CN114062548A - Method for simultaneously detecting multiple advanced glycosylation end products in dairy product - Google Patents
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Abstract
The invention relates to a method for simultaneously detecting multiple advanced glycosylation end products in a dairy product, which comprises the following steps: preparing amino acid standard solutions with different concentrations; digesting a dairy product sample by using a microwave digestion instrument to obtain a digestion solution after digestion is finished; cooling the digestion solution to room temperature, then enabling the digestion solution cooled to the room temperature to flow through a filter membrane for filtration, collecting filtrate, and diluting the filtrate to obtain a processed dairy product sample; and respectively injecting the amino acid standard solution and the processed dairy product sample into a liquid chromatography-tandem mass spectrometer for detection. The invention provides a method for simultaneously detecting multiple advanced glycosylation end products in a dairy product, which can detect multiple amino acids of the advanced glycosylation end products in the dairy product and solves the problems of long detection time, high cost and the like in the prior art.
Description
Technical Field
The invention relates to the technical field of food quality detection, in particular to a method for simultaneously detecting multiple advanced glycosylation end products in a dairy product.
Background
The dairy product contains amino acids such as lysine, arginine, methionine and the like which are beneficial to human bodies, and in the processing and storage processes of the dairy product, Maillard reaction can occur between carbonyl-containing compounds and amino-containing compounds (amino acids and proteins) in the dairy product to generate non-digestible and non-utilizable advanced glycosylation end products (such as furfuryl acid, carboxymethyl lysine, carboxyethyl lysine and the like). Maillard reactions in dairy products reduce the nutritional content of the dairy product and produce harmful products. Therefore, various advanced glycosylation end products in the dairy product can be used as the quality monitoring index of the dairy product.
At present, most of the pretreatment of dairy products adopts oven hydrolysis, the detection method using the oven hydrolysis needs hydrolysis for 20-24 hours, and hydrolysate needs extraction and purification operation through a solid phase extraction column, so that the pretreatment of the dairy products has the problems of low efficiency, complex operation and the like.
Disclosure of Invention
Based on the method, the method for simultaneously detecting multiple advanced glycosylation end products in the dairy product is provided, and the problems of low efficiency, complex operation and the like of the existing pretreatment of the dairy product are solved while the detection of multiple amino acids of the advanced glycosylation end products in the dairy product is realized.
A method for simultaneously detecting multiple advanced glycosylation end products in a dairy product comprises the following steps:
preparing amino acid standard solutions with different concentrations;
digesting a dairy product sample by using a microwave digestion instrument to hydrolyze amino acid molecules of an advanced glycosylation end product combined with protein in the dairy product into free amino acid micromolecules, and obtaining a digestion solution after digestion;
filtering the digestion solution by a filter membrane, collecting filtrate, and diluting the filtrate to obtain a processed dairy product sample;
and respectively injecting the amino acid standard solution and the processed dairy product sample into a liquid chromatography-tandem mass spectrometer for detection.
Preferably, the amino acid standard solution includes a solvent including ultrapure water and solutes including lysine, furfuryl acid, carboxymethyl lysine and carboxyethyl lysine.
Preferably, the step of digesting comprises:
adding the processed dairy product sample and 6mol/L hydrochloric acid into a microwave digestion tank, filling nitrogen into the microwave digestion tank to remove oxygen in the microwave digestion tank, placing the microwave digestion tank in a microwave digestion instrument for digestion, and obtaining digestion liquid after digestion.
Preferably, the digestion temperature is 150 ℃, and the digestion time is 15-20 min.
Preferably, the hydrochloric acid used is 0.267mL per 1mg protein equivalent (1mg Pro) of the treated dairy sample.
Preferably, the pore size of the filter is 0.22 μm.
Preferably, the specific step of diluting the filtrate to obtain the processed dairy product sample comprises:
diluting the filtrate by 100 times with ultrapure water to obtain a treated dairy product sample.
