HPLC-DAD双波长法测定护肝片中5个柴胡皂苷的含量

目的：建立测定护肝片中柴胡皂苷a、柴胡皂苷b₁、柴胡皂苷b₂、柴胡皂苷c、柴胡皂苷d的定量分析方法。方法：采用Agilent Eclipse Plus C₁₈（250 mm×4.6 mm，5 μm）色谱柱，流动相为乙腈（A）-水（B），梯度洗脱（0~40 min，30% A→53% A；40~48 min，53% A→98% A），检测波长210 nm（柴胡皂苷a、柴胡皂苷c、柴胡皂苷d）、254 nm（柴胡皂苷b₁、b₂），流速1 mL·min^-1，柱温25℃。结果：5个柴胡皂苷分离度良好；柴胡皂苷a、柴胡皂苷b₁、柴胡皂苷b₂、柴胡皂苷c、柴胡皂苷d进样量分别在0.013 5~0.674、0.015 6~0.781、0.015 2~0.762、0.015 1~0.757和0.013 1~0.653 μg范围内线性关系良好（r≥0.999 5）；稳定性及重复性试验RSD均低于2.0%；平均回收率在99.7%~102.9%，RSD均小于2.0%（n=6）。12个生产厂家50批样品中均未检测到柴胡皂苷a、柴胡皂苷c、柴胡皂苷d，除2个厂家外的其余厂家样品中均检测到柴胡皂苷b₁、柴胡皂苷b₂。结论：所建立的方法简便、快捷、重复性好，可用于护肝片中5个柴胡皂苷的含量测定，为护肝片的全面质量控制提供科学依据。

Objective: To establish a quantitative method for determination of saikosaponin a,saikosaponin b₁,saikosaponin b₂,saikosaponin c and saikosaponin d in Hugan tablets. Methods: HPLC experiment was performed on an Agilent Eclipse Plus C₁₈ column (250 mm×4.6 mm,5.0 μm). The mobile phase consisted of acetonitrile (A) -water (B) in gradient mode (0-40 min,30%A→53%A;40-48 min,53%A→98%A). The detection wavelength was set at 210 nm for saikosaponin a,saikosaponin c,saikosaponin d and 254 nm for saikosaponin b₁,saikosaponin b₂. The flow rate was 1 mL·min^-1. The column temperature was maintained at 25℃. Results: The results showed that the separation of the five saikosaponins was good. Saikosaponin a,saikosaponin b₁,saikosaponin b₂,saikosaponinc and saikosaponin d showed good linear relationship within the range of 0.0135-0.674,0.0156-0.781,0.0152-0.762,0.0151-0.757 and 0.0131-0.653 μg,respectively (r ≥ 0.9995). Good precision,stability and reproducibility was achieved with the RSDs below 2.0%. The average recoveries of saikosaponin a,saikosaponin b₁,saikosaponin b₂,saikosaponinc and saikosaponin d were among 99.7%-102.9% with RSDs below 2.0%. Saikosaponin a,saikosaponin c and saikosaponin d was not detected in fifty batches of samples from twelve companies. Saikosaponin b₁ and saikosaponin b₂ were detected in most samples except samples from two companies. Conclusion: The method is simple,rapid and reproducible. It can be used for the determination of the five saikosaponins in Hugan Tables,providing a scientific basis for the comprehensive quality control of Hugan Tables.

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