Method for detecting cyclodextrin in fermented grains based on UHPLC-CAD technology

Abstract

The invention relates to the technical field of cyclodextrin detection, and discloses a method for detecting cyclodextrin in fermented grains based on a UHPLC-CAD (Ultra High Performance Liquid Chromatography-Computer Aided Design) technology, which comprises the following steps of: optimizing chromatographic column selection and detection parameters of a high performance liquid chromatograph and detection parameters of an electric fog type detector by adopting an ultra high performance liquid chromatograph and electric fog type detector combined system; in cooperation with pretreatment of a sample in the fermented grains, qualitative and quantitative detection of alpha cyclodextrin, beta cyclodextrin and gamma cyclodextrin in the fermented grains at the same time is successfully realized; the separation effect is excellent, interference is small, the detection speed is high, and the internal relation between the starch content, the dextrin content and the gelatinization degree in the fermented grains can be deeply excavated.

Description

Technical Field

[0001] This invention relates to the field of cyclodextrin detection technology, specifically to a method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology. Background Technology

[0002] The statements in this section provide only background information relevant to the disclosure of this application and may not constitute prior art.

[0003] Optimizing key technologies in baijiu brewing is crucial. In baijiu brewing, the mash (fermentation mash) is the core intermediate product, and its starch content is a key raw material for the production of cyclodextrin. Starch gelatinization is a crucial early stage in the conversion of starch into cyclodextrin, and its degree directly affects cyclodextrin formation. However, the current baijiu industry lacks detection technology for cyclodextrin in the mash. This limits the exploration of the intrinsic correlation between starch, dextrin content, and the degree of gelatinization in the mash. Consequently, brewers lack crucial data support during process control, relying heavily on experience and making it difficult to achieve precise and scientific operation.

[0004] The existing technology, "2-Hydroxypropyl-β-Cyclodextrin Aggregates: Identification and Development of Analytical Techniques," discloses a method for detecting β-cyclodextrin polymers using ultra-high performance liquid chromatography (UHPLC) and an electro-cavitation detector (CAD), achieving a detection correlation of 0.9993. However, this reaction detects a single component of β-cyclodextrin polymers in water. Cyclodextrins distributed in fermented mash are a mixed system, and there is detection of multiple structurally highly similar cyclodextrins, such as α, β, and γ cyclodextrins. Therefore, it is necessary to improve the existing detection method to obtain a specific method suitable for the separate detection of α, β, and γ cyclodextrins in fermented mash. Summary of the Invention

[0005] The purpose of this invention is to address the current difficulty in detecting cyclodextrins in fermented mash by providing a method for detecting cyclodextrins in fermented mash based on UHPLC-CAD technology. This method uses ultra-high performance liquid chromatography (UHPLC) and an electro-cavitation detector (CAD) as the core detection tools, giving full play to their advantages of high resolution and high sensitivity, and can simultaneously perform accurate detection and quantitative analysis of three key cyclodextrins (α, β, γ) in fermented mash.

[0006] This invention provides a method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology, employing a system combining an ultra-high performance liquid chromatograph and an electrospray detector. The detection conditions of the ultra-high performance liquid chromatograph are as follows: Column: XBridge C18 4.6mm×250mm 5μm; or Phenomenex Gemini NX C18 4.6×250 mm, 5μm, with comparable pH tolerance in the range of 1-12, maximum pressure of 6000psi, carbon loading >16%, and selectivity close to that of XBridge.

[0007] Column temperature: 20℃; Mobile phase: 8%-9% methanol, 91%-92% water; Flow rate: 1.0 mL / min; The detection conditions for the electro-fog detector are: The electrofogging temperature was 50.0℃, the sampling frequency was 4.5Hz-6Hz, and the filtration constant was 3s-4s.

