Method for simultaneously detecting 11 organic acids in white spirit fermented grains
By combining ethanol extraction, low-temperature settling, centrifugation, and solid-phase extraction column purification with high-performance liquid chromatography (HPLC) detection, the problem of separating and quantifying multiple highly polar organic acids in baijiu mash was solved, achieving highly sensitive and accurate detection results.
Patent Information
- Application Number
- CN202511480720.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-16
- Publication Date
- 2025-11-25
AI Technical Summary
Existing technologies struggle to simultaneously and accurately detect multiple highly polar organic acids in baijiu mash, and the interference of complex components affects the stability and accuracy of the detection.
After extraction with 60–75% ethanol aqueous solution, low-temperature standing and centrifugation, purification was performed using a Cleanert IC-NANO-V solid-phase extraction column, combined with high-performance liquid chromatography detection, using a 0.02 mol/L potassium dihydrogen phosphate solution at pH=2.7 as the mobile phase, to achieve the separation and quantification of 11 organic acids.
It achieves high sensitivity and accurate detection of 11 organic acids, effectively removes interference from macromolecules and moderately polar impurities, and ensures the stability of detection results and the service life of the chromatographic column.
Smart Images
Figure CN121007991A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of chemical analysis and detection technology, specifically relating to a method for simultaneously detecting 11 organic acids in baijiu mash. Background Technology
[0002] Organic acids in fermented mash are metabolic products of microorganisms and a source of important flavor compounds in baijiu (Chinese liquor). They are also precursors to corresponding esters, significantly influencing the types and amounts of esters, and consequently affecting the flavor and quality of baijiu. The concentration of organic acids in the mash also affects its acidity. Suitable acidity not only inhibits some harmful bacteria but also promotes the growth and metabolism of beneficial bacteria, thereby facilitating the targeted synthesis of flavor compounds. Therefore, it is necessary to establish a method for detecting organic acid compounds in fermented mash, monitoring changes in the types and amounts of organic acids during fermentation, reflecting the changes in microorganisms and the synthesis of important flavor compounds, and optimizing and controlling fermentation process parameters based on these changes.
[0003] There are many types of organic acids in liquor mash. Acetic acid, lactic acid, formic acid, and succinic acid are the main organic acids in liquor mash. Among them, acetic acid and formic acid are volatile organic acids, and are mainly analyzed and detected by gas chromatography / mass spectrometry (GC-MS) and gas chromatography (GC). Lactic acid is a thermally unstable organic acid, while succinic acid and malic acid are non-volatile acids. When using the above two methods for detection, derivatization techniques are required, which is cumbersome and time-consuming. High-performance liquid chromatography (HPLC) can be used to detect organic acids in liquor mash, enabling the determination of thermally unstable organic acids such as lactic acid, as well as non-volatile and some volatile organic acids. This method is rapid, sensitive, relatively low-cost, and simple to operate. However, organic acids with strong polar characteristics in liquor mash show weak retention on the chromatographic column when detected by HPLC, making effective separation difficult. Therefore, the simultaneous qualitative and quantitative detection of strongly polar organic acids in fermented mash is a major challenge in fermented mash analysis technology.
[0004] Moreover, the composition of fermented mash is exceptionally complex, including macromolecular components such as proteins, polysaccharides, and lipids, as well as flavor components such as alcohols and esters. These components significantly interfere with the separation of organic acids, affecting the stability and accuracy of organic acid detection. How to eliminate the interference of these substances is another technical challenge for the separation and detection of organic acids in fermented mash.
[0005] Currently, there are few reported methods for the simultaneous detection of multiple highly polar organic acids in fermented mash. Providing a method that can simultaneously and accurately determine multiple organic acids in fermented mash is of great value for the control of the baijiu brewing process. Summary of the Invention
[0006] To address the problem that the complex components in fermented mash greatly interfere with the separation of organic acids, affecting the stability and accuracy of organic acid detection, and that it is difficult to simultaneously and accurately detect multiple highly polar organic acids qualitatively and quantitatively, this invention provides a method for the simultaneous detection of 11 highly polar organic acids in fermented mash. This method has high sensitivity and accurate detection results, and is an effective method for detecting the types and contents of organic acids in fermented mash.
