Tagawa N, Tsuruta H, Fujinami A, Kobayashi Y
Clinical Chemistry Laboratory, Kobe Pharmaceutical University, Japan.
Biol Pharm Bull. 1998 Nov;21(11):1211-4. doi: 10.1248/bpb.21.1211.
A method to prevent co-elution of steroid sulfates with proteins in serum from the pre-column in column-switching HPLC was developed. The pre-column, a polymer-coated mixed function column, was used for ion-pair chromatography with 5 mM tetra-n-butylammonium (TBA) ion. As steroid sulfates, estriol 3-sulfate, dehydroepiandrosterone 3-sulfate and pregnenolone 3-sulfate were used. Human serum (25 microl) was diluted with mobile phases including 5, 100 and 500 mM TBA ion, and then injected directly into the pre-column. The peak areas of the steroid sulfates in serum samples were compared with those of the steroid standards without serum. When 25/microl of serum was diluted with mobile phase including 100 or 500 mM TBA ion, the steroid sulfates in serum were retained in the pre-column; however, the steroid sulfates from the same sample diluted with mobile phase containing 5 mM TBA ion were not retained in the pre-column. Addition of an excess amount of counter ion (TBA ion) into the serum sample made it possible to retain the steroid sulfates in the pre-column. This method was applied to column-switching HPLC for measurement of steroid sulfates in serum using a semi-microcolumn as the analytical column.
开发了一种在柱切换高效液相色谱法中防止血清中甾体硫酸盐与蛋白质在预柱上共洗脱的方法。预柱是一种聚合物涂层混合功能柱，用于与5 mM四丁基铵（TBA）离子进行离子对色谱分析。使用雌三醇3-硫酸盐、脱氢表雄酮3-硫酸盐和孕烯醇酮3-硫酸盐作为甾体硫酸盐。将人血清（25微升）用含有5、100和500 mM TBA离子的流动相稀释，然后直接注入预柱。将血清样品中甾体硫酸盐的峰面积与不含血清的甾体标准品的峰面积进行比较。当25微升血清用含有100或500 mM TBA离子的流动相稀释时，血清中的甾体硫酸盐保留在预柱中；然而，用含有5 mM TBA离子的流动相稀释的同一样品中的甾体硫酸盐没有保留在预柱中。向血清样品中加入过量的抗衡离子（TBA离子）使得甾体硫酸盐能够保留在预柱中。该方法应用于柱切换高效液相色谱法，使用半微柱作为分析柱测定血清中的甾体硫酸盐。
