A method for extracting acrylamide in rhizoma polygonati and its application in liquid chromatography detection

Abstract

This invention belongs to the field of traditional Chinese medicine processing and testing technology, and relates to an ultrasonic-assisted extraction method and content determination method for acrylamide in processed Polygonatum sibiricum. The optimal extraction process for acrylamide from processed Polygonatum sibiricum is as follows: 1.0 g of Polygonatum sibiricum, 6 mL of a mixed solution of ethyl acetate and methanol as the extractant (volume ratio 6:4), ultrasonic time of 15 min, and ultrasonic power of 180 W. After purification using a C18 solid-phase extraction column, the acrylamide content was found to be 0.498 ± 0.0015 μg / g. This method is stable and reliable. Compared with a single water extractant, using a mixed solution of ethyl acetate and methanol as the extractant significantly improves the extraction efficiency, and the operation is simple, showing good market application prospects and industrial applicability.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine extraction and testing technology, specifically relating to a method for detecting acrylamide in wine-processed Polygonatum odoratum using liquid chromatography. Background Technology

[0002] The 2025 revised edition of the Chinese Pharmacopoeia defines Polygonatum as the plant Polygonatum yunnanensis, belonging to the Liliaceae family. Polygonatum kingianum Coll.et Hemsl., Polygonatum Polygonatum sibiricum Red. or Polygonatum multiflorum Polygonatum The dried rhizome of *Cyrtonema Hua.* is commonly known as "Dahuangjing," "Jitouhuangjing," or "Jiangxinghuangjing" depending on its shape. Fresh *Cyrtonema Hua.* is sourced from Liuhe County, Tonghua City, Jilin Province. *Cyrtonema Hua.* is sweet and neutral in nature, entering the spleen, kidney, and lung meridians. It has functions such as tonifying qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys. It is used for tonifying and strengthening the body, treating kidney deficiency and essence depletion, lung deficiency and dry cough, and spleen and stomach weakness. It is a commonly used tonic and strengthening medicine in traditional Chinese medicine. Studies have found that *Cyrtonema Hua.* has pharmacological effects such as lowering blood pressure, lowering blood sugar, lowering blood lipids, and anti-tumor activity.

[0003] Raw Polygonatum has a numbing taste and irritates the throat, so it usually needs to be processed before being used in medicine. Traditional Chinese medicine believes that processing can enhance the efficacy and reduce toxic side effects. During the wine-processing of Polygonatum, the temperature is high and the time is long. The reducing sugar and asparagine undergo a high-temperature dehydration condensation reaction to produce Schiff bases. The Schiff bases are then heated to obtain decarboxylated Schiff bases, which further generate acrylamide.

[0004] Literature reports the following methods for extracting acrylamide: Zhan Xumei et al. extracted acrylamide from foods processed by high-temperature frying and baking (biscuits, instant noodles, fried dough twists, bread, potato chips, etc.): 5.0 g of sample was added to 40 mL of ultrapure water, magnetically stirred for 30 min, and centrifuged for 20 min. The supernatant was taken and 10 mL of n-hexane was added, shaken thoroughly, allowed to stand and separate into layers, the lower layer of water was collected, centrifuged at 8000 r / min for 10 min, and the supernatant was collected in a 5 mL volumetric flask and diluted to volume with ultrapure water; the acrylamide content in biscuits was 220 μg / kg, the content in instant noodles was 423 μg / kg, the content in bread was 81 μg / kg, the content in fried dough twists was 172 μg / kg, and the content in potato chips was 688 μg / kg. [Zhan Xumei, Liu Ping, Qi Xingpu et al. Determination of acrylamide in high-temperature processed foods by high performance liquid chromatography [J]. Journal of Food Safety and Quality Inspection, 2020, 11(22): 8496-8501]. Sun Zhen et al. reported that fried chicken leg meat was minced using a meat grinder, 5 g was placed in a 50 mL centrifuge tube, a certain amount of n-hexane was added to remove fat, the mixture was dried with a nitrogen blower, then 20 mL of ultrapure water was added for ultrasonic extraction for 15 min, then 500 μL of Carrez I and Carrez II solution was added and shaken for 5 min, centrifuged at 10000 r / min, 4℃ for 10 min, the middle layer was filtered through a 0.22 μm aqueous filter membrane and set aside. The results showed that the acrylamide content in chicken leg meat from 5 supermarkets was 5.21±1.21 μg / g, 9.28±0.73, 3.43±0.08, 6.66±0.92, and 4.52±0.43 μg / g, respectively. [Sun Zhen, Zhou Guanghong. Determination of acrylamide content in fried chicken legs by high performance liquid chromatography [J]. Food Industry Technology, 2012, 22(016):67-70.].

