Fukunaga K, Suzuki T, Takama K
Department of Food Science and Technology, Faculty of Fisheries, Hokkaido University, Japan.
J Chromatogr. 1993 Nov 17;621(1):77-81. doi: 10.1016/0378-4347(93)80078-i.
A highly sensitive method for the measurement of malondialdehyde as thiobarbituric acid-malondialdehyde complex by reversed-phase high-performance liquid chromatography with fluorescence detection in biological samples is described. As samples, 20 microliters of rat plasma or 10% (w/v) liver homogenate mixed with 2.0% thiobarbituric acid in 2 M sodium acetate buffer containing 0.05% butyl hydroxytoluene (pH 3.5) were heated at 95 degrees C for 45 min to give the complex. The complex, extracted with n-butanol, was chromatographed on a system equipped with a reversed-phase column, and the eluted peak was monitored with a fluorescence detector (excitation wavelength 515 nm, emission wavelength 553 nm). The mobile phase was a acetonitrile-water (2:8, v/v) under isocratic conditions at ambient temperature, and a single analysis was done in ca. 4 min. The minimum detection level for malondialdehyde was as low as 0.05 pmol. The n-butanol extract was stable at least for 3 days. The simple mobile phase, the extremely sensitive detection limit, and the stability of the complex make this system applicable to routine clinical analysis with a small amount of tissue or biopsy sample.
本文描述了一种通过反相高效液相色谱结合荧光检测法测量生物样品中丙二醛（以硫代巴比妥酸 - 丙二醛复合物形式）的高灵敏度方法。取20微升大鼠血浆或10%（w/v）肝脏匀浆作为样品，与含0.05%丁基羟基甲苯（pH 3.5）的2M醋酸钠缓冲液中的2.0%硫代巴比妥酸混合，在95℃加热45分钟生成复合物。用正丁醇萃取该复合物，在配备反相柱的系统上进行色谱分析，并用荧光检测器（激发波长515nm，发射波长553nm）监测洗脱峰。流动相为室温等度条件下的乙腈 - 水（2:8，v/v），单次分析约需4分钟。丙二醛的最低检测限低至0.05皮摩尔。正丁醇提取物至少稳定3天。简单的流动相、极高的检测限以及复合物的稳定性使得该系统适用于使用少量组织或活检样品的常规临床分析。
