猪牛羊胆汁HPLC指纹图谱研究

HPLC fingerprints of pig,cattle and sheep biles

为有效控制猪、牛和羊胆汁质量,采用高效液相色谱法（HPLC）,以AcclaimTMRSLC 120 C18色谱柱（3.0 mm×100mm,2.2μm,120）;在柱温35℃下,以乙腈-1%高氯酸为流动相体系,0.5 m L·min-1流速梯度洗脱,200 nm波长检测,以中药色谱指纹图谱相似度评价软件（2004A版）生成指纹图谱。以对照品归属各共有指纹峰,并采用蒸发光散射检测器（ELSD）和液相色谱-质谱联用（LC-MS/MS）验证。结果显示,猪、牛、羊胆汁色谱图分别有14,9,8个共有指纹峰,各相似度均大于0.92。以甘氨猪脱氧胆酸（GHDCA）、牛磺胆酸（TCA）分别作为猪胆汁和牛、羊胆汁HPLC指纹图谱的参照物,方法学考察结果符合指纹图谱建立要求,各共有指纹峰相对保留时间RSD小于1.5%,相对峰面积RSD均小于5.0%。归属猪胆汁共有指纹峰有牛磺猪脱氧胆酸（THDCA）、牛磺鹅脱氧胆酸（TCDCA）、甘氨猪脱氧胆酸（GHDCA）和甘氨鹅脱氧胆酸（GCDCA）,牛、羊胆汁共有指纹峰有牛磺胆酸（TCA）、牛磺鹅脱氧胆酸（TCDCA）、甘氨胆酸（GCA）、甘氨鹅脱氧胆酸（GCDCA）和甘氨脱氧胆酸（GDCA）。研究结果表明,猪牛羊胆汁主要成分为结合型胆汁酸,猪胆汁与牛羊胆汁中的胆汁酸类成分存在显著性差异;三者的HPLC指纹图谱,特征性强、重复性好,可用于鉴别猪牛羊胆汁,控制胆汁质量。以快速液相色谱柱建立指纹图谱,可在HPLC系统分析,简便快速,具有可操作性。

To establish the fingerprints of biles of pig,cattle and sheep,HPLC was used with AcclaimTMRSLC 120 C18 column（ 3. 0 mm × 100 mm,2. 2 μm,120 ）,the column temperature 35 ℃,acetonitrile-1% perchloric acid as mobile phase,gradient elution,0. 5 m L·min-1 flow rate,and detection wavelength at 200 nm. The fingerprint was generated by using Similarity Evaluation Software of Chromatographic Fingerprint of Chinese Medicine（ 2004 A Edition）. The fingerprint peaks were identified by reference substances and verified by ELSD and LC-MS/MS. Then,the biles of pig,cattle and sheep were detected to contain 14,9 and 8 common fingerprint peaks respectively,and the similarity was greater than 0. 92. To analyze each technical parameter,GHDCA in pig bile and TCA in cattle and sheep bile were selected as reference peak. The precision,repeatability and stability all meet the requirements of fingerprint establishment. The RSD of the relative retention time of the fingerprint peaks was less than 1. 5%,and the RSD of the relative peak area was less than 5%. The fingerprint peaks in pig bile were THDCA,TCDCA,GHDCA and GCDCA,and TCA,TCDCA,GCA,GCDCA and GDCA in cattle and sheep bile. The main components of pig,cattle and sheep bile were conjugated bile acids,but there were significant differences in bile acids between pig bile and cattle,sheep biles. The HPLC method established in this paper is simple,rapid and reproducible,and could be applied to the identification and quality control of biles.