A headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products

Abstract

The present invention relates to the technical field of detection of furfural compounds in dairy products, and in particular to a headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products. The detection method comprises the following steps: mixing a dairy sample with oxalic acid, heating, cooling, and obtaining a pretreatment liquid; mixing the pretreatment liquid with trichloroacetic acid solution A, centrifuging, and obtaining a supernatant one and a solid; mixing the solid with trichloroacetic acid solution B, centrifuging, and obtaining a supernatant two; mixing supernatant one and supernatant two, and using trichloroacetic acid solution B to make a constant volume to obtain a mixed solution; placing the mixed solution in a headspace bottle, and then performing headspace-gas chromatography-mass spectrometry analysis to determine the content of volatile furfural components in dairy products. The present invention is convenient for simultaneously detecting furfural, 2-furan methyl ketone, and 5-methyl furfural in dairy samples, can realize low-content furfural compound detection, and can extend the use time of a gas chromatographic column.

Description

Technical Field

[0001] The invention relates to the technical field of detection of furfural compounds in dairy products, and in particular to a headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products. Background Art

[0002] Dairy products include pasteurized milk, sterilized milk, and reconstituted milk, as well as milk powders such as whole milk powder, skim milk powder, partially skimmed milk powder, reconstituted milk powder, and colostrum powder. There are also condensed milk, cheese, milk fat (cream), as well as casein, whey powder, reconstituted milk, fermented milk, specialty dairy products (such as milk skin, milk cake, and milk fan), etc. Dairy products are various foods made from cow's milk or goat's milk and their processed products as the main raw materials, with or without appropriate amounts of vitamins, minerals, and other auxiliary materials, in accordance with the conditions required by laws, regulations, and standards.

[0003] After the reducing sugars and proteins in dairy products are heat-treated, non-enzymatic browning reactions, also known as Maillard reactions, often occur, and different potentially harmful substances are produced in the early, middle, and late stages of the reaction. Furfural compounds, represented by 5-hydroxymethylfurfural (5-HMF), furfural (F), furanyl methyl ketone (FMC), and methylfurfural (MF), are products of the Maillard reaction, and are distributed in different amounts in various foods such as dairy products. The European Food Safety Authority recommends that the daily upper limit of hydroxymethylfurfural (HMF) intake per person is 1.6 mg. A small amount of furfural compounds can enhance the flavor and color of food, but excessive intake of furfural compounds has also been reported to have the risk of damaging organs such as the heart and inducing chronic diseases such as diabetes.

[0004] In recent years, the Chinese people's awareness of food safety has been increasing, and the quality requirements for dairy products have become higher and higher. In terms of dairy product quality control, the Maillard reaction is an important part that cannot be ignored. As a non-enzymatic browning reaction between reducing sugars and lysine during the processing of dairy products, its initial product furosine is used by the European Union as an indicator for monitoring the quality damage of pasteurized milk and UHT milk. Furfural compounds produced in the middle stage of the Maillard reaction can also be used as an indicator of heat damage to dairy products.

[0005] So far, the research on furfural compounds in dairy products has mainly focused on: 5-hydroxymethylfurfural, furfural, 2-furanmethyl ketone and 5-methylfurfural. Existing detection methods include spectroscopy and chromatography: spectroscopy has the advantages of simplicity and low cost, but the method has poor selectivity and is prone to matrix interference; chromatography mainly includes liquid chromatography and gas chromatography.

[0006] Existing liquid chromatography detection methods for furfural compounds include high performance liquid chromatography-ultraviolet detector (HPLC-UV) and gas chromatography-mass spectrometry (GC-MS). Liquid chromatography-ultraviolet detector uses direct injection analysis, but due to the poor specificity of the ultraviolet detector, this method can only detect 5-hydroxymethylfurfural and furfural. Gas chromatography uses direct injection analysis, during which complex samples directly enter the chromatographic column, which causes great damage to the chromatographic column. Usually, each gas chromatography column can only complete quantitative analysis of 10 samples.

[0007] Therefore, there is an urgent need to provide a detection method for simultaneously detecting furfural, 2-furanmethyl ketone and 5-methylfurfural in dairy samples with less impact on the chromatographic column. Summary of the invention

[0008] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products, which is used to solve the problem that the detection of furfural, 2-furanmethyl ketone and 5-methylfurfural in dairy products in the prior art is relatively difficult and has a significant impact on the chromatographic column during the detection process.

