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目的:对比不同溶剂提取对黑顺片中6种酯型生物碱含量的影响,建立HPLC同时测定其含量的方法。方法:采用4种不同溶剂提取制备供试品溶液,以6种成分含量归一化法处理后综合评分为评价指标,比较4种提取溶剂对含量的影响。色谱条件:采用Zorbax SB-C18(4.6 mm×250 mm,5μm)色谱柱,以乙腈-40 mmol·L~(-1)醋酸铵为流动相,梯度洗脱,流速1.0 m L·min~(-1),检测波长235 nm,柱温30℃。结果:6种成分含量综合评分显示,氨试液-乙醚法依次优于水、氨试液-异丙醇-乙酸乙酯和酸性甲醇法,确定氨试液-乙醚为最佳提取溶剂。苯甲酰新乌头碱、苯甲酰乌头原碱、苯甲酰次乌头碱、新乌头碱、乌头碱、次乌头碱进样量分别在0.437 5~8.750μg(r=0.999 8),0.101~2.020μg(r=0.999 8),0.062 5~1.250μg(r=0.999 9),0.010 4~0.208μg(r=0.999 8),0.014 2~0.284μg(r=0.999 8),0.052 8~1.056μg(r=0.999 8)范围与色谱峰面积呈良好的线性关系;加样回收率(n=6)均在95.99%~104.6%,RSD均小于3%。结论:氨试液-乙醚为6种生物碱最佳提取溶剂,建立的含量测定方法简单、准确、重复性好,为黑顺片的质量评价奠定了基础。
OBJECTIVE: To compare the effects of different solvent extraction on the content of six kinds of ester alkaloids in Heshun tablet, and to establish a method for the simultaneous determination of its content by HPLC. Methods: Four kinds of different solvents were used to prepare the test solution. After the normalized method of six components was used to evaluate the comprehensive evaluation index, the effects of four kinds of extraction solvents on the content were compared. Chromatographic conditions: A mobile phase of acetonitrile-40 mmol·L -1 ammonium acetate was used as mobile phase on a Zorbax SB-C18 (4.6 mm × 250 mm, 5 μm) column with a gradient elution at a flow rate of 1.0 mL · min -1 -1), detection wavelength 235 nm, column temperature 30 ℃. Results: The comprehensive scores of the six components showed that the ammonia test solution - ether method was superior to water, ammonia test solution - isopropanol - ethyl acetate and acid methanol method in turn to determine the ammonia test solution - ether as the best extraction solvent. Benzoyl mesaconitine, benzoyl aconitine base, benzoyl hypaconitine, mesaconitine, aconitine, hypaconitine injection volume were 0.437 5 ~ 8.750μg (r = 0.999 to 2.08 μg (r = 0.999 8), 0.062 2 to 0.284 μg (r = 0.999 8), 0.062 to 1.250 μg (r = 0.999 9), 0.010 4 to 0.208 μg , 0.052 8 ~ 1.056μg (r = 0.999 8) and the chromatographic peak area showed a good linear relationship. The recoveries (n = 6) ranged from 95.99% to 104.6% with RSD less than 3%. CONCLUSION: Ammonia test solution - ether is the best extraction solvent for six kinds of alkaloids. The determination method of the established alkaloids is simple, accurate and reproducible, which lays the foundation for the quality evaluation of heishun tablets.