Method for determining content of biogenic amine in marine fish

Abstract

The invention discloses a method for determining biogenic amines in marine fishes, which is characterized in that a sample is purified and analyzed by liquid chromatography-tandem mass spectrometry. The method has the advantages that the method is easy to operate, the analysis speed is high, the sensitivity is high, and a new effective solution is provided for detection of the biogenic amines in the marine fishes.

Description

Technical Field

[0001] The invention relates to the field of food analysis, and in particular to a method for determining the content of biogenic amines in marine fish. Background Art

[0002] Biogenic amines (BA) are a general term for a class of low-molecular-weight organic compounds with biological activity and containing amino groups. They are mainly formed by the decarboxylation of the corresponding amino acids by microorganisms or produced by aldehydes and ketones under the action of amino acid transaminases. Biogenic amines can be divided into two categories according to their composition: monoamines and polyamines. Monoamines mainly include tyramine, histamine, putrescine, cadaverine, 2-phenylethylamine, tryptamine, etc.; polyamines mainly include spermine and spermidine. Trace biogenic amines are normal active ingredients in organisms (including humans), and play important physiological functions in active cells of animals, plants and microorganisms. The human body itself also synthesizes some biogenic amines to meet the needs of the body's metabolism, but excessive intake of biogenic amines will cause symptoms of poisoning such as dyspnea, cramps, and headaches, and in severe cases, it can endanger life. Biogenic amines are widely present in protein-rich foods such as meat and meat products, aquatic products, and cheese. Unreasonable environmental hygiene, food processing technology, and food storage methods will cause the production of a large number of biogenic amines. The content of biogenic amines can be used as an indicator of the freshness of food. It is reported that aquatic products and their products are among the products with the highest content of biogenic amines. The content of biogenic amines in spoiled aquatic products will increase significantly. Even high-temperature treatment can only destroy a small part of them. If people eat such food, they may suffer from food poisoning. Therefore, controlling the content of biogenic amines is an important way to ensure food safety. It is crucial to establish a rapid, simple and sensitive method for detecting the content of biogenic amines.

[0003] At present, the detection methods of biogenic amines include gas chromatography-tandem mass spectrometry, ion chromatography, high performance liquid chromatography, and high performance liquid chromatography-tandem mass spectrometry. Gas chromatography-tandem mass spectrometry often detects volatile biogenic amines, most of which are not stable at high temperatures; ion chromatography is a liquid phase method for analyzing ions, which can separate and analyze biogenic amines without derivatization, but the sensitivity is not high; high performance liquid chromatography requires derivatization treatment, which is time-consuming. Summary of the invention

[0004] The purpose of the present invention is to provide a method for determining the content of biogenic amines in marine fish in view of the shortcomings of the prior art. The method uses liquid chromatography-triple quadrupole tandem mass spectrometry analysis to improve sensitivity and ensure that trace amounts of biogenic amines in complex food matrices can be detected.

[0005] To achieve the above object, the present invention provides a method for determining the content of biogenic amines in marine fish, comprising the following steps:

[0006] S100. Extracting samples: crushing and mixing the samples and adding them into a plastic centrifuge tube, adding an extractant and vortex mixing, centrifuging to obtain a sample supernatant, and blowing with nitrogen to obtain a test solution;

[0007] S200. Liquid chromatography detection: The sample solution obtained above is detected by liquid chromatography-triple quadrupole tandem mass spectrometry.

[0008] The chromatographic conditions are:

[0009] Chromatographic column: Agilent Poroshell Hilic (2.1×150 mm, 2.7 μm);

[0010] Flow rate: 0.3 mL / min;

[0011] Column temperature: 35°C;

[0012] Injection volume: 2 μL;

[0013] Mobile phase: 5mmol / L ammonium acetate (0.5% formic acid) and acetonitrile;

[0014] S300. Qualitative analysis: qualitative analysis is performed by comparing the retention time of the results obtained by the biogenic amine and the sample test solution;

[0015] S400. Quantitative analysis: By adding different concentrations of biogenic amine standard solutions to the marine fish blank matrix, the corresponding matrix standard curve is obtained to remove matrix interference and avoid the influence of interfering substances on the quantitative results. The biogenic amine content is calculated by the following formula:

[0016]

[0017] Where: X is the content of biogenic amines in the sample (mg / kg); V is the final volume of the sample solution (mL); m is the mass of the sample (g); c is the concentration of the solution measured by the instrument (μg / L).

