Aijin Shen

Hydrophilic interaction chromatography for selective separation of isomeric saponins

A method based on a Click XIon zwitterionic stationary phase was developed to separate isomeric saponins efficiently in hydrophilic interaction chromatography (HILIC) mode using methanol as a weak eluent. The retention times of a set of isomeric saponins, ginsenoside Rc (S1), ginsenoside Rb2 (S2), and ginsenoside Rb3 (S3) on five kinds of HILIC columns were compared using acetonitrile/water or methanol/water as mobile phase. All results indicated that the Click XIon column showed the highest retention times of all these isomeric saponins. Then the retention behaviors of these isomeric saponins on the Click XIon column were investigated. The results demonstrated that the retention behaviors of saponins on Click XIon in methanol/water mobile phase were different from that in acetonitrile/water. Methanol/water binary eluent offered enhanced selectivity to these isomeric saponins (αS2/S3(MeOH)=1.16>αS3/S2(ACN)=1.04), and the elution order of ginsenoside Rb2 (S2) and ginsenoside Rb3 (S3) was reversed compared with acetonitrile/water. Moreover, methanol/water solvent has better solubility for saponins than acetonitrile/water, resulting in improved preparative peak shape, which is greatly beneficial for saponin purification. Application to the preparative separation of saponins from leaves of P. notoginseng was also investigated, and eleven saponins, including three sets of isomeric saponins with one new saponin were isolated and identified. All these results indicated that this method was efficient for analytical and preparative separation of saponins, especially for isomeric saponins, containing xylopyranosyl, arabinofuranosyl or arabinopyranosyl units.

N-Glycosylation site analysis of proteins from Saccharomyces cerevisiae by using hydrophilic interaction liquid chromatography-based enrichment, parallel deglycosylation, and mass spectrometry.

N-Glycosylation site analysis of bakers yeast Saccharomyces cerevisiae is of fundamental significance to elucidate the molecular mechanism of human congenital disorders of glycosylation (CDG). Here we present a mass spectrometry (MS)-based workflow for the profiling of N-glycosylated sites in S. cerevisiae proteins. In this workflow, proteolytic glycopeptides were enriched by using a hydrophilic material named Click TE-Cys to improve the glycopeptide selectivity and coverage. To enhance the reliability of the identified results, the enriched glycopeptides were subjected to parallel deglycosylation by using two endoglycosidases (i.e., PNGase F and Endo Hf), respectively, prior to LC-MS/MS analysis. On the basis of the workflow, a total of 135 N-glycosylated sites including 6 known, 93 potential, and 36 novel sites were identified and mapped to 79 proteins. Among the novel-type sites, nine sites from eight proteins, which were simultaneously identified via PNGase F and Endo Hf deglycosylation, are believed to possess high confidence. The established workflow, together with the profile of N-glycosylated sites, will contribute to the improvement of S. cerevisiae model for revealing the pathogenesis of CDG.

Synthesis and evaluation of sulfobetaine zwitterionic polymer bonded stationary phase

Zwitterionic polymer stationary phases have attracted increasing attention in hydrophilic interaction chromatography (HILIC). In this work, a zwitterionic sulfobetaine functionalized polyacrylamide stationary phase (named TENS) based on porous silica particles was prepared via controlled surface initiated reversible addition-fragmentation transfer (RAFT) polymerization. Instead of traditional methacrylate type sulfobetaine monomer, acrylamide type sulfobetaine monomer, which has higher chemical stability and hydrophicility, was employed in this work. The characterization of elemental analysis and solid-state 13C cross polarization/magic-angle-spinning nuclear magnetic resonance indicated the successful preparation of TENS stationary phase. Meanwhile, scanning electron microscope (SEM), nitrogen adsorption experiment and study of size exclusion performance were conducted, revealing that the surface initiated polymerization was well controlled. For better understanding of TENS material under HILIC mode, chromatographic evaluation of TENS material was performed, among which, TENS material exhibited good hydrophilicity and chemical stability. To further study the applicability of TENS material, saccharides which were considered as challenging targets in HILIC, were chosen as tested analytes. Various saccharide samples, including fructooligosaccharide, trisaccharide isomers and ginsenosides, were well separated on TENS material. Moreover, TENS material displayed good selectivity for the enrichment of glycopeptides. These results demonstrated the capability of TENS as a promising material in glycomics and glycoproteomics.

