Determination of 13 kinds of free and bound phenolic acids in fruits by high-performance liquid chromatography-mass spectrometry
OBJECTIVE To establish a high-performance liquid chromatography-mass spectrometry(HPLC-MS/MS)method for detecting 13 kind of free and bound phenolic acids(chlorogenic acid,protocatechuic acid,ferulic acid,p-coumaric acid,gallic acid,gentisic acid,vanillic acid,caffeic acid,syringic acid,sinapic acid,rosmarinic acid,salicylic acid,p-hydroxybenzoic acid)in fruits,and optimize the pre-treatment conditions to meet the detection requirements for phenolic acid content in various types of fruits.METHODS Free phenolic acids in fruits were extracted using methanol through ultrasonic extraction.Conjugated phenolic acids in the centrifuged residue were released by alkaline hydrolysis and extracted with ethyl acetate.The two extracts were combined,concentrated,and analyzed using HPLC-MS/MS.Separation was achieved using an Agilent ZORBAX SB-C18 chromatography column(3.0 mm×100 mm,3.5 μm),and detection was performed in multiple reaction monitoring(MRM)mode.RESULTS All 13 standard phenolic acids achieved complete separation within 10 minutes,with linear correlation coefficients greater than 0.998 and detection limits ranging from 0.172 to 3.471 ng/mL.After optimization of the pre-treatment method,the recovery rates of the method for four types of fruits-apples,strawberries,oranges,and peaches-ranged from 80.0％to 119.4％,and the precision were lower than 7.00％(n=6).The result of testing on four categories of twelve types of fruits demonstrated significant variations in the content of phenolic acids among different fruits,and within the same category,the composition of phenolic acids did not exhibit consistency.CONCLUSION The HPLC-MS/MS method exhibits high sensitivity,precision,and accuracy.It is suitable for the detection of both free and bound phenolic acids in various types of fruits.
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