Establishment And Application Of Liquid Chromatography Technology In The Field Of Food Safety And Biopharmaceuticals

In proteomics,pharmaceutical analysis,food safety and other fields,traditional chromatography is often difficult to provide sufficient analytical capacity due to the lack of peak capacity,low resolution and sensitivity,or differences in separation characteristics.Due to the flexible combination of different selective chromatography techniques,two-dimensional chromatography(2D-LC)can produce excellent peak capacity,selectivity and separation degree,and at the same time,automatic pretreatment capacity,which can meet the requirements of automatic and high-throughput analysis of large quantities of complex samples.Hydrophilic chromatography(HILIC)is an important complementary chromatographic technique because it uses strong polar materials as the stationary phase and a high proportion of organic dissolvent-water system as the mobile phase to separate strong polar substances,such as sugars,peptides and amino acids,which can't be analyzed by reverse-phase chromatography(RP-LC)and normal phase chromatography(NP-LC).In this paper,the applications of 2D-LC and HILIC in food safety and biological pharmaceutical quality control were explored.The First Part:Because of the simple production process,low cost and large profit,bean sprout vegetables attract a large number of vendors to produce and grow.However,due to the market supervision is not comprehensive,bean sprouts supply confusion.To obtain higher profits,some vendors illegally used a variety of plant growth regulators(PGRs)in the production of bean sprouts,which caused great harm to the health of consumers.Therefore,it is very important to monitor the added PGRs in bean sprouts.In this paper,a method for simultaneous determination of gibberellin acid(GAs),6-benzyl adenine(6-BA),4-chlorophenoxyacetic acid(4-CPA)and 2,4-dichlorophenoxyacetic acid(2,4-D)in mung bean sprouts was established by 2D-LC.The separation was first performed on the first-dimension column ZB-10 C18(ODS-AP,10×10 mm).The analysts were trapped on this column for purification and the mobile phase used here was V(formic acid):V(methanol):V(water)=1:10:89 in isocratic elution mode.Then,the trapped analysts were transferred to the second-dimension column(Supersil ODS 2.5 ?m,4.6×150 mm)using valve-switching technique,methanol and 0.1%formic acid water solutions were used as mobile phase in gradient elution mode.Thus,they could be determined at 254 nm with a UV detector.The results showed that the linear ranges of GAs,6-BA,4-CPA and 2,4-D acid were 0.004?2,0.0001?0.2,0.005?2 and 0.008?2 mg/mL,respectively;and their detection limits were 1.4,0.03,1.6 and 2.4?g/mL respectively.The RSD values were 2%?3%and the recoveries ranged from 95%?104%.When those plant hormone residues were purified by the first-dimension column,they could be successfully detected in mung bean sprouts at the level of 0.018 mg/g for GAs and 0.006 mg/g for 2,4-D.This research shows that,the first dimension was configured as solid phase extraction column,and samples of complex matrix could be directly injected.By means of automatic on-line purification and enrichment of low concentration analytes,the matrix effect and residual phenomenon were significantly improved,the sensitivity and reproducibility of the analysis were improved,and the requirements for automatic and high-throughput analysis of large quantities of samples were satisfied.The Second Part:The N-glycosylation modification of Fc plays an important role in the regulation of the functional characteristics of immunoglobulin G(IgG),and the detection and control of glycosylation is also the key to the quality of antibody preparation.Therefore,it is of great significance to carry out the analysis and research on the glycosylation of monoclonal antibodies(mAbs).To this end,a quantitative analysis technique of N-sugar chains was established by HILIC-LC coupled with fluorescence detection.For the three common N-sugar chains GOF,G1F,G2F,their linear ranges were 0.075?15.0,0.045?9.0,and 0.075?4.5 pmol/mL while the detection limits were 0.028,0.025,0.026 pmol/mL,respectively.The RSD values of reserved time and peak area were all found less than 3%,and their recoveries were greater than 95%.It meant that the established method was sufficient for the content analysis of these three N-sugar chains in a real sample,which were released by digesting cetuximab and further labeled with 2-aminobenzamide(2-AB).Further results indicated they were 86.0,85.1,16.6 ?g/g protein,respectively.Besides,we also provided an UPLC-ESI-MS method to detection and analysis technology,combined with GlycoWorkbench 2 analysis software and The Consortium for Functional Glycomics(CFG,http://www.functionalglycomics.org/)database,to achieve the analysis of 7 kinds of N-sugar chains with higher relative abundance of cetuximab.These studies can be not only applied for quality control analysis of the N-sugar chains of mAbs,but also determination of the types of sugar chains for further biological research.