Method for rapidly determining various phenolic acid substances in fermentation product

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 determination method comprises the following steps: 1. Taking a to-be-detected substance, and extracting phenolic acid substances in the to-be-detected substance through an alcohol precipitation-SAX solid-phase extraction combined technology to obtain a to-be-detected sample; analyzing the to-be-detected sample by adopting ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry to obtain the content of the target substance in the to-be-detected substance; the target substance is a non-volatile phenolic acid substance. Accurate quantification of 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 fermentation products within 8-15 min is achieved by combining impurity removal and concentration pretreatment with ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry. According to the method, the pretreatment mode is convenient to operate, the detection process is fast, and the detection result is accurate.

Description

Technical Field

[0001] This invention relates to a method for rapidly determining various phenolic acids in fermentation products, belonging to the field of fermentation product detection technology. Background Technology

[0002] Phenolic acids are a class of organic acids containing phenolic hydroxyl groups. They are widely found in plants and have many positive effects on human health. For example, ferulic acid and caffeic acid can reduce the risk of oxidative stress-related diseases. Salicylic acid and its derivatives have good anti-inflammatory effects. Gallic acid has a certain inhibitory effect on common pathogens such as Escherichia coli and Staphylococcus aureus. Many phenolic acids can protect the cardiovascular system through multiple pathways.

[0003] High-performance liquid chromatography (HPLC) is currently the most commonly used method for the detection of phenolic acids, characterized by high resolution, high sensitivity, and high accuracy. By optimizing the chromatographic column, mobile phase, and detection wavelength, HPLC can simultaneously separate and quantify multiple phenolic acids. Despite its significant advantages in the detection of phenolic acids, HPLC also has considerable drawbacks, including complex sample pretreatment, high instrument costs, long analysis times, limited sensitivity, complex method development, environmental concerns, and high operator skill requirements. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a rapid method for determining various phenolic acids in fermentation products. This method employs alcohol precipitation-SAX solid-phase extraction coupled with other techniques to filter impurities in the sample, reducing interference from other substances in the detection of phenolic acids. This enables accurate quantitative detection of trace and ultra-trace amounts of caffeic acid, dihydrocaffeic acid, ferulic acid, chlorogenic acid, gallic acid, vanillic acid, syringic acid, p-coumaric acid, salicylic acid, and (±)-catechins in the sample.

[0005] This method eliminates the need for lengthy pretreatment, significantly improving detection efficiency. It also boasts advantages such as accuracy, speed, high sensitivity, and strong stability, providing a reliable basis for the accurate detection of phenolic acids in fermentation products.

[0006] This invention provides a method for rapidly determining multiple phenolic acids in fermentation products, comprising the following steps:

[0007] Step 1: Pre-processing

[0008] Add the analyte to an appropriate amount of ethanol, refrigerate at 0-6℃ for 8-12 hours to extract the non-volatile phenolic acids, and enrich the phenolic acids in the solvent phase; centrifuge the extract, take the supernatant and pass it through an SAX strong anion exchange solid phase extraction column to adsorb the phenolic acids, discard the eluent; rinse with ultrapure water to remove impurities, then elute the target phenolic acids with an acidic solution, collect the eluent; dilute the eluent to a fixed volume and filter through a 0.22µm organic filter membrane to obtain the analyte sample.

[0009] Step 2: Plotting the standard curve

[0010] Prepare standard mixed stock solutions of each target substance with a concentration of 10 mg / L; accurately pipette a certain amount of the standard mixed stock solution and dilute it with ultrapure water by different factors to prepare standard gradient working solutions of different concentrations; use ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry to detect the obtained standard gradient working solutions, and use the instrument's built-in quantitative software for analysis. Plot a standard working curve with the quantitative ion peak area of ​​the target substance as the ordinate and the content of the target substance as the abscissa.

[0011] Step 3: Sample Measurement

[0012] The sample obtained in step 1 was analyzed by ultra-high performance liquid chromatography in tandem triple quadrupole mass spectrometry. Based on the standard working curve and the quantitative ion peak area of ​​the target substance in step 2, the content of the target substance in the liquid was calculated.

[0013] The substance to be tested includes fermentation products, which include fermentation broth, esterification broth, fermentation starter, etc.

[0014] The target substance is a non-volatile phenolic acid.

[0015] Furthermore, the target substance includes 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.

