Method for detecting deoxynivalenol in milk and milk products

Abstract

The application discloses a method for detecting deoxy-najinmycin in milk and dairy products. The method comprises the following steps: extracting a sample solution to be derivatized by using an extracting agent, adding a buffer solution and a derivatization reagent to the sample solution to be derivatized to perform derivatization, performing liquid chromatography test on the sample to be tested after the derivatization, and qualitatively and quantitatively determining the sample according to the retention time and external standard method. The content of deoxy-najinmycin in the sample to be tested is obtained according to the test result. The extracting agent is an acetonitrile aqueous solution, the buffer solution is a potassium borate buffer solution, and the derivatization reagent comprises a fluorenylmethyloxycarbonyl chloride acetonitrile solution, a glycine solution and an acetic acid aqueous solution. The precision, repeatability, recovery rate and standard curve linearity of the detection method meet the industry requirements. The method has good extraction effect, high sensitivity, stable and accurate results, and is simple to operate and convenient to popularize.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, specifically relating to a method for detecting deoxynojirimycin in milk and dairy products. Background Technology

[0002] Deoxynojirimycin, chemically known as (2R,3R,4R,5S)-2-hydroxymethylpiperidine-3,4,5-triol (DNJ), is a piperidine alkaloid. DNJ is not unique to mulberry trees; it is also found in plants, animals, and microorganisms. Its structure shares some similarities with the natural substrate of glucosidase, and it can inhibit the activity of various enzymes involved in glucose conversion. It exhibits particularly strong inhibitory activity against α-glucosidase, and thus plays important roles including lowering blood sugar for diabetes treatment, inhibiting tumor metastasis and viral activity, and anti-HIV activity.

[0003] Modern diets, characterized by high calories and fat, coupled with reduced physical activity and an aging population, have led to a quiet increase in the incidence of diseases such as diabetes and cancer. Developing food-derived active ingredients with low blood sugar and anti-cancer effects and minimal side effects, and researching their application in food and pharmaceuticals, has become a research hotspot. Deoxynojirimycin, as an important functional factor in pharmaceuticals and food, is being widely used in the food industry. Establishing detection methods for deoxynojirimycin can provide data support for food research and development, while also helping to ensure product quality and safety.

[0004] Research has found that the main methods for determining deoxynojirimycin include spectrophotometry, gas chromatography, liquid chromatography-mass spectrometry, and high-performance liquid chromatography (HPLC). Spectrophotometry has fewer steps, readily available reagents, and low instrument requirements, but suffers from significant matrix interference and poor precision. Gas chromatography is cumbersome and time-consuming, while HPLC is costly. HPLC has become the primary method for determining deoxynojirimycin. The existing method for deoxynojirimycin detection, GB / T40642-2021, "Detection of 1-Deoxynojirimycin in Mulberry Leaf Extract - HPLC," primarily targets the detection of DNJ content in mulberry leaf extracts.

[0005] However, current research on milk and dairy products is limited, and applying the aforementioned method for detecting deoxynojirimycin in mulberry leaf extract to the detection of milk and dairy products presents the following problems: 1. Because milk and dairy products contain substances such as fat and protein, this method cannot obtain a clear solution, making it unsuitable for instrumental analysis; 2. Using the elution procedure and column type mentioned in the literature results in numerous extraneous peaks at the baseline, making it impossible to identify the target peak. This indicates that the mulberry leaf detection method is not suitable for the detection of milk and dairy products.

[0006] Therefore, it is necessary to optimize or develop new detection methods to meet the requirements for the detection of deoxynojirimycin in milk and dairy products. Summary of the Invention

[0007] To address the aforementioned problems, the present invention aims to provide a method for detecting deoxynojirimycin in milk and dairy products. This method is capable of detecting deoxynojirimycin in milk and dairy products, and features good extraction efficiency, short detection time, and stable and accurate results.

[0008] Based on this, the present invention provides a method for detecting deoxynojirimycin in milk and dairy products, comprising:

[0009] (1) Add the extractant to the sample to be tested and make up the volume. The ratio of the sample volume to the volume of the final volume is (2-10).

