Ion Chromatographic Determination Method for 12 Biogenic Amines

Abstract

This invention discloses an ion chromatography method for the determination of 12 biogenic amines. The method utilizes ion chromatography with a mixed mobile phase of nitric acid aqueous solution and acetonitrile via a cation exchange column to simultaneously determine the content of 12 biogenic amines. The 12 biogenic amines can be accurately qualitatively and quantitatively analyzed simultaneously within 35 minutes. The linear range of the ion chromatography method for the determination of 12 biogenic amines established in this invention is 0.5–100 mg / L, the recoveries of each biogenic amine component are between 85.2% and 116.7%, and the precision is 0.2%–2.4%. The ion chromatography method established in this invention features simple operation, a wide linear range, good repeatability, high accuracy, broad applicability, and good stability, and shows promising application prospects in the simultaneous determination and analysis of biogenic amines in food waste.

Description

Technical Field

[0001] This invention relates to a method for the simultaneous determination of the content of multiple biogenic amines, and more particularly to an ion chromatography method for the determination of the content of 12 biogenic amines in leftover food, which belongs to the field of biogenic amine content determination. Background Technology

[0002] Food proteins, under the action of enzymes in the food itself and microbial enzymes, break down into amino acids. These amino acids further decompose to produce low-molecular-weight substances with putrefactive characteristics. Firstly, amino acids are decarboxylated by decarboxylases to form amines, such as histidine to histamine, ornithine to putrescine, lysine to cadaverine, and tryptophan to tryptophan. Secondly, amino acids are deaminoked by deaminases to form ammonia. Ammonia reacts with methyl groups to produce monomethylamine, dimethylamine, trimethylamine, etc.

[0003] Biogenic amines (BA) are a class of small-molecule nitrogen-containing organic compounds with biological activity. Based on their chemical structure, they can be classified into: aliphatic amines, including cadaverine and putrescine; aromatic amines, including tyramine and phenylethylamine; and heterocyclic amines, including histamine and tryptamine. Biogenic amines are an important indicator of food spoilage and are widely used to evaluate food freshness.

[0004] The methods reported in the literature for the determination of biogenic amines mainly include liquid chromatography (LC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), and ion chromatography-tandem mass spectrometry (I-MS / MS). LC is the most widely reported method for determining biogenic amines, but it requires derivatization for pretreatment, is cumbersome, and has poor method stability. LC-MS / MS and I-MS / MS require sophisticated equipment, have narrow linear detection ranges, and are not suitable for determining high levels of biogenic amines in complex matrix samples. Summary of the Invention

[0005] The main objective of this invention is to provide an ion chromatography method for the determination of the content of 12 biogenic amines. This method is simple to operate, has a wide linear range, good repeatability, high accuracy, wide applicability, and good stability.

[0006] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0007] This invention provides an ion chromatography method for the determination of the content of 12 biogenic amines, wherein the 12 biogenic amines are methylamine, ethylamine, octopamine, trimethylamine, tyramine, benzylamine, β-phenylethylamine, putrescine, cadaverine, histamine, tryptamine, and guanidine; the ion chromatography method includes:

[0008] (1) Prepare standard working stock solutions of methylamine, ethylamine, octopamine, trimethylamine, tyramine, benzylamine, β-phenylethylamine, putrescine, cadaverine, histamine, tryptamine and guanidine respectively; mix the standard working stock solutions and make up to volume to obtain a mixed standard working solution;

[0009] (2) The mixed standard working solution was analyzed by ion chromatography to obtain ion chromatograms of methylamine, ethylamine, octopamine, trimethylamine, tyramine, benzylamine, β-phenylethylamine, putrescine, cadaverine, histamine, tryptamine and guanidine.

[0010] (3) Based on the chromatographic peak area of ​​each biogenic amine in the ion chromatogram, the standard curves of each biogenic amine in the standard working stock solution were plotted respectively for methylamine, ethylamine, octopamine, trimethylamine, tyramine, benzylamine, β-phenylethylamine, putrescine, cadaverine, histamine, tryptamine and guanidine.

[0011] (4) After pretreatment of the sample to be tested, the sample is analyzed by ion chromatography to obtain the ion chromatogram of the sample solution to be tested;

[0012] (5) The content of each biogenic amine in the sample solution to be tested is calculated by using the chromatographic peak area corresponding to each biogenic amine in the ion chromatogram of the sample solution to be tested and the corresponding standard curve as a benchmark.