[0008] The advantages of this setup are: (1) The mobile phase is a combination of 8%-9% methanol and 91%-92% water, which provides a suitable polar environment for the separation of cyclodextrin in the ultra-high performance liquid chromatography column, allowing cyclodextrin to be better separated from other possible coexisting components in the mash, avoiding peak overlap, and improving the accuracy and precision of detection. (2) The electro-atomization temperature of the electro-atomization detector is 50.0℃, ensuring that cyclodextrin can be atomized uniformly and efficiently during atomization and ionization, improving the response sensitivity, thereby achieving accurate qualitative and quantitative determination of cyclodextrin.

[0009] According to a preferred embodiment, the analysis time of the electro-fogging detector is 13.00 min to 17.00 min.

[0010] According to a preferred embodiment, the injection volume is 5 μl.

[0011] According to a preferred embodiment, the specific steps include: preparing a reference solution and a test solution; detecting the reference solution and the test solution under the aforementioned detection conditions and recording the corresponding CAD detection signal peak areas to obtain the standard curve equation and the detection peak area of ​​the test sample; substituting the detection peak area of ​​the mash sample to be tested into the standard curve equation to calculate the cyclodextrin content in the mash.

[0012] According to a preferred embodiment, the method for preparing the test solution is as follows: Mix the fermented mash sample with the solvent at a ratio of 1-3:4-6 (g / mL), then extract by ultrasonication. After ultrasonication, remove the sample solution, cool it to room temperature, centrifuge, filter, and take the supernatant as the test solution.

[0013] This ratio of mash sample to solvent ensures that the solvent and sample are in full contact and wetted to achieve optimal solubility, thereby improving extraction efficiency and degree, while reducing the dissolution of non-target substances and affecting the accuracy of the results.

[0014] According to a preferred embodiment, the solvent is a 10% methanol aqueous solution. Using a 10% methanol aqueous solution as a mixed solvent system provides moderate polarity, good solubility for the target analyte, and effectively extracts cyclodextrin from the fermentation mash, reducing the dissolution of non-target analytes and facilitating subsequent analysis.

[0015] According to a preferred embodiment, the ultrasonic extraction conditions are: ultrasonic extraction at 50℃-60℃ for 20-30 minutes. Considering the solubility and stability of cyclodextrin in a 10% methanol aqueous solution and the influence of impurities in the sample, a temperature of 60℃ and ultrasonic extraction for 20 minutes were ultimately determined to be the optimal combination. Under these conditions, cyclodextrin can be extracted from the mash to the maximum extent, achieving a better balance between extraction efficiency and extraction yield.

[0016] According to a preferred embodiment, the centrifugation is performed at 4000 rpm for 5 minutes.

[0017] According to a preferred embodiment, the filtration is performed using a 0.45 filter membrane.

[0018] According to a preferred embodiment, α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin standards are diluted with a 10% methanol aqueous solution to prepare mixed standard solutions of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin with concentrations of 100.00 mg / L, 50.00 mg / L, 25.00 mg / L, 10.00 mg / L, and 5.00 mg / L, respectively.

[0019] Compared with existing technologies, the advantages of this invention are: 1. A method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology. This method uses a combined system of ultra-high performance liquid chromatography (UHPLC) and electro-cavitation detector (CAD) to accurately detect three key cyclodextrins (α, β, γ) simultaneously. The sample pretreatment steps are simple and easy to perform, reducing excessive operation procedures and sources of error. 2. A method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology can rapidly determine the cyclodextrin content in fermented mash and deeply explore the intrinsic relationship between starch, dextrin content, and gelatinization degree in fermented mash. This helps brewing enterprises accurately control the degree of starch gelatinization in fermented mash, and optimize production process parameters such as cooking time, temperature, and fermentation conditions in a timely manner during the brewing process. This leads to a steady improvement in product quality, enhances the competitiveness of enterprises in the liquor market, and promotes the entire liquor industry towards a more scientific and refined direction. 3. A method for detecting cyclodextrin in brewing mash based on UHPLC-CAD technology. This method reduces interfering substances through optimized sample pretreatment and combines the precise instrumental analysis conditions of ultra-high performance liquid chromatography (UHPLC) with an electro-fogging detector (CAD), effectively improving the accuracy of cyclodextrin detection and providing reliable data support for the control of brewing process parameters. The method features low detection limits: α-cyclodextrin detection limit 2.07 mg / L, quantitation limit 6.21 mg / L; β-cyclodextrin detection limit 2.49 mg / L, quantitation limit 7.47 mg / L; γ-cyclodextrin detection limit 2.10 mg / L, quantitation limit 6.30 mg / L. 4. A method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology. The high separation efficiency of ultra-high performance liquid chromatography (UHPLC) and the high sensitivity of the electro-fogging detector (CAD) make the entire detection process fast and efficient, which can meet the needs of large-scale fermented mash sample detection and improve production detection efficiency. 5. A method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology. This method is applicable to the detection of cyclodextrin in fermented mash during the brewing process in the brewing industry. It has broad application prospects and helps to promote the technological development and quality improvement of the brewing industry. Attached Figure Description