[0009] To achieve the above objectives and other related objectives, the present invention is achieved through the following technical solutions.

[0010] The present invention provides a headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products, the detection method comprising the following steps:

[0011] (1) mixing a dairy product sample with oxalic acid, heating, and cooling to obtain a pre-treated solution;

[0012] (2) mixing the pretreated solution with trichloroacetic acid solution A, centrifuging, and obtaining a supernatant and a solid;

[0013] (3) mixing the solid with trichloroacetic acid solution B, centrifuging to obtain supernatant 2; mixing supernatant 1 and supernatant 2, and fixing the volume with trichloroacetic acid solution B to obtain a mixed solution;

[0014] (4) placing the mixed solution obtained in step (3) into a headspace bottle, and then performing headspace-gas chromatography-mass spectrometry analysis to determine the content of volatile furfural components in the dairy product.

[0015] As described above, the headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products of the present invention has the following beneficial effects:

[0016] The present invention uses the headspace injection mode to complete the extraction of three volatile furfurals, namely furfural, 2-furanmethyl ketone and 5-methylfurfural, in dairy products, and combines the gas chromatography-mass spectrometry method to complete the qualitative analysis. Compared with the traditional HPLC-UV and GC-MS detection methods, the operation is simple and easy to implement, and low-content furfural compound detection can be achieved, which can prolong the use time of the gas chromatography column. DETAILED DESCRIPTION

[0017] In order to make the invention objectives, technical solutions and beneficial technical effects of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0018] The present invention provides a headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products, the detection method comprising the following steps:

[0019] (1) mixing a dairy product sample with oxalic acid, heating, and cooling to obtain a pre-treated solution;

[0020] (2) mixing the pretreated solution with trichloroacetic acid solution A, centrifuging, and obtaining a supernatant and a solid;

[0021] (3) mixing the solid with trichloroacetic acid solution B, centrifuging to obtain supernatant 2; mixing supernatant 1 and supernatant 2, and fixing the volume with trichloroacetic acid solution B to obtain a mixed solution;

[0022] (4) placing the mixed solution obtained in step (3) into a headspace bottle, and then performing headspace-gas chromatography-mass spectrometry analysis to determine the content of volatile furfural components in the dairy product.

[0023] The present invention first processes the dairy sample, then extracts furfural, 2-furanmethyl ketone and 5-methylfurfural in the dairy sample by headspace injection, reduces interference from other components in the dairy sample, simplifies the components of the dairy sample, and then analyzes the dairy sample by gas chromatography-mass spectrometry, so as to facilitate the detection of low-content furfural compounds. At the same time, after the sample components entering the gas chromatograph are simplified, the number of samples that can be detected is greatly increased from 10 samples before to 100-200 samples now, thereby extending the service life of the gas chromatograph column. Headspace injection can avoid complex sample pretreatment and has certain convenience. At the same time, compared with direct liquid injection, it can reduce the influence of non-volatile components on the instrument, chromatographic column and the influence on the results. The analysis using gas chromatography-tandem mass spectrometry multiple reaction monitoring mode can eliminate interference and improve sensitivity.

[0024] In some embodiments of the present invention, the volatile furfural substances are selected from at least one of furfural, 2-furanmethyl ketone or 5-methylfurfural; preferably, the volatile furfural substances include furfural, 2-furanmethyl ketone and 5-methylfurfural.

[0025] In some embodiments of the present invention, the mass ratio of the dairy sample to oxalic acid in step (1) is 1:0.3-0.5 g / ml. Specifically, the mass ratio of the dairy sample to oxalic acid can be 1:0.3-0.4 g / ml or 1:0.4-0.5 g / ml.

[0026] In some embodiments of the present invention, the molar concentration of oxalic acid in step (1) is 0.1-0.2 mol / L; specifically, the molar concentration of oxalic acid may be 0.1-0.15 mol / L, 0.15-0.2 mol / L; preferably, the molar concentration of oxalic acid is 0.15 mol / L.