[0018] As a further improvement of the above solution, the mass spectrometry method in step S200 adopts a multiple reaction monitoring mode.

[0019] As a further improvement of the above scheme, in step S100, the mixture is vortexed and shaken for at least 20 minutes, and then centrifuged at a speed of 5000 rpm. The supernatant is transferred to a test tube, dried with nitrogen, and the mobile phase solution is re-dissolved to obtain the sample solution.

[0020] As a further improvement of the above scheme, the mass spectrometry parameters in step S2 are: ion source: ESI source; scanning mode: positive ion mode; ion source temperature: 450°C; ion detection mode: multiple ion reaction monitoring (MRM); IS voltage: 2500V; curtain gas CUR: 30psi; nebulizer gas GS1: 50psi; auxiliary gas GS2: 55psi; collision gas CAD: High.

[0021] As a further improvement of the above scheme, the centrifugal separation environment temperature is 4° C. and the centrifugal separation time is 5 minutes.

[0022] As a further improvement of the above scheme, after the sample in step S100 is crushed and mixed, 2 g is weighed and added to a 50 mL plastic centrifuge tube, and 5 mL of 0.2% acidified acetonitrile is added, and vortex mixing is performed for 20 minutes.

[0023] The beneficial effects of the present invention are:

[0024] 1. The present invention uses liquid chromatography-triple quadrupole tandem mass spectrometer for detection, and adopts multiple reaction ion monitoring mode (MRM), which can greatly reduce the influence of matrix interference on detection accuracy and detection limit, improve sensitivity, ensure that trace amounts of biogenic amines in complex food matrices can be detected, and speed up the detection speed;

[0025] 2. The sample is vortex-extracted using an acidified acetonitrile extractant, and then centrifuged, dried with nitrogen, and then redissolved as a pre-treatment method, which has the advantages of simple operation and low detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The MRM total ion chromatogram of the 7 biogenic amine standard solutions in the present invention;

[0027] Figure 2 This is a calibration curve diagram of 7 biogenic amine standards in the present invention;

[0028] Figure 3 The chromatogram of the present invention for detecting the content of biogenic amines in yellow croaker;

[0029] Figure 4 The chromatogram of the present invention for detecting the content of biogenic amines in silver pomfret;

[0030] Figure 5 The chromatogram is a chromatogram of detecting the content of biogenic amines in hairtail according to the present invention. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings of the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.

[0032] The present invention provides a method for determining the content of biogenic amines in marine fish, comprising the following steps:

[0033] S100. Extract the sample: grind and mix the sample and add it into a plastic centrifuge tube, add the extractant, vortex mix and shake for at least 20 minutes, then centrifuge at 5000 rpm and 4°C for 5 minutes, transfer the supernatant to a test tube, blow dry with nitrogen, and re-dissolve the sample in the mobile phase solution to obtain the sample solution;

[0034] S200. Liquid chromatography detection: The sample solution obtained above is detected by liquid chromatography-triple quadrupole tandem mass spectrometry, and the mass spectrometry method adopts multiple reaction monitoring mode.

[0035] The chromatographic conditions are:

[0036] Chromatographic column: Agilent Poroshell Hilic (2.1×150 mm, 2.7 μm); flow rate: 0.3 mL / min; column temperature: 35°C; injection volume: 2 μL; mobile phase: 5 mmol / L ammonium acetate (0.5% formic acid) and acetonitrile;

[0037] The mass spectrometry parameters were as follows: ion source: ESI source; scan mode: positive ion mode; ion source temperature: 450°C; ion detection mode: multiple ion reaction monitoring (MRM); IS voltage: 2500V; curtain gas CUR: 30psi; nebulizer gas GS1: 50psi; auxiliary gas GS2: 55psi; collision gas CAD: High.