[0010] g: (25-100) mL, precipitate proteins and fats, centrifuge and take the supernatant to obtain the sample solution to be derivatized;

[0011] (2) Add buffer solution and derivatizing agent to the sample solution to be derivatized to obtain the derivatized sample solution;

[0012] (3) Perform liquid chromatography on the derivatized sample solution to obtain the content of deoxynojirimycin in the sample to be tested;

[0013] The extractant is a 40%–80% aqueous solution of acetonitrile.

[0014] In a preferred embodiment, the extractant is a 60% aqueous acetonitrile solution.

[0015] In some implementations, the sample to be tested is milk and dairy products, preferably modified milk, fermented dairy products or milk beverages.

[0016] In some embodiments, the buffer solution is a 0.2–0.6 mol / L potassium borate buffer solution (pH = 8.5). As a preferred embodiment, the buffer solution is a 0.4 mol / L potassium borate buffer solution (pH = 8.5).

[0017] In some embodiments, the derivatizing agent includes a 3-10 mmol / L fluorenyl methoxycarbonyl chloride (FMOC-CL) acetonitrile solution, a 0.5-5 mol / L glycine solution, and a 0.5%-10% aqueous acetic acid solution.

[0018] As a preferred embodiment, the derivatizing agent comprises a 5 mmol / L fluorenyl methoxycarbonyl chloride (FMOC-CL) acetonitrile solution, a 1 mol / L glycine solution, and a 1% aqueous acetic acid solution.

[0019] In some implementations, step (2) includes:

[0020] Add potassium borate buffer solution (pH=8.5) and fluorenemethyloxycarbonyl chloride acetonitrile solution to the sample solution to be derivatized, mix and react for 15-35 min under constant temperature water bath conditions of 20-30℃. Then add glycine solution and react for 20-30 min. Then add acetic acid aqueous solution and water, mix well and filter with organic phase filter membrane to obtain derivatized sample solution.

[0021] In some implementations, the volume ratio of the sample solution to be derived, potassium borate buffer solution (pH=8.5), fluorenemethyloxycarbonyl chloride acetonitrile solution, and glycine is 50-200:100-200:200-300:50-200.

[0022] In some implementations, the volume ratio of the sample solution to be derived to the aqueous acetic acid solution is 50-200:50-200.

[0023] In a preferred embodiment, the volume ratio of the sample solution to be derived, potassium borate buffer solution (pH=8.5), fluorenemethyloxycarbonyl chloride acetonitrile solution, and glycine is 100:175:250:100.

[0024] In a preferred embodiment, the volume ratio of the sample solution to be derived to the aqueous acetic acid solution is 100:75.

[0025] In some implementations, step (2) includes:

[0026] Add 100-200 μL of potassium borate buffer solution (pH=8.5) and 200-300 μL of fluorene methoxycarbonyl chloride (FMOC-CL) acetonitrile solution to 50-200 μL of the sample solution to be derivatized. Mix and react in a constant temperature water bath at 20-30℃ for 15-30 min to carry out derivatization. Then add 50-200 μL of glycine solution and react for 10-30 min to neutralize excess fluorene methoxycarbonyl chloride. Finally, add 50-200 μL of acetic acid aqueous solution. Water is used to replenish the derivatization system so that the final derivatization system is 1-2 ml, thus obtaining the derivatized sample solution.

[0027] In some implementations, step (2) includes:

[0028] Add 100-200 μL of potassium borate buffer solution (pH=8.5) and 200-300 μL of fluorene methoxycarbonyl chloride (FMOC-CL) acetonitrile solution to 50-200 μL of the sample solution to be derivatized. Mix and react in a constant temperature water bath at 25℃ for 25 min to carry out derivatization. Then add 50-200 μL of glycine solution and react for 20 min to neutralize excess fluorene methoxycarbonyl chloride. Then add 50-200 μL of acetic acid aqueous solution and water. Water is used to replenish the derivatization system so that the final derivatization system is 1-2 ml. Filter with a 0.45 μm organic phase filter membrane to obtain the derivatized sample solution.

