Department of Analytical Chemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Japan; Laboratory of Bioanalytical and Environmental Chemistry, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Japan.
Department of Analytical Chemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Japan.
J Pharm Biomed Anal. 2020 Jul 15;186:113253. doi: 10.1016/j.jpba.2020.113253. Epub 2020 Mar 14.
A highly sensitive method for determining urine homogentisic acid (HGA) is required to provide adequate diagnosis and therapy for alkaptonuria in early stages. In this study, we developed a highly sensitive high-performance liquid chromatography with electrochemical detection (HPLC-ECD) for determining HGA in urine. In order to obtain a chromatogram of HGA by HPLC-ECD, an oxidation current was monitored at +0.5 V vs. Ag/AgCl. The peak heights of HGA showed linearity (r = 0.999) ranging from 4.2 ng/mL to 168 ng/mL, and the detection limit was 1.2 ng/mL (signal-to-noise ratio, S/N = 3). In recovery tests using human control urine spiked with an HGA standard, the recoveries of HGA were more than 93.2 %, and the relative standard deviations (n = 6) were less than 1.9 %. As an in vivo application using male Wistar rats, the level of urine HGA, which was metabolized from tyrosine in tyrosine-enriched food, was determined by this HPLC-ECD method. The determination of HGA in urine by this HPLC-ECD method requires only 0.1 mL of a rat urine specimen and simple sample preparation consisting of dilution and filtration.
需要一种高灵敏度的方法来测定尿液中的同型尿黑酸（HGA），以便在早期为尿黑酸症提供充分的诊断和治疗。本研究开发了一种高灵敏度的高效液相色谱电化学检测法（HPLC-ECD），用于测定尿液中的 HGA。为了通过 HPLC-ECD 获得 HGA 的色谱图，在 +0.5 V 相对于 Ag/AgCl 监测氧化电流。HGA 的峰高呈现出线性关系（r=0.999），范围从 4.2 ng/mL 到 168 ng/mL，检测限为 1.2 ng/mL（信噪比，S/N=3）。在使用人对照尿样中加入 HGA 标准品进行回收试验中，HGA 的回收率超过 93.2%，相对标准偏差（n=6）小于 1.9%。作为使用富含酪氨酸的食物代谢酪氨酸的雄性 Wistar 大鼠的体内应用，用这种 HPLC-ECD 方法测定了尿液中的 HGA 水平。该 HPLC-ECD 方法测定尿液中的 HGA 仅需 0.1 mL 大鼠尿液标本，且样品制备简单，只需稀释和过滤。
