反相高效液相色谱法测定鼠胆汁中游离与结合型胆汁酸

Determination of Free and Conjugated Bile Acids in Bile of Rat by High Performance Liquid Chromatography

中文关键词:  高效液相色谱,荧光检测,柱前衍生,胆汁酸

英文关键词:HPLC,fluorescence detection,pre-column derivatization,bile acids

中文摘要:

      建立了一种柱前衍生反相高效液相色谱荧光检测大鼠胆汁中游离与结合型胆汁酸的方法.以4-溴甲基-7-甲氧基香豆素为衍生剂，月桂酸为內标，Waters Nova C18 色谱柱分离， 采用甲醇/乙腈和水梯度洗脱，荧光激发波长（λ_ex）330 nm，发射波长（λ_em）400 nm，內标标准曲线法定量.各胆汁酸在5~2 400 μmol/L范围内线性良好，r为0.998 9~0.999 7.最低检出限为1.2~2.5 μmol/L.精密度日内在1.05%~3.67%之间，日间在3.16%~6.83%之间.平均回收率为87.8%~106.7%.该方法灵敏、重复性好，分析时间短，线性范围宽，适合于科研工作中对鼠胆汁中各胆汁酸进行定量检测.

英文摘要:

      A method for the determination of free and conjugated bile acids in rat bile by RP-HPLC was established. Bile acids were derivatized with 4-bromomethyl-7-methoxycoumarin and eluted using an acetonitrile/ methanol/ water gradient on a Waters Nova C18 column. Concentrations of each derivatized bile acid were determined by RP-HPLC with fluorescence detection. The assay was linear in the range 5~2 400 μmol/L. The linear regression coefficients ranged from 0.9989 to 0.9997. Recovery was 87.8%~106.7%. The detection limit ranged from 1.2 to 2.5 μmol/L. This method, with wide linear range, is sensitive, reproducible, rapid, and suitable for the determination of bile acids in rat bile in scientific research.