[0029] In some embodiments, the organic phase filter membrane has a specification of 0.45 μm or 0.22 μm.

[0030] In a specific embodiment of the present invention, the organic phase filter membrane has a specification of 0.45 μm.

[0031] In some embodiments, the detector for the liquid chromatograph is an ultraviolet detector.

[0032] In some implementations, the liquid chromatography conditions are as follows: Eclipse XDB-C18 5μm column.

[0033] The column thermometer was 4.6*250mm, the detection wavelength was 254nm, the column oven temperature was 30℃, the injection volume was 10μL, the flow rate was 1.0ml / min, the mobile phase A was acetonitrile, the mobile phase B was 0.1% acetic acid water, and the elution method was gradient elution.

[0034] 10. The detection method according to any one of claims 1-6, wherein the gradient elution procedure is as follows:

[0035] 0.00 min: Mobile phase A is 32%, mobile phase B is 68%;

[0036] 12.00 min: Mobile phase A is 32%, mobile phase B is 68%;

[0037] 16.00 min: Mobile phase A is 80%, mobile phase B is 20%;

[0038] 24.00 min: Mobile phase A is 80%, mobile phase B is 20%;

[0039] 29.00 min: Mobile phase A is 32%, mobile phase B is 68%;

[0040] 35.00 min: Mobile phase A is 32%, mobile phase B is 68%.

[0041] In some implementations, the detection method further includes plotting a standard working curve.

[0042] In some embodiments, the standard working curve plotting includes the preparation of standard solutions; the standard solutions include stock solutions and standard curve working solutions. In some embodiments, the stock solution and standard curve working solution of deoxynojirimycin are prepared using acetonitrile aqueous solution.

[0043] Beneficial effects:

[0044] This invention provides a method for detecting deoxynojirimycin in milk and dairy products. The method involves extracting a sample solution to be derivatized using an extractant, then adding a buffer solution and a derivatizing reagent to the sample solution for derivatization. After derivatization, the sample is subjected to high-performance liquid chromatography (HPLC), with retention time for qualitative analysis and external standard method for quantitative analysis. The content of deoxynojirimycin in the sample is determined based on the test results. The extractant is an aqueous acetonitrile solution, the buffer solution is a potassium borate buffer solution, and the derivatizing reagents include fluorenemethyloxycarbonyl chloride acetonitrile solution, glycine solution, and aqueous acetic acid solution. The precision, repeatability, recovery rate, and linearity of the standard curve provided by this invention all meet industry requirements. This method has good extraction efficiency, high sensitivity, stable and accurate results, is simple to operate, and is easy to promote.

[0045] The method for determining deoxynojirimycin in milk and dairy products of the present invention is simple and easy to perform, has good extraction effect of deoxynojirimycin, and the detection results are stable and accurate. Attached Figure Description

[0046] Figure 1 The results of using deoxynojirimycin with different detectors are shown.

[0047] Figure 2 The graph shows a comparison of the recovery rates of different extractants.

[0048] Figure 3 The recovery rates corresponding to different acetonitrile concentrations are shown.

[0049] Figure 4 The recoveries and chromatograms for different derivatization systems are shown.

[0050] Figure 5 The graph shows a comparison of the detection effects of different gradient elution procedures. Detailed Implementation

[0051] To provide a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below.

[0052] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0053] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0054] Unless otherwise stated, the experimental methods, detection methods, and preparation methods not described in detail in this invention all adopt conventional techniques in this technical field.

[0055] The method for detecting deoxynojirimycin provided by the present invention includes extracting the sample to be tested, precipitating proteins and fats, derivatizing the extract, performing liquid chromatography on the derivatized sample solution, and analyzing the content of deoxynojirimycin in the sample to be tested based on the test results.

[0056] The sample tested in this invention is a modified milk containing raw milk, milk mineral salts, lactase, etc.

