Qiqin Wang

Enantioseparation of N-derivatized amino acids by micro-liquid chromatography using carbamoylated quinidine functionalized monolithic stationary phase.

In order to obtain satisfactory column permeability, efficiency and selectivity for micro-HPLC, a capillary monolithic column containing O-9-[2-(methacryloyloxy)-ethylcarbamoyl]-10,11-dihydroquinidine (MQD) as chiral selector was re-optimized. The monolithic column was used to successfully enantioresolve a wide range of N-derivatized amino acids including alanine, leucine, methionine, threonine, phenylalanine, valine, serine, isoleucine, tryptophan, and cysteine. The influence of mobile phase parameters, such as the organic solvent type and concentration, the apparent pH, and buffer concentration, on retention and enantioseparation of N-derivatized amino acids has been investigated. 3,5-dinitrobenzoyl-amino acids and 3,5-dichlorobenzoyl-amino acids were resolved into enantiomers with exceptionally high selectivity and resolution. The chemoselectivity of the monolithic column for a multicomponent mixture of N-derivatized amino acids was also investigated. A mixture of three pairs of 3,5-dichlorobenzoyl-amino acids could be fully resolved in 22.5 min.

Preparation and evaluation of a novel monolithic column containing double octadecyl chains for reverse-phase micro high performance liquid chromatography.

A novel reverse-phase monolithic stationary phase containing double long alkyl chains was prepared based on the thermally initiated co-polymerization of 3-methylacryloyl-3-oxapropyl-3-(N,N-dioctadecylcarbamoyl)-propionate (AOD) and ethylene glycol dimethacrylate (EDMA) in the presence of 2-methyl-1-propanol and 1,4-butanediol as the selected porogens. The polymerization was carefully optimized and good permeability, stability and column efficiency were observed for the final monolithic columns. The column also showed good long term stability and reproducibility. The methylene selectivity demonstrates typical reversed phase characteristics. The optimized poly (AOD-co-EDMA) monolith exhibited good selectivity for a range of non-polar test analytes such as PAHs, tocopherols and alkylphenones. A good separation of intact proteins was also observed.

Separation of N-derivatized di- and tri-peptide stereoisomers by micro-liquid chromatography using a quinidine-based monolithic column - Analysis of l-carnosine in dietary supplements.

In the present study, a new analytical methodology was developed enabling the enantiomeric determination of N-derivatized di- and tri-peptides in dietary supplements using chiral micro-LC on a monolithic column consisting of poly(O-9-[2-(methacryloyloxy)-ethylcarbamoyl]-10,11-dihydroquinidine-co-2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (poly(MQD-co-HEMA-co-EDMA)). After optimization of the mobile phase conditions, a baseline resolution of the stereoisomers of 24 out of 53 N-derivatized di- and tri-peptides was obtained. 3,5-Dinitrobenzoyl- and 3,5-dichlorobenzoyl-peptide stereoisomers were separated with exceptionally high selectivity and resolution. The monolithic column was then applied to the quantitative analysis of l-carnosine and its enantiomeric impurity in three different commercial dietary supplements. Method validation demonstrated satisfactory results in terms of linearity, precision, selectivity, accuracy and limits of detection and quantification. The determined amounts of l-carnosine in commercial formulations were in agreement with the labeled content for all analyzed samples, and the enantiomeric impurity was found to be below the limit of detection (LOD), showing the potential of the poly(MQD-co-HEMA-co-EDMA) monolithic column as a reliable tool for the quality control of l-carnosine in dietary supplements by micro-LC.

Enantioseparation of N-derivatized amino acids by micro-liquid chromatography/laser induced fluorescence detection using quinidine-based monolithic columns☆

A novel carbamoylated quinidine based monolith, namely poly(O-9-[2-(methacryloyloxy)-ethylcarbamoyl]-10,11-dihydroquinidine-co-ethylene dimethacrylate (poly(MQD-co-EDMA)), was prepared for the micro-LC enantioseparation of N-derivatized amino acids. The influence of the mobile phase composition, including the organic modifier proportion, the apparent pH and the buffer concentration, on the enantioresolution of N-derivatized amino acids was systematically investigated. Satisfactory column performance in terms of permeability, efficiency and reproducibility was obtained in most cases. The majority of the enantiomers of the tested N-protected amino acids, including 3,5-DNB, 3,5-DClB, FMOC, 3,5-DMB, p-NB, m-ClB, p-ClB and B derivatives, could be baseline separated on the poly(MQD-co-EDMA) monolithic column within 25min. A self-assembled laser induced fluorescence (LIF) detector was employed to improve sensitivity when analyzing 7-nitro-2,1,3-benzoxadiazole (NBD) derivatives of amino acids. Ten NBD-derivatized amino acids, including arginine and histidine whose enantioseparation on quinidine carbamate based CSPs has not been reported so far, were enantioresolved on the poly(MQD-co-EDMA) monolith column. It is worth noting that the d-enantiomers of NBD-derivatized amino acids eluted first, except in the case of glutamic acid. The LOD values obtained with the LIF detector were comparable to those reported using conventional LC-FL methods. The prepared poly(MQD-co-EDMA) monolithic column coupled with the LIF detector opens up interesting perspectives to the determination of trace D-amino acids in biological samples.

