Gaowa Jin

Study on the retention equation in hydrophilic interaction liquid chromatography.

Hydrophilic interaction liquid chromatography (HILIC) is an effective technique for separating polar compounds. But its retention equation has not been studied systematically yet. In this study, an appropriate retention equation was established by using eight nucleosides as model analytes and by comparing four retention models on six different HILIC columns. As a result, retention equation Ink = a + b x InC(B) + cxC(B) could be quantitatively described the retention factors with good accuracy in HILIC mode. Based on this equation, the retention times of eight nucleosides under the conditions by other mobile phase can be predicted on each column. All of the predicted relative errors of retention times were smaller than 5%. The established retention model was also successfully applied to predict retention times of the real Traditional Chinese Medicine-Carthaus tinctorius L. sample.

Synthesis of molecularly imprinted polymer sorbents and application for the determination of aminoglycosides antibiotics in honey.

Aminoglycoside antibiotic (AA)-selective molecularly imprinted polymer (MIP) sorbent was synthesized by polymerization of methacrylic acid and ethylene glycol dimethacrylate in the presence of streptomycin as template molecule. The MIP sorbent was in detail characterized and its performance was evaluated by selecting four model AAs including streptomycin, gentamicin, spectinomycin and dihydrostreptomycin. Relative to non-imprinted polymer (NIP), the MIPs exhibited much higher recognizable capacity and specificity to the AAs and negligible adsorption for non-AAs as well. In combination of hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS), the MIP sorbent-based solid phase extraction (SPE) was able to effectively determine the residue of four model AAs in honey samples with recovery ranging from 90% to 110%. The limit of detection (and the limit of quantitation) of streptomycin, gentamicin, spectinomycin and dihydrostreptomycin was 4.5 (15.0), 2.4 (8.0), 6.0 (20.0) and 1.8 (6.0) μg/kg, respectively. Relative standard deviations of intraday and inter-day assay under three spiked levels were 4.4-12.0% and 6.8-14.6%, respectively.

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.

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.

2-D RP/RPLC method to separate components in Fructus schisandrae chinensis.

An off-line 2-D RP/RPLC method was developed to separate components in extracts of Fructus schisandrae chinensis, which is a traditional Chinese medicine. In this method, Click beta-CD stationary phase was proposed for constructing 2-D RP/RPLC system combined with C18 column. Beta-CD column showed great potential to separate compounds in traditional Chinese medicines under RP mode. Collected fractions from Click beta-CD column used in first dimension were resolved into many additional components, indicating that the 2-D LC system based on Click beta-CD and C18 phases is orthogonal. The mobile phase was same on both columns. This 2-D LC method facilitates us not only to separate components that cannot be resolved by unidimensional chromatography, but also to achieve much more efficient detection of minor sample components.

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.

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.

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.

Chemically Bonded Polyacrylamide via Thiol-Ene Click Chemistry as Separation Materials for Hydrophilic Interaction Liquid Chromatography

Abstract Thiol-ene click chemistry was applied to immobilize polyacrylamide onto alkenyl-modified silica to prepare a novel stationary phase for hydrophilic interaction liquid chromatography. Polyacrylamide was synthesized by reversible addition-fragmentation chain transfer reaction. Alkenyl functionalities were introduced onto the silica by the reaction of silica with the reaction product of 3-isocyanate opropyl trimethoxy silane, allylamine and pyridine in toluene to obtain alkenyl-modified silica (EUS, E-thiol-ene, U-urea, S-silica). The alkenyl-modified silica was then reacted with polyacrylamide in methanol at 55 °C for 48 h in the presence of 2,2-azo diisobutyl amidine hydrochloride to prepare a novel stationary phase (TE-UPAM, TE-thiol-ene, U-urea, PAM-polyacrylamide), for hydrophilic interaction liquid chromatography. This new stationary phase “TE-UPAM” was characterized by element analysis and infrared spectra, and the results showed that, compared with EUS, the carbon content of TE-UPAM increased and the characteristic peaks of multiple amide existed at 1636 and 1570 cm −1 . The effects of water content, salt concentration and pH of the mobile phase on the retention time of polar compounds were investigated. The results showed that the retention time of polar compounds decreased with the increase of water content. In contrast with acidic polar compounds, the retention time of neutral and basic polar compounds increased with the increase of salt concentration, and the retention time of basic polar compounds decreased with the decrease of pH value in the range of 3.3–4.8. The novel stationary phase behaved better than traditional silica stationary phase in the separation of uridine and bases. In addition, good separation of oligosaccharides by the novel stationary phase was also achieved, indicating great potential in the separation of polar compounds.