[0057] The extraction step includes weighing 5.0g of sample into a 50mL volumetric flask, adding 60% acetonitrile aqueous solution to dilute to the mark, and mixing well.

[0058] The centrifugation procedure includes transferring the sample solution to a 50 mL centrifuge tube and centrifuging at 10,000 r / min for 10 min at 4 °C.

[0059] The derivatization procedure involved transferring 100 μL of the supernatant from centrifugation into a 1.5 mL centrifuge tube. Then, 175 μL of 0.4 mol / L potassium borate buffer solution (pH = 8.5) and 250 μL of 5 mmol / L fluorenemethyloxycarbonyl chloride acetonitrile solution were added sequentially. The mixture was thoroughly mixed for 30 seconds and reacted in a 25°C water bath for 25 minutes. Next, 100 μL of 1 mol / L glycine solution was added, and the reaction was continued for 20 minutes to neutralize excess fluorenemethyloxycarbonyl chloride. Finally, 75 μL of 1% acetic acid aqueous solution and 300 μL of deionized water were added, and the mixture was thoroughly mixed. The solution was then filtered through a 0.45 μm organic phase filter membrane to obtain the derivatized sample solution. The sample solution was stored protected from light, and the standard working solution was derivatized simultaneously.

[0060] Liquid chromatography conditions include:

[0061] Column: Eclipse XDB-C18 5μm 4.6*250mm

[0062] Detection wavelength: 254nm

[0063] Detector: Ultraviolet detector

[0064] Column oven: 30℃

[0065] Injection volume: 10 μL

[0066] Flow rate: 1.0 mL / min

[0067] Mobile phase: acetonitrile, 0.1% acetic acid water; gradient elution times are shown in Table 1.

[0068] Table 1 Gradient elution program

[0069] time min Acetonitrile (%) 0.1% acetic acid in water (%) 0.000 32 68 12.00 32 68 16.00 80 20 24.00 80 20 29.00 32 68 35.00 32 68

[0070] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0071] Unless otherwise specified, the methods used in the following examples are conventional methods, and the reagents used are commercially available reagents.

[0072] Example 1

[0073] (1) Reagents

[0074] Fluorenemethyloxycarbonyl chloride (FMOC-CL), acetonitrile, glycine, glacial acetic acid, potassium borate, potassium chloride, sodium hydroxide, and boric acid.

[0075] 5 mmol / L fluorenyl methoxycarbonyl chloride (FMOC-CL) acetonitrile solution: Weigh 0.0130 g of fluorenyl methoxycarbonyl chloride (FMOC-CL) and dilute to 10 mL in a volumetric flask with acetonitrile and mix well;

[0076] 1 mol / L glycine solution: Weigh 7.507 g of glycine into a 100 mL volumetric flask, and dilute to volume with water until well mixed;

[0077] 1% acetic acid solution: Pipette 0.1 mL of glacial acetic acid and dilute to 10 mL in a volumetric flask and mix well;

[0078] 0.1% acetic acid solution: Pipette 1 mL of glacial acetic acid and dilute to 1000 mL in a volumetric flask and mix well;

[0079] 0.8 mol / L boric acid solution: Weigh 24.74 g of boric acid, add water to a final volume of 500 mL, dissolve, and mix well;

[0080] 0.8 mol / L potassium chloride solution: Weigh 30.22 g of potassium chloride, add water to a final volume of 500 mL, dissolve, and mix well;

[0081] 0.4 mol / L potassium borate buffer solution (pH=8.5): Mix 0.8 mol / L boric acid solution with 0.8 mol / L potassium chloride solution, and then adjust the pH to 8.5 with 0.2 mol / L sodium hydroxide solution.

