with the growth of Bifidobacterium spp. and Lactobacillus spp. in the gut of infants during early and later lactation, respectively. Enriched expression of genes encoding glycoside hydrolases, glycosyl transferases, ATP-binding cassette (ABC) transporters, and permeases in infants fed by secretor CMs contributed to the promotion of these bacteria in infants. Our data highlight the important role of fucosylated milk glycans in shaping the gut microbiome of infants and provide a solid foundation for development of “personalized” nutrition for Chinese infants. IMPORTANCE Human milk glycans provide a broad range of carbon sources for gut microbes in infants. Levels of protein glycosylation in human milk vary during lactation and may also be affected by the stages of gestation and lactation and by the secretor status of the mother. This was the first study to evaluate systematically dynamic changes in human milk oligosaccharides and fucosylated N-glycans in the milk of Chinese mothers with different secretor statuses during 6 months of lactation. Given the unique single nucleotide polymorphism site (rs1047781, A385T) on the fucosyltransferase 2 gene among Chinese populations, our report provides a specific insight into the milk glycobiome of Chinese mothers, which may exert effects on the gut microbiota of infants that differ from findings from other study cohorts.

Since the content of alkaloids is usually low in plants and they are easily co-eluted with other constituents, enrichment of alkaloids is essential in the discovery of bioactive lead compounds from natural products. In this paper, an easy SPE enrichment method was developed in a buffer-free solvent system based on electrostatic repulsion mechanism. The feasibility of the new method was verified by successful enrichment of alkaloids from Scopolia tangutica (S. tangutica) with an optimized eluting condition. Then this developed method was applied to other representative plants in different families, including Przewalskia tangutica and Peganum harmala L, Lycoris radiata and Menispermum dauricum DC, which enlarged the scope of applicability. Additionally, the new SPE procedure avoided possible structural change destruction caused by pH change. Simple solvent system, including formic acid (FA) and methanol, would benefit subsequent mass analysis, quantity determination and bioactivity screening, and so on.

In this study, a novel two dimensional liquid chromatography – mass spectrometry (2D-LC-MS) method with use of a weak anion exchange column between the 1st DLC RP column and the 2nd DLC RP column (RP1-WAX-RP2) was developed and applied to identify drug impurities from MS incompatible mobile phases containing sodium 1-octanesulfonate and non-volatile buffer. The 1st DLC conditions follow exactly the original standard HPLC method recorded in Chinese Pharmacopeia (ChP), European Pharmacopeia (EP) or US Pharmacopeia (USP). An impurity fraction was collected with a built-in sample loop (100 μL) and transferred to the WAX column where 1-octanesulfonate and phosphate were trapped and removed. While, the impurity and other cations were eluted to the 2nd D column (RP2) for separation and identification by connected IT-TOF MS. Methods were programmed and applied to identify impurities in two generic drugs, sulpiride (hydrophilic drug with logP 0.57) and dobutamine (hydrophobic drug with logP 3.6). The results indicate that the methods based on RP1-WAX-RP2 column configuration offer a feasible solution for direct impurity identification in generic drug product or API without needs of off-line desalting from the MS incompatible mobile phases containing ion-pairing reagent and non-volatile buffer.