花红胶囊中咖啡酰奎宁酸类成分的识别和含量测定

目的：分析花红胶囊醇提物中的咖啡酰奎宁酸类成分，并建立同时测定花红胶囊中新绿原酸、绿原酸和隐绿原酸含量的高效液相色谱法。方法：超高效液相色谱与串联四极杆飞行时间质谱仪联用技术（UPLC/Q-TOF-MS/MS）：采用Thermo BEH C₁₈（2.1 mm×100 mm，1.7 μm） 色谱柱，以0.1%甲酸乙腈溶液（A）-0.1%甲酸水溶液（B）为流动相，梯度洗脱，流速0.4 μL·min^-1，柱温40 ℃；运用电喷雾离子源（ESI源），负离子模式检测（m/z 100~1500），毛细管电压2.5 kV，锥孔电压40 kV，离子源温度120 ℃，脱溶剂温度450 ℃，碰撞能量15~40 eV，对花红胶囊中的咖啡酰奎宁酸类成分进行快速表征、鉴定。高效液相色谱法：采用Phenomenex C₁₈（250 mm×4.6 mm，4 μm） 色谱柱，以0.1%甲酸乙腈-0.1%甲酸水溶液为流动相，梯度洗脱（0~7 min，5%A→8%A；7~17 min，8%A→15%A；17~45 min，15%A→30%A；45~55 min，30%A→50%A；55~60 min，50%A→100%A），流速1.0 mL·min^-1，检测波长326 nm，柱温30 ℃。结果：采用UPLC/Q-TOF-MS/MS对该制剂的甲醇提取物进行分析，鉴定了4个咖啡酰奎宁酸类化合物，分别为新绿原酸、绿原酸、隐绿原酸和异绿原酸A。采用HPLC法测定新绿原酸、绿原酸和隐绿原酸，它们的浓度分别在3.54~176.80 μg·mL^-1（r=0.9999）、6.98~348.80 μg·mL^-1 （r=0.9999）、4.06~202.80 μg·mL^-1 （r =0.9999）范围内与峰面积呈良好的线性关系；方法的平均加样回收率（n =6）为101.5%~102.2%，RSD为1.9%~2.4%。结论：该方法为花红胶囊化学成分的快速鉴定奠定了基础，为花红胶囊的质量控制提供参考依据。

Objective: To analyze the caffeoylquinic acids and to develop an HPLC method for simultaneous determination of neochlorogenic acid,chlorogenic acid and cryptochlorogenin acid in Huahong capsules. Methods: A UPLC/Q-TOF-MS/MS method was developed to rapidly identify the caffeoylquinic acids in Huahong capsules.And the UPLC separation was performed on a Thermo BEH C₁₈ column (2.1 mm×100 mm,1.7 μm) with 0.1% formic acid acetonitrile-0.1% formic acid as the mobile phase in gradient elution at a flow rate of 0.4 μL·min^-1.The column temperature was set at 40 ℃.The MS spectrometry detection was performed with electrospray ionization in negative ion mode,and using the following operating parameters:scan range,100-1500 m/z;capillary voltage,2.5 kV;skimmer voltage,40 kV;ion source temperature,120 ℃;drying gas temperature,450 ℃ and sample collision energy,15-40 eV.Besides,HPLC was performed on a Phenomenex C₁₈ (250 mm×4.6 mm,4 μm) column with 0.1% formic acid acetonitrile-0.1% formic acid as the mobile phase in gradient elution (0-7 min,5%→8%A;7-17 min,8%→15%A;17-45 min,15%→30%A;45-55 min,30%→50%A;55-60 min,50%→100%A) at a flow rate of 1.0 mL·min^-1.The detect wavelength was set at 326 nm,and the column temperature was set at 30 ℃. Results: Based on the UPLC/Q-TOF-MS/MS analysis,four caffeoylquinic acids,neochlorogenic acid,chlorogenic acid and cryptochlorogenin acid and isochlorogenic acid A,were identified from Huahong capsules.In addition,by using HPLC method,the correlation between the concentration and the chromatographic peak area of neochlorogenic acid,chlorogenic acid and cryptochlorogenin acid was linear in ranges of 3.54-176.80 μg·mL^-1 (r=0.9999),6.98-348.80 μg·mL^-1 (r =0.9999),4.06-202.80 μg·mL^-1 (r=0.9999),respectively.The average recoveries of the three caffeoylquinic acids were in the range of 101.5%-102.2%,and the relative standard deviations (RSD) were in the range of 1.9%-2.4%. Conclusion: The UPLC/Q-TOF-MS/MS and HPLC methods do not only pave a way for further understanding of the chemical constituents in Huahong capsules,but also provide scientific evidences for the quality control of Huahong capsules.