改性苯乙烯-马来酸酐共聚物色谱固定相用于磷脂分离分析

Modified styrene-maleic anhydride copolymer-based chromatographic stationary phase for phospholipid separation and analysis

磷脂是重要的信号分子,磷脂的代谢与多种疾病密切相关。因此,开展磷脂的分离分析研究至关重要。苯乙烯-马来酸酐共聚物(SMA)作为一种新型两亲性交替共聚物可以插入生物膜的磷脂双分子层中,形成以膜蛋白质为中心的脂质纳米盘,对膜蛋白质和磷脂具有良好的增溶作用。本文基于“点击”反应和自由基聚合反应,将对磷脂具有良好增溶性能的SMA接枝到硅胶表面,然后以蛋氨酸甲酯盐酸盐(MME·HCl)为开环试剂,通过亲核开环反应对SMA进行修饰,制备了新型的改性SMA修饰色谱固定相(Sil-SMA-MME)。结合高效液相色谱-紫外检测法,利用酰胺类和核苷/核酸碱基类以及苯酚类3类小分子物质对填充Sil-SMA-MME色谱柱的保留机制和分离性能进行了系统评价,Sil-SMA-MME色谱柱具有典型的亲水作用保留机制,其柱效最高可达90900 N/m,并显示了良好的分离选择性。进一步结合高效液相色谱-蒸发光散射检测法,考察了Sil-SMA-MME色谱柱对磷脂样品的分离性能。二棕榈酰磷脂酰丝氨酸钠(DPPS)、二油酰磷脂酰胆碱(DOPC)、二棕榈酰磷脂酰乙醇胺(DPPE)和4种磷脂酰胆碱(PC)类标准品溶血卵磷脂(LysoPC)、二肉豆蔻酰磷脂酰胆碱(DMPC)、二硬脂酰磷脂酰胆碱(DSPC)、二棕榈酰磷脂酰胆碱(DPPC)均可实现基线分离,并且成功地实现了南极磷虾油和人血清磷脂提取物的分离分析。以上结果表明,所制备的Sil-SMA-MME色谱柱在磷脂类物质分离分析中具有良好的应用潜力。

Phospholipids are important signaling molecules,and their metabolism is closely related to various diseases,such as neurodegenerative diseases and cancers.Phospholipids are typically characterized with extreme complexity and structural diversity.For example,phospholipids present in many different forms,such as sn position isomers,double-bond position isomers,double-bond stereochemical isomers,and enantiomers.Therefore,further research on novel separation and analytical techniques for phospholipids is of great importance.As an amphiphilic alternating copolymer,styrene-maleic anhydride copolymer(SMA)can be inserted into the phospholipid bilayer of biofilms to form lipid nanodisks with membrane proteins as the centers,thereby solubilizing membrane proteins and phospholipids.Thus,the introduction of SMA into a chromatographic stationary phase can potentially improve the separation and analysis of phospholipids.In this paper,SMA was successfully grafted onto the surface of silica gel via the“click”reaction and free radical polymerization.After further ring-opening modification of SMA with methyl methionine hydrochloride(MME·HCl),a novel SMA-modified stationary phase material(Sil-SMA-MME)was fabricated.The Sil-SMA-MME stationary phase was characterized using thermogravimetric analysis and Fourier transform infrared spectroscopy(FT-IR),and the results indicated the successful fabrication of the target material.The retention mechanism of the packed Sil-SMA-MME chromatographic column was investigated using hydrophilic nucleosides and nucleic acid bases via high performance liquid chromatography(HPLC)and UV detection.According to the retention characteristics of the nucleosides and nucleic acid bases in different mobile phases,the Sil-SMA-MME chromatographic column exhibited a typical hydrophilic-interaction-based retention mechanism,similar to that of a commercially available amino(SiO 2-NH 2)column.The separation performance of the Sil-SMA-MME column was evaluated using three types of small-molecule substances,including amides,nucleoside/nucleic acid bases,and phenols.Cyanoacetamide,2-iodoacetamide,benzamide,p-aminobenzamide,and nicotinamide were used to evaluate the chromatographic performance of the developed Sil-SMA-MME column.When acetonitrile-H 2O(96∶4,v/v)was used as the mobile phase,the five compounds exhibited good peak shapes and could be baseline-separated within 8 min.The highest column efficiency achieved was 90900 N/m.By contrast,under the same chromatographic conditions,the test substances were not separated effectively on the SiO 2-NH 2 column.Regardless of the mobile phase ratio,the peaks of benzamide and 2-iodoacetamide overlapped.These results demonstrate that the developed Sil-SMA-MME column has good separation selectivity.The separation performance of the Sil-SMA-MME column for phospholipid samples was also investigated by HPLC and evaporative light scattering detection(ELSD)to explore its feasibility for phospholipid separation and analysis.Different phospholipid standards were used to evaluate the separation performance of the column.Under certain mobile phase conditions,baseline separation could be achieved for dipalmityl phosphatidyl serine sodium(DPPS),diolyl phosphatidyl choline(DOPC),and dipalmityl phosphatidyl ethanolamine(DPPE),as well as four phosphatidyl choline(PC)standards,namely,lysophosphatidylcholine(LysoPC),dimyristoyl phosphatidyl choline(DMPC),distearyl phosphatidyl choline(DSPC),and dipalmitoyl phosphatidyl choline(DPPC).The separation potential of the developed Sil-SMA-MME column was further evaluated by separating and analyzing phospholipid extracts from Antarctic krill oil and human serum.The results showed that the developed Sil-SMA-MME column has good potential for phospholipid separation and analysis.