[0007] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: This invention provides a method for simultaneously detecting 11 organic acids in baijiu mash, comprising the following steps: S1: Extract the baijiu mash with 60-75 v / v% ethanol aqueous solution, sonicate, let stand and centrifuge, take the supernatant, dilute it, and then purify and filter it through a Cleanert IC-NANO-V solid phase extraction column as the sample to be tested. S2: The sample to be tested in step S1 is subjected to qualitative and quantitative detection by high performance liquid chromatography (HPLC); the HPLC is reversed-phase chromatography, and the mobile phase is a 0.02 mol / L potassium dihydrogen phosphate solution with pH=2.7; the 11 organic acids are oxalic acid, tartaric acid, formic acid, malic acid, lactic acid, acetic acid, citric acid, L-pyroglutamic acid, succinic acid, fumaric acid, and propionic acid.
[0008] Furthermore, the ethanol-water solution is a 60 v / v% ethanol-water solution.
[0009] Furthermore, the amount of extraction solvent used is 2 to 6 times the weight of the fermented mash. Preferably, the amount of extraction solvent used is 4 times the weight of the fermented mash.
[0010] Furthermore, the ultrasonic temperature is 5–15°C, and the ultrasonic time is 20–45 min. Preferably, the ultrasonic temperature is 5°C, and the ultrasonic time is 30 min.
[0011] Furthermore, the settling temperature is 0–4°C, and the settling time is 12–24 h. Preferably, the settling temperature is 4°C, and the settling time is 12 h.
[0012] Furthermore, the centrifugation temperature is 4°C, the centrifugation rate is 8000 r / min, and the centrifugation time is 3–15 min. Preferably, the centrifugation time is 3 min.
[0013] Furthermore, the organic acid extract is diluted with ultrapure water, and the volume ratio of organic acid extract to ultrapure water is 1:4 to 1:9. Preferably, the volume ratio of organic acid extract to ultrapure water is 1:4.
[0014] Furthermore, the packing adsorbent of the Cleanert IC-NANO-V solid-phase extraction column is a functionalized carbon nanomaterial.
[0015] Furthermore, the purification process of the Cleanert IC-NANO-V solid-phase extraction column is as follows: the diluent is loaded into the column and pushed out, the effluent is collected, and the sample is obtained after filtration through a 0.22 μm filter membrane.
[0016] Furthermore, the high-performance liquid chromatography employs isocratic elution with an elution time of 30 min.
[0017] Further, the chromatographic column of the high-performance liquid chromatography (HPLC) is a C18 column. Preferably, the HPLC column is a ZORBAX SB-Aq C18 column. More preferably, the ZORBAX SB-Aq C18 column has a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.
[0018] Further, the high-performance liquid chromatography column temperature is 20–40 °C, the flow rate is 0.8–1.2 mL / min, and the injection volume is 1–100 μL. Preferably, the column temperature is 25 °C, the flow rate is 1.0 mL / min, and the injection volume is 10 μL.
[0019] Furthermore, each 1 mL of sample injected by high performance liquid chromatography corresponds to 0.05 g of fermented mash.
[0020] Furthermore, the detector in the high-performance liquid chromatography is an ultraviolet detector.
[0021] Furthermore, the detection conditions for the ultraviolet detector are: detection wavelength 214 nm.
[0022] Furthermore, the UV detector signal peaks of organic acids in the fermented mash are determined based on standard reference materials. Using the detection method described above, the elution times of the UV detector signal peaks of the 11 organic acids and their corresponding standard reference materials are consistent.
[0023] Furthermore, the peak elution times of the UV detector signals for the 11 organic acids are as follows: .
[0024] Furthermore, the method for calculating the content of organic acids includes the following steps: a. Plotting a standard curve: Prepare standard solutions of 11 organic acids, and detect them according to the detection method described above. Plot a concentration-peak area standard curve with the peak area of the organic acid UV detector signal peak and the concentration of the standard solution as the x and y axes. b. Detect and calculate the content: Dilute the fermented mash extract and inject it for detection according to the detection method described above. Calculate the content based on the peak area of the organic acid UV detector signal peak and the standard curve from step a.