[0038] S300. Qualitative analysis: qualitative analysis is performed by comparing the retention time of the results obtained by the biogenic amine and the sample test solution;

[0039] S400. Quantitative analysis: By adding different concentrations of biogenic amine standard solutions to the marine fish blank matrix, the corresponding matrix standard curve is obtained to remove matrix interference and avoid the influence of interfering substances on the quantitative results. The biogenic amine content is calculated by the following formula:

[0040]

[0041] Where: X is the content of biogenic amines in the sample (mg / kg); V is the final volume of the sample solution (mL); m is the mass of the sample (g); c is the concentration of the solution measured by the instrument (μg / L).

[0042] Example 1

[0043] A method for determining the content of biogenic amines in marine fish comprises the following steps:

[0044] Select fresh yellow croaker, pomfret and hairtail, remove the scales, viscera and skin, grind the edible part of the fish meat to make minced fish, pack the samples, place the samples in a frozen (-20℃) refrigerator and seal them for storage;

[0045] Weigh about 2 g of sample to an accuracy of 0.001 mg, with m representing the mass, add 5 mL of 0.2% acidified acetonitrile solution, vortex shake for 20 min, freeze centrifuge at 8000 rpm for 5 min, take the supernatant into a test tube, repeat the extraction once, collect the supernatant, blow nitrogen at 45°C to dryness, re-dissolve with 1 mL of mobile phase, vortex shake, take the supernatant and filter through a 0.22 μm polytetrafluoroethylene filter membrane, and the filtrate is tested by liquid chromatography-mass spectrometry;

[0046] Liquid chromatography-triple quadrupole tandem mass spectrometer: The biogenic amine standard solution and the sample solution were detected by liquid chromatography-triple quadrupole tandem mass spectrometry. The chromatographic conditions were as follows: chromatographic column: Agilent Poroshell Hilic (2.1×150 mm, 2.7 μm); flow rate: 0.3 mL / min; chromatographic column temperature: 35° C.; injection volume: 2 μL; mobile phase: A: 5 mmol / L ammonium acetate (0.5% formic acid), B: acetonitrile; the gradient elution program is shown in the following table:

[0047]

[0048] The mass spectrometry parameters are:

[0049] Ion source: ESI source; Scan mode: positive ion mode; Ion source temperature: 450°C; Ion detection mode: multiple ion reaction monitoring (MRM); IS voltage: 2500V; Curtain gas CUR: 30psi; Nebulizer gas GS1: 50psi; Auxiliary gas GS2: 55psi; Collision gas CAD: High; 7 biogenic amines multiple reaction monitoring scan mode mass spectrometry parameters are shown in the table:

[0050]

[0051] Note: * indicates quantitative ion.

[0052] Step 3: Qualitative analysis: Qualitative analysis is performed by comparing the retention time of the results obtained from the biogenic amine standard solution and the sample test solution; the retention time of the target chromatographic peak in the sample is compared with the retention time of the corresponding standard chromatographic peak, and the variation range is within ±2.5%. The mass spectrometry qualifier ion of each compound must appear, and the sample mass spectrum after background subtraction should include at least one parent ion and two daughter ions. In addition, for the same test batch, the relative ion abundance ratio of the two daughter ions of the target compound in the sample is compared with the matrix standard solution with equivalent mass concentration. If it meets the requirements of the following table, it can be judged that the corresponding compound exists in the sample.

[0053] Relative ion abundance ＞50％ 20％～50％ 10％～20％ ≤10％ Allowable relative deviation ±20％ ±25％ ±30％ ±50％

[0054] Step 4: Quantitative analysis: Add different concentrations of biogenic amine standard solutions to the blank matrix of aquatic products to obtain the corresponding matrix standard curve. This is to remove the interference of the aquatic product matrix and avoid the influence of interfering substances on the quantitative results. The biogenic amine content is calculated by the following formula:

[0055]

[0056] Where: X is the content of biogenic amines in the sample (mg / kg); V is the final volume of the sample solution (mL); m is the mass of the sample (g); c is the concentration of the solution measured by the instrument (μg / L).

[0057] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0058] Example 2

[0059] Standard Spectrum and Curve Equation of Biogenic Amines

[0060] The biogenic amine standard solution was diluted stepwise into a series of standard working solutions of 5 μg / L, 20 μg / L, 40 μg / L, 50 μg / L, 80 μg / L, 100 μg / L, 150 μg / L, 200 μg / L, 300 μg / L, and 400 μg / L, and analyzed using the chromatographic conditions and mass spectrometry parameters provided by the present invention. The results are shown in the figure. Figure 1 This is the MRM total ion chromatogram of 7 biogenic amine standard solutions. Figure 2 This is a calibration curve diagram for seven biogenic amine standards.