[0020] Figure 1 The spectrum is obtained under the detection conditions of Example 1 for a method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology; Figure 2 The spectrum obtained under the detection conditions of Comparative Example 1 is a method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology. Figure 3 The spectrum obtained under the detection conditions of Comparative Example 2 is a method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology. Figure 4 The spectrum obtained under the detection conditions of Comparative Example 3 is a method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology. Figure 5 The spectrum obtained under the detection conditions of Comparative Example 4 is a method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology. Figure 6 The spectrum obtained under the detection conditions of Comparative Example 5 is a method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology. Figure 7 The spectrum obtained under the detection conditions of Comparative Example 6 is a method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology. Figure 8The linear regression diagram is shown for the α-cyclodextrin standard solution in Example 1. Figure 9 The linear regression diagram is shown for the β-cyclodextrin standard solution in Example 1. Figure 10 The graph shows the linear regression of the γ-cyclodextrin standard solution in Example 1. Detailed Implementation

[0021] The specific embodiments listed in this invention are merely examples, and the invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of this invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of this invention should be covered within its scope. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments whose manufacturers are not specified are commercially available conventional products. To better illustrate this invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this invention can be practiced even without certain specific details. In other embodiments, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of this invention.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production.

[0023] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0024] Example 1: A method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology (a) Sample pretreatment Accurately weigh 10g of the fermented mash sample and place it in a 100mL centrifuge tube. Add 50mL of 10% methanol aqueous solution as the extraction solvent. Vortex mix for 2min, and then sonicate at 60℃ for 20min. After sonication, remove the sample solution, cool to room temperature, centrifuge at 4000 rpm for 5min, filter through a 0.45mm filter membrane, and take the supernatant as the test sample.

[0025] (II) Instrumental Analysis Conditions An ultra-high performance liquid chromatography (UHPLC) system coupled with an electro-cavitation detector (CAD) was used. The selected column was an XBridge C18 4.6 mm × 250 mm 5 μm. The UHPLC conditions were: mobile phase 8.5% methanol, 91.5% water; column temperature 20 °C; flow rate 1.0 mL / min; injection volume 5 μl; and autosampler. The CAD conditions were: atomization temperature 50.0 °C; acquisition frequency 5 Hz; and filtration constant 3.6 s. The analysis time was 15.00 min.

[0026] (III) Solution preparation 1. 10% methanol aqueous solution: Accurately transfer 100 mL of methanol (HPLC grade), dilute to 1000 mL with ultrapure water, mix thoroughly and set aside.

[0027] 2. Preparation of Standard Stock Solutions: Accurately weigh 0.1 g each of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin standards (rounded to 0.0001 g) into 50 mL beakers, dissolve in 10% methanol aqueous solution, and then transfer to 100 mL volumetric flasks respectively. Wash the inner wall of the beakers several times with an appropriate amount of 10% methanol aqueous solution, and add the washings to the 100 mL volumetric flasks. Finally, dilute to the mark with 10% methanol aqueous solution and mix thoroughly. Prepare single-standard stock solutions with a mass concentration of 1000 mg / L and store at 4°C.