Hydrophilic interaction/weak cation-exchange mixed-mode chromatography for chitooligosaccharides separation

A hydrophilic interaction/weak cation-exchange (HILIC/WCX) mixed-mode chromatography was developed for separation of chitooligosaccharides (COS) using a weak cation-exchange column. For better understanding of the retention characteristics of the column, organic solvent content effect was investigated. WCX mode, HILIC mode, and mixed-mode could be performed on this column according to investigating results. COS were separated on the column in mixed-mode. The usage of volatile buffer salt made it favorable for evaporative light-scattering detector (ELSD). Mild elution conditions were economical and environment-friendly relative to other conditions used in early reports. This method may offer a possibility for large-scale COS preparation.

Preparative separation of a challenging anthocyanin from Lycium ruthenicum Murr. by two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography

The preparative separation of anthocyanins by HPLC often suffers from insufficient separation selectivity. In this work, a two-dimensional liquid (LC-LC) method was established to efficiently purify a challenging anthocyanin in Lycium ruthenicum Murray. Reversed phase liquid chromatography (RPLC) was used in the first-dimension preparation to fractionate the sample for its high separation efficiency. After the optimization of second-dimension methods, hydrophilic interaction chromatography (HILIC) was applied to further isolate the anthocyanin for the good orthogonality to RPLC. To improve HILIC separation for anthocyanins, stationary phases and mobile phases were investigated systematically. A satisfactory result was obtained on a zwitterionic Click XIon column with 1% phosphoric acid as an acidic additive. Using the above method, the anthocyanin and three new alkaloids were isolated from L. ruthenicum for the first time. This RPLC/HILIC method solved the coelution problem of anthocyanin and basic non-anthocyanins in one-dimensional HPLC, benefiting from the significantly improved separation resolution.

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.

Preparation of glyco-silica materials via thiol-ene click chemistry for adsorption and separation

Glyco-silica materials were successfully developed based on thiol-ene click chemistry between alkene–saccharides and mercapto-silica, which behaved well in HILIC separation and exhibited high affinity to specific proteins.

Purification of bufadienolides from the skin of Bufo bufo gargarizans Cantor with positively charged C18 column.

As a kind of promising anticancer compounds, the preparation of bufadienolides is a hot study spot. However, due to the complexity of biological sample, the purification of bufadienolides from a crude sample (toad skin) is a tough work. In this paper, we reported a new way based on positively charged C18 material (XCharge C18) to quickly separate and purify bufadienolides from toad skin. By this method, the different ionic feature of the amino acid conjugated bufadienolides (AACBs) and the free form bufadienolides (AAUBs) was firstly utilized to obtain distinct separation selectivity on the XCharge C18 column. Additionally, the peak tailing problem of AACBs on conventional C18 was resolved and better resolutions were achieved on the XCharge C18, thus, two kinds of bufadienolides on one column were successfully purified respectively. Taking F13 as an example, the method was validated by liquid chromatography-mass spectrometry (LC-MS), and then 4 AACBs as well as 4 AAUBs were simultaneously purified by preparative XCharge C18. In addition, the application of this method in other fractions was also validated. The results suggested that the developed method is a practical and promising tool for efficient separation and purification of bufadienolides from toad skin.

Hydrophilic interaction liquid chromatography-solid phase extraction directly combined with protein precipitation for the determination of triptorelin in plasma.

Peptide drugs play a critical role in therapeutic treatment. However, as the complexity of plasma, determination of peptide drugs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a daunting task. To solve this problem, hydrophilic interaction liquid chromatography-solid phase extraction (HILIC-SPE) directly combined with protein precipitation (PPT) was developed for the selective extraction of triptorelin from plasma. The extracts were analyzed by reversed-phase liquid chromatography (RPLC). Proteins, phospholipids and highly polar interferences could be removed from plasma by the efficient combination of PPT, HILIC-SPE and RPLC-MS/MS. This method was evaluated by matrix effect, recovery and process efficiency at different concentration levels (50, 500 and 5,000 ng/mL) of triptorelin. Furthermore, the performance of HILIC-SPE was compared with that of reversed-phase C18 SPE and hydrophilic lipophilic balance (Oasis HLB) SPE. Among them, HILIC-SPE provided the minimum matrix effect (ranging from 96.02% to 103.41%), the maximum recovery (ranging from 80.68% to 90.54%) and the satisfactory process efficiency (ranging from 82.83% to 92.95%). The validated method was successfully applied to determine triptorelin in rat plasma.

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.