摘要
建立反相高效液相色谱法（RP-HPLC）并测定黑斑蛙（Rana nigromaculata）和中华大蟾蜍（Bufo bujfo gargarizans）幼体与成体组织中GSH含量.样本处理后经高氯酸沉淀,采用邻苯二甲醛（OPA）柱前衍生,338nm波长检测,以甲醇-乙酸盐缓冲液为流动相,测得GSH标准液浓度在25～300μmo/L范围内与峰面积有良好线性关系,相关系数达0.999.GSH外标回收率为99.44%±2.22%,日内和5日日间变异系数（CV）为≤2.21和≤2.35,最低检出限1.1μg/g.结果黑斑蛙和中华大蟾蜍幼体与成体GSH均为皮肤含量最高,达213.4、197.3μg/g和189.7、201.9μg/g,其次为肝脏、脑、全血、脾脏,肌肉组织含量最低,为51.3、16.2μg/g和40.3、14.9μg/g.本方法样本处理简单且具有良好检测灵敏度和精密度,能够满足两栖动物组织GSH含量测定要求.
The purpose is to establish a RP-HPLC method to determine the glutathione （GSH） contents in the immature and grown individuals of both Rana nigromaculata and Bufo bujfo gargarizans. Proteins contained in samples were precipitated by 10% perchloric acid before OPA pre-column defvatization, and then were detected with ultraviolet detection at 338nm using a mobile phase of Methanol-Acetate irrigation solution. GSH contents were calculated according to the linear range of calibration curve of GSH concentration related with the peak area. There was a good dynamic linear range from 25μmol/L to 300μmol/L and the correlation index upto 0. 999. The recoveries of added GSH was 99.44 % ± 2.22%, the coefficients of variability were less than 2.21 in a day and that of 2.35 between 5 days. The MDL of GSH was 1.μg/mL. The result shows that both of the immature and grown individuals of the two species get the highest level of GSH content in skin, and then liver, brain, blood and spleen in order, and finally comes the muscle. The maximum contents are 213.4μg/g, 197.3μg/g and 189.7μg/g, 201.9μg/g in skin, the minimum contents are 51.3μg/g, 16.2μg/g and 40.3μg/g, 14.9μg/g in muscle. Using simple treatment, this method can determine the GSH contents of amphibian tissues sensitively and precisely.
出处
《山东科学》
CAS
2006年第2期24-28,共5页
Shandong Science
基金
国家自然科学基金资助(30070586)
