基于UPLC-Q TOF MS/MS和HPLC的祛瘀散结胶囊的化学成分分析及其多成分含量测定

Analysis of chemical components and determination of multi-component contents in Quyusanjie capsules based on UPLC-Q TOF MS/MS and HPLC

目的:应用液质联用法分析祛瘀散结胶囊的化学成分,构建祛瘀散结胶囊中有效成分的含量测定方法。方法:采用超高效液相色谱-三重四极杆飞行时间质谱联用(UPLC-Q TOF MS/MS)技术,色谱柱为Hypersil Gold C_(18)(100 mm×2.1 mm, 1.9μm),以乙腈(A)-0.1%甲酸水(B)为流动相,梯度洗脱,体积流量0.4 mL·min^(-1),柱温40.0℃,质谱数据采集为负离子模式扫描;通过数据库匹配、元素组成和碎片结构分析,鉴定祛瘀散结胶囊中的主要化学成分。另外,采用HPLC,色谱柱为Ultimate?AQ-C_(18)(250 mm×4.6 mm, 5μm),以乙腈(A)-0.1%磷酸水溶液(B)为流动相,梯度洗脱,体积流量1 mL·min^(-1),柱温25℃,检测波长203 nm;以外标法计算,测定了11个不同批次祛瘀散结胶囊中的柚皮苷、新橙皮苷、三七皂苷R_(1)、人参皂苷Rg_(1)、人参皂苷Rb_(1)的含量,并以人参皂苷Rg_(1)为内参物,建立一测多评法。结果:从祛瘀散结胶囊中鉴定出29个化合物;外标法测得柚皮苷、新橙皮苷、三七皂苷R_(1)、人参皂苷Rg_(1)、人参皂苷Rb_(1)的含量分别为0.484~1.097、0.341~0.618、1.685~2.399、5.748~8.386、3.868~5.898 mg·g^(-1),一测多评法测得的柚皮苷、新橙皮苷、三七皂苷R_(1)、人参皂苷Rb_(1)的含量分别为0.516~1.153、0.372~0.667、1.794~2.580、4.373~6.690mg·g^(-1),一测多评法计算值与外标法实测值之间相对误差≤8.9%。结论:UPLC-Q TOF MS/MS法能快速鉴定祛瘀散结胶囊的化学成分;建立的外标法稳定可靠,可用于祛瘀散结胶囊的质量控制;以人参皂苷Rg_(1)为内参物建立的一测多评法有较好的可行性,适用于祛瘀散结胶囊日常生产的含量测定。

Objective:To analyze the chemical constituents of Quyusanjie capsules by LC/MS, and establish a method for the determination of active ingredients in Quyusanjie capsules. Methods: Using UPLC-Q TOF MS/MS technology, the Hypersil Gold C_(18) column(100 mm×2.1 mm, 1.9 μm) was used, the mobile phase was acetonitrile(A) and 0.1% formic acid in water(B) with gradient elution, at a flow rate of 0.4 mL·min^(-1), the the column temperature was 40.0 ℃, and the mass spectrometry data was collected by negative ions mode scanning. Through database matching, elemental composition and fragment structure analysis, the main chemical substances in Quyusanjie capsules were identified. HPLC was used to qualitatively analyze the chemical components of Quyusanjie capsules. The Ultimate AQ-C_(18 )column(250 mm×4.6 mm, 5 μm) was used, the mobile phase was acetonitrile(A)-0.1% phosphoric acid(B) with gradient elution at the flow rate of 1.0 mL·min^(-1), the column temperature was 25 ℃, and the detection wavelength was 203 nm. The content of naringin, neohesperidin, notoginsenoside R_(1), ginsenoside Rg_(1), and ginsenoside Rb_(1) in 11 different batches of Quyusanjie capsules were determined using external standard method. QAMS method was established using ginsenoside Rg_(1) as the internal reference. Results: Twenty-nine compounds were identified from Quyusanjie capsule. The contents of naringin, neohesperidin, notoginsenoside R_(1), ginsenoside Rg_(1) and ginsenoside Rb_(1) measured by external standard method were 0.484-1.097 mg·g^(-1), 0.341-0.618 mg·g^(-1), 1.685-2.399 mg·g^(-1), 5.748-8.386 mg·g^(-1), 3.868-5.898 mg·g ^(-1), respectively. Measured with the QAMS method, the contents of naringin, neohesperidin, notoginsenoside R_(1) and ginsenoside Rb_(1) were 0.516-1.153 mg·g^(-1), 0.372-0.667 mg·g^(-1), 1.794-2.580 mg·g^(-1), 4.373-6.690 mg·g^(-1), respectively. The relative error between the calculated values of the QAMS method and the measured value of the external standard method was less than 8.9%. Conclusion: UPLC-Q TOF MS/MS method can quickly identify the chemical components of Quyusanjie capsules. The established external standard method is stable and reliable, and can be used for the quality control of Quyusanjie capsules. The method of QAMS has good feasibility and is suitable for the determination of the daily production of Quyusanjie capsules.