运用同一波长HPLC法测定大鼠血清色氨酸及其代谢产物

Determination Of Serum Tryptophan And its Metabolites In Rats with HPLC Method

目的:运用高效液相色谱-紫外检测法建立一种简单、快速同时测定大鼠血清中的色氨酸(Trp)及主要代谢产物5-羟色胺(5-HT)和犬尿氨酸(Kyn)的检测方法。方法:以20%高氯酸为血清蛋白质沉淀剂,采用C18(250×4.6 mm,5μm)色谱柱,流动相为0.05mol/l磷酸二氢钾-甲醇(80∶20,v/v),于波长225nm处对大鼠血清进行检测。结果:15min内可以完成本实验分析过程,大鼠血清中Trp、5-HT、Kyn平均回收率分别为90.69%、103.21%、101.15%,RSD分别为1.78%、0.81%、1.12%(n=9);分别在0μg^27.0336μg、0μg^1.47456μg、0μg^1.51552μg的范围内成较好的线性关系,相关系数r均为0.9995。结论:通过对实验中多个环节的考察,建立了大鼠血清中色氨酸及其代谢产物5-羟色胺与犬尿氨酸含量测定的方法,该方法准确性较好,能快速简单地完成检测过程。

Objective： To establish a simple and rapid simultaneous determination of rat serum tryptophan （Trp） using high performance liquid chromatography and detection methods of UV, metabolites of serotonin （5- HT） and kynurenine （Kyn）. Methods： Using 20% serum protein perchlorate as the precipitating agent and C18 （250 × 4. 6 mm, 5m） chromatographic column, the mobile phase is 0. 05 mol/1 of potassium dihydrogen phosphate - methanol （80： 20, v/v）, 225 nm wavelengths to test the serum of rats. Results： The process of this experiment analysis can be completed in only 15 minutes. The average recovery rates of Trp, 5 - HT, Kyn, and Trp were 90. 69% , 103.21% , 101.15%, respectively. RSD was 1.78% , 0. 81%, 1.12% （n = 9）. The correlation coefficient was 0. 9995 in the range of 0μg- 27.0336μg,0μg- 1.47456μg,0μg 1.51552μg respectively. Conclusion： Based on the investigation of multiple links in the experiment, the establishments of the method for the determination of serum tryptophan, its serotonin metabolites, and uric acid in rats, the method has good accuracy and can quickly and simply complete the process.