Isolation of Human Milk Difucosyl Nona- and Decasaccharides by Ultrahigh-Temperature Preparative PGC-HPLC and Identification of Novel Difucosylated Heptaose and Octaose Backbones by Negative-Ion ESI-MSn.

Despite their many important physiological functions, past work on the diverse sequences of human milk oligosaccharides (HMOs) has been focused mainly on the highly abundant HMOs with a relatively low degree of polymerization (DP) due to the lack of efficient methods for separation/purification and high-sensitivity sequencing of large-sized HMOs with DP ≥ 10. Here we established an ultrahigh-temperature preparative HPLC based on a porous graphitized carbon column at up to 145 °C to overcome the anomeric α/ß splitting problem and developed further the negative-ion ESI-CID-MS/MS into multistage MSn using a combined product-ion scanning of singly charged molecular ion and doubly charged fragment ion of the branching Gal and adjacent GlcNAc residues. The separation and sequencing method allows efficient separation of a neutral fraction with DP ≥ 10 into 70 components, among which 17 isomeric difucosylated nona- and decasaccharides were further purified and sequenced. As a result, novel branched difucosyl heptaose and octaose backbones were unambiguously identified in addition to the conventional linear and branched octaose backbones. The novel structures of difucosylated DF-novo-heptaose, DF-novo-LNO I, and DF-novo-LNnO I were corroborated by NMR. The various fucose-containing Lewis epitopes identified on different backbones were confirmed by oligosaccharide microarray analysis.

Carbon dots as a novel photosensitizer for photodynamic therapy of cancer and bacterial infectious diseases: recent advances.

Carbon dots (CDs) are novel carbon-based nanomaterials that have been used as photosensitizer-mediated photodynamic therapy (PDT) in recent years due to their good photosensitizing activity. Photosensitizers (PSs) are main components of PDT that can produce large amounts of reactive oxygen species (ROS) when stimulated by light source, which have the advantages of low drug resistance and high therapeutic efficiency. CDs can generate ROS efficiently under irradiation and therefore have been extensively studied in disease local phototherapy. In tumor therapy, CDs can be used as PSs or PS carriers to participate in PDT and play an extremely important role. In bacterial infectious diseases, CDs exhibit high bactericidal activity as CDs are effective in disrupting bacterial cell membranes leading to bacterial death upon photoactivation. We focus on recent advances in the therapy of cancer and bacteria with CDs, and also briefly summarize the mechanisms and requirements for PSs in PDT of cancer, bacteria and other diseases. We also discuss the role CDs play in combination therapy and the potential for future applications against other pathogens.

GIPS-Mix for Accurate Identification of Isomeric Components in Glycan Mixtures Using Intelligent Group-Opting Strategy.

Accurate identification of glycan structures is highly desirable as they are intimately linked to their different functions. However, glycan samples generally exist as mixtures with multiple isomeric structures, making assignment of individual glycan components very challenging, even with the aid of multistage mass spectrometry (MSn). Here, we present an approach, GIPS-mix, for assignment of isomeric glycans within a mixture using an intelligent group-opting strategy. Our approach enumerates all possible combinations (groupings) of candidate glycans and opts in the best-matched glycan group(s) based on the similarity between the simulated spectra of each glycan group and the acquired experimental spectra of the mixture. In the case that a single group could not be elected, a tie break is performed by additional MSn scanning using intelligently selected precursors. With 11 standard mixtures and 6 human milk oligosaccharide fractions, we demonstrate the application of GIPS-mix in assignment of individual glycans in mixtures with high accuracy and efficiency.

Application of chromatography in purification and structural analysis of natural polysaccharides: A review.

Polysaccharides are widely distributed in natural sources from monocytic microorganisms to higher animals, and are found in a variety of biological activities in recent decades. Natural polysaccharides have the characteristics of large molecular weight, diverse composition, and complex structure, so their purification and structural analysis are difficult issues in research. Chromatography as a powerful separation technique, plays an irreplaceable role in the separation and structural analysis of natural polysaccharides, especially in the purification of polysaccharides, the separation of hydrolysates, and the analysis of monosaccharide composition. The separation mechanisms and application of different chromatographic methods in the studies of polysaccharides were summarized in this review. Moreover, the advantages and drawbacks of various chromatography methods were discussed as well.

