Search

CN-122017093-A - Method for rapidly determining various phenolic acid substances in fermentation product

CN122017093ACN 122017093 ACN122017093 ACN 122017093ACN-122017093-A

Abstract

The invention discloses a method for rapidly determining various phenolic acid substances in a fermentation product, and belongs to the technical field of detection. The method comprises the steps of taking a substance to be measured, extracting phenolic acid substances in the substance to be measured through an alcohol precipitation-SAX solid phase extraction combined technology to obtain a sample to be measured, analyzing the sample to be measured by adopting an ultra-high performance liquid phase tandem triple quadrupole mass spectrum to obtain the content of a target substance in the substance to be measured, wherein the target substance is a phenolic acid substance difficult to volatilize. The accurate quantification of micro and trace caffeic acid, dihydrocaffeic acid, ferulic acid, chlorogenic acid, gallic acid, vanillic acid, syringic acid, p-coumaric acid, salicylic acid and (+/-) -catechin in the fermentation product is realized within 8-15 min by combining impurity removal and concentration pretreatment with ultra-high performance liquid phase tandem triple quadrupole mass spectrometry. The pretreatment mode of the method is convenient to operate, the detection process is quick, and the detection result is accurate.

Inventors

  • YANG ZHEN
  • LU WEI
  • TANG YOUHONG
  • CHEN SHAN
  • CHEN KAI
  • LIANG YING
  • LIU QINGYANG

Assignees

  • 安徽古井贡酒股份有限公司

Dates

Publication Date
20260512
Application Date
20260228

Claims (8)

  1. 1. A method for rapidly determining a plurality of phenolic acid substances in a fermentation product, which is characterized by comprising the following steps: step 1 pretreatment Adding a substance to be detected into ethanol, refrigerating and standing for 8-12 hours at 0-6 ℃, extracting the difficult volatile phenolic acid substances, enriching the phenolic acid substances in a solvent phase, centrifuging the extracting solution, taking supernatant, adsorbing the phenolic acid substances through an SAX strong anion exchange solid phase extraction column, discarding effluent liquid, eluting with ultrapure water to remove impurities, eluting the target phenolic acid substances with an acidic solution, collecting eluent, and filtering the eluent with a 0.22 mu m organic filter membrane after the volume is fixed to obtain a sample to be detected; Step 2, drawing a standard curve Preparing standard mixed mother liquor of each target substance with the concentration of 10mg/L, accurately sucking a certain amount of standard mixed mother liquor, respectively diluting different multiples with ultrapure water, preparing standard gradient working solutions with different concentrations, detecting the obtained standard gradient working solution by adopting an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer, analyzing by means of quantitative software carried by the instrument, and preparing a standard working curve by taking the quantitative ion peak area of the target substance as an ordinate and the content of the target substance as an abscissa; step 3, sample measurement Analyzing the sample to be detected obtained in the step (1) by adopting an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrum, and calculating to obtain the content of the target substance in the liquid to be detected based on the standard working curve drawn in the step (2) and the quantitative ion peak area of the target substance to be detected; the substance to be detected comprises a fermentation product, wherein the fermentation product comprises one or more of fermentation liquor, esterification liquor and fermentation starter; the target substance is a difficult-to-volatilize phenolic acid substance.
  2. 2. The method according to claim 1, characterized in that: the target substance comprises one or more of caffeic acid, dihydrocaffeic acid, ferulic acid, chlorogenic acid, gallic acid, vanillic acid, syringic acid, p-coumaric acid, salicylic acid and (+/-) -catechin.
  3. 3. The method according to claim 1, characterized in that: In step 1, the acidic solution used for elution was a 5% formic acid aqueous solution.
  4. 4. The method according to claim 1, characterized in that: In the step 2, the concentration range of the target substances in the standard gradient working solution with different concentrations is 0-500 mug/L.
  5. 5. The method according to claim 1, characterized in that: In the step 2, the ultra-high performance liquid chromatography Column is CORTECS PREMIER RP18 Column,1.6 μm, 2.1X100 mm.
  6. 6. The method according to claim 5, wherein: The detection conditions of the ultra-high performance liquid chromatography are set to be that the column temperature is 30-40 ℃, the sample injection amount is 1-10 mu L, the flow rate is 0.2-0.5 mL/min, and the analysis time is 8-15 min; The mobile phase of the ultra-high performance liquid chromatography is mobile phase A of 0.1-1 g/L formic acid aqueous solution, mobile phase B of acetonitrile and gradient elution.
  7. 7. The method according to claim 6, wherein: The gradient elution procedure was set as follows: 0-1 min,5% of mobile phase B, 1-8.5 min,5% -40% of mobile phase B, 8.5-8.6 min,40% -5% of mobile phase B, 8.6-10 min,5% of mobile phase B.
  8. 8. The method according to claim 7, wherein: in the step 2, the detection condition of the ultra-high performance liquid tandem triple quadrupole mass spectrum is set to be that an anion electrospray mode is adopted, a multi-reaction monitoring mode is adopted, the capillary voltage is 0.5-3.0 kV, the taper hole voltage is 20-135V, the desolvation gas temperature is 400-500 ℃, and the carrier gas flow rate is 600-1000L/Hr.