[0013] In a preferred embodiment of the present invention, the method for preparing the standard working stock solutions of methylamine, ethylamine, octopamine, trimethylamine, tyramine, benzylamine, β-phenylethylamine, putrescine, cadaverine, histamine, tryptamine and guanidine in step (1) includes: weighing each biological amine standard, adding nitric acid for acidification, and then adjusting the volume with nitric acid solution to obtain their respective standard working stock solutions.

[0014] As a preferred embodiment of the present invention, the chromatographic conditions for analysis using an ion chromatograph in step (2) or step (4) include:

[0015] The mobile phase consists of an aqueous nitric acid solution and acetonitrile; preferably, the volume ratio of the 4.25 mmol / L aqueous nitric acid solution to the acetonitrile is 95:5; more preferably, the aqueous nitric acid solution is an aqueous nitric acid solution with a concentration of 4.25 mmol / L.

[0016] The ion chromatography column is a cation column, wherein the cation column is preferably Metrosep C4 100 / 4.0;

[0017] The preferred flow rate is 0.9 mL / min;

[0018] The preferred injection volume is 20 μL;

[0019] The preferred column temperature is 30℃;

[0020] The detector is preferably a non-suppression-conductivity detector.

[0021] In a preferred embodiment of the present invention, step (4) of pretreatment of the sample to be tested includes: adding an extraction solution to the sample to be tested, shaking and extracting, centrifuging, and passing the supernatant through a microporous filter membrane to obtain the sample; wherein, the extraction solution is preferably a 4.25 mol / L nitric acid aqueous solution.

[0022] As a preferred embodiment of the present invention, the method for calculating the content of each biogenic amine in the sample solution to be tested by comparing the chromatographic peak area corresponding to each biogenic amine in the chromatogram of the sample solution to be tested with the corresponding standard curve in step (5) includes:

[0023] The content of each biogenic amine in the sample solution was calculated using the external standard method based on the standard curve. The calculation formula is as follows:

[0024]

[0025] In the formula:

[0026] The content of biogenic amines in sample X, in mg / g;

[0027] c - The concentration of biogenic amines measured in the sample solution, mg / L;

[0028] V - Extraction volume, mL;

[0029] m - Sample mass, in g

[0030] K - Dilution factor.

[0031] This invention employs an ion chromatography method, using a mixed mobile phase of nitric acid aqueous solution and acetonitrile via a cation exchange column, to simultaneously determine the content of 12 biogenic amines. These 12 biogenic amines can be qualitatively and quantitatively analyzed simultaneously within 35 minutes. The linear range of the simultaneous determination method for the 12 biogenic amines established in this invention is 0.5-100 mg / L; the detection limits for the 12 biogenic amines are as follows: methylamine 0.05 mg·L⁻¹ -1 Ethylamine 0.06 mg·L -1 Octopamine 0.16 mg·L -1 Trimethylamine 0.17 mg·L -1 Tyramine 0.33 mg·L -1 Benzylamine 0.32 mg·L -1 β-Phenylacetamine 0.47 mg·L -1 putrescine 0.25 mg·L -1 0.35 mg·L⁻¹ cadaverine -1 Histamine 0.58 mg·L -1 Tryptophan 0.67 mg·L -1Guanidine 0.59 mg·L -1 The recovery rates of each biogenic amine component ranged from 85.2% to 116.7%, and the precision ranged from 0.2% to 2.4%. This invention features simple operation, wide linear range, good repeatability, high accuracy, wide applicability, and good stability, and has promising applications in the simultaneous determination of biogenic amines in leftover food. Attached Figure Description

[0032] Figure 1 To analyze the mixed standard working solution using ion chromatography, ion chromatograms of methylamine, ethylamine, octopamine, trimethylamine, tyramine, benzylamine, β-phenylethylamine, putrescine, cadaverine, histamine, tryptamine, and guanidine were obtained. From left to right, the ion chromatograms are for methylamine, ethylamine, octopamine, trimethylamine, tyramine, benzylamine, β-phenylethylamine, putrescine, cadaverine, histamine, tryptamine, and guanidine.

[0033] Figure 2 The standard curve for methylamine is shown below; the concentrations are 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 5.0 mg / L, 10.0 mg / L, 20.0 mg / L, 50.0 mg / L and 100.0 mg / L respectively.