Short-Term Effects of Human versus Bovine Sialylated Milk Oligosaccharide Microinjection on Zebrafish Larvae Survival, Locomotor Behavior and Gene Expression.

Milk oligosaccharides are a complex class of carbohydrates that act as bioactive factors in numerous defensive and physiological functions, including brain development. Early nutrition can modulate nervous system development and can lead to epigenetic imprinting. We attempted to increase the sialylated oligosaccharide content of zebrafish yolk reserves, with the aim of evaluating any short-term effects of the treatment on mortality, locomotor behavior, and gene expression. Wild-type embryos were microinjected with saline solution or solutions containing sialylated milk oligosaccharides extracted from human and bovine milk. The results suggest that burst activity and larval survival rates were unaffected by the treatments. Locomotion parameters were found to be similar during the light phase between control and treated larvae; in the dark, however, milk oligosaccharide-treated larvae showed increased test plate exploration. Thigmotaxis results did not reveal significant differences in either the light or the dark conditions. The RNA-seq analysis indicated that both treatments exert an antioxidant effect in developing fish. Moreover, sialylated human milk oligosaccharides seemed to increase the expression of genes related to cell cycle control and chromosomal replication, while bovine-derived oligosaccharides caused an increase in the expression of genes involved in synaptogenesis and neuronal signaling. These data shed some light on this poorly explored research field, showing that both human and bovine oligosaccharides support brain proliferation and maturation.

Comprehensive O-Glycosylation Analysis of the SARS-CoV-2 Spike Protein with Biomimetic Trp-Arg Materials.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a serious public health threat. Most vaccines against SARS-CoV-2 target the highly glycosylated spike protein (S). A good knowledge of the glycosylation profile of this protein is key to successful vaccine development. Unlike the 22 confirmed N-glycosylation sites on SARS-CoV-2 S, only a few O-glycosylation sites on this protein have been reported. This difference is mainly ascribed to the extremely low stoichiometry of O-glycosylation. Herein, we designed the biomimetic materials, Trp-Arg (WR) monomer-grafted silica microspheres (designated as WR-SiO2), and these biomimetic materials can enrich N- and O-linked glycopeptides with high selectivity. And WR-SiO2 can resist the nonglycopeptides' interference with the 100 molar fold of BSA during O-linked glycopeptide enrichment. We utilized WR-SiO2 to comprehensively analyze the O-glycosylation profile of recombinant SARS-CoV-2 S. Twenty-seven O-glycosylation sites including 18 unambiguous sites are identified on SARS-CoV-2 S. Our study demonstrates that the biomimetic polymer can offer specific selectivity for O-linked glycopeptides and pave the way for O-glycosylation research in biological fields. The O-glycosylation profile of SARS-CoV-2 S might supplement the comprehensive glycosylation in addition to N-glycosylation of SARS-CoV-2 S.

High levels of fucosylation and sialylation of milk N-glycans from mothers with gestational diabetes mellitus alter the offspring gut microbiome and immune balance in mice.

Gestational diabetes mellitus (GDM) is significantly associated with allergen sensitization in early childhood, and this may influence the gut microbiome and immune system of the children. In addition to mother-to-child transmission of microbes, milk glycans play a pivotal role in shaping the gut microbiome of infants. A previous study has demonstrated alterations in the major milk N-glycans of mothers with GDM. However, the impact of these changes on the gut microbiome and immune response of the neonates has yet to be studied. Here, we aimed to compare the glycosylation levels of various milk glycans between normal and GDM mice, and to characterize the intestinal microbiome and immune responses of the offspring after weaning. We found that GDM mouse milk contained significantly higher concentrations of fucosylated and sialylated N-glycans than control mice, but there was no difference in the concentration of milk oligosaccharides between the groups. The differences in milk N-glycans had direct effects on the intestinal microbiome of the offspring, which in turn affected their immune response upon challenge with ovalbumin (OVA), with disruptions in the Th1/Th2 and Th17/Treg cell balances. This study lays the foundation for further research and development of specific nutritional care for the offspring of GDM mothers.