[0082] (2) Preparation of standard solutions

[0083] Stock solution (1.0 mg / mL): Weigh 0.0100 g of 1-deoxynojirimycin standard, dissolve in 60% acetonitrile and dilute to 10 mL, mix well;

[0084] Intermediate solution (100 μg / mL): Transfer 1 mL of 1 mg / mL standard stock solution and dilute to 10 mL with water;

[0085] Working solution: Transfer 0.02 mL, 0.1 mL, 0.05 mL, 0.3 mL, and 0.5 mL of 100 μg / mL intermediate solution respectively, and dilute to 10 mL with 60% acetonitrile. The concentrations of the working solutions are 0.2 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 3.0 μg / mL, and 5.0 μg / mL respectively.

[0086] (3) Sample pretreatment

[0087] Weigh 5.0 g of the prepared milk sample into a 50 mL volumetric flask, add 60% acetonitrile aqueous solution to the mark, and mix well. Transfer the sample solution to a 50 mL centrifuge tube, sonicate for 10 min, and centrifuge at 10000 r / min for 10 min at 4℃. Transfer 100 μL of the supernatant after centrifugation to a 1.5 mL centrifuge tube, add 175 μL of 0.4 mol / L potassium borate buffer solution (pH = 8.5) and 250 μL of 5 mmol / L fluorenemethyloxycarbonyl chloride acetonitrile solution, mix thoroughly for 30 s, react in a constant temperature water bath at 25℃ for 25 min, add 100 μL of 1 mol / L glycine solution, and react for 20 min to neutralize excess fluorenemethyloxycarbonyl chloride. Then add 75 μL of 1% acetic acid aqueous solution and 300 μL of deionized water, mix thoroughly, and filter through a 0.45 μm organic phase filter membrane to obtain the derivatized sample solution. The sample solution should be stored away from light, and the standard working solution should be derivatized simultaneously.

[0088] (4) Measurement conditions

[0089] Chromatographic column: Eclipse XDB-C18 5μm 4.6*250mm.

[0090] Mobile phase: acetonitrile, 0.1% acetic acid water; gradient elution times are shown in Table 1.

[0091] (5) Calculation of results

[0092] Substitute the peak area of ​​the extracted and derivatized sample solution into the standard curve in Table 2 to obtain the concentration of the sample in the instrument.

[0093] The content of deoxynojirimycin in the test sample is calculated according to the following formula: X = C * V / m.

[0094] Where X—the content of 1-deoxynojirimycin in the sample, in micrograms per gram (μg / g); C—the concentration of 1-deoxynojirimycin in the sample, in micrograms per milliliter (μg / mL); V—the volume of the sample, in milliliters (mL); m—the amount of the sample, in grams (g); the calculation results are retained to two decimal places.

[0095] In this embodiment, C is the concentration of the sample solution to be tested as determined by liquid chromatography, V is 50 mL, and m is approximately 5 g.

[0096] (6) Method curve

[0097] The standard curve and correlation coefficient of deoxynojirimycin were plotted based on the test results of the standard working solution.

[0098] Table 2 Standard curve of deoxynojirimycin

[0099]

[0100] The results are shown in Table 2. As can be seen from Table 2, the R² of the deoxynojirimycin curve meets the requirement of correlation coefficient ≥ 0.99 in Appendix F.2 of GB27404, where x is the concentration of deoxynojirimycin and y is the peak area. The obtained curve has a good linear relationship.

[0101] (7) Validation of limit of detection and limit of quantitation

[0102] Weigh 5g of the prepared milk sample into a 50mL volumetric flask. Add six sets of deoxynojirimycin standard solutions with a concentration less than 2μg / g to the detection limit and six sets of deoxynojirimycin standard solutions with a concentration less than 5μg / g to the quantitation limit. Dilute to the mark with 60% acetonitrile aqueous solution and mix well. Transfer the sample solution to a 50mL centrifuge tube, sonicate for 10min, and centrifuge at 10000r / min for 10min at 4℃. Aspirate 100 μL of the supernatant solution after centrifugation into a 1.5 mL centrifuge tube, add 175 μL of 0.4 mol / L potassium borate buffer solution (pH = 8.5) and 250 μL of 5 mmol / L fluorenemethyloxycarbonyl chloride acetonitrile solution, mix thoroughly for 30 s, react in a constant temperature water bath at 25 °C for 25 min, then add 100 μL of 1 mol / L glycine solution and react for 20 min to neutralize excess fluorenemethyloxycarbonyl chloride. Then add 75 μL of 1% acetic acid aqueous solution and 300 μL of deionized water, mix thoroughly, and filter through a 0.45 μm organic phase filter membrane to obtain the derivatized sample solution for instrumental analysis.

