Kawaguchi S, Hirachi N, Fukamachi M
Scientific Instrument Division, Tokyo Research Center, Tosoh Corporation, Kanagawa, Japan.
J Chromatogr. 1991 Jun 14;567(1):11-9. doi: 10.1016/0378-4347(91)80304-u.
We have developed a novel analyser for the determination of vanillylmandelic acid, homovanillic acid and creatinine in urine by high-performance liquid chromatography using three different types of column, cation-exchange, anion-exchange and reversed-phase and the column-switching technique. In this procedure, 10 microliters of intact urine were directly injected into the cation-exchange column, and the pass-through fraction, containing vanillylmandelic acid and homovanillic acid was transferred to the anion-exchange column by column switching. The fraction partially purified from endogenous urinary impurities on the anion-exchange column was transferred to the reversed-phase column. Vanillylmandelic acid and homovanillic acid, separated by the solvent-switching technique, were detected fluorimetrically (excitation at 280 nm, emission at 320 nm). Then, creatinine eluted from the cation-exchange column is spectrophotometrically detected (254 nm). Therefore the successive simultaneous analysis of the three could be performed in a 15-min cycle; the within-assay coefficients of variation for normal and patients' urines were less than 1.9%, less than 3.3% and less than 3.0% for vanillylmandelic acid, homovanillic acid and creatinine, respectively; the recoveries averaged 100, 103 and 100%, respectively, for supplemented urines.
我们开发了一种新型分析仪，通过高效液相色谱法，使用三种不同类型的色谱柱（阳离子交换柱、阴离子交换柱和反相柱）以及柱切换技术来测定尿液中的香草扁桃酸、高香草酸和肌酐。在此过程中，将10微升未处理的尿液直接注入阳离子交换柱，通过柱切换将含有香草扁桃酸和高香草酸的穿透组分转移至阴离子交换柱。在阴离子交换柱上部分纯化以去除内源性尿液杂质后的组分被转移至反相柱。通过溶剂切换技术分离的香草扁桃酸和高香草酸采用荧光法检测（激发波长280nm，发射波长320nm）。然后，从阳离子交换柱洗脱的肌酐采用分光光度法检测（254nm）。因此，这三种物质可在15分钟的周期内连续同时进行分析；正常尿液和患者尿液的批内变异系数，香草扁桃酸、高香草酸和肌酐分别小于1.9%、小于3.3%和小于3.0%；添加尿液的回收率平均分别为100%、103%和100%。