[0016] In step 1, the acidic solution used for elution is a 5% formic acid aqueous solution.

[0017] In step 2, the concentration range of the target substance in the standard gradient working solutions of different concentrations is 0~500µg / L.

[0018] In step 2, the ultra-high performance liquid chromatography column is a CORTECS Premier RP18 column, 1.6µm, 2.1×100 mm.

[0019] Furthermore, the detection conditions for the ultra-high performance liquid chromatography are set as follows: column temperature 30~40℃, injection volume 1-10μL, flow rate 0.2-0.5mL / min, and analysis time 8~15min; the mobile phase of the ultra-high performance liquid chromatography is: mobile phase A: 0.1~1g / L formic acid aqueous solution; mobile phase B: acetonitrile; gradient elution.

[0020] Further optimization: column temperature 35℃, injection volume 5μL, flow rate 0.3 mL / min, analysis time 10min; mobile phase A: 0.1g / L formic acid aqueous solution, mobile phase B: acetonitrile.

[0021] Furthermore, the gradient elution program is set as follows: 0~1 min, 5% mobile phase B (the remainder is mobile phase A, volume percentage); 1~8.5 min, 5%→40% mobile phase B (the remainder is mobile phase A, volume percentage); 8.5~8.6 min, 40→5% mobile phase B (the remainder is mobile phase A, volume percentage); 8.6~10 min, 5% mobile phase B (the remainder is mobile phase A, volume percentage).

[0022] In step 2, the detection conditions for ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry were set as follows: negative ion electrospray ionization (ESI-) mode, monitoring mode: multiple reaction monitoring (MRM) mode, capillary voltage 0.5~3.0kV, cone voltage 20~135V, desolvation gas temperature: 400~500℃; carrier gas flow rate: 600~1000L / Hr.

[0023] Further optimizations include a capillary voltage of 2.5kV, a cone voltage of 60V, a desolvation gas temperature of 450℃, and a carrier gas flow rate of 1000L / Hr.

[0024] This invention utilizes alcohol precipitation-SAX solid-phase extraction coupled with ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry to analyze samples. It can accurately and quantitatively detect trace amounts of caffeic acid, dihydrocaffeic acid, ferulic acid, chlorogenic acid, gallic acid, vanillic acid, syringic acid, p-coumaric acid, salicylic acid, and (±)-catechins in samples. The sample is thoroughly purified, the operation is simple, the detection process is rapid, and the detection results are accurate. Attached Figure Description

[0025] Figure 1 The chromatograms are of working solutions of 10 phenolic acid standards. The elution order is: 1 gallic acid, 2 chlorogenic acid, 3 (±)-catechin, 4 dihydrocaffeic acid, 5 caffeic acid, 6 vanillic acid, 7 syringic acid, 8 p-coumaric acid, 9 ferulic acid, 10 salicylic acid. Detailed Implementation

[0026] The following specific embodiments further illustrate the technical solution of the present invention. These specific embodiments do not represent a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0027] (1) Preparation of standard mixed solution:

[0028] Ten phenolic acid compounds (caffeic acid, dihydrocaffeic acid, ferulic acid, chlorogenic acid, gallic acid, vanillic acid, syringic acid, p-coumaric acid, salicylic acid, and (±)-catechin) were dissolved in 60 wt% ethanol to prepare a standard mixed stock solution of 10 mg / L.

[0029] (2) Plotting the standard curve:

[0030] The standard stock solution was diluted to obtain eight standard working solutions with different concentrations. The standard concentrations of the ten phenolic acid compounds in the eight standard solutions were 500 µg / L, 250 µg / L, 125 µg / L, 62.5 µg / L, 31.25 µg / L, 15.63 µg / L, 7.81 µg / L, and 3.91 µg / L, respectively.

[0031] The obtained standard working solution was analyzed using ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UHPLC-MS / MS) under the following conditions:

[0032] Ultra-high performance liquid chromatography (UHPLC) conditions: The HPLC column was a CORTECS Premier RP18 column, 1.6 µm, 2.1 × 100 mm; the column temperature was 35℃, the injection volume was 5 μL, the flow rate was 0.3 mL / min, and the analysis time was 10 min. Mobile phase A: formic acid aqueous solution containing 0.1 g / L formic acid; mobile phase B: acetonitrile.