[0027] In some embodiments of the present invention, the heating temperature in step (1) is 90-110° C.; and the heating time is 20-30 min.

[0028] In some embodiments of the present invention, the cooling temperature in step (1) is 20-30° C. Specifically, the cooling temperature may be 20-25° C., 25-30° C.; typically but not limitingly, for example, 20° C., 25° C., 30° C.

[0029] In some embodiments of the present invention, the concentration of the trichloroacetic acid solution A in step (2) is 30-50% (w / v); preferably, the concentration of the trichloroacetic acid solution A is 40% (w / v).

[0030] In some embodiments of the present invention, the centrifugation time in step (2) is 10-20 min.

[0031] In some embodiments of the present invention, the concentration of the trichloroacetic acid solution B in step (3) is 3-5% (w / v); preferably, the concentration of the trichloroacetic acid solution B is 4% (w / v).

[0032] In some embodiments of the present invention, the centrifugation time in step (3) is 10-20 min. Specifically, the centrifugation time can be 10-15 min, 15-20 min; typically but not limited to, for example, 10 min, 15 min, 20 min.

[0033] In some embodiments of the present invention, the chromatographic column in step (4) is a polar chromatographic column; preferably, the chromatographic column may be SH-PolarWax-MS.

[0034] In some embodiments of the present invention, the specification of the chromatographic column in step (4) is 0.25 mm x 0.25 μm x 30 m.

[0035] In some embodiments of the present invention, the carrier gas in step (4) is high purity helium; the column flow rate is 1-1.4 ml / min; specifically, the column flow rate can be 1-1.01 ml / min, 1.01-1.4 ml / min; typical but non-limiting examples include 1 ml / min, 1.01 ml / min, and 1.4 ml / min.

[0036] In some embodiments of the present invention, the split ratio in step (4) is 10-15: 1. Specifically, the split ratio may be 10-13: 1, 13-15: 1; typically but not limiting, for example, 10: 1, 13: 1, 15: 1.

[0037] In some embodiments of the present invention, the temperature rising procedure in step (4) is as follows: the starting temperature is 30-50°C and maintained for 2-5 min, then the temperature is raised to 140-160°C at 3-7°C / min, and finally the temperature is raised to 240-260°C at 15-30°C / min and maintained for 8-13 min.

[0038] In some embodiments of the present invention, the head space injection conditions in step (4) are: the constant temperature oven temperature is 70-90°C; the quantitative loop temperature is 100-120°C; the transfer line temperature is 110-130°C; the sample equilibration time is 7-10 min; the injection duration is 1-2 min; the injection volume is 1-2 μL; and the GC cycle time is 46-50 min. Specifically, the temperature of the constant temperature furnace can be 70-80°C, 80-90°C; typically but not limitatively, for example, 70°C, 80°C, 90°C; the temperature of the quantitative loop can be 100-110°C, 110-120°C; typically but not limitatively, for example, 100°C, 110°C, 120°C; the temperature of the transmission line can be 110-120°C, 120-130°C; typically but not limitatively, for example, 110°C, 120°C, 130°C; the sample equilibration time can be 7-9min, 9-10min; typically but not limitatively, for example, 7min, 9min, 10min.

[0039] In some embodiments of the present invention, the mass spectrometry conditions in step (4) are: ion source: EI; ion source temperature: 190-210°C; interface temperature: 240-260°C; ionization energy: 70-80ev; scanning mode: Q3 SIM; quadrupole temperature: 240-260°C; solvent delay time: 2-4min.

[0040] In some embodiments of the present invention, the mixed solution in step (3) is filtered through a filter membrane. The pore size of the filter membrane in step (3) is 0.35-0.55 μm; specifically, the pore size of the filter membrane can be 0.35-0.45 μm, 0.45-0.55 μm; typically but not limiting, for example, 0.35 μm, 0.45 μm, 0.55 μm.