[0028] 3. Preparation of mixed standard intermediate solution: Accurately pipette 2 mL of the three single standard stock solutions into the same 10 mL volumetric flask, dilute to the mark with 10% methanol aqueous solution to prepare a mixed standard solution with a mass concentration of 200 mg / L, and store it in a refrigerator at 4℃.

[0029] 4. Preparation of standard working series solutions: Dilute the 200 mg / L mixed standard solution stepwise to prepare a series of solvent standard working solutions with concentrations of 5.00 mg / L to 100.00 mg / L.

[0030] (iv) Qualitative Analysis Following the sample pretreatment method and instrument analysis conditions described above, blank spiked samples containing α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin were analyzed and detected to determine the retention time of each component on the CAD (electro-atomized detector), thus achieving qualitative analysis of cyclodextrin.

[0031] Table 1 Qualitative Confirmation Data and Results

[0032] (V) Standard curve plotting and quantitative analysis A 200 mg / L mixed standard solution was diluted to prepare mixed standard solutions of cyclodextrin (α, β, γ) at concentrations of 100.00 mg / L, 50.00 mg / L, 25.00 mg / L, 10.00 mg / L, and 5.00 mg / L, respectively. The sample pretreatment method and instrument analysis conditions described above were followed to obtain the corresponding CAD detection signal peak areas. A standard curve was plotted with cyclodextrin concentration on the x-axis and peak area on the y-axis. The results are shown below. Figure 8-10 As shown. The content of cyclodextrin in the mash was calculated by substituting the detection peak area of ​​the mash sample into the standard curve equation.

[0033] Table 2. Linearity confirmation data and results for standard solutions

[0034] (vi) Data Analysis The cyclodextrin content in the sample was automatically calculated directly using the instrument's built-in data software, Chromeleon 7.

[0035] Under the parameter settings of this embodiment, the detection results are as follows: Figure 1 As shown, it can achieve good separation of cyclodextrin in the chromatographic column and obtain accurate and stable detection signals through CAD detector, effectively improving the accuracy and precision of detection.

[0036] Experimental conclusions: The spectrum shows a stable baseline with minimal noise interference. A normal peak (γ-cyclodextrin) appears at 4.05 min, a second normal peak (α-cyclodextrin) at 5.45 min, and a third normal peak (β-cyclodextrin) at 8.32 min. The peak shapes are good, separation is complete, and there is no interference. These analytical conditions are basically sufficient for the qualitative identification of cyclodextrin substances.

[0037] (vii) Presentation of analysis results 1. The cyclodextrin content in the sample is calculated using the following formula: ; In the formula: X —The content of cyclodextrin in the sample, in milligrams per kilogram (mg / kg); — The concentration of cyclodextrin in the sample obtained from the standard curve, in milligrams per liter (mg / L). The calculation result is rounded to two decimal places.

[0038] 2. The cyclodextrin content in the sample (when the determination result needs to be converted according to the dilution factor) is calculated according to the following formula:

[0039] In the formula: —The content of cyclodextrin in the sample, in milligrams per kilogram (mg / kg); — The concentration of cyclodextrin in the sample obtained from the standard curve, in milligrams per liter (mg / L). —Concentration of blank sample, in milligrams per liter (mg / L); —The mass of the sample, in grams (g); —The volume of the sample is measured in milliliters (mL); 1000 — Conversion factor.

[0040] The calculation results are expressed as the arithmetic mean of two measurements obtained under repeatability conditions, and the results are rounded to two decimal places.