[0005] Ye Yanfen et al. took 5.00 g of biscuits, added 20 mL of water, stirred magnetically for 20 min, centrifuged for 30 min, took the supernatant, added n-hexane to extract 3 times, then added 5 mL each of Carrez I and Carre II reagents and mixed well. Centrifuged at 4000 r / min for 20 min to remove protein; the levels in the three types of biscuits tested were 1.04, 4.88 and 6.94 μg / kg, respectively. [Ye Yanfen, Xu Liheng. Determination of acrylamide in flour-based foods by solid phase extraction-high performance liquid chromatography [J]. Analytical Laboratory, 2013, 32(07):95-97.].

[0006] Acrylamide is a common organic compound found in high-temperature processed foods, posing various health hazards. Currently, there are few reports on extraction and detection methods for acrylamide generated during the processing of traditional Chinese medicine (TCM) materials, particularly in high-temperature baked and fried foods, and specifically for its extraction and liquid chromatography detection. Because the acrylamide content in food matrices is extremely low (typically in the μg / kg range) and accompanied by numerous coexisting interfering substances, effective enrichment and purification methods are needed to eliminate impurities and ensure more accurate and reliable detection results. This invention utilizes response surface methodology to screen a simple, practical, efficient, and stable extraction process for acrylamide in Polygonatum sibiricum, which can be widely applied to TCM processing and food processing systems, showing promising application prospects. Summary of the Invention

[0007] To address the issues of low acrylamide content and high detection costs in processed Polygonatum sibiricum, this invention provides a method for the extraction and determination of acrylamide in processed Polygonatum sibiricum, solving the problems of high cost and difficulty in detection in the food industry.

[0008] In a first aspect of the present invention, the wine-processed Polygonatum is subjected to ultrasonic extraction using a mixed solution of ethyl acetate and methanol as the extractant.

[0009] Preferably, the volume ratio of the extractant ethyl acetate to methanol is 4:6-8:2, and more preferably 6:4.

[0010] Preferably, the ultrasound duration is 5-25 minutes, more preferably 15 minutes.

[0011] Preferably, the ultrasonic power is 120-240 W, and more preferably 180 W.

[0012] Based on the above-mentioned extraction method, using 6 mL of a mixed solution of ethyl acetate and methanol in a volume ratio of 6:4, an ultrasonic time of 15 min, and an ultrasonic power of 180 W, the acrylamide extraction rate reached 0.498 ± 0.0015 μg / g.

[0013] A second aspect of the present invention provides a method for testing acrylamide in processed Polygonatum sibiricum products, comprising the following steps: Take 1.0 g of processed Polygonatum sibiricum powder, add 6 mL of a mixture of ethyl acetate and methanol (volume ratio 6:4) as extractants, mix for 3 min on a high-speed mixer, and then extract using ultrasonication at room temperature. Centrifuge at 5000 r / min for 5 min and collect the supernatant. Repeat the above steps once, and combine the two extracts for further purification. (C) 18 A solid-phase extraction (SPE) adsorption column (500 mg, 3 mL) was inserted into the vacuum pump system. The column was first activated with 3 mL of methanol, and the supernatant was added to the column. The eluent was collected and evaporated to dryness under nitrogen at 0.4 L / min in a fume hood. The volume was then adjusted to 2 mL with ultrapure water, followed by the addition of 2 mL of n-hexane. The mixture was stirred for 3 min, allowed to stand for separation, and then centrifuged at 3000 rpm for 5 min. The lower layer was collected and filtered through a 0.22 μm microporous membrane to obtain the test solution, which was then used for determination by liquid chromatography.