[0061] With the concentration of the standard solution as the horizontal axis and the corresponding quantitative ion pair peak area as the vertical axis, the corresponding standard curve equation was obtained, as shown in the following table. The concentration of the mixed standard solution of biogenic amines and the corresponding peak area showed a good linear relationship, and the correlation coefficient was greater than 0.99; the detection limit calculated by the quantitative ion signal-to-noise ratio method was 5μg / kg.

[0062]

[0063] Example 3

[0064] Analysis of biogenic amine content in yellow croaker under frozen storage conditions

[0065] On April 15, 2024, fresh yellow croaker was purchased from Hema Fresh, origin: Ningde, Fujian.

[0066] Remove the internal organs of the fresh-frozen yellow croaker, peel the skin, take out the edible part of the fish meat, grind it into minced fish, and pack it into 4 samples, each sample should not be less than 50g. Put them into sealed bags, affix labels, and place the samples in a freezer (-20℃) and store them in a sealed container for 1d, 20d, 50d, and 80d.

[0067] Weigh 2 g of minced fish meat and place it in a 50 mL centrifuge tube, put in a ceramic homogenizer, add 5 mL of 0.2% acidified acetonitrile, vortex in a vortex mixer for 20 min, centrifuge at 4 ° C 8000 r / min for 5 min, transfer the supernatant into a test tube, repeat the extraction once, combine the supernatants, blow to near dryness with nitrogen at 45 ° C, add 1 mL of mobile phase to re-dissolve, vortex mix, take the supernatant and pass it through a 0.22 μm polytetrafluoroethylene filter membrane, and the filtrate is detected by liquid chromatography-mass spectrometry.

[0068] The sample was analyzed by liquid chromatography-mass spectrometry, and the obtained chromatogram was as follows: Figure 3 As shown in the figure, a peak appeared at the biogenic amine target according to the retention time, and one parent ion and two daughter ion qualitative ions appeared, and the relative abundance was within the allowable deviation, so it was judged as a positive result.

[0069] Example 4

[0070] Analysis of biogenic amines content in silver pomfret under frozen storage conditions

[0071] On April 15, 2024, fresh silver pomfret was purchased from HEMA Fresh, origin: Zhoushan, Zhejiang.

[0072] Remove the internal organs of the fresh-frozen silver pomfret, peel the skin, take out the edible part of the fish meat, grind it into surimi, and pack it into 4 samples, each sample should not be less than 50g, put them into sealed bags, affix labels, and place the samples in a freezer (-20℃) and store them in a sealed manner for 1d, 20d, 50d, and 80d.

[0073] Weigh 2 g of minced fish meat and place it in a 50 mL centrifuge tube, put in a ceramic homogenizer, add 5 mL of 0.2% acidified acetonitrile, vortex in a vortex mixer for 20 min, centrifuge at 4 ° C 8000 r / min for 5 min, transfer the supernatant into a test tube, repeat the extraction once, combine the supernatants, blow to near dryness with nitrogen at 45 ° C, add 1 mL of mobile phase to re-dissolve, vortex mix, take the supernatant and pass it through a 0.22 μm polytetrafluoroethylene filter membrane, and the filtrate is detected by liquid chromatography-mass spectrometry.

[0074] The sample was analyzed by liquid chromatography-mass spectrometry, and the obtained chromatogram was as follows: Figure 4 As shown in the figure, a peak appeared at the biogenic amine target according to the retention time, and one parent ion and two daughter ion qualitative ions appeared, and the relative abundance was within the allowable deviation, so it was judged as a positive result.

[0075] Example 5

[0076] Analysis of biogenic amines content in hairtail under frozen storage conditions

[0077] On April 15, 2024, fresh hairtail was purchased from Hema Fresh, origin: Zhoushan, Zhejiang.

[0078] Remove the internal organs of the fresh-frozen hairtail, peel the skin, take out the edible part of the fish meat, grind it into surimi, and pack it into 4 samples, each sample should not be less than 50g, put them into sealed bags, affix labels, and place the samples in a freezer (-20℃) and store them in a sealed manner for 1d, 20d, 50d, and 80d.