[0025] Beneficial Effects: This invention employs extraction ultrasound, low-temperature settling, centrifugation, and carbon nanotube purification column purification to pretreat the baijiu mash. Then, high-performance liquid chromatography (HPLC) is used for qualitative and quantitative analysis to detect 11 strongly polar organic acids. First, based on the principle of "like dissolves like," a 60-75% ethanol-water solution is used as the extraction solvent to effectively extract the 11 strongly polar organic acids from the mash, while simultaneously achieving effective separation of small-molecule organic acids from large-molecule protein and polysaccharide components. Then, after low-temperature settling and centrifugation, the large-molecule components are fully precipitated, thereby removing impurities such as large-molecule polysaccharides and proteins from the extract. Subsequently, a Cleanert IC-NANO-V carbon nanotube solid-phase extraction column was used for a simple and rapid pretreatment of the diluted liquor extract. This removed moderately polar substances such as esters and non-polar impurities (pigments, oils) from the sample, avoiding interference from these impurities in the detection of organic acids. This achieved purification of 11 organic acids and prevented the retention of moderately polar and non-polar impurities in the column during isocratic elution in HPLC, which could lead to instability in sample results and column wear. Finally, the pretreated liquor mash was analyzed by HPLC. By selecting appropriate HPLC conditions and the concentration and pH of potassium dihydrogen phosphate in the mobile phase, the column retention time of organic acids was extended, achieving good separation of strongly polar organic acids without causing excessively high instrument noise or reduced detection sensitivity under HPLC conditions.
[0026] The test results show that the method of the present invention can simultaneously determine 11 strongly polar organic acids, including oxalic acid, tartaric acid, formic acid, malic acid, lactic acid, acetic acid, citric acid, L-pyroglutamic acid, succinic acid, fumaric acid and propionic acid, in baijiu mash. It has high sensitivity and accurate detection results, and is an effective method for detecting the types and contents of organic acids in baijiu mash. Attached Figure Description
[0027] Figure 1 The chromatogram of the standard diluted 20 times is shown for the mobile phase in Example 1, which was potassium dihydrogen phosphate solution. Figure 2 This is a chromatogram of the mash sample pretreated with the Cleanert IC-NANO-V solid-phase extraction column in Example 1; Figure 3 This is a chromatogram of the spiked sample of fermented mash pretreated with a Cleanert IC-NANO-V solid-phase extraction column in Example 1; Figure 4 The chromatogram of the mash sample from Comparative Example 1 that was not pretreated with the Cleanert IC-NANO-V solid-phase extraction column; Figure 5The chromatogram of the standard diluted 20 times is shown for Comparative Example 2, where the mobile phase is phosphoric acid solution. Detailed Implementation
[0028] To make the technical problems, solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with the embodiments. Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.
[0029] This invention provides a method for simultaneously detecting 11 organic acids in baijiu mash, comprising the following steps: S1: Extract the baijiu mash with 60-75 v / v% ethanol aqueous solution, sonicate, let stand and centrifuge, take the supernatant, dilute it, and then purify and filter it through a Cleanert IC-NANO-V solid phase extraction column as the sample to be tested. S2: The sample to be tested in step S1 is subjected to qualitative and quantitative detection by high performance liquid chromatography (HPLC); the HPLC is reversed-phase chromatography, and the mobile phase is a 0.02 mol / L potassium dihydrogen phosphate solution with pH=2.7; the 11 organic acids are oxalic acid, tartaric acid, formic acid, malic acid, lactic acid, acetic acid, citric acid, L-pyroglutamic acid, succinic acid, fumaric acid, and propionic acid.
[0030] In some embodiments of the present invention, using a 60-75% ethanol aqueous solution as the extraction solution, followed by ultrasonication, standing at 4°C for 12-24 hours, and centrifugation, can effectively remove macromolecular proteins and polysaccharide components from the mash, achieving effective extraction of 11 organic acids from the mash and their primary separation from macromolecular impurities. The extraction effect is even better when the ethanol aqueous solution is 60 v / v% ethanol aqueous solution.