[0014] Preferably, the chromatographic detection conditions of the liquid chromatograph are: methanol:water (containing 0.1% formic acid) = 10:90 (v / v), flow rate 1.0 mL / min, chromatographic column: Phenomenex Luna C18, column temperature: 30℃, UV wavelength: 210 nm, injection volume: 20 μL, target acrylamide peak elution time: 4-5 min, elution time: 10 ± 2 min. The methodological requirements for resolution, recovery rate, precision, stability, and limit of detection are all met. The technical solution of the present invention is as follows: 1. Establish a reaction system model (1) Preparation method of Polygonatum sibiricum slices Take fresh Polygonatum medicinal material, remove impurities and roots, wash it clean; steam it for 40 minutes, cut it into 0.4-0.6 cm thick slices, and dry it at 60℃ for 12 hours to obtain Polygonatum slices.

[0015] Brewing and moistening process Take the Polygonatum slices from step (1), add 20 kg of rice wine to every 100 kg of Polygonatum slices, with a wine-to-water ratio of 1:1.875, and stir the Polygonatum evenly. Add the wine evenly according to the number of steaming cycles, let it soak for 80 min, steam it in a water bath for 6 h, and dry it in an oven at 60 ℃ for 12-13 h. One steaming cycle yields one sample; the process can be repeated 1-9 times.

[0016] Ultrasonic-assisted extraction of acrylamide from Polygonatum sibiricum samples The effect of the volume ratio of ethyl acetate to methanol as extractant: with a fixed ultrasonic time of 15 min, ultrasonic power of 180 W, and 6 mL of ethyl acetate and methanol mixed solution, the effects of extractant volume ratios of 4:6, 5:5, 6:4, 7:3, and 8:2 (V:V) were studied.

[0017] Ultrasonic time factor: Under the optimal extraction solvent ethyl acetate to methanol volume ratio of 6 mL, and with a fixed ultrasonic power of 180 W, the effect of ultrasonic time of 5 min, 10 min, 15 min, 20 min and 25 min was studied.

[0018] Ultrasonic power factor: The effect of ultrasonic power at 120 W, 150 W, 180 W, 210 W, and 240 W on 6 mL of mixed extract solution, according to the optimal volume ratio and ultrasonic time.

[0019] Response surface methodology: Based on single-factor experiments, a three-factor, three-level experimental design was conducted using a 6 mL mixture of ethyl acetate and methanol as the extractant, with the volume ratio of ethyl acetate to methanol, ultrasonic time, and ultrasonic power as independent variables, and the acrylamide extraction rate as the response value. A total of 17 experimental groups were measured.

[0020] Accurately weigh 1.0 g of the processed Polygonatum sibiricum product, pulverize it using a universal grinder, add 6 mL of extractant ethyl acetate and methanol, mix well on a high-speed mixer for 3 min, and then extract ultrasonically at room temperature. Centrifuge at 5000 r / min for 5 min, and collect the supernatant. Repeat the above steps once, and combine the two supernatants. (The last sentence appears to be incomplete and possibly refers to C.) 18The solid-phase extraction adsorption column was inserted into the vacuum pump system and activated with 3 mL of methanol. The supernatant was added to the column, and the eluent was collected. The column was then evaporated under nitrogen at 0.4 L / min in a fume hood. The volume was brought to 2 mL with ultrapure water, and 2 mL of n-hexane was added. The mixture was stirred for 3 min, allowed to stand for separation, and then centrifuged at 3000 r / min for 5 min. The lower layer was filtered through a 0.22 μm microporous membrane to obtain the test solution, which was used for determination by liquid chromatography.

[0021] (4) Determine the acrylamide content in the sample using high performance liquid chromatography (HPLC). ① Sample determination: First, determine the peak time of acrylamide standard, and then determine the sample in sequence; ② Set the chromatographic conditions: methanol:water (containing 0.1% formic acid) = 10:90, flow rate of 1.0 mL / min, Phenomenex Luna C18 column, column temperature of 30℃, UV wavelength of 210 nm, and injection volume of 20 μL; use a diode array detector (DAD) to scan the full spectrum of the sample.

[0022] ③ Data processing and analysis: 6 mL of a mixed solution of ethyl acetate and methanol with a volume ratio of 6:4, ultrasonic time of 15 min, ultrasonic power of 180 W, and the optimal extraction rate of acrylamide was 0.498 ± 0.0015 μg / g.

[0023] Compared with the prior art, the present invention has the following advantages: This invention optimizes the extraction of acrylamide using an ultrasound-assisted method. Combined with C 18 Solid-phase extraction column purification + hexane impurity removal effectively eliminates interference from other components of Polygonatum odoratum. The liquid chromatogram shows a sharp target peak without impurity peaks, eliminating the need for Carrez reagent purification steps and simplifying the sample pretreatment process.