Chiral separation of acidic compounds using an O-9-(tert-butylcarbamoyl)quinidine functionalized monolith in micro-liquid chromatography.

An O-9-(tert-butylcarbamoyl) quinidine (t-BuCQD) functionalized polymeric monolithic capillary column was prepared by the in situ copolymerization method. The physicochemical properties of the optimized monolithic column were characterized by scanning electron microscopy and micro-LC. Satisfactory column permeability, efficiency, stability and reproducibility were obtained for this monolithic column. The chiral recognition ability of the resulting monolith was also evaluated using 47 N-derivatized amino acids, eight N-derivatized dipeptides, and two herbicides. Under the selected conditions, the enantiomers of all chiral analytes were baseline separated with exceptionally high selectivity and resolution using micro-LC. It is worth noting that this chiral stationary phase (CSP) containing quinidine with a tert-butyl carbamate residue as chiral selector exhibits much higher enantioselectivity and diastereoselectivity than the previously developed O-9-[2-(methacryloyloxy)-ethylcarbamoyl]-10,11-dihydroquinidine (MQD) based CSP for N-derivatized amino acids and dipeptides. These results indicate that this novel quinidine-based polymeric monolith can be used as an effective tool for the enantioseparation of chiral acidic compounds.

Preparation of a biomimetic polyphosphorylcholine monolithic column for immobilized artificial membrane chromatography

The present work aims to prepare a novel phosphatidylcholine functionalized monolithic stationary phase by in situ co-polymerization of 12-methacryloyl dodecylphosphocholine (MDPC) and ethylene dimethacrylate (EDMA) for immobilized artificial membrane chromatography. Scanning electron microscopy, energy dispersive X-ray spectroscopy, FT-IR spectroscopy, pore size distribution analysis, ζ-potential analysis and micro-HPLC were used to evaluate the monolithic structure and physicochemical properties. Satisfactory morphology, high mechanical stability, good permeability and chromatographic performance were obtained on the optimized monolithic columns. A typical reverse-phase retention mechanism was observed over a wide range of organic solvent content (acetonitrile< 80%). The optimized poly(MDPC-co-EDMA) monolith exhibited good selectivity for proteins and basic drugs. Good correlation was observed between the retention on commercial IAM column (IAM.PC.DD2) and poly(MDPC-co-EDMA) monolith. This novel poly(MDPC-co-EDMA) monolith exhibited good potential for studying the drug-membrane interaction.

Development of double chain phosphatidylcholine functionalized polymeric monoliths for immobilized artificial membrane chromatography

This study described a simple synthetic methodology for preparing biomembrane mimicking monolithic column. The suggested approach not only simplifies the preparation procedure but also improves the stability of double chain phosphatidylcholine (PC) functionalized monolithic column. The physicochemical properties of the optimized monolithic column were characterized by scanning electron microscopy, energy-dispersive X-ray spectrometry, and nano-LC. Satisfactory column permeability, efficiency, stability and reproducibility were obtained on this double chain PC functionalized monolithic column. It is worth noting that the resulting polymeric monolith exhibits great potential as a useful alternative of commercial immobilized artificial membrane (IAM) columns for in vitro predication of drug-membrane interactions. Furthermore, the comparative study of both double chain and single chain PC functionalized monoliths indicates that the presence or absence of glycerol backbone and the number of acyl chains are not decisive for the predictive ability of IAM monoliths on drug-membrane interactions. This novel PC functionalized monolithic column also exhibited good selectivity for a protein mixture and a set of pharmaceutical compounds.

