Study On Novel Approches For Boronate Affinity Based Separation Of Cis-diol Compounds

Objective: The main purpose of this study was to develop methods for the detection and analysis of cis-diol compounds based on boronate affinity.It is divided into the following two aspects: 1.To establish two high-affinity boronic acid-functionalized nanoparticles（PEI-BA-MNPs）assisted enrichment analysis methods for selective analysis of ginsenoside Re and streptomycin in real samples combining with high performance liquid chromatography（HPLC）.2.To develop a novel diboronic acid（DBA）assisted labeling and analysis method for the efficient analysis of saccharides in real samples by the most commonly used chromatographic techniques（RPLC-UV）.Methods: 1.Based on boronate affinity,the branched polyethyleneimine was applied as a scaffold to amplify the number of boronic acid moieties to synthesize high-affinity boronic acid-functionalized nanoparticles（PEI-BA-MNPs）.The prepared PEI-BA-MNPs were characterized and analyzed for nitrogen content by transmission electron microscopy（TEM）and energy dispersive X-ray spectroscopy（EDS）.And the magnetic properties and boronate affinity were investigated.Taking ginsenoside Re as the representative,the magnetic solid-phase extraction conditions were optimized,including extraction time and desorption time.Affinity assays and reusability studies were also examined.（1）The anti-interference ability of PEI-BA-MNPs against competing substances was investigated by HPLC-UV method.A methodological investigation was carried out on the established method.In addition,the direct detection of ginsenoside Re by HPLC-UV and the detection of PEI-BA-MNPs after magnetic solid phase extraction compared the signal-to-noise ratio of the two methods.Finally,the method was applied to the content analysis of ginsenoside Re in Qipi Oral Liquid.（2）MALDI-TOF MS method was used to investigate the anti-interference ability of PEI-BA-MNPs against competing substances and the purification effect on milk matrix.A methodological investigation was carried out on the established method.In addition,the standard addition recovery,precision and signal-to-noise ratio of the direct detection of streptomycin in milk and the detection of PEI-BA-MNPs after magnetic solid-phase extraction were compared by UPLC-MS/MS.Finally,the established method was applied to the detection and analysis of streptomycin in milk samples.2.The synthesized new diboronic acid（DBA）was characterized by 1H NMR and HRMS.The labeling was carried out under normal temperature shaking,and the labeled product was detected by HPLC-UV.The interaction between DBA and glucose was proved by UV-Vis,HPLC-UV and HRMS analysis methods.The DBA concentration,buffer p H and time during labeling were optimized.In addition,optimization of the flow phase ratio was carried out.The established method was investigated and finally applied to the analysis of glycoprotein trypsin glycan and monosaccharide in traditional Chinese medicine.Results: 1.PEI-BA-MNPs can be an ideal magnetic solid phase extraction sorbents for the selective enrichment of cis-diol compounds with their significant advantages,including good magnetic properties,high affinity,high binding capacity,excellent selectivity,easy manipulation and fast binding equilibrium.（1）Selectivity exhibited by PEI-BA-MNPs towards ginsenoside Re in the presence of competing substances.The method exhibited good linearity in the range of 50-800 ng/mL,with a limit of detection（LOD）of 20 ng/mL and a limit of quantification（LOQ）of 50 ng/mL.At three spiked levels,the recoveries were 96.5%-108.9%,and the relative standard deviation（RSD）were 2.7%-11.4%.After enrichment by PEI-BA-MNPs,the detection limit of ginsenoside Re was reduced by about 50 times.Compared with the standard method（Ch P2020）,the developed method exhibit similar results（0.31% vs 0.27%）,but the method established in this paper greatly saves steps and time of the sample pretreatment in actual operation.（2）Selectivity exhibited by PEI-BA-MNPs towards streptomycin in the presence of competing substances.The method showed good linearity in the range of 25-1000 μg/kg,the linear correlation coefficient（R~2）was 0.9986,the LOD was 10 μg/kg and the LOQ was 25 μg/kg.At negative milk matrix three spiked levels,after PEI-BA-MNPs magnetic solid phase extraction compared with the direct injection analysis,the relative standard deviation（7.4%-13.8% vs 4.4%-7.9%）of the small increased,but the recoveries of streptomycin was greatly improved（96.0%-117.7% vs 51.4%-54.6%）.And because of the high affinity of PEI-BA-MNPs for streptomycin,the limit of quantification of streptomycin was effectively reduced,and the interference of other substances in milk can be effectively removed.This method can meet the actual detection requirements of streptomycin in milk.2.The new diboronic acid（DBA）was successfully synthesized and used as chemical labels for saccharides through boronate affinity.The whole labeling procedure was organic solvent-free and could be accomplished about 1 h,the established method provided broad linear dynamics ranges（25-5000 ng/mL）with acceptable correlation coefficients（R2>0.98）.Taking glucose as an example,the established method provided substantial sensitivity（LOD: 5 ng/mL）with high reproducibility（RSD<6.7%）and excellent accuracy（Recoveries: 98.2%-99.8%）.Finally,different saccharides were successfully analyzed in various complex samples,from tryptic glycans of glycoproteins to monosaccharides in traditional Chinese medicine.Conclusion: In this paper,with boronate affinity as the core driving force,two new methods have been developed for the separation and analysis of cis-diol compounds,which can provide new ideas for the detection and analysis of cis-diol compounds and can be applied in real samples.