In the step of respectively injecting the amino acid standard solution and the processed dairy product sample into a liquid chromatography-tandem mass spectrometer for detection, the specific process of liquid chromatography detection is as follows:
placing the processed dairy product sample in a sample injector of a liquid chromatogram, enabling a mobile phase A and a mobile phase B to flow through the sample injector through an infusion pump of the liquid chromatogram, mixing the mobile phase A and the mobile phase B with the processed dairy product sample, enabling a mixed solution obtained after mixing to flow through a liquid chromatogram column so as to enable the liquid chromatogram column to separate amino acids to be analyzed in the processed dairy product sample, and then detecting different amino acids in the processed dairy product sample through a detector of the chromatogram so as to obtain chromatograms of the different amino acids in the processed dairy product sample;
the mobile phase A is a 5mmol/L perfluorovaleric acid aqueous solution, and the mobile phase B is a 5mmol/L perfluorovaleric acid acetonitrile solution.
Preferably, in the step of injecting the amino acid standard solution and the processed dairy product sample into a liquid chromatography-tandem mass spectrometer for detection, the liquid chromatography conditions are as follows:
flow rate of mobile phase: 0.2 mL/min;
sample introduction amount: 2 mu L of the solution;
preferably, in the step of injecting the amino acid standard solution and the processed dairy product sample into a liquid chromatography-tandem mass spectrometer for detection, the specific process of mass spectrometry is as follows:
the method comprises the steps that a dairy product sample subjected to liquid chromatography separation enters a mass spectrum, molecules in the dairy product sample are ionized by atomization gas sprayed by an ion source of the mass spectrum and loaded high voltage, ions for quantification and qualification are further formed on amino acid molecules to be analyzed under certain collision energy through collision gas, the ions are separated into ions with different masses through a mass analyzer of the mass spectrum according to the mass-to-charge ratio of the ions, and then the ions are detected through an ion detector of the mass spectrum, so that the mass spectrogram of different amino acids in the dairy product sample is obtained.
Compared with the prior art, the invention has the following beneficial effects:
according to the method, the traditional oven hydrolysis is replaced by the microwave digestion mode to carry out pretreatment on the dairy product sample, so that the hydrolysis of the advanced glycosylation end product amino acid molecules combined with the protein in the dairy product sample is achieved, and extraction and purification operations are not required to be carried out through a solid phase extraction column after digestion, so that the method simplifies the steps of pretreatment of the dairy product, improves the detection efficiency and reduces the detection cost.
In addition, the liquid chromatography-tandem mass spectrometry detection method adopted by the invention combines liquid chromatography separation and mass spectrometry detection, a target object enters a mass spectrometry ion source to be ionized in a soft ionization mode to form a molecular ion peak, a mass spectrometry mass analyzer is used for screening a proper mass-to-charge ratio of an analyte, and impurity ion interference is removed, so that the response value of the analyte can be greatly improved, and the method can simultaneously detect four amino acids of advanced glycosylation end products in a dairy product.
Drawings
FIG. 1 is a chromatogram of advanced glycation end product Lysine (LYS) in a sample;
FIG. 2 shows a chromatogram of the advanced glycosylation end product, furfuryl acid (FUR), in a sample;
FIG. 3 chromatogram of the advanced glycosylation end product carboxymethyl lysine (CML) in the sample;
FIG. 4 is a chromatogram of carboxyethyl lysine (CEL) as an advanced glycosylation end product in a sample;
FIG. 5 is a graph comparing conventional hydrolysis and microwave digestion of a sample;
FIG. 6 is a comparison of a lysine over solid phase extraction cartridge with a non-over solid phase extraction cartridge;
FIG. 7 is a comparison of a furfuryl acid column with a solid phase extraction column without a solid phase extraction column;
FIG. 8 is a comparison of a carboxymethyl lysine column over and without a solid phase extraction column;
FIG. 9 is a comparison of a carboxyethyl lysine solid phase extraction cartridge with a non-solid phase extraction cartridge.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The experimental procedures in the following examples are conventional unless otherwise specified. Test materials, reagents and the like used in the following examples are commercially available unless otherwise specified. In the quantitative tests in the following examples, three replicates were set, and the data are the mean or the mean ± standard deviation of the three replicates.