[0041] In some embodiments of the present invention, the dairy product sample in step (1) is selected from any one of low-temperature fermented milk, room-temperature fermented milk, ultra-high temperature sterilized milk, and milk powder. The present invention has no special limitation on the source of low-temperature fermented milk, and commercially available products of low-temperature fermented milk frequently used by those skilled in the art can be used. The present invention has no special limitation on the source of room-temperature fermented milk, and commercially available products of room-temperature fermented milk frequently used by those skilled in the art can be used. The present invention has no special limitation on the source of ultra-high temperature sterilized milk, and commercially available products of ultra-high temperature sterilized milk frequently used by those skilled in the art can be used. The present invention has no special limitation on the source of milk powder, and commercially available products of milk powder frequently used by those skilled in the art can be used.

[0042] The present invention is further described below by way of examples, but the scope of the present invention is not limited thereto.

[0043] When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. If specific conditions are not specified in the embodiment, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for all reagents or instruments, they are conventional products that can be purchased commercially. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any method, equipment, and material of the prior art similar or equivalent to the method, equipment, and material in the embodiments of the present invention can also be used to realize the present invention.

[0044] Instrument: Gas chromatography-mass spectrometry, model: 7890B-5977B, purchased from Agilent Technologies, USA.

[0045] The headspace sampling instrument is a headspace autosampler.

[0046] Furfural is abbreviated as F in the present invention.

[0047] The 2-furanmethyl ketone of the present invention is abbreviated as FMC.

[0048] The abbreviation of 5-methylfurfural in the present invention is MF.

[0049] The present application provides a headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products, comprising the following steps:

[0050] (1) mixing a dairy product sample with oxalic acid, heating, and cooling to obtain a pre-treated solution;

[0051] (2) mixing the pretreated solution with trichloroacetic acid solution A, centrifuging, and obtaining a supernatant and a solid;

[0052] (3) mixing the solid with trichloroacetic acid solution B, centrifuging to obtain supernatant 2; mixing supernatant 1 and supernatant 2, and fixing the volume with trichloroacetic acid solution B to obtain a mixed solution;

[0053] (4) placing the mixed solution obtained in step (3) into a headspace bottle, and then performing headspace-gas chromatography-mass spectrometry analysis to determine the content of volatile furfural components in the dairy product.

[0054] The specific preparation method of the mixed solution of this application is:

[0055] (1) Weigh 15 g of dairy product sample, add 5 mL of 0.15 mol / L oxalic acid prepared on the day, heat in a 100°C water bath for 25 min, and cool to room temperature;

[0056] (2) Add 3 mL of 40% (w / v) trichloroacetic acid solution A, shake for 10 min, and centrifuge at 4.355 G for 15 min to obtain a supernatant and a solid;

[0057] (3) Pour supernatant 1 into a 25 mL volumetric flask, add 5 mL of 4% (w / v) trichloroacetic acid solution B to the remaining solid, shake for 10 min, and centrifuge at 4.355 G for 15 min to obtain supernatant 2; combine supernatant 1 and supernatant 2, add 4% (w / v) trichloroacetic acid solution B to the mark to obtain a mixed solution. Take an appropriate amount of the liquid and filter it through a 0.45 μm filter membrane for testing.

[0058] The parameters of the headspace injection in this application are set as follows: injection is performed using a headspace automatic sampler, and the conditions are: constant temperature oven temperature: 80°C; quantitative loop temperature: 110°C; transfer line temperature: 120°C; sample equilibration time: 10 min; injection duration: 1 min; GC cycle time: 47 min; and the injection volume is 1 μL.

[0059] The gas chromatography parameters of this application are set as follows: chromatographic column: SH-PolarWax-MS, 0.25mmx0.25μmx30m; heating program: initial temperature 50°C maintained for 2min, then heated to 150°C at 5°C / min, and finally heated to 250°C at 20°C / min, maintained for 10min; carrier gas: high-purity helium; constant linear velocity mode, column flow: 1.01mL / min; split ratio 10:1.

[0060] The mass spectrometry parameters in this application were set as follows: ion source: EI; ion source temperature: 200°C; interface temperature: 250°C; ionization energy: 70ev; scan mode: Q3 SIM; quadrupole temperature: 250°C; solvent delay time: 3min; monitoring modes F(96), FMC(95), MF(110).

[0061] Standard stock solution: Accurately weigh 20 mg of furfural, furan methyl ketone and methyl furfural standards into a 100 mL volumetric flask, add water to make up to 100 mL, and store at 4 °C for later use.