[0024] The mixed solvent of ethyl acetate and methanol exhibits rapid evaporation, low residue, and high concentration efficiency during nitrogen blowing. Conventional water extraction has low nitrogen blowing efficiency, and if not completely evaporated, it affects the accuracy of subsequent volume adjustment and filtration steps.

[0025] After using the extraction method described above, the extract was pale yellow, indicating that the amount of colored impurities extracted from the Polygonatum odoratum was extremely small. In contrast, the aqueous extract obtained using the literature method was dark red, indicating that water-soluble pigments and other impurities were dissolved, affecting the accurate quantification of acrylamide.

[0026] Comparative studies showed that multi-wavelength testing significantly increased background absorption and baseline noise during UV detection, resulting in strong background interference. This further demonstrates that the extraction method described in the comparative studies affects the uncertainty and accuracy of content determination.

[0027] The chromatographic conditions of this invention are: methanol:0.1% formic acid water = 10:90, detection wavelength 210 nm. Acrylamide elutes at 4-5 min, with no interfering peaks within 10 min. This invention eliminates the need for a mass spectrometer detector, achieving accurate quantification using only a conventional liquid chromatograph with a DAD detector, significantly reducing instrument costs while ensuring detection quality.

[0028] The pretreatment process is simple to operate and suitable for routine quality control in the processing of Chinese medicinal materials and food processing systems. It has good market application prospects and industrial applicability. Attached Figure Description

[0029] Figure 1 Acrylamide standard curve; Figure 2 The curve showing the effect of the volume ratio of ethyl acetate and methanol as extractants on the extraction rate; Figure 3 The curve showing the effect of ultrasonic time on the extraction rate; Figure 4 The curve showing the effect of ultrasonic power on extraction rate; Figure 5 Three-dimensional response surface plot of extraction rate to volume ratio of ethyl acetate to methanol, ultrasonic time, and ultrasonic temperature; Figure 6 A liquid chromatogram of acrylamide produced from Polygonatum sibiricum through nine distillations and nine sun-drying processes; Figure 7 The curve showing the change in the content of Polygonatum sibiricum in wine produced through nine distillations and nine sun-d Figure 8 A comparison diagram of the extraction solutions obtained by the method of this patent and those obtained by methods in prior literature; Figure 9 Acrylamide standard, liquid chromatography two-dimensional imaging isograph of the method of this patent and the method of comparative literature. Detailed Implementation

[0030] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments: Example

[0031] This embodiment provides a method for determining acrylamide content using HPLC. Five different concentrations of acrylamide reference standards were accurately weighed. The liquid chromatography conditions were: methanol:water (containing 0.1% formic acid) = 10:90 (v / v), Phenomenex Luna C18 column, flow rate 1.0 mL / min, column temperature 30℃, UV wavelength set to 210 nm, injection volume 20 μL. The standard curve is shown below. Figure 1The result, Y = 3145.1x - 0.019 (r = 0.9997), shows a good linear relationship with peak area in the range of 0.625 μg / mL to 10 μg / mL. The recovery, precision, and stability tests all meet the methodological requirements, with RSD < 3%. Example

[0032] This embodiment provides a method for preparing Polygonatum sibiricum. Preparation method of Polygonatum sibiricum slices: Take fresh Polygonatum sibiricum, remove impurities and roots, and wash it; steam it in a water bath for 40 min, cut it into 0.4-0.6 cm thick slices, and dry it at 60℃ for 12 h to obtain Polygonatum sibiricum slices. Add 20 kg of rice wine to every 100 kg of Polygonatum sibiricum slices, and add the rice wine to the sample at a mass ratio of 1:1.875. Stir the Polygonatum sibiricum evenly, and add the rice wine evenly according to the number of steaming cycles. Let it soak for 80 min; steam it in a water bath for 6 h, and dry it in an oven at 60℃ for 12-13 h. Steam it once to obtain a sample that has been steamed once; repeat the steaming process 1-9 times. Refer to the teachings in the prior art CN202110840274.1 for the preparation of Polygonatum sibiricum slices.

[0033] Example 3: Single-factor experiment on the extraction of acrylamide using ultrasound-assisted method This embodiment provides a method for extracting acrylamide from processed Polygonatum sibiricum.

