A method for simultaneously detecting multiple biogenic amines in food using high performance liquid chromatography

Abstract

The invention discloses a method for simultaneously detecting multiple biogenic amines in food by high performance liquid chromatography, comprising the following steps: taking a biogenic amine standard curve solution and a food sample or its biogenic amine extract, adding an internal standard working solution, a sodium hydroxide solution, a saturated sodium bicarbonate solution, a FNBT derivative reagent, and N,N-diisopropylethylamine respectively before the derivatization reaction, mixing and performing a derivatization reaction at 40-80°C in the dark for 30-80 minutes; taking it out and letting it stand, cooling it to room temperature, centrifuging it, taking the bottom droplets and dissolving them in methanol, filtering and then conducting high performance liquid chromatography detection. The method of the invention is simple to operate, has a low detection limit, a short detection time, and good reproducibility, and is suitable for conventional detection of biogenic amines in food.

Description

Technical Field

[0001] The invention belongs to the field of food detection, and in particular relates to a method for simultaneously detecting multiple biogenic amines in food by using high performance liquid chromatography. Background Art

[0002] Biogenic amines are a class of nitrogen-containing low-molecular organic bases that have important physiological functions such as promoting the body's growth and metabolism, regulating nerve activity, etc. However, excessive intake of biogenic amines can cause problems such as headaches, chest tightness, and low blood pressure. In severe cases, it can even damage the cardiovascular and nervous systems. This shows that it is particularly important to accurately measure the content of biogenic amines in food.

[0003] There are many methods used at home and abroad to detect biogenic amines in fermented foods, such as high performance liquid chromatography (HPLC), gas chromatography (GC), thin layer chromatography (TlC), capillary electrophoresis (CE), biosensor method, etc.

[0004] Gas chromatography has high separation efficiency and accurate quantification, and is often used to detect biogenic amines in volatile foods such as rice wine, grape wine and vinegar. Thin layer chromatography is easy to operate and low in cost, and can be used for qualitative and semi-quantitative analysis of biogenic amines. Capillary electrophoresis consumes little organic solvent and has a fast detection speed, and is particularly suitable for the analysis of aromatic and heterocyclic biogenic amines.

[0005] HPLC has become a commonly used tool for biogenic amine analysis and detection due to its advantages of high column efficiency, accurate quantitative analysis, and high sensitivity. In addition, most of the biogenic amine detection standards issued in my country use high performance liquid chromatography, among which dansyl chloride is used for derivatization. However, there are some shortcomings in using dansyl chloride as a derivatization reagent, mainly because the derivatization conditions are complicated. Summary of the invention

[0006] The object of the present invention is to provide a method for simultaneously detecting multiple biogenic amines in food by high performance liquid chromatography, which has a low detection limit, a short detection time and good reproducibility.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A method for simultaneously detecting multiple biogenic amines in food using high performance liquid chromatography comprises the following steps:

[0009] Take the biogenic amine standard curve solution and the food sample or its biogenic amine extract, add the internal standard working solution, sodium hydroxide solution, saturated sodium bicarbonate solution, FNBT derivatization reagent, and catalyst DIPEA (N,N-diisopropylethylamine) respectively before the derivatization reaction, mix well, and perform the derivatization reaction at 40-80° C. (preferably 60° C.) in the dark for 30-80 minutes (preferably 30 minutes); take out and let stand, cool to room temperature, centrifuge (8000 r / min for 10 minutes), take the bottom droplets and dissolve them in methanol, filter and perform high performance liquid chromatography detection, record the chromatogram, and calculate according to the internal standard method;

[0010] The biogenic amines include putrescine, cadaverine, spermine, spermidine, tryptamine, β-phenylethylamine, histamine, tyramine and octopamine;

[0011] The biogenic amine standard curve solution is prepared by the following steps:

[0012] Prepare a 1000 mg / L stock solution of each biogenic amine standard with 0.1 mol / L hydrochloric acid solution, then take the same volume of each biogenic amine stock solution, mix and dilute to obtain a mixed standard solution with a concentration of 100 mg / L; take 0.10, 0.25, 0.50, 1.00, 1.50, 2.50 and 5.00 mL of the 100 mg / L biogenic amine mixed standard solution, dilute to 10 mL with 0.10 mol / L hydrochloric acid, and prepare biogenic amine standard curve solutions with concentrations of 1.00, 2.50, 5.00, 10.00, 15.00, 25.00 and 50.00 mg / L;

[0013] When the food is alcoholic beverages (wine, beer, rice wine, etc.) or condiments (vinegar, soy sauce), there is no need to extract biogenic amines, and the sample can be directly measured after dilution;