[0079] Weigh 2 g of minced fish meat and place it in a 50 mL centrifuge tube, put in a ceramic homogenizer, add 5 mL of 0.2% acidified acetonitrile, vortex in a vortex mixer for 20 min, centrifuge at 4 ° C 8000 r / min for 5 min, transfer the supernatant into a test tube, repeat the extraction once, combine the supernatants, blow to near dryness with nitrogen at 45 ° C, add 1 mL of mobile phase to re-dissolve, vortex mix, take the supernatant and pass it through a 0.22 μm polytetrafluoroethylene filter membrane, and the filtrate is detected by liquid chromatography-mass spectrometry.

[0080] The sample was analyzed by liquid chromatography-mass spectrometry, and the obtained chromatogram was as follows: Figure 5 As shown in the figure, a peak appeared at the biogenic amine target according to the retention time, and one parent ion and two daughter ion qualitative ions appeared, and the relative abundance was within the allowable deviation, so it was judged as a positive result.

[0081] Analysis of sample spike results

[0082] Low, medium and high concentrations of biogenic amine standard solutions were added to blank matrix samples, and pretreatment and analysis were performed according to the method described in the present invention. The spiked samples with different concentrations were measured multiple times, and the spiked recovery rate was between 79.3% and 107.6%, and the relative standard deviation was less than 5%.

[0083] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0084] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

[0085] The above-described embodiments are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining biogenic amines in marine fish, characterized in that: The steps include: S100. Extracting samples: crushing and mixing the samples and adding them into a plastic centrifuge tube, adding an extractant and vortex mixing, centrifuging to obtain a sample supernatant, and blowing with nitrogen to obtain a test solution; S200. Liquid chromatography detection: The sample solution obtained above is detected by liquid chromatography-triple quadrupole tandem mass spectrometry. The chromatographic conditions are: Chromatographic column: Agilent Poroshell Hilic (2.1×150 mm, 2.7 μm); Flow rate: 0.3 mL / min; Column temperature: 35°C; Injection volume: 2 μL; Mobile phase: 5mmol / L ammonium acetate (0.5% formic acid) and acetonitrile; S300. Qualitative analysis: qualitative analysis is performed by comparing the retention time of the results obtained by the biogenic amine and the sample test solution; S400. Quantitative analysis: By adding different concentrations of biogenic amine standard solutions to the marine fish blank matrix, the corresponding matrix standard curve is obtained to remove matrix interference and avoid the influence of interfering substances on the quantitative results. The biogenic amine content is calculated by the following formula: Where: X is the content of biogenic amines in the sample (mg / kg); V is the final volume of the sample solution (mL); m is the mass of the sample (g); c is the concentration of the solution measured by the instrument (μg / L).

2. The method for determining biogenic amines in marine fish according to claim 1, characterized in that: The mass spectrometry method in step S200 adopts a multiple reaction monitoring mode.

3. The method for determining biogenic amines in marine fish according to claim 1, characterized in that: In step S100, the mixture is vortexed and shaken for at least 20 minutes, and then centrifuged at 5000 rpm. The supernatant is transferred to a test tube, dried with nitrogen, and the mobile phase solution is re-dissolved to obtain the sample solution.

4. The method for determining biogenic amines in marine fish according to claim 1, characterized in that: The mass spectrometry parameters in step S2 are: Ion source: ESI source; Scan mode: positive ion mode; Ion source temperature: 450°C; Ion detection mode: multiple ion reaction monitoring (MRM); IS voltage: 2500V; Air curtain air CUR: 30psi; Atomizing gas GS1: 50psi; Auxiliary gas GS2: 55psi; Collision gas CAD: High.

5. The method for determining biogenic amines in marine fish according to claim 3, characterized in that: The centrifugal separation environment temperature is 4° C. and the centrifugal separation time is 5 minutes.

6. The method for determining the content of biogenic amines in marine fish according to claim 1, characterized in that: After the sample in step S100 is crushed and mixed, 2 g is weighed and added to a 50 mL plastic centrifuge tube, and 5 mL of 0.2% acidified acetonitrile is added, and vortex mixed for 20 minutes.

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