Phosphatidic acid-functionalized monolithic stationary phase for reversed-phase/cation-exchange mixed mode chromatography

A novel phosphatidic acid functionalized polymeric monolithic column was prepared through the thermally initiated co-polymerization of 12-methacryloyl dodecylphosphatidic acid (MDPA) and ethylene glycol dimethacrylate (EDMA) in the presence of 1,4-butanediol and isopropanol as porogens within 100 μm I.D. capillaries. The polymerization conditions of monolithic columns were systematically optimized in order to obtain good permeability, stability and column efficiency. The reproducibility of the optimized monolithic column was also satisfactory. The physicochemical properties of the monolithic column were evaluated by use of instrumental techniques including scanning electron microscopy, Fourier transform infrared spectra, ζ-potential analysis and micro-HPLC. A series of test compounds such as small peptides, alkylphenones, etc., were employed to investigate the retention mechanism on the poly(MDPA-co-EDMA) monolithic column. The results demonstrate that both hydrophobic and cation-exchange interactions could contribute to the overall retention of analytes. Furthermore, the novel reversed-phase/cation-exchange mixed mode monolithic column was applied to the separations of small peptides, phenols, water-soluble vitamins B, and pharmaceutical compounds. The successful applications indicate the potential of the poly(MDPA-co-EDMA) monolithic column in complex sample analysis.

Determination of phenolic acids in extra virgin olive oil using supercritical fluid chromatography coupled with single quadrupole mass spectrometry

HIGHLIGHTSA rapid, sensitive and efficient SFC‐MS method was developed for the separation of 12 phenolic acids.The analysis time with the SFC method was about three times shorter than using HPLC.The SFC‐MS method was successfully applied to the analysis of phenolic acids in extra virgin olive oils. ABSTRACT Phenolic acids represent one third of the total phenolic compounds in extra virgin olive oil (EVOO) and contribute greatly to the biological and sensory properties of EVOO. In the present research, an analytical method based on supercritical fluid chromatography coupled with single quadrupole mass spectrometry (SFC‐MS) was developed for the analysis of phenolic acids in EVOOs. The chromatographic and MS conditions were optimized in terms of selectivity, peak shape and sensitivity. Satisfactory separation of 12 phenolic acids was achieved within 20min on a Platisil CN column (250mm×4.6mm, 5&mgr;m) using 7% (v/v) water and 0.5% (v/v) formic acid in MeOH as mobile phase additives at the backpressure of 140bar and temperature of 60°C. The developed method was validated by evaluating the linearity, sensitivity, repeatability, intermediate precision and accuracy. The limits of detection (LODs) and limits of quantification (LOQs) of the SFC‐MS method for phenolic acids ranged from 0.03 to 5.00&mgr;g/mL and 0.08 to 15.0&mgr;g/mL, respectively. The relative standard deviations (RSDs) for intraday and interday precision were lower than 9.91% and the recoveries ranged from 80.2 to 117% and 80.9 to 118% for low‐level and high‐level concentrations of standards spiked in olive oil, respectively. Compared to an HPLC method, the SFC method was about three times faster and provided better selectivity. Finally, the SFC‐MS method was successfully employed for the quantitative analysis of phenolic acids in EVOOs. Differences in individual and total phenolic acid contents were observed between six varieties.

Online screening of acetylcholinesterase inhibitors in natural products using monolith-based immobilized capillary enzyme reactors combined with liquid chromatography-mass spectrometry

In order to develop a direct and reliable method for discovering lead compounds from traditional Chinese medicines (TCMs), a comparative online ligand fishing platform was developed using immobilized capillary enzyme reactors (ICERs) in combination with liquid chromatography-mass spectrometry (LC-MS). Methacrylate-based monolithic capillaries (400 μm I.D. × 10 cm) containing epoxy reactive groups were used as support to immobilize the target enzyme acetylcholinesterase (AChE). The activity and kinetic parameters of the AChE-ICER were investigated using micro-LC-UV. Subsequently, ligand fishing and identification from mixtures was carried out using the complete AChE-ICER-LC-MS platform. For efficient distinction of true actives from false positives, highly automated comparative analyses were run alternatingly using AChE-ICERs and negative control-ICERs, both online installed in the system. After washing unbound compounds to the waste, bound ligands were eluted from the AChE-ICER to a trapping loop using a denaturing solution. The trapped ligands were further separated and identified using LC-MS. Non-specific binding to the monolith support or non-functional sites of the immobilized enzyme was investigated by exposing analytes to the negative control-ICER. The specificity of the proposed approach was verified by analyzing a known AChE inhibitor in the presence of an inactive compound. The platform was applied to screen for AChE inhibitors in extracts of Corydalis yanhusuo. Eight compounds (columbamine, jatrorrhizine, coptisine, palmatine, berberine, dehydrocorydaline, tetrahydropalmatine and corydaline) with AChE binding affinity were detected and identified, and their AChE inhibitory activities were further verified by an in vitro enzymatic inhibition assay. Experimental results show that the proposed comparative online ligand fishing platform is suitable for rapid screening and mass-selective detection of AChE inhibitors in complex mixtures.