Simple and efficient preparation of high-purity trehalulose from the waste syrup of isomaltulose production using solid-phase extraction followed by hydrophilic interaction chromatography.

A simple and efficient method was developed for the preparation of high-purity trehalulose from the waste syrup of isomaltulose production. The waste syrup was pre-treated with C18 solid-phase extraction, where 98% decolorization and 97% reducing sugar recovery were obtained, followed by hydrophilic interaction liquid chromatography separation on a cysteine-bonded zwitterionic column. Under optimized conditions, trehalulose was separated from isomaltulose isomer and prepared on a semi-preparative scale with >99% purity. The structure of the prepared trehalulose was subsequently confirmed by nuclear magnetic resonance, and three tautomers of trehalulose (α-D-glucosylpyranosyl-1,1-ß-D-fructopyranose, α-D-glucosylpyranosyl-1,1-ß-D-fructofuranose, and α-D-glucosylpyranosyl-1,1-α-D-fructofuranose) were detected and completely characterized by 13 C NMR spectroscopy for the first time in this study. The tautomerization of α-D and ß-D type transition was observed by hydrophilic interaction liquid chromatography on an AdvanceBio Glycan Mapping column, with smaller particle size (2.7 µm). Furthermore, the prepared trehalulose was applied as a standard for trehalulose quantification during the sucrose conversion by Klebsiella sp. LX3. The combination of solid-phase extraction and hydrophilic interaction liquid chromatography offers a new avenue for the preparation of sugar isomers from complex natural or fermentation products.

The Intestinal Dysbiosis of Mothers with Gestational Diabetes Mellitus (GDM) and Its Impact on the Gut Microbiota of Their Newborns.

Gestational diabetes mellitus (GDM) is defined as "diagnosed as impaired glucose tolerance for the first time during pregnancy," which can lead to adverse pregnancy outcomes and produces divergent effects on mothers and newborns. In recent years, with the continuous expansion of obese people, GDM shows an upward trend. The abundant and diverse members of the human gut microbiota exert critical roles in the maintenance of human health. Studies have shown that GDM may be associated with disordered gut microbiota in both mothers and newborns. Taking into account the potential effects on maternal and consequently neonatal health, in this review, we analyzed the available data and discussed the current knowledge about the potential relationship between GDM and intestinal dysbiosis in mothers and newborns. In addition, we also discussed the influencing factors derived from GDM mothers on the gut microbiome of their newborns, including the vertical transmission of microbiota from mothers, the alteration of milk components of GDM mothers, and using of probiotics. Hoping that new insights into the role of the gut microbiota in GDM could lead to the development of integrated strategies to prevent and treat these metabolic disorders.

Human Milk Oligosaccharides Activate Epidermal Growth Factor Receptor and Protect Against Hypoxia-Induced Injuries in the Mouse Intestinal Epithelium and Caco2 Cells.

BACKGROUND: Hypoxia-induced intestinal barrier injuries lead to necrotizing enterocolitis (NEC). Although NEC in preterm neonates is preventable by human milk oligosaccharides (HMOs), the underlying mechanism remains unknown. OBJECTIVE: To reveal the role and mechanism of HMOs in protecting against hypoxia-induced injuries in intestinal epithelium of neonatal mice and cultured Caco2 cells. METHODS: NEC was induced by hypoxia and cold stress. Seventy C57BL/C pups (7-d-old) were divided into 5 groups and fed maternal breast milk (BM), formula alone (FF), or the formula added with HMOs at 5 (LHMO), 10 (MHMO), or 20 mg/mL (HHMO) for 3 d. Ileal hypoxia inducible factor 1α (HIF1α) and cleaved Caspase 3 were determined, along with staining for Ki-67 protein to labeled proliferative cells. In vitro, adherent Caco2 cells (undifferentiated, passage 14) were treated with HMOs, galacto-oligosaccharides, fructo-oligosaccharides, or mixed oligosaccharides at 10 mg/mL for 1 d exposed to 1% O2. Cell proliferation and apoptosis, along with phosphorylated epidermal growth factor receptor (P-EGFR) and 38KD MAPK (P-P38), were assayed in differentiated or undifferentiated Caco2 cells. RESULTS: Compared with the FF-fed mice, those fed MHMO and HHMO had 52% lower (P < 0.05) NEC scores, 60-80% greater (P < 0.05) KI67-positive cell numbers, and 56-71% decreases (P < 0.05) in ileal HIF1α and cleaved Caspase 3 (56-71%). Compared with those untreated, the HMO-treated Caco2 cells displayed 60% greater (P < 0.05) proliferative activity and 19% lower (P < 0.05) apoptotic cells after the hypoxia exposure. The HMO treatment led to 58% or 10-fold increases (P < 0.05) of P-EGFR and 48-89% decreases (P < 0.05) of P-P38 in either differentiated or undifferentiated Caco2 cells compared with the controls. CONCLUSION: Supplementing HMOs at 10-20 mg/mL into the formula for neonatal mice or media for Caco2 cells conferred protection against the hypoxia-induced injuries. The protection in the Caco2 cells was associated with an activation of EGFR.

