Iwaki K, Okumura N, Yamazaki M
School of Pharmacy, Hokuriku University, Ishikawa, Japan.
J Chromatogr. 1992 Oct 9;623(1):153-8. doi: 10.1016/0021-9673(92)85310-p.
A highly sensitive method for the determination of tetracycline antibiotics (TCs) using reversed-phase high-performance liquid chromatography with fluorescence detection is presented. This method was based on the use of disodium ethylenediaminetetraacetate (EDTA) and calcium chloride as fluorescence-increasing reagents in the mobile phase. The concentrations of each reagent in the mobile phase greatly influenced the fluorescence intensity of TCs. When the concentration of EDTA and calcium chloride were 25 and 35 mM, respectively, and the pH of the mobile phase was 6.5, the maximum fluorescence intensity was obtained. The column temperature hardly influenced the fluorescence intensity. At 3.75 ng of TCs injected, the precision (relative standard deviation) ranged from 1.12 to 2.20%. In the range 0.075-37.5 ng for tetracycline and oxytetracycline and 0.225-37.5 ng for chlortetracycline, a linear response was observed. The detection limits of this method were 49-190 pg for three different TCs. The proposed method was applied to the determination of one of the TCs in pharmaceuticals by the internal standard method using other TCs as internal standards and was also applied to determination of TCs added to fish tissue.
介绍了一种采用反相高效液相色谱-荧光检测法测定四环素类抗生素（TCs）的高灵敏度方法。该方法基于在流动相中使用乙二胺四乙酸二钠（EDTA）和氯化钙作为荧光增强试剂。流动相中各试剂的浓度对TCs的荧光强度有很大影响。当EDTA和氯化钙的浓度分别为25和35 mM，且流动相的pH值为6.5时，可获得最大荧光强度。柱温对荧光强度影响很小。进样3.75 ng TCs时，精密度（相对标准偏差）在1.12%至2.20%之间。在四环素和土霉素的0.075 - 37.5 ng以及金霉素的0.225 - 37.5 ng范围内，观察到线性响应。该方法对三种不同TCs的检测限为49 - 190 pg。所提出的方法通过以内标法使用其他TCs作为内标应用于药物中一种TCs的测定，也应用于鱼组织中添加的TCs的测定。