[0034] Investigate the volume ratio of ethyl acetate and methanol as extractants Five groups of processed Polygonatum sibiricum products from Example 2 were taken, and the ultrasonic time was fixed at 15 min, ultrasonic power at 180 W, and 6 mL of a mixed solution of ethyl acetate and methanol were used to study the effects of volume ratios of 4:6, 5:5, 6:4, 7:3, and 8:2. Acrylamide content was determined by HPLC according to the method described in Example 1, and the optimal volume ratio was found to be 6:4.

[0035] (2) Examination of ultrasound time Take 1.0 g of the processed Polygonatum sibiricum from each of the five groups in Example 2. According to the optimal extraction agent ethyl acetate and methanol volume ratio of 6:4 in Example 3 (1), fix the extraction agent volume at 6 mL and the ultrasonic power at 180 W. Study the effect of ultrasonic time at 5 min, 10 min, 15 min, 20 min and 25 min. Determine the acrylamide content according to the HPLC method in Example 1. The optimal ultrasonic time is selected as 15 min.

[0036] (3) Examine the ultrasonic power Take 1.0 g of the processed Polygonatum sibiricum from each of the five groups in Example 2, 6 mL of the extractant with the optimal ethyl acetate to methanol volume ratio of 6:4 selected in Examples 3(1) and 3(2), and sonicate for 15 min. Under the influence of ultrasonic power of 120 W, 150 W, 180 W, 210 W and 240 W, respectively, determine the acrylamide content according to the HPLC method in Example 1, and the ultrasonic power of 180 W is selected.

[0037] Table 1. Effects of different factors on acrylamide extraction rate

[0038] Extraction rate refers to the ratio (μg / g) of acrylamide content in the extract of processed Polygonatum odoratum in a mixed solution of ethyl acetate and methanol to the mass of the processed product.

[0039] Example 4: Response Surface Methodology Experiment for Acrylamide Extraction Using Ultrasonic Assistance Based on the single-factor experiments, a fixed 6 mL mixture of ethyl acetate and methanol was used. The volume ratio of ethyl acetate to methanol, ultrasonic time, and ultrasonic power were selected as independent variables, and the acrylamide extraction rate was selected as the response value. A total of 17 experimental groups were measured. The response surface methodology was designed using the statistical analysis software Design-Expert.13. The measurement method was the same as in Example 1, as shown in Table 2.

[0040] Table 2 Response Surface Experimental Factor Level Design

[0041] A three-factor, three-level experimental design was conducted using Design-Expert.13 software based on the X-Behnken design principle. The experimental design and response values ​​are shown in Table 3, and the model variance analysis is shown in Table 4.