[0103] Table 3 Validation of Limit of Detection and Limit of Quantitation

[0104]

[0105]

[0106] As shown in Table 3, when the theoretical addition amount is <2 μg / g, the signal-to-noise ratio requirement of GB / T 27417-2017 is met, so the detection limit of this method is 2 μg / g; when the theoretical addition amount is <5 μg / g, the signal-to-noise ratio requirement of GB / T27417-2017 is met, so the quantitation limit is 5 μg / g.

[0107] (8) Precision verification

[0108] After weighing the prepared milk, deoxynojirimycin standard solution was added to make the final concentration of deoxynojirimycin in the mixed solution reach about 8 μg / g. Precision was measured 7 times. Table 4 shows the results of the precision measurement.

[0109] Table 4 Precision Measurement Results

[0110]

[0111] As shown in Table 4, according to GB / T 27404-2008, when the sample content is between 1-10 mg / kg, the precision should be less than 7.5%. Based on the data in the table above, the precision of the deoxynojirimycin content detection method meets the national standard requirements.

[0112] (9) Recovery rate verification

[0113] Weigh 5g of the prepared milk sample into a 50mL volumetric flask, add deoxynojirimycin standard solution to achieve a final concentration of approximately 8μg / g in the mixed solution, and perform 3 parallel experiments. Add deoxynojirimycin standard solution to the prepared milk sample to achieve a final concentration of approximately 14μg / g in the mixed solution, and perform 3 parallel experiments. Add deoxynojirimycin standard solution to the prepared milk sample to achieve a final concentration of approximately 20μg / g in the mixed solution, and perform 3 parallel experiments. Add 60% acetonitrile aqueous solution to the mark and mix well. Transfer the sample solution to a 50mL centrifuge tube, sonicate for 10min, and centrifuge at 10000r / min for 10min at 4℃. Aspirate 100 μL of the supernatant solution after centrifugation into a 1.5 mL centrifuge tube, add 175 μL of 0.4 mol / L potassium borate buffer solution (pH = 8.5) and 250 μL of 5 mmol / L fluorenemethyloxycarbonyl chloride acetonitrile solution, mix thoroughly for 30 s, react in a constant temperature water bath at 25 °C for 25 min, then add 100 μL of 1 mol / L glycine solution and react for 20 min to neutralize excess fluorenemethyloxycarbonyl chloride. Then add 75 μL of 1% acetic acid aqueous solution and 300 μL of deionized water, mix thoroughly, and filter through a 0.45 μm organic phase filter membrane to obtain the derivatized sample solution for instrumental analysis.

[0114] Table 5 shows the recovery rate of deoxynojirimycin.

[0115] Table 5. Recovery rate of deoxynojirimycin

[0116]

[0117] As shown in Table 5, according to GB / T27404-2008, when the sample content is greater than 1 mg / kg to 100 mg / kg, the recovery rate ranges from 90% to 110%, and the recovery rate of the deoxynojirimycin content detection method meets the national standard requirements.

[0118] Comparative Example 1

[0119] This test example provides a method for detecting deoxynojirimycin. The method uses deoxynojirimycin standard working solutions of 0.2402 μg / mL, 0.3603 μg / mL, 0.5044 μg / mL, 0.99683 μg / mL, 3.0025 μg / mL, and 5.0042 μg / mL as the test samples. The difference is that different detectors are used in this test example.