[0062] Standard working solution: Use water to prepare mixed standard working solutions with mass concentrations of 10μg / mL, 4μg / mL, 2μg / mL, 1μg / mL, 0.4μg / mL, 0.2μg / mL, and 0.1μg / mL, respectively.

[0063] Detection limit solution: Use water to prepare a mixed standard working solution with a mass concentration of 20 ng / mL.

[0064] Quantitation limit solution: Use water to prepare a mixed standard working solution with a mass concentration of 40 ng / mL.

[0065] According to the above parameter settings, headspace injection-gas chromatography-mass spectrometry was used to detect furfural standard solutions, 2-furanmethyl ketone standard solutions, and 5-methylfurfural standard solutions of different concentrations. The ratio of the chromatographic peak area of ​​each analyte to the internal standard peak area Y was used as the ordinate and the corresponding mass concentration X (μg / mL) was used as the abscissa for regression analysis. The regression equation is shown in Table 1. The working standard solution should cover the sample concentration range and be ready for use. The separation time of the different furfurals is as follows: F, 13.90min; FMC, 14.91min; MF, 16.59min.

[0066] The standard solution used in the calculation process is a 0.1 μg / mL mixed standard working solution, and the calculation formula is as follows:

[0067]

[0068] in:

[0069] A STD represents the peak area of ​​furfural standard;

[0070] W STD represents the weight of furfural standard (mg);

[0071] V STD represents the dilution volume of furfural standard (mL);

[0072] P STD represents the weight content of furfural standard (%, w / w);

[0073] RF STD represents the response factor of the furfural standard;

[0074] A sample Represents the peak area of ​​furfural in the sample solution;

[0075] W sample Represents the weight of the sample (mg);

[0076] V sample represents the dilution volume of the sample (mL);

[0077] Assay furfurals Represents the content of furfural standard in the sample solution (μg / 100g).

[0078] Table 1 Verification of headspace injection-gas chromatography-mass spectrometry detection method parameters for furfural, 2-furanmethyl ketone and 5-methylfurfural corresponding standard solutions

[0079]

[0080] Example

[0081] Example 1 Detection of three volatile furfurals, furfural, 2-furanmethyl ketone and 5-methylfurfural in low-temperature fermented milk

[0082] 1. This embodiment provides a headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products, comprising the following steps:

[0083] (1) Weigh 15 g of dairy product sample, add 5 mL of 0.15 mol / L oxalic acid prepared on the day, heat in a 100°C water bath for 25 min, and cool to room temperature; wherein the dairy product sample is low-temperature fermented milk;

[0084] (2) Add 3 mL of 40% (w / v) trichloroacetic acid solution A, shake for 10 min, and centrifuge at 4.355 G for 15 min to obtain a supernatant and a solid;

[0085] (3) Pour the supernatant 1 into a 25 mL volumetric flask, add 5 mL of 4% (w / v) trichloroacetic acid solution B to the remaining solid, shake for 10 min, and centrifuge at 4.355 G for 15 min to obtain supernatant 2; combine supernatant 1 and supernatant 2, add 4% (w / v) trichloroacetic acid solution B to the mark to obtain a mixed solution. Take an appropriate amount of liquid and filter through a 0.45 μm filter membrane, and analyze it in a chromatograph;

[0086] (4) placing the mixed solution obtained in step (3) into a headspace bottle, and then performing headspace-gas chromatography-mass spectrometry analysis to determine the content of volatile furfural components in the dairy product.

[0087] Among them, the headspace conditions are: constant temperature oven temperature: 80°C; quantitative loop temperature: 110°C; transfer line temperature: 120°C; sample equilibration time: 10 min; injection duration: 1 min; GC cycle time: 47 min; the injection volume is 1 μL;

[0088] Chromatographic conditions: Chromatographic column: SH-PolarWax-MS, 0.25mmx0.25μmx30m; Temperature program: Initial temperature 50℃ for 2min, then increase to 150℃ at 5℃ / min, and finally increase to 250℃ at 20℃ / min, and maintain for 10min; Carrier gas: high-purity helium; Constant linear velocity mode, column flow rate: 1.01mL / min; Split ratio 10:1;

[0089] Mass spectrometry conditions: ion source: EI; ion source temperature: 200°C; interface temperature: 250°C; ionization energy: 70 eV; scan mode: Q3 SIM; quadrupole temperature: 250°C; solvent delay time: 3 min; monitoring mode: F (96), FMC (95), MF (110). The detection results are shown in Table 2.