[0042] Table 3. Response Surface Experiment Scheme and Results

[0043] Table 4. Analysis of Variance for Response Surface Model

[0044] The data in Table 3 were analyzed using Design Expert V.13 software for fitting regression, yielding a quadratic regression equation for the peak area based on three factors: ethyl acetate:methanol (X1), ultrasonic time (X2), and ultrasonic power (X3). An analysis of variance was performed on the model in Table 3, and the results are shown in Table 4. The analysis of variance was also performed on the above regression model. High F-value (54.30) and low... P The value (0.0001) indicates that the model is highly significant. The coefficient of determination (R²) 2The accuracy is 0.9859, indicating that the model has good reliability and can reflect 94.32% of the response value variation. The precision value (18.1904 > 4) indicates that the model has good accuracy. As shown in Table 4, when P When < is 0.05, the linear coefficients (X1, X3, X2, X3, X1) 2 X2 2 X3 2 The conditions had a significant impact, but the interaction term had no significant impact. Therefore, the interaction between the factors was not significant. The order of influence of conditions on acrylamide extraction rate was ultrasonic power (X3), ethyl acetate:methanol (X1), and ultrasonic time (X2). The interaction effects of operating parameters on acrylamide yield were studied using three-dimensional response surface methodology (3D) and two-dimensional (2D) contour plots, as follows. Figure 5 As shown. Three-dimensional plots are used to represent the relationships and interactions between variables and response values. In two-dimensional contour plots, the interactions between variables represented by elliptical contour plots are significant. Conversely, circular contour plots indicate that the interactions are negligible. Contour plots and three-dimensional response surfaces derived from response surfaces show the interactions between two independent factors and the optimal values ​​of each experimental variable. Figure 5 The three-dimensional response surface and contour plot in (A) illustrate the interaction between the extractant volume ratio and the ultrasonic time. In the initial stage of the reaction, increasing the extractant volume ratio and ultrasonic time increases the yield of acrylamide, but after reaching a certain level, the yield no longer increases. Figure 5 (B) and (C) show the relationship between the extractant volume ratio and ultrasonic power, and the relationship between ultrasonic time and ultrasonic power, respectively. The acrylamide yield is consistent with the results of the single-factor experiments, i.e., the yield first increases significantly and then decreases significantly with the increase of the liquid-to-solid ratio. The optimal extraction conditions obtained by Design-Expert 13 are: 6 mL extractant, volume ratio of 6.357:3.643, ultrasonic time of 16.628 min, and ultrasonic power of 180 W. Under these conditions, the predicted acrylamide extraction rate is 0.498 μg / g. The optimal experiment designed with this model was used for verification. For ease of operation, the optimal conditions can be adjusted to: 6 mL extractant with an ethyl acetate to methanol volume ratio of 6:4, ultrasonic time of 15 min, ultrasonic power of 180 W, and a predicted extraction rate of 0.496 μg / g. Therefore, by optimizing the volume ratio of the extractant mixture, ultrasonic time, and ultrasonic power, the acrylamide yield can be effectively improved. The optimal extraction process was validated in three parallel trials, and the results are shown in Table 5. The extraction rate of the three parallel trials was 0.498 ± 0.0015 μg / g, indicating that the process is stable and feasible.

[0045] Table 5 Process Validation

[0046]

[0047] Example 5 This embodiment provides a method for extracting acrylamide from processed Polygonatum sibiricum. Weigh 1.0 g of the obtained processed Polygonatum sibiricum product, pulverize it using a universal pulverizer, and extract it according to the optimal ultrasonic process parameters obtained in Examples 3 and 4: ethyl acetate to methanol volume ratio of 6:4, extractant 6 mL, ultrasonic time 15 min, ultrasonic power 180 W, mix for 3 min on a rapid mixer, and extract ultrasonically at room temperature. After centrifugation at 5000 r / min for 5 min, collect the supernatant. Repeat the above steps once, and combine the two extracts. (The last sentence appears to be incomplete and possibly refers to C.) 18 The solid-phase extraction adsorption column was activated with 3 mL of methanol, and the supernatant was added to the column. The eluent was collected and evaporated to dryness under nitrogen at 0.4 L / min in a fume hood. The volume was then adjusted to 2 mL with ultrapure water, and 2 mL of n-hexane was added. The mixture was stirred for 3 min, allowed to stand for separation, and then centrifuged at 3000 r / min for 5 min. The lower layer was collected and filtered through a 0.22 μm microporous membrane to obtain the test solution, which was used for determination by liquid chromatography.

[0048] Example 6 This embodiment provides a method for determining the acrylamide content in Polygonatum sibiricum. The acrylamide content of Polygonatum sibiricum produced by nine-distillation and nine-sun-drying was detected under the optimal extraction process in Example 4. The liquid chromatogram is shown below. Figure 6 As shown, the results are as follows Figure 7 As shown, the acrylamide content of P0 was 0.058 μg / g. The contents of the first to ninth distillations (P1-P9) were 0.082, 0.252, 0.363, 0.421, 0.472, 0.498, 0.397, 0.38, and 0.348 μg / g, respectively. The content of the acrylamide in the wine products was higher than that in the sliced ​​wine, showing a trend of first increasing and then decreasing, with the highest content at the sixth distillation (P6).

[0049] Example 7 Comparative Test The method described in this patent is as follows: Take 1.0 g of hexadecanoic acid powder (six-distilled), add 6 mL of a mixed solution of ethyl acetate and methanol (volume ratio 6:4), and sonicate for 15 min at an optimal power of 180 W. The chromatographic conditions are: methanol:water (containing 0.1% formic acid) = 10:90, flow rate 1.0 mL / min, column temperature 30℃, UV wavelength set to 210 nm, and injection volume 20 μL. The liquid chromatogram is shown below. Figure 6 As shown.

