Simultaneous Determination Of 8 Phenolic Acids In Plant Root Exudates By SPE-HPLC

Phenolic acids are secondary metabolites that are commonly found in higher plant tissues in nature and closely related to the growth and germination of plants.They are mainly formed by the synthesis pathway of shikimic acid or malonic acid in plants.With continuous research in recent decades,the understanding of phenolic acids is gradually deepened.Phenolic acids have an effect of"low promotion,high-suppression"on plants.They not only affect the cell membrane system and photosynthesis of plants,but also change the composition of microorganisms and the activity of enzymes in soil,etc.Studies have also shown that phenolic acids have certain health effect on the human body,such as anti-oxidation,anti-cardiovascular disease,anti-bacteria,anti-viruse,anti-inflammatory,anti-tumor and so on.Determining of simple and efficient pretreatment of phenolic compounds in root exudates and optimizing the separation and analysis conditions of high performance liquid chromatography are two important tasks of establishing SPE-HPLC to analyse phenolic acids.In this paper,a solid phase extraction device was successfully prepared and the experimental conditions of SPE and HPLC were optimized,respectively.This fast,accurate and effective SPE-HPLC analytical method could analyze not only eight standards of phenolic acids qualitatively and quantitatively,but also actual samples and the analysis results could meet the laboratory requirements.Hopely,this SPE-HPLC could provide assistance or guidance for the research of other phenolic acids in plant root exudates.1.The extraction method and its conditions were determined.A solid phase extraction device was designed and the optimum conditions of the extraction process were determined.By investigating the total extraction amount and their respective extraction efficiency of eight phenolic acids in root exudates and the recovery efficiency of benzoic acid,the extraction method,solvent and time were ultrasonic extraction,50%ethanol-water and 30min,respectively.From several commonly used reverse phase extractants,such as C18,PEP-2,silica gel,DPS-401 and DEAE,etc,the high-extraction-efficiency solid phase materials and their ratios were screened out.Many influence factors,such as the type,composition,volume and proportion of the strippant and eluent and extraction times in SPE were compared and optimized by orthogonal test.By examining the recovery efficiency of gallic acid,chlorogenic acid,p-hydroxybenzoic acid and phloridzin,the optimum type of solid phase extraction sorbent was identified as PEP-2:DEAE=4:1(V/V)and the main factors affecting the extraction effect were the ratio and volume of the strippant.The operation steps of solid phase extraction procedure were as follows.Sample extraction was performed 3 times.Then the extraction column was eluted by 15 m L 10.0%methanol-water(V/V)and 15 mL of 2.0%acetic acid-methanol(V/V),successively.2.The HPLC conditions were determined and optimized from the standpoint of choosing the UV detection wavelength,mobile phase and optimizing the gradient elution procedure.The UV maximum absorption wavelengths of the eight phenolic acids were quite different and 270 nm-290 nm in the second maximum absorption range where every phenolic acid had obvious absorption was determined experimentally as the detection wavelength,the total wavelength of the whole experiment was 280 nm,while the wavelength of catechin was 270nm and that of p-hydroxybenzoic acid was 290 nm.Four different gradient elution procedures were verified and one of them was determined as follows:A-acetonitrile,B-acetic acid-water(pH=2.8);0-16 min,5%-22%A;16-18 min,22%-10%A;18-23 min,10%-29%A;23-30 min,29%-40%A;30-42 min,40%-10%A;42-45 min,10%-5%A.3.The repeatability,linear equations and detection limits of this established sample preparation and analysis methods were verified.The linear correlation coefficient(R~2)of eight phenolic acids all reached 0.99 or more in the concentration range of 0.25?g/m L-50.0?g/m L.The relative standard deviation of their peak area was in 1.63%-3.34%when the repeated injection times was 6.The detection limit of phenolic acids was in 0.012?g/m L-0.032?g/mL(S/N=3)and the limit of quantification was in 0.048?g/mL-0.106?g/mL(S/N=10).The spike recovery ratio was between 83.0%and 91.2%(N=6)with a relative standard deviation of 4.81%.Eight phenolic acids could be detected in root exudates of cucumber seedlings.