Description

Method for rapidly determining various phenolic acid substances in fermentation product Technical Field The invention relates to a method for rapidly determining various phenolic acid substances in a fermentation product, and belongs to the technical field of fermentation product detection. Background Phenolic acid substances are organic acids containing phenolic hydroxyl groups, are widely existing in plants, have various positive effects on human health, such as ferulic acid and caffeic acid can reduce the occurrence risk of diseases related to oxidative stress, salicylic acid and derivatives thereof have good anti-inflammatory effects, gallic acid has certain inhibition effects on common pathogenic bacteria such as escherichia coli, staphylococcus aureus and the like, and various phenolic acid substances can have protective effects on cardiovascular systems through various ways. High Performance Liquid Chromatography (HPLC) is the most commonly used phenolic acid substance detection method at present, and has the characteristics of high resolution, high sensitivity and high accuracy. By optimizing the chromatographic column, mobile phase and detection wavelength, HPLC can simultaneously separate and quantify various phenolic acids. Although HPLC has significant advantages in phenolic acid detection, its disadvantages are not negligible, including complex sample pretreatment, high instrument cost, long analysis time, limited sensitivity, complex method development, environmental problems, and high demands on operators. Disclosure of Invention In order to solve the problems, the invention provides a method for rapidly determining various phenolic acid substances in a fermentation product. The method adopts an alcohol precipitation-SAX solid phase extraction combined technology to filter impurities in a sample to be detected, reduces interference of other substances on phenolic acid detection, and can accurately realize quantitative detection on trace and trace caffeic acid, dihydrocaffeic acid, ferulic acid, chlorogenic acid, gallic acid, vanillic acid, syringic acid, p-coumaric acid, salicylic acid and (+/-) -catechin in the sample. The method does not need long-time pretreatment, can greatly improve the detection efficiency, has the advantages of accuracy, rapidness, high sensitivity and strong stability, and provides a reliable basis for accurately detecting phenolic acid substances in fermentation products. The invention relates to a method for rapidly determining various phenolic acid substances in a fermentation product, which comprises the following steps: step 1 pretreatment Adding a substance to be detected into a proper amount of ethanol, refrigerating and standing for 8-12 hours at 0-6 ℃, extracting the difficult volatile phenolic acid substances, enriching the phenolic acid substances in a solvent phase, centrifuging the extracting solution, taking supernatant, adsorbing the phenolic acid substances by a SAX strong anion exchange solid phase extraction column, discarding effluent liquid, eluting by using ultrapure water to remove impurities, eluting the target phenolic acid substances by using an acidic solution, collecting eluent, and filtering the eluent with an organic filter membrane of 0.22 mu m after the volume is fixed to obtain the sample to be detected. Step 2, drawing a standard curve Preparing standard mixed mother liquor of each target substance with the concentration of 10mg/L, accurately sucking a certain amount of standard mixed mother liquor, respectively diluting with ultrapure water to different multiples, preparing standard gradient working solutions with different concentrations, detecting the obtained standard gradient working solutions by adopting an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer, analyzing by means of quantitative software carried by the instrument, and preparing a standard working curve by taking the quantitative ion peak area of the target substance as an ordinate and the content of the target substance as an abscissa. Step 3, sample measurement And (3) analyzing the sample to be detected obtained in the step (1) by adopting an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrum, and calculating the content of the target substance in the liquid to be detected based on the standard working curve drawn in the step (2) and the quantitative ion peak area of the target substance to be detected. The substance to be detected comprises a fermentation product, wherein the fermentation product comprises fermentation liquor, esterified liquor, fermented yeast and the like. The target substance is a difficult-to-volatilize phenolic acid substance. Further, the target substance comprises one or more of caffeic acid, dihydrocaffeic acid, ferulic acid, chlorogenic acid, gallic acid, vanillic acid, syringic acid, p-coumaric acid, salicylic acid and (+/-) -catechin. In step 1, the acidic solution u