Li Yao, Yu Hong-Li, Wang Wei, Mao Shan-Hu, Wang Kui-Long, Wu Hao
College of Pharmacy, Nanjing University of Traditional Chinese Medicine, Nanjing 210023, China.
Zhongguo Zhong Yao Za Zhi. 2018 Jun;43(12):2580-2585. doi: 10.19540/j.cnki.cjcmm.20170905.007.
To establish the fingerprints of biles of pig, cattle and sheep, HPLC was used with Acclaim™ RSLC 120 C₁₈ column (3.0 mm×100 mm, 2.2 μm, 120 Å), the column temperature 35 °C, acetonitrile-1% perchloric acid as mobile phase, gradient elution, 0.5 mL·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 (2004A 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.
为建立猪、牛、羊胆汁的指纹图谱，采用高效液相色谱法，使用Acclaim™ RSLC 120 C₁₈色谱柱（3.0 mm×100 mm，2.2 μm，120 Å），柱温35℃，以乙腈 - 1%高氯酸为流动相，梯度洗脱，流速0.5 mL·min⁻¹，检测波长200 nm。采用《中药色谱指纹图谱相似度评价系统》（2004A版）生成指纹图谱。通过对照品对指纹图谱峰进行鉴定，并经蒸发光散射检测器（ELSD）和液相色谱 - 串联质谱（LC - MS/MS）验证。结果表明，猪、牛、羊胆汁分别含有14、9和8个共有指纹峰，相似度均大于0.92。以猪胆汁中的甘氨脱氧胆酸（GHDCA）和牛、羊胆汁中的牛磺胆酸（TCA）为参照峰，对各项技术参数进行分析，精密度、重复性和稳定性均符合指纹图谱建立要求。指纹图谱峰相对保留时间的相对标准偏差（RSD）小于1.5%，相对峰面积的RSD小于5%。猪胆汁中的指纹图谱峰为猪去氧胆酸（THDCA）、鹅去氧胆酸（TCDCA）、甘氨脱氧胆酸（GHDCA）和甘氨胆酸（GCDCA），牛、羊胆汁中的指纹图谱峰为牛磺胆酸（TCA）、鹅去氧胆酸（TCDCA）、甘氨胆酸（GCA）、甘氨胆酸（GCDCA）和甘氨脱氧胆酸（GDCA）。猪、牛、羊胆汁的主要成分均为结合型胆汁酸，但猪胆汁与牛、羊胆汁中的胆汁酸存在显著差异。本文建立的高效液相色谱法简便、快速、重现性好，可用于胆汁的鉴别和质量控制。
