Analysis Of Phenolic Acids In Six Type Teas By UPLC-PDA And UHPLC-Q-TOF-MS

Phenolic acid is a class of aromatic compounds with carboxyl acid and phenolic group.They have high biological activities.At present,the research on phenolic acid is mainly focused on Chinese herbal medicine,fruits,vegetables,grains,etc.The distribution and content of phenolic acids in tea are not systematic studied.In order to quantitatively analyze the phenolic acids in the six type,175 tea samples from six types were collected.The extraction conditions of the phenolic compounds in teas were optimized.Subsequently,the high-performance liquid chromatography(UPLC-PDA)was used to determine gallic acid(GA),chlorogenic acid(CA)and p-coumaric acid(P-Co A),caffeic acid(Caf A),ellagic acid(EA)in teas.Moreover,a liquid chromatography-mass spectrometry(UHPLC-Q-TOF-MS)was used to quantify the main phenolic acids and their derivatives in teas.The main results are the following:(1)Optimization of phenolic acid extraction in teas:formic acid:methanol(5:95,v/v)was the best solvent for tea phenolic acid extraction compared with pure water,methanol.70%acetone,5%formic acid water,5%acetic acid water,acetic acid:methanol(5:95,v/v)and 95%ethanol.Combined with response surface optimization,the optimal extraction was that 0.2g tea powder was extracted by 9mL formic acid:methanol solvent(5:95,v/v)for 65min at 50℃.(2)Establishment of UPLC quantitative method for five phenolic acids in teas:ACQUITY UPLC HSS T3 column(1.8pm 2.1 X 150mm);mobile phase A,water:acetonitrile(95:5,v/v)with 0.1%formic acid;mobile phase B,acetonitrile:water(95:5,v/v)with 0.1%formic acid;flow rate:0.4 mL/min;column temperature:30℃;sample pan temperature:20℃;injection volume:2 μL;ultraviolet dual wavelength detection:280 nm and 326 nm.The stability of the sample was 0.286～2.559%within 24h,the RSD for repeatability was 1.975～2.826%,and the recovery rate was 82.37～108.33%.(3)The contents of GA,CA,Caf A,p-Co A and EA in 175 tea samples of six types quantified by UPLC was 0.16～0.69 mg/g,0.07～0.16 mg/g,0.03～0.05 mg/g,0.09～0.16 mg/g and 0.41-0.79 mg/g,respectively.(4)Establishment of UHPLC-Q-TOF-MS method for tea phenolic acids and their derivatives:ACQUITY UPLC HSS T3 column(1.8μm 2.1×50mm);Mobile phase A:acetonitrile with 0.1%formic acid:water system(5:95,v/v),mobile phase B:water with 0.1%formic acid:acetonitrile system(5:95,v/v);flow rate:0.3mL/min;column temperature:30℃;injection volume:2μL.11 phenolic acids and their derivatives and quinic acid were identified by targeted MS/MS.(5)The contents of GA,CA,p-Co A,Caf A,EA and quinic acid in 30 tea samples quantified with external standards by UHPLC-Q-TOF-MS were 0.099～2.027 mg/g,0.036～0.253 mg/g,0.012～0.558mg/g,0.015～0.090 mg/g,0.141～1.393 mg/g and 0～0.379 mg/g,respectively;The contents of methyl gallate,galloylglucose isomer,digalloylglucose,theogallin,p-coumarylquinic acid and strictinin quantifed as the equivalent of gallic acid were 0～0.07 mg/g,0～0.63 mg/g,0～0.275 mg/g,0～0.054 mg/g,0～0.005 mg/g and 0～0.282 mg/g,respectively.(6)The principal component analysis and Bayes Discrimination by SIMCA-P 13.0 and SPASS 19.0 were applied to the discrimination of six tea types with the indicators of the contents of phenolic acids.The initial discriminant rate was only 62.3%when GA,CA,p-Co A,Caf A and EA as indicators from 175 tea samples.However,the initial discrimination rate was up to 100%when 11 phenolic acids and quinic acid as indicators from 30 tea samples.The results showed that the six type teas,namely black tea,dark tea,oolong tea,white tea,yellow tea and green tea,had different amount of phenolic acids.This difference might be related to tea varieties,origins and processing methods.It indicated that the content of phenolic acids in teas could be used as one of the indicators for the chemical classification of tea.