RRLC-MS/MS法同时测定骨刺片中14个成分的含量

目的：建立RRLC-MS/MS法同时测定10个厂家生产的骨刺片中14个成分的含量。方法：采用Agilent ZORBAX SB-C₁₈色谱柱（2.1 mm×50 mm，1.8 μm），以0.1%甲酸水溶液（A）-乙腈（B）为流动相，梯度洗脱，流速为0.3 mL·min^-1，柱温30℃，进样量2 μL；质谱采用负离子扫描，多反应监测（MRM）模式，脱溶剂温度350℃，脱溶剂气流（N₂）600 L·h^-1，雾化器压力0.2 MPa，毛细管电压4.0 kV，皂苷类成分采用积雪草苷为内标，其他类成分采用水杨酸为内标。结果：新绿原酸、马钱苷酸、绿原酸、隐绿原酸、芍药内酯苷、芍药苷、新北美圣草苷、柚皮苷、党参炔苷、三七皂苷R₁、人参皂苷Rg₁、人参皂苷Re、人参皂苷Rb₁和人参皂苷Rd分别在0.008~8.208、0.004~4.084、0.009~9.312、0.008~8.368、0.005~5.416、0.004~4.344、0.001~3.928、0.002~8.512、0.004~1.864、0.008~4.104、0.009~4.560、0.008~4.120、0.010~4.932、0.008~2.188 μg·mL^-1范围内线性关系良好，R² ≥ 0.999 3，定量下限为0.8~9.9 ng·mL^-1，加样回收率为95.7%~106.8%。采用建立的方法对骨刺片样品进行含量测定，结果显示，新绿原酸、马钱苷酸、绿原酸、隐绿原酸、芍药内酯苷、芍药苷、新北美圣草苷、柚皮苷、党参炔苷、三七皂苷R₁、人参皂苷Rg₁、人参皂苷Re、人参皂苷Rb₁和人参皂苷Rd在骨刺片样品中的含量分别为10.73~663.41、235.40~995.78、62.16~1315.00、9.74~873.45、43.15~576.11、14.69~1337.46、0.52~194.73、0~428.61、0~9.969、0~240.51、0~1045.38、0~109.53、0~866.82、0~197.84 μg·片^-1，可以看出，除马钱苷酸外，其他成分在不同厂家生产的骨刺片中含量差异非常大，提示部分厂家在白芍、杜仲叶、骨碎补、三七及党参等药材的投料上可能存在问题。结论：本实验建立的多指标含量测定方法灵敏、准确，可用于全面评价骨刺片的质量优劣，对于提高骨刺片的质量标准、规范厂家的生产行为具有重要意义。

Objective:To establish an RRLC-MS/MS method for simultaneous determination of 14 compounds in Guci tablets from 10 manufacturers.Methods:Chromatographic separation was performed on an Agilent ZORBAX SB-C₁₈ column (2.1 mm×50 mm, 1.8 μm) at a column temperature of 30.0.1% aqueous formic acid (A) -acetonitrile (B) were adopted as the mobile phase with a gradient elution.The flow rate was set at 0.3 mL·min^-1, and the injection volume was 2 μL.Detection was carried out on a triple quadrupole mass spectrometer in negative ion mode using an electrospray source.Multiple reaction monitoring (MRM) mode was employed. The operating conditions were as follows:desolvation temperature, 350℃;desolvation gas flow, 600 L·h^-1;nebulizer pressure, 0.2 MPa;capillary voltage, 4.0 kV.Asiaticoside was used as the internal standard (IS) for analysis of saponins, and salicylic acid was used as internal standard for analysis of other compounds.Results:The developed method showed good linearity in the range of 0.008-8.208 μg·mL^-1 for neochlorogenic acid, 0.004-4.084 μg·mL^-1 for loganic acid, 0.009-9.312 μg·mL^-1 for chlorogenic acid, 0.008-8.368 μg·mL^-1 for cryptochlorogenic acid, 0.005-5.416 μg·mL^-1 for albiflorin, 0.004-4.344 μg·mL^-1 for paeoniflorin, 0.001-3.928 μg·mL^-1 for neoeriocitrin, 0.002-8.512 μg·mL^-1 for naringin, 0.004-1.864 μg·mL^-1 for lobetyolin, 0.008-4.104 μg·mL^-1 for notoginsenoside R₁, 0.009-4.560 μg·mL^-1 for ginsenoside Rg₁, 0.008-4.120 μg·mL^-1 for ginsenoside Re, 0.010-4.932 μg·mL^-1 for ginsenoside Rb₁, 0.008-2.188 μg·mL^-1 for ginsenoside Rd with R² ≥ 0.999 3.The limits of quantification (LOQs) ranged from 0.8 to 9.9 ng·mL^-1.The recoveries varied between 95.7% and 106.8%.Quantitative data showed that the contents of neochlorogenic acid, loganic acid, chlorogenic acid, cryptochlorogenic acid, albiflorin, paeoniflorin, neoeriocitrin, naringin, lobetyolin, notoginsenoside R₁, ginsenoside Rg₁, ginsenoside Re, ginsenoside Rb₁ and ginsenoside Rd in samples were 10.73-663.41, 235.40-995.78, 62.16-1315.00, 9.74-873.45, 43.15-576.11, 14.69-1337.46, 0.52-194.73, 0-428.61, 0-9.969, 0-240.51, 0-1045.38, 0-109.53, 0-866.82, and 0-197.84 μg per tablet, respectively.Except for loganic acid, these tested compounds varied significantly in samples produced by different manufacturers, suggesting that there might be some feeding problems about Paeoniae Radix Alba, Eucommiae Folium, Drynariae Rhizoma, Notoginseng Radix et Rhizoma, and Codonopsis Radix in some manufacturers.Conclusion:The established multi-component analysis method is proved to be accurate and sensitive enough.It can be used for overall quality evaluation of Guci tablets, and is valuable for improvement of the quality standard of this preparation, as well as regulation of the production behaviors of the manufacturers.

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