In addition, "and/or" in the whole text includes three schemes, taking a and/or B as an example, including a technical scheme, and a technical scheme that a and B meet simultaneously; in addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present claims.
A method for simultaneously detecting multiple advanced glycosylation end products in a dairy product comprises the following steps:
preparing amino acid standard solutions with different concentrations;
digesting a dairy product sample by using a microwave digestion instrument to hydrolyze amino acid molecules of an advanced glycosylation end product combined with protein in the dairy product into free amino acid micromolecules, and obtaining a digestion solution after digestion;
filtering the digestion solution by a filter membrane, collecting filtrate, and diluting the filtrate to obtain a processed dairy product sample;
and respectively injecting the amino acid standard solution and the processed dairy product sample into a liquid chromatography-tandem mass spectrometer for detection.
According to the method, the traditional oven hydrolysis is replaced by the microwave digestion mode to carry out pretreatment on the dairy product sample, so that the hydrolysis of the advanced glycosylation end product amino acid molecules combined with the protein in the dairy product sample solution is achieved, and extraction and purification operations are not required to be carried out through a solid phase extraction column after digestion, so that the method simplifies the pretreatment steps of the dairy product, improves the detection efficiency and reduces the detection cost.
In addition, the liquid chromatography-tandem mass spectrometry detection method adopted by the invention combines liquid chromatography separation and mass spectrometry detection, a target object enters a mass spectrometry ion source to be ionized in a soft ionization mode to form a molecular ion peak, a mass spectrometry mass analyzer is used for screening a proper mass-to-charge ratio of an analyte, and impurity ion interference is removed, so that the response value of the analyte can be greatly improved, and the method can simultaneously detect four amino acids of advanced glycosylation end products in a dairy product.
Specifically, the digestion solution is cooled to room temperature and then filtered, the filtering operation is not suitable when the temperature of the digestion solution is too high, and the digestion solution with accurate volume is not easy to be transferred during later dilution;
filtering the digestion solution to remove particles larger than 0.22 mu m in the digestion solution, and preventing impurity particles in the dairy product sample subjected to subsequent treatment from entering a chromatographic system to cause blockage;
and (3) making a trend line according to the relationship formed by the peak areas and the concentrations of the peak areas obtained by the amino acid standard solutions at a plurality of concentration points, forming a correlation relationship between a horizontal axis X (amino acid concentration) and a vertical axis Y (peak area), obtaining a standard curve, and calculating the content of the amino acid in the sample according to the standard curve.
In some embodiments, the amino acid standard solution comprises a solvent comprising ultrapure water and a solute comprising lysine, furfuryl acid, carboxymethyl lysine, and carboxyethyl lysine.
In some embodiments, the step of digesting comprises:
adding the processed dairy product sample and 6mol/L hydrochloric acid into a microwave digestion tank, filling nitrogen into the microwave digestion tank to remove oxygen in the microwave digestion tank, placing the microwave digestion tank in a microwave digestion instrument for digestion, and obtaining a digestion solution after digestion.
Specifically, hydrochloric acid is added for hydrolysis, and the advanced glycosylation end product amino acid combined with protein is hydrolyzed into the target amino acid small molecule to be detected by the method;
the Maillard reaction of the dairy product sample can be accelerated when meeting oxygen, and in order to ensure the authenticity of experimental data, nitrogen is filled into the microwave digestion tank to remove the oxygen in the microwave digestion tank, so that the oxidation of protein is avoided.
In some embodiments, the digestion temperature is 150 ℃ and the digestion time is 15-20 min.
The microwave digestion method is used, the digestion time is 15-20 min and is far shorter than the hydrolysis time (20-24 h) of the oven, the pretreatment time of the dairy product sample is greatly reduced, and the detection efficiency is improved.
In some embodiments, the hydrochloric acid used per 1mg protein equivalent (1mg Pro) of the dairy sample is 0.267mL, wherein the sample mass corresponding to 1mg protein equivalent can be determined from the nutritional label of the dairy sample.