[0014] When the food is aquatic products (fish and its products, shrimp and its products), meat, dairy products (yogurt, cheese) and other fermented foods (pickles, pickled eggs, bean paste, etc.), extraction and purification shall be performed according to steps 5.1.2-5.1.3 of the national standard GB5009.208-2016 "Determination of biogenic amines in food, national food safety standard" to obtain the biogenic amine extract of the food sample;

[0015] The concentration of the sodium hydroxide solution is 2 mol / L;

[0016] The FNBT derivatization reagent is a 3-5% (v / v) 4-fluoro-3-nitrotrifluorotoluene methanol solution, with a preferred concentration of 4% (v / v);

[0017] The internal standard working solution is prepared by the following steps:

[0018] Take 100 mg of 1,7-diaminoheptane, dissolve it in 0.1 mol / L hydrochloric acid solution and dilute it to 100 mL to prepare a 1000 mg / L internal standard stock solution; take 1.0 mL of the internal standard stock solution and dilute it to 10 mL with 0.1 mol / L hydrochloric acid solution to prepare a 100 mg / L internal standard working solution;

[0019] The filtration preferably uses a filter membrane with a pore size of 0.22-0.45 μm;

[0020] The HPLC detection was performed using an octadecylsilane bonded silica gel column (4.6×250 mm, 5 μm), a column temperature of 30° C., a UV detector or a diode array detector, a UV detection wavelength of 254 nm, mobile phase A of ultrapure water, mobile phase B of acetonitrile, a flow rate of 1.0 mL / min, and a gradient elution procedure as follows:

[0021] Time / minute 0 10 17 25 30 35 Mobile phase A% 65 8 25 30 65 65 Mobile phase B% 35 92 75 70 35 35

[0022] Compared with the prior art, the present invention has the following advantages and effects:

[0023] 1. In the present invention, 4-fluoro-3-nitrotrifluorotoluene (FNBT) can react with monoamines and polyamines, and the derivative products are stable and have high ultraviolet response values. Compared with dansyl chloride, it has the advantages of low toxicity, easy storage, and low price.

[0024] 2. The method of the present invention is simple to operate, has a low detection limit, a short detection time, and good reproducibility, and is suitable for routine detection of biogenic amines in food; sodium hydroxide solution and saturated sodium bicarbonate solution are used to maintain a stable pH value of the reaction system. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the relationship between the reaction temperature and the response values ​​of nine biogenic amines;

[0026] Figure 2 is a graph showing the relationship between reaction time and the response values ​​of nine biogenic amines;

[0027] Different letters in the same column indicate different differences, P < 0.05.

[0028] Figure 3 It is the liquid chromatogram of 9 biogenic amines. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0030] Preparation of required reagents:

[0031] (1) FNBT derivatization reagent: Pipette 4 mL of FNBT and dilute to 100 mL with methanol to prepare a 4% (v / v) FNBT derivatization reagent. Store in a brown reagent bottle at 4°C away from light.

[0032] (2) Single-standard stock solutions of biogenic amines: Weigh 9 biogenic amine standards (histamine hydrochloride, β-phenylethylamine hydrochloride, tyramine hydrochloride, putrescine hydrochloride, cadaverine hydrochloride, tryptamine hydrochloride, spermine hydrochloride, spermidine hydrochloride, and octopamine hydrochloride) respectively, prepare single-standard stock solutions of 1000 mg / L (based on each biogenic amine monomer) with 0.1 mol / L hydrochloric acid solution, and store in a -20°C refrigerator away from light.

[0033] (3) Biogenic amine mixed standard solution: Pipette 1.00 mL of each of the nine single-standard stock solutions into a 10 mL brown volumetric flask, dilute to the mark with 0.1 mol / L hydrochloric acid to prepare a 100 mg / L biogenic amine mixed standard solution, and store in a -20°C refrigerator away from light.

[0034] (4) Biogenic amine standard curve solution: Take 0.10, 0.25, 0.50, 1.00, 1.50, 2.50 and 5.00 mL of the biogenic amine mixed standard solution (100 mg / L) respectively, place them in a 10 mL brown volumetric flask, dilute to the mark with 0.10 mol / L hydrochloric acid, and prepare a series of biogenic amine mixed standard gradient solutions with concentrations of 1.00, 2.50, 5.00, 10.00, 15.00, 25.00 and 50.00 mg / L. Prepare them before use.

[0035] Example

[0036] Samples: Commercially available soy sauce samples, numbered 1Hjbs, 2Hxws, 3Hjj, 4H365, 5Hysj.

[0037] Soy sauce pretreatment: Take 1 mL of soy sauce and dilute to 10 mL to prepare the soy sauce sample.

