高效液相色谱-荧光检测法测定大鼠骨骼肌组织内微量三甲基组氨酸  

Determination of 3-methylhistidine levels in skeletal muscle of rats by high performance liquid chromatography

作　　者：申传安[1] 柴家科[1] 廖杰[2] 盛志勇[1] 

机构地区：[1]解放军第304医院全军烧伤研究所临床部,北京100037 [2]解放军总医院仪器中心,北京100853

出　　处：《感染．炎症．修复》2003年第1期32-34,共3页Infection Inflammation Repair

基　　金：国家自然科学基金项目(39970716);"十五"军队医药卫生科研基金重点项目(01Z095)

摘　　要：目的:建立快速检测肌肉组织内微量三甲基组氨酸(3-MH)含量的方法。方法:利用荧光胺与3-MH 在酸性和高温条件下发生柱前衍生反应,在 ZORBAX SB-C18(4.6×150mmol/L,5μm)柱上,用10mmol/L磷酸钠缓冲液(含30%乙腈,pH7.50)作流动相,以1.0ml/min 的流速进行等度洗脱后,荧光(激发波长365nm/发射波长460nm)检测3-MH 含量。结果:应用此方法检测骨骼肌组织内的氨基酸,仅见到3-MH 和组氨酸的色谱峰,色谱保留时间分别约为(4.45±0.02)min 和(6.92±0.05)min,分离度>16;线性检测范围是0.005～10.0nmol/ml(r=0.9999,P=0.00);衍生回收率为92.6%～96.8%;3-MH 荧光衍生后15d 分析与衍生后立即检测相比结果无显著差异(P=0.9095,n=24)。结论:本方法测定骨骼肌组织内3-MH,衍生步骤简单,重复性好,检测灵敏度高,适合快速、准确地大批量检测肌组织内的微量3-MH,为开展骨骼肌肌纤维蛋白分解代谢研究提供了可行的方法。Objective:To establish a fast and sensitive method for the determination of trace amount of 3-meth ylhisdine(3-MH)in skeletal muscle of rats.Methods:After the formation of heat-treated and stable fluorscanmine derivatives of 3-MH in rat skeletal muscle,high performance liquid chromatographic separation and fluorescent de- tection (E_x=365nm,E_m=460nm)were used to determine the 3-MH levels in samples.The solvent system was 10 mmol/L sodium phosphate (pH7.5)and acetonitrile (30%)with a flow rate of 1.0ml/min.Results:No amino acid peaks but two peaks,coeluting with histidine and 3-MH,were noted when standard sample and skeletal mus- cle sample were chromatographed.The 3-MH was eluted completely within 5 minutes and its peak could be easily distinguished from that of histidine (separation rate>16).The assay was linear over a range of 0.005 10.0nmol/ml (r=0.9999,P=0.0000).Conclusion:This method is not only very sensitive and accurate,but also quick and economical for determining the 3-MH amount in skeletal muscle of rats.It is especially suitable for ana- lyzing samples in batches or with low concentration of 3-MH.

关 键 词：高效液相色谱法 荧光检测 三甲基组氨酸 骨骼肌 大鼠 

分 类 号：R[医药卫生]