In some embodiments, the filter has a pore size of 0.22 μm.
In some embodiments, the specific step of diluting the filtrate into a treated dairy sample solution comprises:
diluting the filtrate by 100 times with ultrapure water to obtain the treated dairy product sample solution.
Specifically, the concentration of the amino acid to be analyzed in the dairy product sample is too high to exceed the bearing range of the chromatographic column or an interfering substance exists, which may cause separation problems such as "flat head" or peak bifurcation, shoulder, poor symmetry, etc., and further affect the accuracy of detection, so that the filtrate needs to be diluted to obtain the processed dairy product sample.
In the step of respectively injecting the amino acid standard solution and the processed dairy product sample solution into a liquid chromatography-tandem mass spectrometer for detection, the specific process of liquid chromatography detection is as follows:
placing the processed dairy product sample in a sample injector of a liquid chromatogram, enabling a mobile phase A and a mobile phase B to flow through the sample injector through an infusion pump of the liquid chromatogram, mixing the mobile phases A and B with the dairy product sample, enabling a mixed solution obtained after mixing to flow through a liquid chromatogram column so as to enable the liquid chromatogram column to separate amino acids to be analyzed in the dairy product sample, and then detecting different amino acids in the dairy product sample through a detector of the chromatogram to obtain a chromatogram of different amino acids in the dairy product sample;
wherein, the conditions of the liquid chromatographic column are as follows:
liquid chromatography column: c18, 100X 2.1mm, 2.6 μm;
the mobile phase A is a 5mmol/L perfluorovaleric acid aqueous solution, and the mobile phase B is a 5mmol/L perfluorovaleric acid acetonitrile solution;
elution gradient: based on the volume percentage content, the content of the mobile phase A is 90 percent and the content of the mobile phase B is 10 percent for 0 min; 2min, the mobile phase A is 90%, and the mobile phase B is 10%; 5min, the mobile phase A is 30%, and the mobile phase B is 70%; 7min, the mobile phase A is 30%, and the mobile phase B is 70%; 9min, the mobile phase A is 10%, and the mobile phase B is 90%; 10min, the mobile phase A is 10%, and the mobile phase B is 90%; 12min, wherein the mobile phase A is 90% and the mobile phase B is 10%; for 15min, the mobile phase A is 90%, and the mobile phase B is 10%.
Specifically, the mobile phase A and the mobile phase B are two mutually soluble solvents, the mobile phase B has strong elution capability, and the mobile phase A has poor elution capability, so that mixed mobile phases with different elution capabilities are obtained by adjusting the proportion of the mobile phase A and the mobile phase B, and the separation of the processed dairy product sample on a chromatographic column is realized.
In some embodiments, in the step of injecting the amino acid standard solution and the processed dairy product sample into the liquid chromatography-tandem mass spectrometer for detection, the liquid chromatography conditions are as follows:
flow rate of mobile phase: 0.2 mL/min;
sample introduction amount: 2 mu L of the solution;
column temperature of liquid chromatography column: 33 to 37 ℃.
In some embodiments, in the step of injecting the amino acid standard solution and the processed dairy product sample into the liquid chromatography-tandem mass spectrometer for detection, the specific process of mass spectrometry is as follows:
enabling a dairy product sample subjected to liquid chromatography separation to enter a mass spectrum, ionizing molecules in the dairy product sample by atomized gas ejected by an ion source of the mass spectrum and loaded high voltage, further forming ions for quantification and qualification for amino acid molecules to be analyzed under certain collision energy through collision gas, separating the ions into ions with different masses according to the mass-to-charge ratio of the ions by a mass analyzer of the mass spectrum, and then detecting each ion through an ion detector of the mass spectrum to obtain mass spectrograms of different amino acids in the dairy product sample;
wherein, the mass spectrum conditions are as follows:
the ion source is an electrospray ion source and is in a positive ion mode, the spraying voltage is 5kV, the atomizing temperature is 350 ℃, the residence time is 100ms, and the collision gas is 45psi high-purity nitrogen;
the detection mode of the ion detector is multi-reaction monitoring.
