高效液相色谱法同时测定猪胆粉中4种结合型胆酸含量及其特征图谱研究

Simultaneous determination of 4 conjugated cholic acids and their specific chromatograms by HPLC

目的建立HPLC法同时测定猪胆粉中4种结合型胆酸含量的方法并研究其特征图谱。方法采用Thermo Ultimate 3000高效液相色谱仪,Ultimate 3000可变波长检测器,Acclaim 120 C18色谱柱(4.6mm×250 mm,5μm);流速1.0 mL·min^-1;检测波长200 nm;柱温25℃;进样量10μL。流动相为乙腈-0.03 mol·L^-1磷酸二氢钠(磷酸调pH=4.4),梯度洗脱。结果牛磺猪去氧胆酸、甘氨猪去氧胆酸、牛磺鹅去氧胆酸和甘氨鹅去氧胆酸钠盐在0.01~0.5 mg·mL^-1内与峰面积线性关系良好,方法回收率在95%~105%。使用该方法分析12批样品,确定了7个共有峰,从而建立了猪胆粉中结合型胆酸的HPLC特征图谱。结论该方法简便合理,可作为猪胆粉的质量评价的依据之一。

Objective To establish an HPLC method to simultaneously determine 4 conjugated cholic acids in pulvis fellis suis and their specific chromatograms. Methods Thermo Ultimate 3000 HPLC, Ultimate 3000 variable wavelength detector, and Acclaim 120 C18 column (4.6 mm×250 mm, 5 μm) were used. Gradient elution was performed with the mobile phase of acetonitrile-0.03 mol·L^-1 sodium dihydrogen phosphate (phosphoric acid adjust pH = 4.4), detection wavelength of 200 nm, flow rate of 1.0 mL·min^-1 , column temperature at 25℃, and injection volume of 10 μL. Results Taurohyodeoxycholic acid, glycohyodeoxycholic acid, taurochenodeoxycholic acid and glycyrrhizin deoxycholic acid sodium salt had a good linearity at 0.01 - 0.5 mg·mL^-1 . The average recoveries were at 95%- 105%. Seven common peaks were identified and the HPLC specific chromatograms of conjugated cholic acid in pulvis fellis suis were established by analyzing the 12 batches of samples. Conclusion The method is simple and reasonable, which can be used in the quality evaluation of pulvis fellis suis.