Fukunaga K, Yoshida M, Nakazono N
Laboratory of Public Health, Kansai Medical University, Moriguchi, Osaka, Japan.
Biomed Chromatogr. 1998 Sep-Oct;12(5):300-3. doi: 10.1002/(SICI)1099-0801(199809/10)12:5<300::AID-BMC751>3.0.CO;2-#.
A simple, rapid, highly sensitive and reproducible quantification method for plasma malondialdehyde (MDA) was developed using high-performance liquid chromatography (HPLC). The present method is based on the thiobarbituric acid (TBA) reaction and reversed-phase HPLC separation with fluorescence detection. For sample preparation, an aliquot of 5 microL plasma was mixed with 0.2% (w/v) TBA in 0.1 M sodium acetate buffer, pH3.5. After heating at 95 degrees C for 60 min, the reaction solution was centrifuged and an aliquot of 10 microL supernatant was injected into the HPLC system without neutralization or extraction procedures. The TBA-MDA adduct was separated on a reversed-phase column and quantified by a fluorescence detection (lambda ex = 515 nm; lambda em = 553 nm). The mobile phase was a mixture of acetonitrile: water (7:3, v/v) under isocratic conditions at ambient temperature. These procedures gave good results with regard to sensitivity (detection limit S/N = 3, 0.5 fmol per injection), linearity (0.5-500 fmol per injection), precision (between-assay C.V = 3.1%, within-assay C.V = 1.2%) and recovery (98.6%). The simple sample procedure, the highly sensitive detection limit and the stability of the TBA-MDA adduct make the present method applicable to numerous clinical samples. As many as 250-300 samples per day can be assayed using an autosampler.
利用高效液相色谱法（HPLC）开发了一种简单、快速、高灵敏度且可重复的血浆丙二醛（MDA）定量方法。本方法基于硫代巴比妥酸（TBA）反应以及反相HPLC分离并结合荧光检测。样品制备时，取5微升血浆 aliquots与0.1 M乙酸钠缓冲液（pH3.5）中0.2%（w/v）的TBA混合。在95℃加热60分钟后，将反应溶液离心，取10微升上清液 aliquots直接注入HPLC系统，无需中和或萃取步骤。TBA-MDA加合物在反相柱上分离，并通过荧光检测（激发波长λex = 515 nm；发射波长λem = 553 nm）进行定量。流动相为乙腈与水的混合物（7:3，v/v），在室温等度条件下。这些步骤在灵敏度（检测限S/N = 3，每次进样0.5 fmol）、线性（每次进样0.5 - 500 fmol）、精密度（批间变异系数C.V = 3.1%，批内变异系数C.V = 1.2%）和回收率（98.6%）方面均取得了良好结果。简单的样品处理程序、高灵敏度的检测限以及TBA-MDA加合物的稳定性使得本方法适用于众多临床样品。使用自动进样器每天可检测多达250 - 300个样品。