The mass spectrum is triple quadrupole mass spectrum, triple quadrupole mass spectrum is provided with an electrospray ion source, the electrospray ion source generates charged liquid drops by an electric field in the ionization process, and then forms ions to be analyzed through solvent evaporation. Considering that the target compound is four amino acids, the mobile phase can be made to combine protons and have positive charges by adjusting the pH value of the mobile phase. Therefore, the experiment selected an electrospray ion source and the positive ion mode for ion scanning.
Example 1
Preparing an amino acid standard solution: lysine (LYS), Furnine (FUR), carboxymethyl lysine (CML) and carboxyethyl lysine (CEL) were formulated with ultrapure water to the following 12 concentration gradients, respectively:
0.25ng/mL, 0.5ng/mL, 1ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 250ng/mL, 500ng/mL, 1000ng/mL, and 2000 ng/mL;
taking 100mg infant milk powder into a 50mL microwave digestion tank, adding 4mL of hydrochloric acid with the concentration of 6mol/L, uniformly mixing, filling nitrogen into the microwave digestion tank for 3min to remove oxygen in a sample, placing the microwave digestion tank into a microwave digestion instrument for digestion, setting the digestion temperature at 150 ℃, the digestion time at 15min, and obtaining a digestion solution after digestion;
after the digestion solution is cooled to room temperature, filtering the digestion solution by using a 0.22 mu m filter head, collecting filtrate, diluting the filtrate by using ultrapure water to 100 times of the filtrate in an injection bottle, and obtaining a treated dairy product sample solution;
and respectively injecting the amino acid standard solution and the processed dairy product sample solution into a liquid chromatography-tandem mass spectrometer for detection.
The chromatographic and mass spectrometric conditions of the detection were as follows:
(1) chromatographic conditions
A chromatographic column: c18, 100X 2.1mm, 2.6 μm;
flow rate of mobile phase: 0.2 mL/min;
sample introduction amount: 2 mu L of the solution;
column temperature: 35 ℃;
mobile phase A: 5mM perfluorovaleric acid in water;
mobile phase B: 5mM perfluoropentanoic acid acetonitrile solution;
gradient elution procedure: based on the volume percentage content, the content of the mobile phase A is 90 percent and the content of the mobile phase B is 10 percent for 0 min; 2min, the mobile phase A is 90%, and the mobile phase B is 10%; 5min, the mobile phase A is 30%, and the mobile phase B is 70%; 7min, the mobile phase A is 30%, and the mobile phase B is 70%; 9min, the mobile phase A is 10%, and the mobile phase B is 90%; 10min, the mobile phase A is 10%, and the mobile phase B is 90%; 12min, wherein the mobile phase A is 90% and the mobile phase B is 10%; for 15min, the mobile phase A is 90%, and the mobile phase B is 10%.
(2) Conditions of Mass Spectrometry
The ion source is an electrospray ion source and is in a positive ion mode, the spraying voltage is 5kV, the atomizing temperature is 350 ℃, the residence time is 100ms, and the collision gas is 45psi high-purity nitrogen; the detection mode of the ion detector is multi-reaction monitoring; other mass spectral conditions are shown in table 1.
TABLE 1 Mass Spectrometry parameter setting Table
FIGS. 1 to 4 are chromatograms of Lysine (LYS), furfuryl acid (FUR), carboxymethyl lysine (CML) and carboxyethyl lysine (CEL) in a dairy product sample, respectively.
As can be seen from FIGS. 1 to 4, the four substances have good separation degree, no obvious interference peak is near each substance, the four amino acids have different peak-out times and sharp peak shapes, which indicates that the four amino acids are qualified, the peak area is calculated according to software, and the concentration is obtained according to the peak area corresponding to the working curve of the standard solution so as to achieve the quantitative analysis of the four amino acids. Therefore, the liquid chromatogram-tandem mass spectrum combined method can meet the requirements of qualitative and quantitative detection on four amino acids.
Example 2
This example is used to verify the stability and recovery rate of the test method, and the detection steps are consistent with those in example 1, and are not described again.