[0050] Literature comparison method: Take 5 g of six-times-distilled Polygonatum sibiricum, place it in a 50 mL centrifuge tube, add a certain amount of n-hexane, defatt it twice, and dry it in a fume hood using a nitrogen blower; then add 20 mL of ultrapure water, and extract it by sonication for 15 min; then add Carrez I and Carrez II solutions, shake for 5 min, centrifuge at 4℃ and 10000 r / min for 10 min, take the middle layer liquid, filter it through a 0.22 μm aqueous filter membrane, and set it aside. [Sun Zhen, Zhou Guanghong, Xu Xinglian, et al. Determination of acrylamide content in fried chicken legs by high performance liquid chromatography [J]. Food Industry Technology, 2012, 33(22):67-70.]. Ultrasonic power 180 W. Chromatographic conditions were the same as above.

[0051] like Figure 8 As shown, this invention uses an ethyl acetate-methanol mixed solvent to extract acrylamide, and the resulting extract is pale yellow. In contrast, the aqueous extract from the literature method is dark red, indicating a significant color difference. This suggests that a large amount of water-soluble pigments and other impurities are dissolved, which will affect the accuracy of subsequent purification and quantitative testing.

[0052] Two-dimensional spectral contour maps were generated using full-wavelength data acquired by DAD. Figure 9 The full-spectral absorption distribution characteristics in the 190–295 nm wavelength range at different retention times are visually presented in contour form. At a detection wavelength of 210 nm, the signal peak at 4.8 min is clear, and the separation meets the requirements. Impurity peaks in the 3.0–5.0 min range only show weak responses in non-characteristic wavelength regions, indicating that the absorption of impurities is weak and does not affect content determination. Compared with literature methods, the baseline noise difference is significant in the 240–295 nm range, with denser background noise, reflecting that impurities in the sample matrix have strong absorption in the 240–295 nm range, affecting the uncertainty and accuracy of sample detection wavelength quantification.

[0053] This patent effectively reduces the extraction of colored substances in the Polygonatum matrix and the background absorption in the ultraviolet region, thereby improving the accuracy of the test.

[0054] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for extracting acrylamide from Polygonatum sibiricum processed into wine, characterized by: Ultrasonic extraction was used, with a mixed solution of ethyl acetate and methanol as the extractant, and C was used. 18 The acrylamide content was determined by HPLC after purification using a solid-phase extraction column, nitrogen blowing, redissolution, extraction, and detection.

2. The method according to claim 1, wherein the extractant ethyl acetate and methanol mixed solution is 6 mL in volume ratio of 4:6 to 8:2, preferably 6:

4.

3. The method according to claim 2, wherein the ultrasound time is 5-25 min, preferably 15 min; and the ultrasound power is 120 W-240 W, preferably 180 W.

4. The method according to claim 1, C 18 Solid-phase extraction column parameters are: particle size 40-60 μm, pore size 60 Å, specific surface area 300 m². 2 / g, C content 16-19%, packing weight 500 mg, column capacity 3 mL.

5. The application of the method according to claim 1 in liquid chromatography detection, characterized in that: Take 1.0 g of Polygonatum odoratum powder, add 6 mL of a mixed solution of ethyl acetate and methanol (volume ratio 6:4), mix for 3 min on a rapid mixer, and then extract with ultrasound at room temperature. Centrifuge at 5000 r / min for 5 min and collect the supernatant. Repeat the above steps once and combine the supernatants from both extractions. Insert a C18 solid-phase extraction adsorption column into the vacuum pump system, activate it with 3 mL of methanol, add the supernatant to the column, and collect the eluent. Blow with nitrogen at 0.4 L / min in a fume hood until dry, bring the volume to 2 mL with ultrapure water, add 2 mL of n-hexane, mix for 3 min on a mixer, allow to stand for separation, and then centrifuge at 3000 r / min for 5 min. Take out the lower layer after centrifugation, filter it through a 0.22 μm microporous membrane to obtain the test solution, which is used for determination by liquid chromatography.

6. The application of the method according to claim 5 in liquid chromatography detection, characterized in that: The mobile phase volume ratio was methanol:water (containing 0.1% formic acid) = 10:90, the flow rate was 1.0 mL / min, the chromatographic column was Phenomenex Luna C18, the column temperature was 30℃, the UV wavelength was set to 210 nm, the injection volume was 20 μL, the acrylamide peak elution time was 4-5 min, the elution time was 10 ± 2 min, and the resolution met the requirements.

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