Chemoenzymatic Synthesis of O-Mannose Glycans Containing Sulfated or Nonsulfated HNK-1 Epitope.

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.

Profiling of Human Milk Oligosaccharides for Lewis Epitopes and Secretor Status by Electrostatic Repulsion Hydrophilic Interaction Chromatography Coupled with Negative-Ion Electrospray Tandem Mass Spectrometry.

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.

Profiling of Sialylated Oligosaccharides in Mammalian Milk Using Online Solid Phase Extraction-Hydrophilic Interaction Chromatography Coupled with Negative-Ion Electrospray Mass Spectrometry.

Sialylated oligosaccharides are important components in mammalian milk. They play a key role in the health and growth of infants by helping to shape up infant's gastrointestinal microbiota and defense against infection by various pathogenic agents. A detailed knowledge of the structures, compositions, and quantities of the sialylated milk oligosaccharides (SMOs) is a prerequisite for understanding their biological roles. However, because of the presence of very large amounts of lactose and neutral oligosaccharides, accurate analysis of SMOs is difficult. A pretreatment step is required to remove lactose and neutral oligosaccharides but conventional off-line pretreatment methods are time-consuming and of poor reproducibility. In this presentation, we linked solid-phase extraction (SPE) with hydrophilic interaction chromatography (HILIC) followed by mass spectrometry (MS) identification for the analysis of SMOs. A SPE column with electrostatic repulsion function was used for removal of lactose and neutral oligosaccharides, a HILIC analytical column for separation of the SMOs, and negative-ion electrospray ionization tandem MS was used for their identification and sequencing. The success of the established online SPE-HILIC-MS method was demonstrated by profiling of SMOs in human to investigate detailed SMO changes during lactation period and in animals to compare the difference in SMO contents among the different species.

Synthesis and chromatographic evaluation of phenyl/tetrazole bonded stationary phase based on thiol-epoxy ring opening reaction.

A silica-based reversed-phase stationary phase bonding with phenyl and tetrazole groups was synthesized by thiol-epoxy ring opening reaction. The bonded groups could not only provide hydrophobic interaction, but also π-π, hydrogen bonding, electrostatic interactions, and so on. The results of characterization with elemental analysis and solid-state 13 C cross-polarization magic-angle-spinning NMR spectroscopy indicated the successful preparation of phenyl/tetrazole sulfoether bonded stationary phase. Chromatographic evaluation revealed that phenyl/tetrazole sulfoether bonded stationary phase behaved well under the reversed-phase mode. The column parameters (H, S*, A, B, and C) showed different selectivity compared with some typical commercial columns, and it was validated by the separation of estrogen, ginsenoside, alkaloid samples. Based on the different selectivity between phenyl/tetrazole sulfoether bonded stationary phase and C18 columns, phenyl/tetrazole sulfoether bonded stationary phase also showed potential to construct a 2D reversed-phase liquid chromatography system with C18. And it was verified by the separation of corydalis tuber and curcuma zedoary extracts.

In-Depth Analysis of Glycoprotein Sialylation in Serum Using a Dual-Functional Material with Superior Hydrophilicity and Switchable Surface Charge.