Carrying out quantitative analysis on the prepared amino acid standard solution, and drawing a standard curve by using least square linear regression with the peak area response value of the amino acid as the ordinate and the concentration of the standard solution as the abscissa respectively; and obtaining the concentration corresponding to the signal-to-noise ratio (S/N) of 3 and the detection limit of the method, obtaining the concentration corresponding to the signal-to-noise ratio (S/N) of 10 and the quantification limit of the method, wherein the relative standard deviation is obtained by parallelly measuring 5 times at the level of the quantification limit, the recovery rate is obtained by respectively adding 4 mu g of lysine, 0.4 mu g of furaldehyde, 120 mu g of carboxymethyl lysine and 120 mu g of carboxyethyl lysine into a sample and parallelly measuring 5 times, and the results listed in the following table are the average value +/-standard deviation. The specific performance index of the method is shown in Table 2.
TABLE 2 four amino acid test Performance index
According to the data in the table 2, the relative standard deviation of the detection performance indexes of the four amino acids is within 5 percent, which shows that the method has better stability;
the recovery rate is 85-115%, which shows that the method meets the basic requirements of analysis and detection.
Example 3
This example is used to compare microwave digestion and conventional hydrolysis (oven hydrolysis) of a dairy product in the test method, and the detection steps are consistent with those in example 1 and will not be described again.
FIG. 5 is a comparison of the results of conventional hydrolysis and microwave digestion of samples.
As can be seen from FIG. 5, according to the comparison of the results of conventional hydrolysis in an oven for 24 hours and microwave digestion in 15min on the actual milk powder sample, the ratio of the peak area of the amino acid detected by using microwave hydrolysis to the peak area of the amino acid detected by conventional hydrolysis is close to 1, and therefore, the microwave digestion can reach the level of conventional hydrolysis. The pretreatment of the dairy product sample can adopt a microwave digestion mode to replace the conventional hydrolysis to obtain the free target amino acid compound.
Example 4
This example is used to compare the solid phase extraction column (HLB) after microwave digestion with the solid phase extraction column (No-HLB) in the test method, and the detection steps are consistent with those in example 1 and will not be described again.
Fig. 6 to 9 are graphs showing comparison of experimental results of passing through a solid phase extraction column (HLB) and not passing through the solid phase extraction column (No-HLB) when Lysine (LYS), furamic acid (FUR), carboxymethyl lysine (CML) and carboxyethyl lysine (CEL) are simultaneously detected in an actual milk powder sample, and three sets of parallel tests are performed.
As can be seen from fig. 6 to 9, the peak area response values of the solid phase extraction column are more stable, and in general, the peak area values of the four amino acids are higher when the solid phase extraction column is not used, which indicates that the target amino acid does not have a certain loss compared with the solid phase extraction column. In summary, it is considered that the process of passing through the solid phase extraction cartridge can be omitted.
Example 5
This example is used to detect the content of lysine, furfuryl acid, carboxymethyl lysine and carboxyethyl lysine in the sample, and the detection steps are the same as those in example 1, and are not described again.
The contents of lysine, furfuryl acid, carboxymethyl lysine and carboxyethyl lysine in 100mg of infant formula (paragraph 3) in which the protein content was 15mg and 460. mu.L of ultra-high-temperature sterilized milk in which the protein content was 16.56mg were measured by the method of the present invention (the calculation results are converted to the amino acid content per 100g of Pro, and the results of three parallel tests were averaged), and the results are as follows:
TABLE 3 infant formula milk powder (3 paragraphs) and ultra-high temperature sterilized milk determination results
As can be seen from Table 3, the method of the present invention can be used for simultaneously detecting the contents of lysine, furfuryl acid, carboxymethyl lysine and carboxyethyl lysine which are advanced glycosylation end products in the dairy product.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A method for simultaneously detecting multiple advanced glycosylation end products in a dairy product is characterized by comprising the following steps:
preparing amino acid standard solutions with different concentrations;
digesting a dairy product sample by using a microwave digestion instrument to hydrolyze amino acid molecules of an advanced glycosylation end product combined with protein in the dairy product into free amino acid micromolecules, and obtaining a digestion solution after digestion;
filtering the digestion solution by a filter membrane, collecting filtrate, and diluting the filtrate to obtain a processed dairy product sample;
and respectively injecting the amino acid standard solution and the processed dairy product sample into a liquid chromatography-tandem mass spectrometer for detection.