[0031] The applicant discovered that if the sample is directly injected for detection after extraction, the moderately polar and non-polar impurities in the fermented mash interfere with the detection of organic acids, causing some organic acid peaks to be indistinguishable or overlapping. Therefore, in some specific embodiments of the present invention, a carbon nanotube purification column (Cleanert IC-NANO-V) is used to pretreat the diluted extract of the fermented mash before injection for detection. After loading the Cleanert IC-NANO-V solid-phase extraction column, the moderately polar and non-polar impurities are adsorbed and removed. At this point, the target organic acid compounds to be detected are not adsorbed. The eluent from the loading is collected, achieving separation of the target compounds from the impurities in the sample, avoiding interference from impurities during the detection process, and achieving purification of 11 organic acids. This avoids the instability of sample detection results and column wear caused by the residual moderately polar and non-polar impurities in the chromatographic column under isocratic elution in high-performance liquid chromatography (HPLC).
[0032] Further investigation by the applicant revealed that the carbon nanomaterials in the Cleanert IC-NANO-V solid-phase extraction column have a typical layered hollow structure, providing a large specific surface area that can accommodate more impurity molecules through physical adsorption. The basic framework of the carbon nanomaterials is hydrophobic, enabling strong hydrophobic interactions with non-polar impurities. Special functional groups bonded to the surface of the carbon nanomaterials exhibit strong selective adsorption capacity for moderately polar components through polar interactions, such as hydrogen bonds. The Cleanert IC-NANO-V solid-phase extraction column adsorbs moderately polar and non-polar impurities in the mash through these three mechanisms. Organic acids in the sample cannot be retained on the column; by collecting the effluent from the Cleanert IC-NANO-V purified column, organic acids are effectively separated from impurities, avoiding interference from impurities during high-performance liquid chromatography (HPLC) detection and achieving accurate detection.
[0033] In some specific embodiments of the present invention, high performance liquid chromatography is used to detect the organic acids, and a 0.02 mol / L potassium dihydrogen phosphate solution with pH=2.7 is further used as the mobile phase. This mobile phase affects the column retention time of organic acids, thereby achieving good separation of strongly polar organic acids and solving the problem of simultaneous qualitative and quantitative analysis of strongly polar organic acids in fermented mash.
[0034] The following specific embodiments will be provided to explain the solution of the present invention. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.
[0035] The following examples utilize an Agilent 1100 high-performance liquid chromatograph and an Agilent Cleanert IC-NANO-V solid-phase extraction column. Taking the 11 major strongly polar organic acids in fermented mash as examples, the methods of this invention demonstrate that they can simultaneously detect all 11 organic acids with high sensitivity and accurate results, making it an effective method for detecting the types and content of organic acids in fermented mash.
[0036] Example 1: Detection of organic acids in fermented mash using the method of the present invention The fermented mash samples were processed and then analyzed by high-performance liquid chromatography.
[0037] High performance liquid chromatography (HPLC) detection conditions: Mobile phase: 0.02 mol / L potassium dihydrogen phosphate solution, pH=2.7; isocratic elution: 0–30 min: 100% v / v B; elution stopped.
[0038] The chromatographic column was a ZORBAX SB-Aq C18 column with a length of 250 mm, an inner diameter of 4.6 mm, a particle size of 5 μm, a column temperature of 25℃, a flow rate of 1 mL / min, and an injection volume of 10 μL.
[0039] Ultraviolet detector: detection wavelength 214 nm.