[0033] Gradient elution program: 0-1 min, 5% mobile phase B (balance mobile phase A, volume percentage); 1-8.5 min, 5% increasing to 40% mobile phase B (balance mobile phase A, volume percentage); 8.5-8.6 min, 40% decreasing to 5% mobile phase B (balance mobile phase A, volume percentage); 8.6-10 min, 5% mobile phase B (balance mobile phase A, volume percentage).

[0034] The mass spectrometry conditions were as follows: ionization mode: negative ion electrospray (ESI-); monitoring mode: multiple reaction monitoring (MRM); capillary voltage: 2.50 kV; desolvation gas temperature: 450 °C; carrier gas flow rate: 1000 L / Hr.

[0035] The instrument's built-in quantitative software was used for analysis. A standard working curve was constructed with the quantitative ion peak area of ​​the target analyte as the ordinate and the content of the target substance as the abscissa. The obtained linear regression equation is shown in Table 1 below.

[0036]

[0037] (3) Spike recovery and precision tests

[0038] Two blanks were prepared from the same sample and spiked with a mixed standard working solution for recovery experiments. The recovery rate was calculated. Six samples were prepared using the same pretreatment method and tested separately. The reproducibility of the analytical method was determined by calculating the range of the relative standard deviation (RSD). The accuracy of the method is expressed as the recovery rate (Table 2); the reproducibility of the method is expressed as the relative standard deviation (RSD) (Table 3). It can be seen that the recovery rate is between 80% and 120%, and the RSD is <10%.

[0039]

[0040]

[0041] Example 1: Detection and analysis of multiple phenolic acids in fermentation broth

[0042] 1. Accurately measure 20 mL of fermentation broth sample into a 500 mL beaker, add excess 80 mL of ethanol, stir well, let stand and refrigerate for 12 hours, centrifuge, and collect the supernatant for later use; take 10 mL of the supernatant and load it onto an activated SAX strong anion exchange solid phase extraction column, control the flow rate ≤0.6 mL / min, and discard the eluent; rinse with 5 mL of ultrapure water to remove neutral and weakly polar impurities, and discard the eluent; add 6 mL of 5% formic acid aqueous solution at a flow rate of 0.6 mL / min, collect the eluent and make up to 10 mL, filter with a 0.22 µm organic filter membrane, and analyze by UPLC-MS / MS.

[0043] 2. Ultra-high performance liquid chromatography (UHPLC) conditions: The HPLC column was a CORTECS Premier RP18 column, 1.6 µm, 2.1 × 100 mm; the column temperature was 35℃, the injection volume was 5 μL, the flow rate was 0.3 mL / min, and the analysis time was 10 min. Mobile phase A: formic acid aqueous solution containing 0.1 g / L formic acid; mobile phase B: acetonitrile.

[0044] Gradient elution program: 0~1 min, 5% B; 1~8.5 min, 5%→40% B; 8.5~8.6 min, 40→5% B; 8.6~10 min, 5% B.

[0045] The mass spectrometry conditions were as follows: ionization mode: negative ion electrospray (ESI-); monitoring mode: multiple reaction monitoring (MRM); capillary voltage: 2.50 kV; desolvation gas temperature: 450 °C; carrier gas flow rate: 1000 L / Hr.

[0046] 3. The results of the detection of the content of 10 phenolic acids in the fermentation broth are shown in Table 4 below:

[0047]

[0048] Example 2: Detection and analysis of various phenolic acids in fermentation koji

[0049] 1. Accurately weigh 5g of fermentation koji sample, add 20mL of 60% ethanol, sonicate for 30min, let stand and refrigerate for 12 hours, centrifuge and collect the supernatant for later use; take 10mL of the supernatant and load it onto an activated SAX strong anion exchange solid phase extraction column, control the flow rate ≤0.6mL / min, and discard the eluent; rinse with 5mL of ultrapure water to remove neutral and weakly polar impurities, and discard the eluent; add 6mL of 5% formic acid aqueous solution at a flow rate of 0.6mL / min, collect the eluent and make up to 10mL, filter with a 0.22µm organic filter membrane, and analyze by UPLC-MS / MS.

[0050] 2. Ultra-high performance liquid chromatography (UHPLC) conditions: The HPLC column was a CORTECS Premier RP18 column, 1.6 µm, 2.1 × 100 mm; the column temperature was 35℃, the injection volume was 5 μL, the flow rate was 0.3 mL / min, and the analysis time was 10 min. Mobile phase A: formic acid aqueous solution containing 0.1 g / L formic acid; mobile phase B: acetonitrile.