[0041] (viii) Recovery rate For sample solutions containing α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin (i.e., "spiked sample content" in the table), accurately add known fixed values ​​(40.00 mg / L in the table) to obtain spiked sample solutions. Six parallel experiments were then performed following the procedure outlined in this method. The results are shown in Tables 3, 4, and 5. The average recoveries of the analytes α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin were 100.55%, 97.65%, and 96.28%, respectively, with relative standard deviations of 1.53%, 1.81%, and 1.13%, respectively. This indicates that the recovery rate of this method is stable.

[0042] Table 3 Results of α-cyclodextrin recovery determination

[0043] Table 4 Results of β-cyclodextrin recovery determination

[0044] Table 5 Results of γ-cyclodextrin recovery rate determination

[0045] (ix) Precision Samples spiked with α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin at different concentrations (40.00 mg / L, 50.00 mg / L, and 60.00 mg / L) were processed. Each spiked sample was measured in triplicate according to the procedure described herein, and the corresponding values ​​were obtained. The results are shown in Table 6. The RSD was less than 5%, indicating good precision and meeting the requirements for quantitative analysis.

[0046] Table 6. Method precision verification data and results

[0047] (x) Limit of detection and limit of quantitation Prepare a 10% methanol aqueous solution as a blank sample. Perform 10 repeated determinations on the blank sample according to the procedure of this method, and record the signal-to-noise ratio (S / N) data of each determination. The results are shown in Tables 7, 8, and 9. The average signal-to-noise ratios (S / N) of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin were calculated to be 0.69, 0.83, and 0.70, respectively, with standard deviations of 0.07, 0.08, and 0.08, respectively. The limits of detection (LOD) of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin were found to be 2.07 mg / L, 2.49 mg / L, and 2.10 mg / L, respectively. The limits of quantitation were determined by multiplying the LOD by three times. The limits of quantitation for α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin were found to be 6.21 mg / L, 7.47 mg / L, and 6.30 mg / L, respectively.

[0048] Table 7. Confirmation data and results for the limit of detection and limit of quantitation of α-cyclodextrin.

[0049] Table 8. Confirmation data and results for the limit of detection and limit of quantitation of β-cyclodextrin.

[0050] Table 9. Confirmation data and results for the limit of detection and limit of quantitation of γ-cyclodextrin.

[0051] Table 10. Repeatability test confirmation data and results

[0052] Table 11. Method validation and analysis results

[0053] As shown in the table above, the detection method of this application has good detection limit and precision, and can accurately detect cyclodextrin in fermented mash.

[0054] Comparative Example 1: The Influence of Changes in Mobile Phase and Instrument Parameters on Detection Results Change the mobile phase and detection parameters of Example 1: (a) Instrumental analysis conditions An ultra-high performance liquid chromatography (UHPLC) system coupled with an electro-cavitation detector (CAD) was used. The selected column was an XBridge C18 4.6 mm × 250 mm 5 μm. UHPLC conditions were as follows: mobile phase: 10% methanol, 90% water; column temperature: 25 °C; flow rate: 1.0 mL / min; injection volume: 5 μl; injection mode: autosampler. CAD conditions were as follows: atomization temperature: 50.0 °C; acquisition frequency: 10 Hz; filtration constant: 5 s. Analysis time: 20.00 min. 1 mL of each of the three single-standard stock solutions was accurately pipetted into a 10 mL volumetric flask and diluted to the mark with ultrapure water to prepare a 100 mg / L mixed standard solution of α, β, and γ cyclodextrin, which was directly analyzed using the system.

[0055] (II) Qualitative Analysis Following the sample pretreatment method and instrument analysis conditions described above, blank spiked samples containing α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin were analyzed and detected to determine the retention time of each component on the CAD (electro-atomized detector), thus achieving qualitative analysis of cyclodextrin.

[0056] Experimental conclusion: The results are as follows Figure 2 As shown in the spectrum, the baseline is stable with minimal noise interference. The first cross-shaped peak appears at 4.05 min, the second at 4.80 min, and the third at approximately 6.50 min. Only three cross-shaped peaks are observed, with no other extraneous peaks. The analysis can be completed within 10 min. This demonstrates that the mobile phase concentration setting significantly impacts the accuracy of the analytical results.