[0034] Figure 3 This is the standard curve for ethylamine; the concentrations are 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 5.0 mg / L, 10.0 mg / L, 20.0 mg / L, 50.0 mg / L and 100.0 mg / L respectively.

[0035] Figure 4 The standard curve for octopamine is shown below; the concentrations are 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 5.0 mg / L, 10.0 mg / L, 20.0 mg / L, 50.0 mg / L and 100.0 mg / L respectively.

[0036] Figure 5 The standard curve for trimethylamine is shown below; the concentrations are 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 5.0 mg / L, 10.0 mg / L, 20.0 mg / L, 50.0 mg / L and 100.0 mg / L respectively.

[0037] Figure 6 The standard curve for tyramine is shown, with concentrations of 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 5.0 mg / L, 10.0 mg / L, 20.0 mg / L, 50.0 mg / L, and 100.0 mg / L, respectively.

[0038] Figure 7The standard curve for aniline is shown, with concentrations of 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 5.0 mg / L, 10.0 mg / L, 20.0 mg / L, 50.0 mg / L, and 100.0 mg / L, respectively.

[0039] Figure 8 The standard curve for β-phenylethylamine is shown, with concentrations of 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 5.0 mg / L, 10.0 mg / L, 20.0 mg / L, 50.0 mg / L, and 100.0 mg / L, respectively.

[0040] Figure 9 The standard curve for putrescine is shown, with concentrations of 5 mg / L, 10 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 400 mg / L, 800 mg / L, and 1000 mg / L, respectively.

[0041] Figure 10 The standard curve for cadaverine is shown, with concentrations of 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 5.0 mg / L, 10.0 mg / L, 20.0 mg / L, 50.0 mg / L, and 100.0 mg / L, respectively.

[0042] Figure 11 The standard curve for histamine is shown, with concentrations of 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 5.0 mg / L, 10.0 mg / L, 20.0 mg / L, 50.0 mg / L, and 100.0 mg / L, respectively.

[0043] Figure 12 The standard curve for tryptophan was prepared with concentrations of 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 5.0 mg / L, 10.0 mg / L, 20.0 mg / L, 50.0 mg / L, and 100.0 mg / L.

[0044] Figure 13 The standard curve for guanidine is shown, with concentrations of 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 5.0 mg / L, 10.0 mg / L, 20.0 mg / L, 50.0 mg / L, and 100.0 mg / L, respectively.

[0045] Figure 14 Ion chromatograms of 12 biogenic amines determined from leftover food samples. Detailed Implementation

[0046] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0047] Example 1: Establishment of a method for simultaneous determination of 12 biogenic amines in leftover food.

[0048] 1. Experimental Methods

[0049] 1.1 Preparation of mobile phase solution

[0050] Mobile phase preparation: 4.25 mmol / L nitric acid aqueous solution: acetonitrile = 95:5

[0051] 1.1.1 Preparation of 0.32 mol / L nitric acid aqueous solution: Add 25 mL of primary water to a 50 mL volumetric flask, slowly add 1.00 mL of concentrated nitric acid, and dilute to the mark with primary water.

[0052] 1.1.2 Preparation of 4.25 mmol / L nitric acid aqueous solution: Accurately transfer 13.32 mL of 0.32 mol / L nitric acid aqueous solution into a 1000 mL volumetric flask, and dilute to the mark with primary water.

[0053] 1.1.3 Preparation of mobile phase: Add 50 mL of acetonitrile and 950 mL of 4.25 mmol / L nitric acid aqueous solution to a 1 L solvent bottle, and mix by sonication.

[0054] 2. Preparation of standard solutions

[0055] Standards for 12 biogenic amines: Accurately weigh the biogenic amines or biogenic amine hydrochloride / sulfate standards into volumetric flasks, acidify with 0.32 mol / L nitric acid aqueous solution, and dilute to the mark with 4.25 mmol / L nitric acid aqueous solution to prepare the biogenic amine standard solution stock solution. Accurately transfer the corresponding volume of the biogenic amine standard solution stock solution into the same 50 mL volumetric flask, and dilute to the mark with 4.25 mmol / L nitric acid aqueous solution to prepare a mixed standard solution.