[0090] 2. This embodiment provides a method for detecting volatile furfural substances in dairy products by using a liquid chromatography tandem detector. The detection method refers to the [0037-0042] section of the specification of the patent CN 109580820A, wherein the detection sample is low-temperature fermented milk.

[0091] Table 2 Comparison of the results of the detection of volatile furfural content in low-temperature fermented milk in Example 1

[0092]

[0093] ND: not detected; 1-1, 1-2, 1-3 are three samples from the same batch and were treated in the same way.

[0094] Example 1 uses the HS-GC-MS detection method to detect furfural, 2-furanmethyl ketone and 5-methylfurfural in low-temperature fermented milk. The content of 2-furanmethyl ketone and 5-methylfurfural in low-temperature fermented milk is low. The HS-GC-MS detection method of the present invention can detect 2-furanmethyl ketone as low as 0.73ug / 100g. It can be seen that the present application uses the HS-GC-MS detection method to detect furfural, 2-furanmethyl ketone and 5-methylfurfural in low-temperature fermented milk, which has high sensitivity and can achieve the detection of low-content furfural compounds. When HPLC-DAD is used to detect furfural, 2-furanmethyl ketone and 5-methylfurfural in low-temperature fermented milk, only furfural can be detected, but 2-furanmethyl ketone and 5-methylfurfural cannot be detected.

[0095] Example 2 Detection of three volatile furfurals, furfural, 2-furanmethyl ketone and 5-methylfurfural in room temperature fermented milk

[0096] 1. This embodiment provides a headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products, which is different from Example 1 in that the dairy product sample is room temperature fermented milk. The detection results are shown in Table 3.

[0097] 2. This embodiment provides a method for detecting volatile furfural substances in dairy products by using a liquid chromatography tandem detector. The detection method refers to the [0037-0042] section of the specification of the patent CN 109580820A, wherein the detection sample is room temperature fermented milk.

[0098] Table 3 Comparison of the results of volatile furfural content detection in room temperature fermented milk

[0099]

[0100]

[0101] ND: not detected; 1-1, 1-2, 1-3 are three samples from the same batch and were treated in the same way.

[0102] Example 2 uses the HS-GC-MS detection method to detect furfural, 2-furanmethyl ketone and 5-methylfurfural in room temperature fermented milk. The content of 2-furanmethyl ketone and 5-methylfurfural in room temperature fermented milk is low. The HS-GC-MS detection method of the present invention can detect 2-furanmethyl ketone as low as 1.68ug / 100g. It can be seen that the present application uses the HS-GC-MS detection method to detect furfural, 2-furanmethyl ketone and 5-methylfurfural in room temperature fermented milk, which has high sensitivity and can achieve the detection of low-content furfural compounds. When HPLC-DAD is used to detect furfural, 2-furanmethyl ketone and 5-methylfurfural in room temperature fermented milk, only furfural can be detected, but 2-furanmethyl ketone and 5-methylfurfural cannot be detected.

[0103] Example 3 Detection of three volatile furfurals: furfural, 2-furanmethyl ketone and 5-methylfurfural in ultra-high temperature sterilized milk

[0104] 1. This embodiment provides a headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products, which is different from Example 1 in that the dairy product sample is ultra-high temperature sterilized milk. The detection results are shown in Table 4.

[0105] 2. This embodiment provides a method for detecting volatile furfural substances in dairy products by liquid chromatography-tandem detector. The detection method refers to the [0037-0042] section of the specification of the patent CN 109580820A, wherein the detection sample is ultra-high temperature sterilized milk.

[0106] Table 4 Comparison of the test results of volatile furfural content in ultra-high temperature sterilized milk

[0107]

[0108] ND: not detected; 1-1, 1-2, 1-3 are three samples from the same batch and were treated in the same way.

