柱前衍生高效液相色谱法测定肝纤维化兔给药后尿羟脯氨酸含量  

Determination of Urinary Hydroxyproline in Rabbits of Hepatic Fibrosis after Administration by High Performance Liquid Chromatography with Pre-column Derivatization

作　　者：夏莉[1] 梁逸曾[1] 王平[1] 

机构地区：[1]中南大学化学化工学院中药现代化研究中心,长沙410083

出　　处：《分析化学》2008年第6期811-814,共4页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(No.20235020);科技部国际科技合作项目(No.2007DFA41090)资助

摘　　要：建立了一种快速测定羟脯氨酸的反相高效液相色谱法（RP-HPLC），并测定了血吸虫病肝纤维化兔给药中药复方口服液（HDS）前后尿液中羟脯氨酸含量变化。采用9-芴基甲氧基羰酰氯（FMOC）为衍生试剂，以3，4-脱羟脯氨酸为内标，在Zorbax SB-C18柱上用甲醇和醋酸钠缓冲溶液为流动相进行梯度洗脱，二极管阵列检测器（DAD）262nm处检测。羟脯氨酸在5．0～200μmol／L浓度范围内呈良好线性相关系（r=0．9999），方法检出限为2．0μmol／L（S／N=3），回收率为96．5％～99．0％。本方法具有样品处理步骤简单、准确度高、重现性好、分离时间短等优点，适合大批量样品测定。A method was developed for the determination of hydroxyproline （Hyp） in urine by reversed phase high performance liquid chromatography （RP-HPLC）, and the variance in concentration of urinary hydroxypro-line in rabbit models of schistosomiasis hepatic fibrosis before and after oral administration of the HDS（ a compound recipe of Chinese medicine ） was measured. After derivatization by 9-fluorenylmethyl chloroformate （FMOC）, a baseline separation of hydroxyproline and 3,4-dehydro-L-proline （the internal standard） was achieved on a Zorbax SB-C18 column by gradient elution with methanol and sodium acetate buffer. The detection was carried out with diode array detector（DAD） by absorbance at 262 nm. The relationship between the peak area and the concentration had a good linearity （ correlation coefficient was 0. 9999） in the range of 5.0-200 μmol/L. While the signal-noise ratio was 3, the detection limits for hydroxyproline was 2.0 μmol/L. The recovery of urinary hydroxyproline was 96.5% -99.0%. The method is simple, accurate, rapid and suitable for the analysis of great mass of samples.

关 键 词：羟脯氨酸 9-芴基甲氧基羰酰氯 柱前衍生 高效液相色谱法 肝纤维化 

分 类 号：R285[医药卫生—中药学]