[0040] a. Constructing Standard Curves: Using water as the solvent, prepare 10 mL of a mixed organic acid standard stock solution (oxalic acid, tartaric acid, formic acid, malic acid, lactic acid, acetic acid, citric acid, L-pyroglutamic acid, succinic acid, fumaric acid, and propionic acid concentrations of 100 mg / L, 320 mg / L, 2570 mg / L, 1140 mg / L, 15780 mg / L, 2450 mg / L, 2130 mg / L, 620 mg / L, 4480 mg / L, 110 mg / L, and 3280 mg / L, respectively). Dilute the stock solutions with water, and prepare five standard curve concentration points by dilution at 1, 2, 10, 20, and 100, respectively. Inject the mixtures into a high-performance liquid chromatograph (HPLC). Obtain the peak area of each organic acid signal peak using an organic acid UV detector. Calculate the concentration-peak area standard curve based on the concentration of each solution and its corresponding peak area. Determine the linear range, linear equation, and R0 for each organic acid compound. 2 Values, detection limits, and detection sensitivity (RSD) are shown in Table 1 below: Table 1 Standard curves for 11 organic acids
[0041] The chromatogram of the above standard substances diluted 20 times is shown below. Figure 1 (The horizontal axis in the figure represents time / min, and the vertical axis represents electrical signal / mAU). The peak area and elution time of each organic acid compound are shown in Table 2 below: Table 2. Elution times of 11 organic acids
[0042] b. Sample preparation of fermented mash: Accurately weigh two 5 g portions of fermented mash and place them in separate 50 mL centrifuge tubes. Add 20 mL of 60% ethanol aqueous solution to one tube, and add 5 mL of a mixed standard prepared with 60% ethanol aqueous solution and 15 mL of 60% ethanol aqueous solution to the other tube. After sonicating at 5℃ for 30 min, place the samples in a 4℃ incubator for 12 h, and centrifuge at 8000 r / min for 3 min at 4℃ to obtain the supernatant. Dilute the supernatant to 1 / 5 of the original concentration, and then purify it using a Cleanert IC-NANO-V solid-phase extraction column. The adsorbent in the solid-phase extraction column is a functionalized carbon nanomaterial. After filtration through a 0.22 µm filter membrane, collect the eluent as the sample to be tested. Analyze the sample using high-performance liquid chromatography (HPLC). The obtained chromatograms are as follows: Figure 2 , Figure 3 (The horizontal axis in the figure represents time / min, and the vertical axis represents electrical signal / mAU).
[0043] c. Analyze the chromatogram and calculate the organic acid content: In the chromatogram, obtain the peak start time and peak end time of 11 organic acids, integrate them, obtain the peak area, and then quantify each organic acid compound according to the standard curve.
[0044] The recovery rate of the method was determined using the fermented mash sample after step b. The results are shown in Table 3 below: Table 3. Results of recovery rate determination of fermented mash samples
[0045] The following comparative examples further demonstrate the beneficial effects of the present invention.
[0046] Comparative Example 1: Effect of Pretreatment and Isocratic Elution Conditions on Detection Results Samples of fermented mash were taken and analyzed using the same method and conditions as in Example 1, but without pretreatment with a Cleanert IC-NANO-V solid-phase extraction column before injection. The results are shown below. Figure 4 (The horizontal axis in the figure represents time / min, and the vertical axis represents electrical signal / mAU).
[0047] As can be seen from the figure, with Figure 2 In the comparison of separation results, the target compound to be detected had a high baseline and many impurity peaks, making accurate detection impossible.
[0048] The above experimental results show that pretreatment of the fermentation mash extract with the Cleanert IC-NANO-V solid-phase extraction column before sample injection can effectively remove moderately polar and non-polar impurities from the sample, avoid interference from these impurities on the detection of organic acids, and achieve purification of 11 organic acids. This avoids the instability of sample detection results and column wear caused by the residual moderately polar and non-polar impurities in the chromatographic column under isocratic elution mode in high-performance liquid chromatography.
[0049] Comparative Example 2: The Influence of Different Flow Rates on Detection Performance The same method and conditions as in Example 1 were used to detect the mixed standard of the same concentration, but the mobile phase was different: a 1% phosphoric acid solution. The detection results are shown below. Figure 5 (The horizontal axis in the figure represents time / min, and the vertical axis represents electrical signal / mAU).
[0050] As can be seen from the figure, with Figure 1 In a comparison of separation results, using a 1% phosphoric acid solution as the mobile phase, it was difficult to separate some compounds in the mixed standard.
[0051] The above experimental results show that, by using the mobile phase conditions of the present invention, the interference of impurities can be minimized, and the separation of the target compounds can be achieved by affecting the column retention time of organic acids. If a 1% phosphoric acid solution is used, it will affect the separation degree between the target compounds, and the interference of impurities on the detected compounds will be more obvious.