[0109] Example 3 HS-GC-MS detection method is used to detect furfural, 2-furan methyl ketone and 5-methyl furfural in ultra-high temperature sterilized milk. The content of 2-furan methyl ketone and 5-methyl furfural in ultra-high temperature sterilized milk is low. The HS-GC-MS detection method of the present invention can detect 2-furan methyl ketone as low as 0.75ug / 100g and 5-methyl furfural as low as 0.71ug / 100g. It can be seen that the present application uses HS-GC-MS detection method to detect furfural, 2-furan methyl ketone and 5-methyl furfural in ultra-high temperature sterilized milk, with high sensitivity, and can achieve the detection of low-content furfural compounds. When HPLC-DAD is used to detect furfural, 2-furan methyl ketone and 5-methyl furfural in ultra-high temperature sterilized milk, only furfural can be detected, but 2-furan methyl ketone and 5-methyl furfural cannot be detected.

[0110] Example 4 Detection of three volatile furfurals: furfural, 2-furanmethyl ketone and 5-methylfurfural in milk powder

[0111] 1. This embodiment provides a headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products, comprising the following steps:

[0112] (1) Weigh 2.25 g of dairy product sample, add 10 ml of water to dissolve, add 5 mL of 0.15 mol / L oxalic acid prepared on the day, heat in a 100°C water bath for 25 min, and cool to room temperature; wherein the dairy product sample is milk powder;

[0113] (2) Add 3 mL of 40% (w / v) trichloroacetic acid solution A, shake for 10 min, and centrifuge at 4.355 G for 15 min to obtain a supernatant and a solid;

[0114] (3) Pour the supernatant 1 into a 25 mL volumetric flask, add 5 mL of 4% (w / v) trichloroacetic acid solution B to the remaining solid, shake for 10 min, and centrifuge at 4.355 G for 15 min to obtain supernatant 2; combine supernatant 1 and supernatant 2, add 4% (w / v) trichloroacetic acid solution B to the mark to obtain a mixed solution. Take an appropriate amount of liquid and filter through a 0.45 μm filter membrane, and analyze it in a chromatograph;

[0115] (4) placing the mixed solution obtained in step (3) into a headspace bottle, and then performing headspace-gas chromatography-mass spectrometry analysis to determine the content of volatile furfural components in the dairy product.

[0116] Headspace conditions: constant temperature oven temperature: 80°C; quantitative loop temperature: 110°C; transfer line temperature: 120°C; sample equilibration time: 10 min; injection duration: 1 min; GC cycle time: 47 min;

[0117] Chromatographic conditions: Chromatographic column: SH-PolarWax-MS, 0.25mmx0.25μmx30m; Temperature program: Initial temperature 50℃ for 2min, then increase to 150℃ at 5℃ / min, and finally increase to 250℃ at 20℃ / min, and maintain for 10min; Carrier gas: high-purity helium; Constant linear velocity mode, column flow: 1.01mL / min; Split ratio 10:1;

[0118] Mass spectrometry conditions: ion source temperature: 200°C; interface temperature: 250°C; ionization energy: 70 eV; scanning mode: Q3SIM monitoring mode F (96), FMC (95), MF (110). The detection results are shown in Table 5.

[0119] 2. This embodiment provides a method for detecting volatile furfural substances in dairy products by liquid chromatography-tandem detector. The detection method refers to the specification [0037-0042] of the patent CN 109580820A, wherein the detection sample is milk powder.

[0120] Table 5 Comparison of the test results of volatile furfural content in milk powder

[0121]

[0122] ND: not detected; 1-1, 1-2, 1-3 are three samples from the same batch and were treated in the same way.

[0123] Embodiment 4 adopts HS-GC-MS detection method to detect furfural, 2-furan methyl ketone and 5-methyl furfural in milk powder, and the content of 2-furan methyl ketone and 5-methyl furfural in milk powder is low. The HS-GC-MS detection method of the present invention can detect 2-furan methyl ketone as low as 5.16ug / 100g, from which it can be seen that the present application adopts HS-GC-MS detection method to detect furfural, 2-furan methyl ketone and 5-methyl furfural in milk powder, with high sensitivity, and can realize the detection of low-content furfural compounds. When HPLC-DAD is used to detect furfural, 2-furan methyl ketone and 5-methyl furfural in milk powder, only furfural can be detected, while 2-furan methyl ketone and 5-methyl furfural cannot be detected.