[0057] Comparative Example 2: The Influence of Mobile Phase Selection and Instrument Parameter Variation on Detection Results Change the mobile phase and detection parameters of Example 1: (a) Instrumental analysis conditions An ultra-high performance liquid chromatography (UHPLC) system coupled with an electro-cavitation detector (CAD) was used. The selected column was an XBridge C18 4.6 mm × 250 mm 5 μm. UHPLC conditions were: mobile phase acetonitrile:water (10:90), column temperature 25℃, flow rate 1.0 mL / min, injection volume 5 μl, and autosampler. CAD conditions were: atomization temperature 50.0℃, acquisition frequency 5 Hz, and filtration constant 3.6 s. The analysis time was 15.00 min. 1 mL of each of the three single-standard stock solutions was accurately pipetted into a 10 mL volumetric flask, and diluted to the mark with ultrapure water to prepare a 100 mg / L mixed standard solution of α, β, and γ cyclodextrin, which was directly analyzed using the system.

[0058] (II) Qualitative Analysis According to the instrumental analysis conditions, blank spiked samples containing α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin were analyzed and detected to determine the retention time of each component on the CAD (electro-atomized detector), thereby achieving qualitative analysis of cyclodextrin.

[0059] Experimental conclusion: Changing the mobile phase yielded the following results. Figure 3 As shown in the spectrum, the baseline is stable with little noise interference. The first peak appears at approximately 0.08 min, the second at 2.44 min, and the third at 2.55 min. The peak elution time is very fast, and the peak separation effect is not obvious. The analysis can be completed within 3 min.

[0060] Comparative Example 3: Effect of Cyclodextrin Pretreatment on Quantitative Analysis in Distilled Grains The difference between this comparative example and Example 1 lies in the sample pretreatment steps: (a) Sample pretreatment Accurately weigh 5g of fermented mash sample and place it in a 50mL centrifuge tube. Add 25mL of ultrapure water to dissolve the mash, vortex to mix for 2min, and then sonicate at 40℃ for 30min. After sonication, remove the sample solution, cool it to room temperature, centrifuge at 4000 rpm for 5min, filter it through a 0.45μm aqueous filter membrane, and take the supernatant as the test sample.

[0061] The instrumental analysis conditions were the same as in Example 1, and the experimental results are as follows: Figure 4 As shown, Figure 4 The spectrum shows a stable baseline, with peaks appearing around 2 minutes in. However, there are many interfering peaks, making it impossible to identify the target peak.

[0062] Comparative Example 4: Effect of Cyclodextrin Pretreatment on Quantitative Analysis in Fermentation Mash The difference between this comparative example and Example 1 lies in the sample pretreatment steps: (a) Sample pretreatment Accurately weigh 5g of fermented mash sample and place it in a 50mL centrifuge tube. Add 50mL of ultrapure water to dissolve the mash, vortex to mix for 2min, and then sonicate at 60℃ for 30min. After sonication, remove the sample solution, cool it to room temperature, centrifuge at 4000 rpm for 5min, filter the solution to be tested, transfer it to an evaporating dish, heat it in a water bath at 60℃ until it is nearly dry, then add 2mL of methanol to the evaporating dish and shake it slowly. Filter the supernatant through a 0.45μm aqueous filter membrane and use the supernatant as the test sample.

[0063] The instrumental analysis conditions were the same as in Example 1, and the experimental results are as follows: Figure 5 As shown, Figure 5The spectrum shows a stable baseline, with peaks emerging around 2 minutes. There are many extraneous peaks, and by 3 minutes, these extraneous peaks merge with the target peaks (γ and α cyclodextrin), indicating poor separation.

[0064] Comparative Example 5: Effect of Cyclodextrin Pretreatment on Quantitative Analysis in Distilled Grains Using the same pretreatment method as Comparative Example 4, the chromatographic conditions were optimized, and the gradient program was set as follows:

[0065] Experimental results are as follows Figure 6 As shown, Figure 6 The spectrum shows a stable baseline, with peaks appearing around 3 minutes later. The separation of the interference peak from the target peak is not significant.