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.

Two-dimensional solid-phase extraction strategy for the selective enrichment of aminoglycosides in milk.

An orthogonal two-dimensional solid-phase extraction strategy was established for the selective enrichment of three aminoglycosides including spectinomycin, streptomycin, and dihydrostreptomycin in milk. A reversed-phase liquid chromatography material (C18 ) and a weak cation-exchange material (TGA) were integrated in a single solid-phase extraction cartridge. The feasibility of two-dimensional clean-up procedure that experienced two-step adsorption, two-step rinsing, and two-step elution was systematically investigated. Based on the orthogonality of reversed-phase and weak cation-exchange procedures, the two-dimensional solid-phase extraction strategy could minimize the interference from the hydrophobic matrix existing in traditional reversed-phase solid-phase extraction. In addition, high ionic strength in the extracts could be effectively removed before the second dimension of weak cation-exchange solid-phase extraction. Combined with liquid chromatography and tandem mass spectrometry, the optimized procedure was validated according to the European Union Commission directive 2002/657/EC. A good performance was achieved in terms of linearity, recovery, precision, decision limit, and detection capability in milk. Finally, the optimized two-dimensional clean-up procedure incorporated with liquid chromatography and tandem mass spectrometry was successfully applied to the rapid monitoring of aminoglycoside residues in milk.

Dysregulated hepatic bile acids collaboratively promote liver carcinogenesis.

Dysregulated bile acids (BAs) are closely associated with liver diseases and attributed to altered gut microbiota. Here, we show that the intrahepatic retention of hydrophobic BAs including deoxycholate (DCA), taurocholate (TCA), taurochenodeoxycholate (TCDCA), and taurolithocholate (TLCA) were substantially increased in a streptozotocin and high fat diet (HFD) induced nonalcoholic steatohepatitis-hepatocellular carcinoma (NASH-HCC) mouse model. Additionally chronic HFD-fed mice spontaneously developed liver tumors with significantly increased hepatic BA levels. Enhancing intestinal excretion of hydrophobic BAs in the NASH-HCC model mice by a 2% cholestyramine feeding significantly prevented HCC development. The gut microbiota alterations were closely correlated with altered BA levels in liver and feces. HFD-induced inflammation inhibited key BA transporters, resulting in sustained increases in intrahepatic BA concentrations. Our study also showed a significantly increased cell proliferation in BA treated normal human hepatic cell lines and a down-regulated expression of tumor suppressor gene CEBPα in TCDCA treated HepG2 cell line, suggesting that several hydrophobic BAs may collaboratively promote liver carcinogenesis.

Separation analysis of macrolide antibiotics with good performance on a positively charged C18HCE column.

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.

Practical method for the definition of chromatographic peak parameters in preparative liquid chromatography.

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/cm3 . The parameters and methods could be used for the evaluation and selection of stationary phases in preparative chromatography.

Highly selective separation of aminoglycoside antibiotics on a zwitterionic Click TE-Cys column.

Hydrophilic interaction liquid chromatography has emerged as a valuable alternative approach to ion-pair chromatography for the separation of aminoglycoside antibiotics in recent years. However, the resolution of structurally related aminoglycosides is a great challenge owing to the limited selectivity. In this work, a cysteine-based zwitterionic stationary phase (named Click TE-Cys) was utilized and compared with five commonly used hydrophilic interaction liquid chromatography columns. Click TE-Cys displayed much better selectivity for structurally similar aminoglycosides. The retention behaviors of aminoglycosides were investigated in detail, revealing that low pH (2.7 or 3.0) and high buffer concentration (≥50 mM) were preferable for achieving good peak shape and selectivity. Effective resolution of ten aminoglycosides including spectinomycin, dihydrostreptomycin, streptomycin, gentamicin C1, gentamicin C2/C2a, gentamicin C1a, kanamycin, paromonycin, tobramycin, and neomycin was realized at optimized conditions. Additionally, spectinomycin and its related impurities were successfully resolved. The results indicated the great potential of the Click TE-Cys column in the separation of aminoglycoside mixtures and related impurities.