摘要
目的建立一种检测人绒毛滋养细胞上清培养液中L-色氨酸(L-tryptophan,L-Trp)的高效液相色谱-紫外检测法。方法采用的色谱柱为Eclipse XDB-C18柱(4.6mm×150mm,5μm),流动相为0.05mmol·L-1磷酸二氢钾溶液-甲醇(92∶8),流速为1.3mL·min-1。紫外检测波长为225nm。滋养细胞上清培养液标本经5.0%(体积分数)高氯酸溶液去除蛋白质后取上清液过滤后直接进样分析测定。结果L-Trp保留时间为7.5min,线性范围为1.0～100.0μmol·L-1,最低检出浓度为0.5μmol·L-1,回收率为95.9%～102.0%。日内、日间测定的相对标准偏差均小于3%。加入1-甲基色氨酸(1-methyl tryptophan,1-MT)的上清培养液中L-Trp的浓度高于未加1-MT的上清培养液。结论该方法简便、快速、稳定、可行,不同条件下L-Trp的浓度变化可间接证实人绒毛滋养细胞自身存在其他类似吲哚胺2,3-二氧化酶(indoleamine2,3-dioxygenase,IDO)的方式调节T淋巴细胞免疫功能。
OBJECTIVE To establish a method for the determination of L-tryptophan（L-Trp） by I-IPLC-UV. METHODS The separation was performed on a Eclipse XDB-CI8 column （4.6 mm×150 mm, 5 pro） with 0.05 mmol.L-1 potassium dihydrogan phosphate - methanol （92 ： 8） as the mobile phase. The detection wavelength was 225 um and the flow rate was 1.3 mL,min-1. The samples of villous cytotrophoblast substrate were first precipitated with 5.0% perchloric acid solution, then centrifuged to remove protein residue and finally analyzed by HPLC. RESULTS The retention time of L-Trp was 7.5 rain, the linear range of the method was from 1.0 to 100.0 μmol.L-1, and the detection limit was 0.5 μmol.L-1. The recoveries of L-Trp were from 95.9%-102%, the intraday and interday variations were less than 3%. The concertration of L-Trp in vitro villous cytotrophoblast with 1-MT was more than the samples without 1-MT. CONCLUSION The method is simple, fast, accurate, and suitable for routine analysis. The concentrations of L-Trp at different condition suggest the different mechanism by which trophoblasts inhibit T cells proliferation in vitro.
出处
《中国药学杂志》
CAS
CSCD
北大核心
2009年第24期1919-1921,共3页
Chinese Pharmaceutical Journal