[0120] Different detectors were used for instrumental measurements, and the instrument spectra are shown below. Figure 1As shown, the fluorescence detector exhibits high sensitivity to deoxynojirimycin after derivatization. Therefore, when the working solution concentration reaches above 0.3 μg / mL, the target peak response saturates, resulting in a flat-topped peak. The ultraviolet detector showed the best response, with no flat-topped peaks observed at any of the five concentration points, and the correlation coefficient met the requirement of 0.99.

[0121] Comparative Example 2

[0122] This test example provides a method for the detection of deoxynojirimycin. The method uses five different extractants—0.01 mol / L hydrochloric acid, 0.05 mol / L hydrochloric acid, 70% methanol, 70% acetonitrile, and 70% ethanol—to extract milk spiked samples (a deoxynojirimycin standard solution is added to the prepared milk sample to achieve a final concentration of approximately 5 μg / g in the mixed solution). The process is basically the same as the detection method in Example 1, except that different extractants are used in this test example.

[0123] Using 0.01 mol / L HCl and 0.05 mol / L HCl failed to destroy fats and proteins, resulting in insufficient extraction of deoxynojirimycin and turbidity in the extracted sample, rendering it unusable for instrumentation. Methanol, acetonitrile, and ethanol all yielded clear supernatants, but the extraction efficiency of deoxynojirimycin varied among different reagents. Figure 2 As shown, acetonitrile has the highest extraction efficiency for deoxynojirimycin, therefore acetonitrile was selected as the extraction agent in this experiment.

[0124] Comparative Example 3

[0125] This test example provides a method for detecting deoxynojirimycin. The method uses aqueous solutions of 40% acetonitrile, 60% acetonitrile, 70% acetonitrile, and 80% acetonitrile to extract from spiked milk samples (a standard solution of deoxynojirimycin is added to the prepared milk sample to achieve a final concentration of approximately 5 μg / g in the mixed solution). The process is basically the same as the detection method in Example 1, except that different concentrations of acetonitrile are used for extraction in this test example.

[0126] like Figure 3 As shown, the recovery rates of different acetonitrile concentrations range from 95% to 103%, all of which meet the recovery rate requirements. However, a 60% acetonitrile concentration not only meets the requirements for clarity and recovery rate of the supernatant, but also facilitates the flow of the sample solution in the mobile phase. The use of 60% acetonitrile aqueous solution for extraction in this invention is beneficial to improving the accuracy of the detection results.

[0127] Comparative Example 4

[0128] This test example provides a method for the detection of deoxynojirimycin. The method involves derivatizing extracted milk spiked samples (deoxynojirimycin standard solution was added to the prepared milk sample to achieve a final concentration of approximately 5 μg / g of deoxynojirimycin in the mixed solution) using different amounts of potassium borate, FMOC-CL acetonitrile solution, glycine, acetic acid, and reaction conditions. The process is basically the same as the detection method in Example 1, except that different amounts of derivatizing reagents and reaction conditions are used in this test example.

[0129] like Figure 4 As shown, the recoveries of derivatization systems 1-2 were only around 80%, and the peak shapes were poor. Derivatization systems 3-6 showed better peak shapes and were all normally distributed. Only derivatization system 6 had a recovery rate of 94%, meeting the recovery requirements, while the recoveries of the remaining derivatization systems 3-5 were all low. Therefore, this invention uses derivatization system 6 for derivatization experiments, demonstrating that the detection method provided by this invention has higher accuracy.

[0130] Comparative Example 5

[0131] This test example provides a method for detecting deoxynojirimycin. The method uses spiked milk samples (deoxynojirimycin standard solution is added to the prepared milk sample to make the final concentration of deoxynojirimycin in the mixed solution reach about 5 μg / g) as samples. The process is basically the same as the detection method in Example 1, except that the method in this test example uses a different mobile phase elution program for detection.

