基于DPPH-HPLC-Q-TOF-MS/MS技术的安化黑茶发酵前后抗氧化活性物质基础研究

Antioxidant components in Anhua dark tea before and after fermentation:A basic research based on DPPH-HPLC-Q-TOF-MS/MS

目的:建立基于高效液相色谱串联四级杆飞行时间质谱技术(HPLC-Q-TOF-MS/MS)的安化黑茶抗氧化活性成分快速筛选和鉴别的方法,并对安化黑茶生茶及熟茶的抗氧化活性成分分别进行检测和分析。方法:采用Inertsil ODS-2C18色谱柱(250mm×4. 6mm,5μm)反相HPLC梯度洗脱法分离各主要成分,在负离子模式下,利用四级杆-飞行时间串联质谱对各主要活性成分进行检测;进而在安化黑茶生茶及熟茶乙酸乙酯及正丁醇部位分别加入适量1,1-二苯基-2-苦基苯肼(DPPH)作为实验组,避光室温反应30min后经液质联用分析,并与不加入DPPH的空白组物质进行比较,快速发现其活性成分;并采用对照品、文献比对、质谱裂解规律分析鉴定生茶、熟茶的抗氧化活性成分。结果:依据质谱裂解规律及文献检索,从安化黑茶生茶乙酸乙酯部位中共鉴定出32个抗氧化活性成分,从生茶正丁醇部位中共鉴定出28个抗氧化活性成分;从熟茶乙酸乙酯部位中共鉴定出31个抗氧化活性成分,从熟茶正丁醇部位中共鉴定出26个抗氧化活性成分。结论:利用DPPHHPLC-Q-TOF-MS/MS技术快速筛选和鉴别安化黑茶中抗氧化活性成分的有效方法,为其质量控制及物质基础研究提供了技术支持。

Objective:To establish the method for rapid screening and identification of antioxidant components in Anhua dark tea based on high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry(HPLC-Q-TOF-MS/MS),and to investigate the antioxidant components in raw and fermented Anhua dark tea.Methods:Reversed-phase HPLC was performed on an Inertsil ODS-2 C18 column(250 mm×4.6 mm,5μm),and gradient elution was performed to separate the main components.Q-TOF-MS/MS was used to detect major active components in the negative ion mode.1,1-Diphenyl-2-picrylhydrazyl-hydrate(DPPH)was added to the ethyl acetate extract and the n-butanol extract of raw and fermented Anhua dark tea,which was established as experimental group,and then liquid chromatography-mass spectrometry was performed after 30 minutes of reaction in a dark place at room temperature.The substances without the addition of DPPH were established as blank group,and the experimental group was compared with the blank group to rapidly identify active components.Reference substance,literature review,and fragmentation pattern in mass spectrometry were used to analyze the antioxidant components of raw and fermented Anhua dark tea.Results:Based on the fragmentation pattern in mass spectrometry and literature search,32 antioxidant components were identified in the ethyl acetate extract of raw Anhua dark tea,and 28 antioxidant components were identified in the nbutanol extract of raw Anhua dark tea;31 antioxidant components were identified in the ethyl acetate extract of fermented Anhua dark tea,and 26 antioxidant components were identified in the n-butanol extract of fermented Anhua dark tea.Conclusion:The DPPH-HPLC-Q-TOF-MS/MS method is an effective method for rapid screening and identification of antioxidant components in Anhua dark tea,which provides technical support for quality control and basic research.