[0051] Gradient elution program: 0~1 min, 5% B; 1~8.5 min, 5%→40% B; 8.5~8.6 min, 40→5% B; 8.6~10 min, 5% B.

[0052] The mass spectrometry conditions were as follows: ionization mode: negative ion electrospray (ESI-); monitoring mode: multiple reaction monitoring (MRM); capillary voltage: 2.50 kV; desolvation gas temperature: 450 °C; carrier gas flow rate: 1000 L / Hr.

[0053] 3. The results of the detection of 10 phenolic acid substances in the fermentation koji are shown in Table 5 below:

[0054]

[0055] Note: ND. indicates not detected.

[0056] Example 3: Detection and analysis of multiple phenolic acids in fermentation broth with extended fermentation time

[0057] The method for rapid determination of multiple phenolic acids in fermentation products in this embodiment differs from that in Example 1 in that the sample to be tested is a fermentation broth with an extended fermentation time. The detection results of the contents of 10 phenolic acids in the extended fermentation broth are shown in Table 6 below:

[0058]

Claims

1. A method for rapidly determining multiple phenolic acids in fermentation products, characterized in that... Includes the following steps: Step 1: Pre-processing Add the analyte to ethanol and refrigerate at 0-6℃ for 8-12 hours to extract the non-volatile phenolic acids, enriching them in the solvent phase. Centrifuge the extract and pass the supernatant through an SAX strong anion exchange solid-phase extraction column to adsorb the phenolic acids, discarding the eluent. Rinse with ultrapure water to remove impurities, then elute the target phenolic acids with an acidic solution, collecting the eluent. Adjust the volume of the eluent and filter it through a 0.22µm organic filter membrane to obtain the analyte sample. Step 2: Plotting the standard curve Prepare standard mixed stock solutions of each target substance with a concentration of 10 mg / L; accurately pipette a certain amount of the standard mixed stock solution and dilute it with ultrapure water by different factors to prepare standard gradient working solutions of different concentrations; use ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry to detect the obtained standard gradient working solutions, and use the instrument's built-in quantitative software for analysis. Plot a standard working curve with the quantitative ion peak area of ​​the target substance as the ordinate and the content of the target substance as the abscissa. Step 3: Sample Measurement The sample obtained in step 1 was analyzed by ultra-high performance liquid chromatography in tandem triple quadrupole mass spectrometry. Based on the standard working curve and the quantitative ion peak area of ​​the target substance in step 2, the content of the target substance in the liquid was calculated. The substance to be tested includes fermentation products, which include one or more of fermentation broth, esterification broth, and fermentation starter. The target substance is a non-volatile phenolic acid.

2. The method according to claim 1, characterized in that: The target substances include 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. The method according to claim 1, characterized in that: In step 1, the acidic solution used for elution is a 5% formic acid aqueous solution.

4. The method according to claim 1, characterized in that: In step 2, the concentration range of the target substance in the standard gradient working solutions of different concentrations is 0~500µg / L.

5. The method according to claim 1, characterized in that: In step 2, the ultra-high performance liquid chromatography column is a CORTECS Premier RP18 column, 1.6µm, 2.1×100mm.

6. The method according to claim 5, characterized in that: The detection conditions for the ultra-high performance liquid chromatography were set as follows: column temperature 30-40℃, injection volume 1-10μL, flow rate 0.2-0.5mL / min, and analysis time 8-15min. The mobile phase of the ultra-high performance liquid chromatography is: mobile phase A: 0.1~1 g / L formic acid aqueous solution; mobile phase B: acetonitrile; gradient elution.

7. The method according to claim 6, characterized in that: The gradient elution procedure is set as follows: 0~1 min, 5% mobile phase B; 1~8.5 min, 5%→40% mobile phase B; 8.5~8.6 min, 40%→5% mobile phase B; 8.6~10 min, 5% mobile phase B.

8. The method according to claim 7, characterized in that: In step 2, the detection conditions for ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry are set as follows: negative ion electrospray mode, monitoring method: multiple reaction monitoring mode, capillary voltage 0.5~3.0kV, cone voltage 20~135V, desolvation gas temperature: 400~500℃; carrier gas flow rate: 600~1000L / Hr.

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