[0132] The detection was performed using elution programs 1, 2, and 3 mentioned in the table. The instrument was run with the mobile phase elution program and the peak shape of the deoxynojirimycin target peak was as follows: Figure 5 As shown, gradient elution program 1, with 45 minutes of isocratic elution, resulted in the appearance of impurity peaks (as shown in the figure) after more than 6 runs, preventing the target peak from being separated. Gradient elution program 2, with 30 minutes of mobile phase gradient elution, still failed to completely separate the deoxynojirimycin target peak from the impurity peaks, and the baselines on both sides of the target peak were not stable. Gradient elution program 3, with 35 minutes of mobile phase gradient elution, achieved the best peak shape for the deoxynojirimycin target peak, and the retention time remained stable after multiple instrument runs.

[0133] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for detecting deoxynojirimycin in milk and dairy products, comprising: (1) Add extractant to the sample to be tested and make up to volume. The ratio of the sample to the volume is (2-10)g:(25-100)mL. Precipitate protein and fat, centrifuge and take the supernatant to obtain the sample solution to be derivatized. (2) Add buffer solution and derivatizing agent to the sample solution to be derivatized to obtain the derivatized sample solution; (3) Perform liquid chromatography on the derivatized sample solution to obtain the content of deoxynojirimycin in the sample to be tested; The extractant is a 40%–80% aqueous solution of acetonitrile.

2. The detection method according to claim 1, wherein, The extractant is a 60% acetonitrile aqueous solution.

3. The detection method according to claim 1, wherein, The sample to be tested is milk and dairy products. Preferably, the milk and dairy products include modified milk, fermented dairy products, or milk beverages.

4. The detection method according to claim 1, wherein, The buffer solution is a 0.2–0.6 mol / L potassium borate buffer solution (pH = 8.5); Preferably, the buffer solution is a 0.4 mol / L potassium borate buffer solution (pH = 8.5).

5. The detection method according to claim 1, wherein, The derivatizing agents include a 3–10 mmol / L fluorenemethyloxycarbonyl chloride (FMOC-CL) acetonitrile solution, a 0.5–5 mol / L glycine solution, and a 0.1%–5% acetic acid aqueous solution. Preferably, the derivatizing agent comprises a 5 mmol / L fluorenyl methoxycarbonyl chloride (FMOC-CL) acetonitrile solution, a 1 mol / L glycine solution, and a 1% aqueous acetic acid solution.

6. The detection method according to any one of claims 1-5, wherein, Step (2) includes: Add potassium borate buffer solution (pH=8.5) and fluorenemethyloxycarbonyl chloride acetonitrile solution to the sample solution to be derivatized, mix and react for 15-35 min under constant temperature water bath conditions of 20-30℃. Then add glycine solution and react for 20-30 min. Then add acetic acid aqueous solution and water, mix well and filter with organic phase filter membrane to obtain derivatized sample solution.

7. The detection method according to claim 6, wherein, The volume ratio of the sample solution to be derivatized, potassium borate buffer solution (pH=8.5), fluorenemethyloxycarbonyl chloride acetonitrile solution, and glycine is 50-200:100-200:200-300:50-200; Preferably, the volume ratio of the sample solution to be derived to the aqueous acetic acid solution is 50-200:50-200.

8. The detection method according to any one of claims 1-5, wherein, The detector used in the liquid chromatography is an ultraviolet detector.

9. The detection method according to any one of claims 1-5, wherein, The conditions for liquid chromatography were as follows: the column was an Eclipse XDB-C18 5μm 4.6*250mm, the detection wavelength was 254nm, the column oven temperature was 30℃, the injection volume was 10μL, the flow rate was 1.0ml / min, the mobile phase A was acetonitrile, the mobile phase B was 0.1% acetic acid water, and the elution method was gradient elution.

10. The detection method according to any one of claims 1-5, wherein, The gradient elution procedure is as follows: 0.00 min: Mobile phase A is 32%, mobile phase B is 68%; 12.00 min: Mobile phase A is 32%, mobile phase B is 68%; 16.00 min: Mobile phase A is 80%, mobile phase B is 20%; 24.00 min: Mobile phase A is 80%, mobile phase B is 20%; 29.00 min: Mobile phase A is 32%, mobile phase B is 68%; 35.00 min: Mobile phase A is 32%, mobile phase B is 68%.

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