Kiguchi M, Sudo J
Department of Toxicology and Clinical Pharmacology, Faculty of Pharmaceutical Sciences, Higashi-Nippon-Gakuen University, Hokkaido, Japan.
J Toxicol Sci. 1987 Aug;12(3):301-7. doi: 10.2131/jts.12.301.
In this study, colorimetric method and high-performance liquid chromatographic (HPLC) method were improved and established, respectively, in order to minimize analytical errors in determination of para-aminohippuric acid (PAH) in rat urine and plasma. In terms of the colorimetric method, an operative step following addition of Tsuda reagent was modified as follows: after the addition of Tsuda reagent, reaction mixture was kept at 40 degrees C for 70 min before spectrophotometry. Linearities were observed both in the higher range of 0 and 2.5 to 12.5 micrograms and in the lower range of 0 and 100 to 1,000 ng per test tube, and its practical detection limit was 100 ng per test tube. In terms of HPLC method, using a reversed-phase column (Nucleosil 5 C18), PAH was separated by a mobile phase of acetonitrile/50 mM KH2PO4 (pH 2.8) = 9/95. Linearities were observed in the higher range of 0 and 10 ng to 2 micrograms and in the lower range of 0 and 1 to 10 ng per injection, and its practical detection limit was 1 ng per injection. These results denote that the above two methods are applicable to routine PAH determination. In addition, our HPLC method is considered to be applicable to microassay of PAH, because its sensitivity is more sensitive and minimization of volume system is more easily achieved as compared with the colorimetric method.
在本研究中，分别对比色法和高效液相色谱（HPLC）法进行了改进并建立，以尽量减少大鼠尿液和血浆中对氨基马尿酸（PAH）测定中的分析误差。就比色法而言，加入津田试剂后的操作步骤修改如下：加入津田试剂后，反应混合物在40℃保持70分钟，然后进行分光光度测定。在每支试管0至2.5微克至12.5微克的较高范围内以及0至100纳克至1000纳克的较低范围内均观察到线性关系，其实际检测限为每支试管100纳克。就HPLC法而言，使用反相柱（Nucleosil 5 C18），PAH通过乙腈/50 mM KH2PO4（pH 2.8）= 9/95的流动相进行分离。在每次进样0至10纳克至2微克的较高范围内以及0至1至10纳克的较低范围内均观察到线性关系，其实际检测限为每次进样1纳克。这些结果表明上述两种方法适用于常规PAH测定。此外，我们的HPLC法被认为适用于PAH的微量测定，因为与比色法相比，其灵敏度更高，并且更易于实现体积系统的最小化。
