RAM柱对生物体液中分析物在线富集及其保留机理研究

发布时间：2018-05-11 10:23

本文选题：限进性填料 + 高效液相色谱　；参考：《兰州大学》2014年硕士论文

【摘要】：生物样品的前处理方法对体内药物分析来说是至关重要的步骤,传统的生物样品前处理方法有蛋白沉淀法,离线固相萃取法(SPE),液液萃取法(LLE)等,这些方法不仅需要大量的有机试剂,还会因为在离线状态下操作,导致生物样品中的待测物被稀释,损失和污染,使得分析结果不准确。此外,生物样品因为基质复杂,含有大量的蛋白质等生物大分子,所以对固相萃取柱污染严重,使分析费用加大,不常用于色谱分析中。限进性填料(RAM)是一种新型的样品前处理材料,它具有两个不同的表面,外表面兼具分子排阻功能和亲水性,内表面键合有不同的功能基团,因此,生物样品中只有小分子物质可以进入内表面并与内表面上的功能基团相互作用,进而保留在限进性填料柱上,而蛋白质等大分子物质无法进入RAM的内表面,又因为外表面的亲水性,这些大分子物质也不会吸附沉积在外表面上,而是被流动相洗脱除去,因此,RAM通过柱切换技术与高效液相色谱(HPLC)或高效液相色谱-质谱(HPLC-MS)联用,可实现生物样品的在线直接进样,相比于蛋白沉淀,SPE等方法,RAM能够避免待测物的损失和污染,也能避免生物大分子物质对萃取柱的损害。此外,RAM还能作为预富集柱富集生物样品中的小分子物质。生物样品中待测物的浓度通常较低,这是体内药物分析面临的又一问题,通常是利用HPLC-MS的高灵敏度来解决的,但是HPLC-MS存在价格昂贵,分析条件严格的问题,所以,性能稳定,易于操作的HPLC依然在体内药物分析中占有重要地位,但其灵敏度无法完全满足体内药物分析的要求,因此,本文建立了RAM-HPLC法,通过大体积直接进样(LVI)的方式,考察了RAM柱对血浆、尿液、脑脊液中小分子药物的富集能力,并在线富集检测这些生物体液中的药物浓度,有效提高了HPLC的检测灵敏度,为生物样品的测定提供简单、快速、准确的在线分析方法。此外,我们还探讨了RAM对药物的保留机理,为更加有效利用RAM柱及合成新型RAM提供一定的实验依据。
[Abstract]:The pretreatment of biological samples is an important step for drug analysis in vivo. The traditional pretreatment methods of biological samples include protein precipitation, off-line solid phase extraction (SPE), liquid-liquid extraction (Lle) and so on. These methods not only require a large number of organic reagents, but also lead to dilution, loss and contamination of the substances to be tested in biological samples due to off-line operation, which makes the analytical results inaccurate. In addition, because of the complex matrix and the large amount of protein and other biological macromolecules, biological samples contaminate the solid phase extraction column seriously and increase the cost of analysis, so it is not often used in chromatographic analysis. Ram is a new kind of sample pretreatment material. It has two different surfaces. The outer surface has both molecular resistance and hydrophilicity, and the internal surface bonding has different functional groups. In biological samples, only small molecules can enter the inner surface and interact with the functional groups on the inner surface, and then remain on the confined packed column, while the protein and other macromolecular substances cannot enter the inner surface of RAM. Because of the hydrophilicity of the outer surface, these macromolecular substances are not adsorbed and deposited on the outer surface, but are eluted by mobile phase, so RAM is used in combination with HPLC or HPLC-MS by means of column switching technique. Compared with other methods such as protein precipitation and SPE, RAM can avoid the loss and pollution of the material to be tested, and the damage to the extraction column caused by biomolecules. In addition, Ram can also be used as a preconcentration column for the enrichment of small molecules in biological samples. The concentration of the substances to be tested in biological samples is usually low, which is another problem in drug analysis in vivo, which is usually solved by using the high sensitivity of HPLC-MS. However, HPLC-MS has the problems of high price and strict analytical conditions, so its performance is stable. The easy-to-operate HPLC still plays an important role in drug analysis in vivo, but its sensitivity can not fully meet the requirements of drug analysis in vivo. Therefore, the method of RAM-HPLC is established in this paper. The enrichment ability of RAM column on small molecular drugs in plasma, urine and cerebrospinal fluid was investigated. The concentration of these drugs in biological body fluid was detected online. The detection sensitivity of HPLC was improved effectively, which provided a simple and rapid method for the determination of biological samples. Accurate online analysis method. In addition, we also discussed the retention mechanism of RAM on drugs, which provided some experimental basis for the more efficient use of RAM column and the synthesis of new RAM.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R917

【参考文献】