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目的:建立夏天无提取物的超高效液相色谱(UPLC)指纹图谱,并采用电喷雾(ESI)-飞行时间质谱(TOF-MSE)对夏天无提取物指纹图谱中特征化学成分进行鉴定。方法:采用ACQUITY UPLCBEH-C18色谱柱(2.1 mm×100 mm,1.7μm),以乙腈(A)-乙酸铵甲酸水溶液(每1000 mL水中含1 mL甲酸和10 mmol乙酸铵)(B)为流动相,梯度洗脱(0~2 min,5%A;2~30 min,5%A→23%A;30~37 min,23%A→60%A),流速0.3 mL·min-1,检测波长280 nm,柱温35℃,进样量1μL;质谱采用电喷雾正离子模式采集数据,毛细管电压为3.5 kV,锥孔电压为35 kV,离子源温度为150℃,扫描范围m/z 100~800,碰撞低能量为6 eV,碰撞高能量15~45 eV。结果:37 min内完成夏天无提取物的UPLC指纹图谱分析,各主要成分色谱峰分离良好,方法精密度、重复性、稳定性的RSD值均小于1.0%。高分辨质谱数据结合对照品信息及相关文献,鉴定了夏天无提取物UPLC指纹图谱中12个主要色谱峰,分别认定为hydrohydrastinine、延胡索单酚碱、球紫堇碱、比枯枯灵、四氢药根碱、原阿片碱、α-别隐品碱、延胡索乙素、药根碱、非洲防己碱、巴马汀和去氢紫堇碱。结论:本方法为夏天无提取物的质量控制及成分的全面阐述奠定了基础。
OBJECTIVE: To establish an HPLC-UPLC fingerprinting of summer without extract and identify the chemical constituents of the fingerprints of summer without extract using electrospray ionization-time of flight mass spectrometry (TOF-MSE). Methods: Aqueous acetonitrile (A) - ammonium acetate solution (1 mL formic acid and 10 mmol ammonium acetate per 1000 mL water) (B) was eluted with ACQUITY UPLC BEH-C18 column (2.1 mm × 100 mm, (0-2 min, 5% A; 2-30 min, 5% A → 23% A; 30-37 min, 23% A → 60% A) at a flow rate of 0.3 mL · min- 1, the detection wavelength was 280 nm, the column temperature was 35 ℃, and the injection volume was 1μL. The data were acquired by electrospray positive ion mode with a capillary voltage of 3.5 kV, a cone voltage of 35 kV and an ion source temperature of 150 ℃. / z 100 ~ 800, the collision energy is 6 eV, and the collision energy is 15 ~ 45 eV. Results: UPLC fingerprinting of summer without extract was completed within 37 min. The chromatographic peaks of all the major components were well separated. The RSD values of precision, repeatability and stability were less than 1.0%. High-resolution mass spectrometry data combined with the reference material and related literature identified 12 major chromatographic peaks in the UPLC fingerprinting of the summer extract, identified as hydrohydrastinine, corydaline, corydaline, Jatrorrhizine, protoporphyrin, alpha-allosteric base, tetrahydropalmatine, jatrorrhizine, tetrandrine, palmatine and dehydro-corydaline. Conclusion: This method lays the foundation for the quality control and composition of summer extract-free.