2. The method for simultaneously detecting multiple advanced glycosylation end products in a milk product according to claim 1, wherein the amino acid standard solution comprises a solvent and solutes, the solvent comprises ultrapure water, and the solutes comprise lysine, furfuryl acid, carboxymethyl lysine and carboxyethyl lysine.
3. The method for simultaneously detecting multiple advanced glycosylation end products in a milk product of claim 1, wherein the digesting step comprises:
adding the processed dairy product sample and 6mol/L hydrochloric acid into a microwave digestion tank, filling nitrogen into the microwave digestion tank to remove oxygen in the microwave digestion tank, placing the microwave digestion tank in a microwave digestion instrument for digestion, and obtaining a digestion solution after digestion.
4. The method for simultaneously detecting multiple advanced glycosylation end products in a dairy product according to claim 3, wherein the digestion temperature is 150 ℃ and the digestion time is 15-20 min.
5. The method for simultaneously detecting multiple advanced glycosylation end products in a dairy product according to claim 3, wherein the amount of hydrochloric acid used per 1mg protein equivalent of the dairy product sample is 0.267 mL.
6. The method for simultaneously detecting multiple advanced glycosylation end products of a dairy product of claim 1, wherein the pore size of the filter is 0.22 μm.
7. The method for simultaneously detecting multiple advanced glycosylation end products in a dairy product according to claim 1, wherein the specific step of diluting the filtrate into a processed dairy product sample comprises:
diluting the filtrate by 100 times with ultrapure water to obtain a treated dairy product sample.
8. The method for simultaneously detecting multiple advanced glycosylation end products in a dairy product according to claim 1, wherein in the step of respectively injecting the amino acid standard solution and the processed dairy product sample solution into a liquid chromatography-tandem mass spectrometer for detection, the specific process of liquid chromatography detection is as follows:
placing the processed dairy product sample in a sample injector of a liquid chromatogram, enabling a mobile phase A and a mobile phase B to flow through the sample injector through an infusion pump of the liquid chromatogram, mixing the mobile phase A and the mobile phase B with the processed dairy product sample, enabling a mixed solution obtained after mixing to flow through a liquid chromatogram column so as to enable the liquid chromatogram column to separate amino acids to be analyzed in the dairy product sample, and then detecting different amino acids in the processed dairy product sample through a mass spectrum detector so as to obtain a chromatogram of the different amino acids in the processed dairy product sample;
the mobile phase A is a 5mmol/L perfluorovaleric acid aqueous solution, and the mobile phase B is a 5mmol/L perfluorovaleric acid acetonitrile solution.
9. The method for simultaneously detecting multiple advanced glycosylation end products in a dairy product according to claim 8, wherein in the step of respectively injecting the amino acid standard solution and the processed dairy product sample into a liquid chromatography-tandem mass spectrometer for detection, the liquid chromatography conditions are as follows:
flow rate of mobile phase: 0.2 mL/min;
sample introduction amount: 2 μ L.
10. The method for simultaneously detecting multiple advanced glycosylation end products in a dairy product according to claim 1, wherein in the step of respectively injecting the amino acid standard solution and the processed dairy product sample into a liquid chromatography-tandem mass spectrometer for detection, the specific process of mass spectrometry detection is as follows:
the method comprises the steps of enabling a dairy product sample subjected to liquid chromatography separation to enter a mass spectrum, ionizing the dairy product sample by using an electrospray ion source of the mass spectrum, separating ions into ions with different masses according to the mass-to-charge ratio of the ions through a mass analyzer of the mass spectrum, and detecting each ion through an ion detector of the mass spectrum to obtain mass spectrograms of different amino acids in the dairy product sample.
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