Claims
1. A method for simultaneously detecting 11 organic acids in baijiu mash, characterized in that, Includes the following steps: S1: Extract the baijiu mash with 60-75 v / v% ethanol aqueous solution, sonicate, let stand and centrifuge, take the supernatant, dilute it, and then purify and filter it through a Cleanert IC-NANO-V solid phase extraction column as the sample to be tested. S2: The sample to be tested in step S1 is subjected to qualitative and quantitative detection by high performance liquid chromatography (HPLC); the HPLC is reversed-phase chromatography, and the mobile phase is a 0.02 mol / L potassium dihydrogen phosphate solution with pH=2.7; the 11 organic acids are oxalic acid, tartaric acid, formic acid, malic acid, lactic acid, acetic acid, citric acid, L-pyroglutamic acid, succinic acid, fumaric acid, and propionic acid.
2. The method for simultaneously detecting 11 organic acids in baijiu mash according to claim 1, characterized in that, The ethanol-water solution is a 60 v / v% ethanol-water solution.
3. The method for simultaneously detecting 11 organic acids in baijiu mash according to claim 1, characterized in that, In step S1, at least one of the following conditions must be met: The amount of extraction solvent used is 2 to 6 times the weight of the mash; preferably, the amount of extraction solvent used is 4 times the weight of the mash. The ultrasonic temperature is 5–15°C, and the ultrasonic time is 20–45 min; preferably, the ultrasonic temperature is 5°C, and the ultrasonic time is 30 min. The settling temperature is 0–4°C, and the settling time is 12–24 h; preferably, the settling temperature is 4°C, and the settling time is 12 h. The centrifugation temperature is 4℃, the centrifugation rate is 8000 r / min, and the centrifugation time is 3 to 15 min; preferably, the centrifugation time is 3 min. The organic acid extract is diluted with ultrapure water, and the volume ratio of organic acid extract to ultrapure water is 1:4 to 1:9; preferably, the volume ratio of organic acid extract to ultrapure water is 1:
4.
4. The method for simultaneously detecting 11 organic acids in baijiu mash according to claim 1, characterized in that, The packing adsorbent of the Cleanert IC-NANO-V solid-phase extraction column is a functionalized carbon nanomaterial.
5. The method for simultaneously detecting 11 organic acids in baijiu mash according to claim 1, characterized in that, The purification process of the Cleanert IC-NANO-V solid-phase extraction column is as follows: the diluent is loaded into the column and pushed out, the effluent is collected, and the sample is obtained after filtration through a 0.22 μm filter membrane.
6. The method for simultaneously detecting 11 organic acids in baijiu mash according to claim 1, characterized in that, The high-performance liquid chromatography employed isocratic elution for 30 minutes.
7. The method for simultaneously detecting 11 organic acids in baijiu mash according to claim 1, characterized in that, High-performance liquid chromatography (HPLC) detection conditions must meet at least one of the following: The high-performance liquid chromatography column is a C18 column; preferably, the high-performance liquid chromatography column is a ZORBAXSB-Aq C18 column. The high-performance liquid chromatography column temperature is 20–40 °C, the flow rate is 0.8–1.2 mL / min, and the injection volume is 1–100 μL; preferably, the column temperature is 25 °C, the flow rate is 1.0 mL / min, and the injection volume is 10 μL. The detector for the high-performance liquid chromatography is an ultraviolet detector; preferably, the detection wavelength is 214 nm.
8. The method for simultaneously detecting 11 organic acids in baijiu mash according to claim 1, characterized in that, The UV detector signal peaks of the organic acids in the fermented mash were determined based on the standard reference material. Using the detection method described above, the elution times of the UV detector signal peaks of the 11 organic acids and their corresponding standard reference materials were consistent.
9. The method for simultaneously detecting 11 organic acids in baijiu mash according to claim 1, characterized in that, The peak elution times of the UV detector signals for the 11 organic acids are as follows: 。 10. The method for simultaneously detecting 11 organic acids in baijiu mash according to claim 1, characterized in that, The method for calculating the content of organic acids includes the following steps: a. Plotting a standard curve: Prepare standard solutions of 11 organic acids, and detect them according to the detection method described above. Plot a concentration-peak area standard curve with the peak area of the organic acid UV detector signal peak and the concentration of the standard solution as the x and y axes. b. Detect and calculate the content: Dilute the fermented mash extract and inject it for detection according to the detection method described above. Calculate the content based on the peak area of the organic acid UV detector signal peak and the standard curve from step a.