[0056] 3 Sample pretreatment methods

[0057] Accurately weigh 0.5g-2g (accuracy 0.1mg) of sample into a 50mL centrifuge tube, add 25mL of 4.25mol / L nitric acid aqueous solution, shake to extract for 20min, centrifuge at 4000r / min for 10min, and filter the supernatant through a 0.22μm microporous membrane.

[0058] 4 Ion Chromatography Analysis Conditions

[0059] Mobile phase: 4.25 mmol / L nitric acid aqueous solution - acetonitrile = 95:5.

[0060] Ion chromatography column: Metrosep C4 100 / 4.0.

[0061] Flow rate: 0.9 mL / min.

[0062] Injection volume: 20 μL.

[0063] Column temperature: 30℃.

[0064] Detector: Non-suppressor - conductivity detector.

[0065] 5. Quantitative Analysis

[0066] The standard working solution was measured according to the instrument reference conditions. A standard curve was plotted with concentration (mg / L) on the x-axis and response chromatographic peak area ((μS / cm) x min) on the y-axis. The content of each biogenic amine was calculated by using the corresponding peak area in the chromatogram of the sample solution through the standard curve.

[0067] The content of the target biogenic amine in the sample solution was quantified using the external standard method based on a standard curve. The calculation formula is as follows:

[0068]

[0069] In the formula:

[0070] The content of biogenic amines in sample X, in mg / g;

[0071] c - Concentration of biogenic amines measured in the sample solution, in mg / L;

[0072] V - Extraction volume, in mL;

[0073] m - Sample mass, in g;

[0074] K - Dilution factor.

[0075] The calculation result should be rounded to three significant figures.

[0076] 2. Test Results

[0077] Figure 1 To analyze the mixed standard working solution using ion chromatography, ion chromatograms of methylamine, ethylamine, octopamine, trimethylamine, tyramine, benzylamine, β-phenylethylamine, putrescine, cadaverine, histamine, tryptamine, and guanidine were obtained.

[0078] The standard working solutions were analyzed by ion chromatography under the above chromatographic conditions to obtain ion chromatograms. Based on the peak areas of each biogenic amine in the ion chromatograms and their corresponding concentrations in the standard working solutions, standard curves were plotted with concentration (mg / L) on the x-axis and peak area ((μS / cm) x min) on the y-axis for methylamine, ethylamine, octopamine, trimethylamine, tyramine, aniline, β-phenylethylamine, putrescine, cadaverine, histamine, tryptamine, and guanidine. The standard curves for methylamine, ethylamine, octopamine, trimethylamine, tyramine, aniline, β-phenylethylamine, putrescine, cadaverine, histamine, tryptamine, and guanidine were respectively... Figures 2-13 As shown.

[0079] Example 2: Linearity and Limit of Detection Tests of the Simultaneous Determination Method for 12 Biogenic Amines in Leftover Food Established in This Invention

[0080] 1. Test Methods

[0081] A series of mixed standard solutions containing 12 biogenic amine components were prepared and injected sequentially under optimized experimental chromatographic conditions, repeated twice. A standard curve was established with peak area as the ordinate (y) and mass concentration as the abscissa (x). Within their respective linear ranges, the coefficient of determination (R²) was calculated. 2 All values ​​were greater than 0.9995. The detection limit of the method was set at 3 times the signal-to-noise ratio (S / N).

[0082] 2. Test Results

[0083] Figure 14 Ion chromatograms of 12 biogenic amines determined from leftover food samples. The linear equations, linear correlation coefficients, linear ranges, and limits of detection for the 12 biogenic amines are shown in Table 1.

[0084] Table 1. Linear equations, linear correlation coefficients, linear ranges, and limits of detection for 12 biogenic amines.

[0085]

[0086] The experimental results show that the linear range of the simultaneous determination method for 12 biogenic amines established in this invention is 0.5–100 mg / L.

[0087] The detection limits of the simultaneous determination methods for 12 feed-grade biogenic amines established in this invention are as follows: methylamine 0.05 mg·L⁻¹ -1 Ethylamine 0.06 mg·L -1 Octopamine 0.16 mg·L -1 Trimethylamine 0.17 mg·L -1 Tyramine 0.33 mg·L -1 Benzylamine 0.32 mg·L -1β-Phenylacetamine 0.47 mg·L -1 putrescine 0.25 mg·L -1 0.35 mg·L⁻¹ cadaverine -1 Histamine 0.58 mg·L -1 Tryptophan 0.67 mg·L -1 Guanidine 0.59 mg·L -1 .