[0124] In Examples 1-4, the HS-GC-MS detection method was used to detect low-temperature fermented milk, room-temperature fermented milk, ultra-high-temperature fermented milk, and milk powder, respectively, and furfural, 2-furanmethyl ketone, and 5-methylfurfural were all effectively detected.

[0125] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products, characterized in that: The detection method comprises the following steps: (1) mixing a dairy product sample with oxalic acid, heating, and cooling to obtain a pre-treated solution; (2) mixing the pretreated solution with trichloroacetic acid solution A, centrifuging, and obtaining a supernatant and a solid; (3) mixing the solid with trichloroacetic acid solution B, centrifuging to obtain supernatant 2; mixing supernatant 1 and supernatant 2, and fixing the volume with trichloroacetic acid solution B to obtain a mixed solution; (4) placing the mixed solution obtained in step (3) into a headspace bottle, and then performing headspace-gas chromatography-mass spectrometry analysis to determine the content of volatile furfural components in the dairy product.

2. The headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products according to claim 1, characterized in that: The volatile furfural substances are selected from at least one of furfural, 2-furanmethyl ketone or 5-methylfurfural; preferably, the volatile furfural substances include furfural, 2-furanmethyl ketone and 5-methylfurfural.

3. The headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products according to claim 1, characterized in that: The step (1) further comprises one or more of the following features: 11) The mass ratio of the dairy product sample to oxalic acid is 1:0.3-0.5 g / ml; 12) The molar concentration of the oxalic acid is 0.1-0.2 mol / L; preferably, the molar concentration of the oxalic acid is 0.15 mol / L; 13) The heating temperature is 90-110° C. and the heating time is 20-30 min; 14) The cooling temperature is 20-30°C.

4. The headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products according to claim 1, characterized in that: The step (2) further includes one or more of the following features: 21) The concentration of the trichloroacetic acid solution A is 30-50% (w / v); preferably, the concentration of the trichloroacetic acid solution A is 40% (w / v); 22) The centrifugation time is 10-20 min.

5. The headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products according to claim 1, characterized in that: The step (3) further includes one or more of the following features: 31) The concentration of the trichloroacetic acid solution B is 3-5% (w / v); preferably, the concentration of the trichloroacetic acid solution B is 4% (w / v); 32) The centrifugation time is 10-20 min.

6. The headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products according to claim 1, characterized in that: The step (4) further includes one or more of the following features: 41) The chromatographic column is a polar chromatographic column; preferably, the chromatographic column is SH-PolarWax-MS; 42) The specification of the chromatographic column is 0.25mmx0.25μmx30m; 43) The carrier gas is high-purity helium; the column flow rate is 1-1.4 ml / min; preferably, the column flow rate is 1.01 ml / min; 44) the split ratio is 10-15:1; 45) Heating program: Initial temperature 30-50°C, maintain for 2-5 min, then increase to 140-160°C at 3-7°C / min, and finally increase to 240-260°C at 15-30°C / min, maintain for 8-13 min.

7. The headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products according to claim 1, characterized in that: The headspace injection conditions are as follows: the constant temperature oven is 70-90°C; the quantitative loop temperature is 100-120°C; the transfer line temperature is 110-130°C; the sample equilibration time is 7-10min; the injection duration is 1-2min; the injection volume is 1-2μL; and the GC cycle time is 46-50min.

8. The headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products according to claim 1, characterized in that: The mass spectrometry conditions in step (4) are: ion source: EI; ion source temperature: 190-210° C.; Interface temperature: 240-260℃; ionization energy: 70-80ev; scan mode: Q3 SIM; quadrupole temperature: 240-260℃; Solvent delay time: 2-4min.

9. The headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products according to claim 1, characterized in that: The mixed solution in step (3) is filtered through a filter membrane; the pore size of the filter membrane in step (3) is 0.35-0.55 μm; preferably, the pore size of the filter membrane is 0.45 μm.

10. The headspace injection tandem gas chromatography-mass spectrometry detection method for volatile furfural substances in dairy products according to claim 1, characterized in that: In the step (1), the dairy product sample is selected from any one of low-temperature fermented milk, room-temperature fermented milk, ultra-high temperature sterilized milk, and milk powder.

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