[0066] Comparative Example 6: Effect of Cyclodextrin Pretreatment on Quantitative Analysis in Distilled Grains Accurately weigh 5-10g of fermented mash sample and place it in a centrifuge tube. Add 50-100mL of 10% methanol aqueous solution as the extraction solvent, vortex to mix for 2 minutes, and then sonicate at 50-60℃ for 20-30 minutes. After sonication, remove the sample solution, cool to room temperature, centrifuge at 4000 rpm for 5 minutes, and filter the supernatant through a 0.45mm filter membrane. Use the supernatant as the test sample. After filtration, take the supernatant and analyze it according to the chromatographic conditions of the qualitative test method in Example 1.

[0067] Experimental conclusions: Adjusting the sample and solvent amounts, with a target analyte to solvent ratio ranging from 1-3:4-6, is sufficient for target analyte extraction. Optimizing the ultrasonic temperature and frequency, with a temperature of 50-60℃ and an ultrasonic time of 25-35 min, is sufficient to dissolve the cyclodextrin in the sample. The spectrum shows a stable baseline, with the target peak appearing at 4 min, 5 min, and 8 min, respectively. Figure 7 As shown, the influence of interference peaks on the target peak is reduced, and the separation effect is good.

[0068] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. A method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology, characterized in that, A system combining ultra-high performance liquid chromatography (UHPLC) with an electrospray detector was used. The detection conditions for the UHPLC were as follows: Chromatographic column: XBridge C18 4.6mm×250mm 5μm; or Phenomenex Gemini NX C18 4.6×250mm 5μm; Column temperature: 20℃; Mobile phase: 8%-9% methanol, 91%-92% water; Flow rate: 1.0 mL / min; The detection conditions for the electro-fog detector are: The electrofogging temperature was 50.0℃, the sampling frequency was 4.5Hz-6Hz, and the filtration constant was 3s-4s.

2. The method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology according to claim 1, characterized in that, The analysis time of the electro-fog detector is 13.00 min to 17.00 min.

3. The method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology according to claim 1, characterized in that, The injection volume was 5 μl.

4. The method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology according to claim 1, characterized in that, Specifically, the steps include: preparing a reference solution and a test solution; testing the reference solution and the test solution according to the detection conditions of claim 1 and recording the corresponding CAD detection signal peak areas to obtain the standard curve equation and the detection peak area of ​​the test sample; substituting the detection peak area of ​​the mash sample to be tested into the standard curve equation to calculate the cyclodextrin content in the mash.

5. The method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology according to claim 4, characterized in that, The preparation method of the test solution is as follows: Mix the fermented mash sample with the solvent at a ratio of 1-3:4-6 (g / mL), then extract by ultrasonication. After ultrasonication, remove the sample solution, cool it to room temperature, centrifuge, filter, and take the supernatant as the test solution.

6. The method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology according to claim 5, characterized in that, The solvent is a 10% aqueous methanol solution.

7. The method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology according to claim 5, characterized in that, The conditions for ultrasonic extraction are: 50℃-60℃ and ultrasonic extraction for 20min-30min.

8. The method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology according to claim 5, characterized in that, The centrifugation was performed at 4000 rpm for 5 minutes.

9. The method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology according to claim 5, characterized in that, The filtration process uses a 0.45 filter membrane.

10. The method for detecting cyclodextrin in fermented mash based on UHPLC-CAD technology according to claim 1, characterized in that, The reference solution was prepared by diluting the three standards of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin with 10% methanol aqueous solution to prepare mixed standard solutions of cyclodextrin, cyclodextrin, and cyclodextrin with concentrations of 100.00 mg / L, 50.00 mg / L, 25.00 mg / L, 10.00 mg / L, and 5.00 mg / L, respectively.

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