[0088] Example 3: Recovery and precision test of the method for simultaneous determination of 12 feed-grade biogenic amines in leftover food established in this invention.

[0089] 1. Test Methods

[0090] Using the mixed bio-amine acidifying agent product as a representative sample, 12 bio-amine standard solutions with 3 different concentrations (see Table 2 for specific concentrations) were added respectively. Each addition level was measured 6 times, and the average spiked recovery rate (R) and precision (RSD) were calculated.

[0091] 2. Test Results

[0092] The test results are shown in Table 2.

[0093] Table 2. Recovery and precision test results of the simultaneous determination methods for 12 biogenic amines.

[0094]

[0095] As can be seen from Table 2, the recovery rates of each biogenic amine component ranged from 85.2% to 116.7%, and the precision ranged from 0.2% to 1.9%, indicating that the ion chromatography detection method for the 12 biogenic amines established in this invention has good repeatability and high accuracy.

Claims

1. An ion chromatography method for determining the content of 12 biogenic amines, wherein the 12 biogenic amines are methylamine, ethylamine, octopamine, trimethylamine, tyramine, benzylamine, β-phenylethylamine, putrescine, cadaverine, histamine, tryptamine, and guanidine; characterized in that, include: (1) Prepare standard working stock solutions of methylamine, ethylamine, octopamine, trimethylamine, tyramine, benzylamine, β-phenylethylamine, putrescine, cadaverine, histamine, tryptamine and guanidine respectively; mix the standard working stock solutions and make up to volume to obtain mixed standard working solution; (2) The mixed standard working solution was analyzed by ion chromatography to obtain the respective ion chromatograms of methylamine, ethylamine, octopamine, trimethylamine, tyramine, benzylamine, β-phenylethylamine, putrescine, cadaverine, histamine, tryptamine and guanidine. (3) Based on the chromatographic peak area of ​​each biogenic amine in the ion chromatogram, draw standard curves for each biogenic amine in the standard working stock solution according to the concentration of each biogenic amine. (4) After pretreatment of the sample to be tested, the sample is analyzed by ion chromatography to obtain the ion chromatogram of the sample solution to be tested; (5) Calculate the content of each biogenic amine in the sample solution by comparing the peak area of ​​each biogenic amine in the ion chromatogram of the sample solution to the corresponding standard curve. The chromatographic conditions for analysis using an ion chromatograph in step (2) or step (4) include: the mobile phase consists of an aqueous nitric acid solution and acetonitrile, with a volume ratio of 95:5; the aqueous nitric acid solution is an aqueous nitric acid solution with a concentration of 4.25 mmol / L. The ion chromatography column was a Metrosep C4 100 / 4.

0. The flow rate was 0.9 mL / min; The injection volume was 20 μL; The column temperature is 30 ℃; The detector is a non-suppression-conductance detector.

2. The ion chromatography determination method according to claim 1, characterized in that, The preparation method of the standard working stock solution of methylamine, ethylamine, octopamine, trimethylamine, tyramine, benzylamine, β-phenylethylamine, putrescine, cadaverine, histamine, tryptamine and guanidine in step (1) includes: weighing each biological amine standard, adding nitric acid for acidification, and then making up to volume with nitric acid aqueous solution to obtain their respective standard working stock solutions.

3. The ion chromatography determination method according to claim 1, characterized in that, Step (4) involves pre-treating the sample to be tested by adding an extraction solution to the sample, shaking and extracting, centrifuging, and then passing the supernatant through a microporous filter membrane.

4. The ion chromatography determination method according to claim 3, characterized in that, The extract is a 4.25 mol / L nitric acid aqueous solution.

5. The ion chromatography determination method according to claim 1, characterized in that, The method for calculating the content of each biogenic amine in the sample solution by comparing the peak area corresponding to each biogenic amine in the chromatogram of the sample solution to the corresponding standard curve in step (5) includes: The content of each biogenic amine in the sample solution was calculated using the external standard method based on the standard curve. The calculation formula is as follows: In the formula: X - Content of biogenic amines in the sample, unit: mg / g; c - Concentration of biogenic amines measured in the sample solution, in mg / L; V - Extraction volume, unit: mL; m- Sample mass, unit: g K - Dilution factor.

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