[0038] Take 1 mL of soy sauce sample and biogenic amine standard curve solution, add 100 μL of internal standard working solution, 100 μL of 2 mol / L sodium hydroxide solution, 200 μL of saturated sodium bicarbonate solution, 1 mL of FNBT derivatization reagent, 50 μL of catalyst DIPEA (N, N-diisopropylethylamine) and vortex for 60 seconds, then react in a 60°C water bath in the dark for 30 minutes. After taking out, let it stand, cool to room temperature, centrifuge at 8000 r / min for 10 minutes, take 20 μL of the bottom droplet and dissolve it in 1 mL of methanol, filter it through a 0.45 μm filter membrane into a liquid phase vial for testing, and all samples are in triplicate.

[0039] Agilent ZORBAX Eclipse XDB-C18 (4.6×250mm, 5μm) chromatographic column was used, the UV detection wavelength was 254nm, the column temperature was 30℃, and the injection volume was 20μL. Mobile phase A: ultrapure water, mobile phase B: acetonitrile, flow rate was 1.0mL / min

[0040] Time / minute 0 10 17 25 30 35 Mobile phase A% 65 8 25 30 65 65 Mobile phase B% 35 92 75 70 35 35

[0041] Take 1 mL of a 100 mg / L mixed standard solution of biogenic amines and perform derivatization according to the above method. The derivatization time is 60 minutes, and the selected derivatization temperatures are 30, 40, 50, 60, 70 and 80 °C. After the derivatization is completed, the sample is injected, and the peak area of ​​each biogenic amine obtained represents the actual derivatization efficiency. The results are shown in Figure 1 As shown in the figure, when the temperature is below 60℃, the efficiency of biogenic amine derivatization increases with the increase of temperature. When the derivatization temperature continues to rise to 80℃, the peak area of ​​biogenic amines decreases to varying degrees, among which the peak area of ​​tyramine decreases most significantly. The response values ​​of some biogenic amines do not change significantly with temperature after the derivatization temperature reaches 40℃. For the determination of several specific biogenic amines, the reaction temperature can be selected within the range of 40-80℃. The derivatization effect of the nine biogenic amines is the best at 60℃ (P<0.05), so 60℃ is the preferred derivatization reaction temperature.

[0042] Take 1 mL of the mixed standard solution of biogenic amines with a concentration of 100 mg / L and perform derivatization according to the above method. The derivatization temperature is 60°C, and the selected derivatization times are 20, 30, 40, 50, 60 and 80 minutes. The results are as follows Figure 2 As shown, the peak area of ​​putrescine increases with time, but there is no significant difference compared with the reaction time of 30 minutes (P<0.05), and the response values ​​of other biogenic amines remain basically unchanged. Therefore, the derivatization reaction time can be 30-80 minutes, preferably 30 minutes.

[0043] Using concentrations of 0.5, 1.0, 5.0, 10.0, 25.0, 50.0 and 100.0 mg / L biogenic amine standard curve solutions, derivatization injection was performed according to the above method. The average peak area of ​​the 9 biogenic amines was used as the y value, and the concentration of the corresponding biogenic amine standard curve solution was used as the x value. Linear regression analysis was performed using the internal standard method to obtain a linear equation and determine the linear range. The detection limit of each biogenic amine was determined by the signal-to-noise ratio (S / N=3), and the quantification limit of each amine was determined by the signal-to-noise ratio (S / N=10). The liquid chromatograms of the 9 biogenic amine standards are shown in Figure 2. Figure 3 shown.

[0044] The regression equations, correlation coefficients, linear ranges and detection limits of the nine biogenic amines are shown in Table 1.

[0045] Table 1 Regression equation, correlation coefficient, linear range and detection limit of the present invention for the determination of 9 biogenic amines

[0046]

[0047] The contents of nine biogenic amines in five soy sauce samples are shown in Table 2.

[0048] Table 2 Contents of 9 biogenic amines in 5 soy sauce samples of the present invention

[0049]

[0050] Comparative Example

[0051] The main operating process differences between the method of the present invention and the liquid chromatography method in the national standard "GB 5009.208-2016 Determination of biogenic amines in food" are shown in the following table.

[0052] Table 3 Differences between the operation process of the present invention patent and the national standard method

[0053]

[0054] The linear regression equations of 9 biogenic amines were obtained by HPLC detection using 0.5, 10.0, 5.0, 10.0, 25.0, 50.0 and 100.0 mg / L biogenic amine series standard solutions, respectively. The detection limits of each amine were determined by the signal-to-noise ratio (S / N=3), and the quantitative limits of each biogenic amine were determined by the signal-to-noise ratio (S / N=10). The detection limit of the method of the present invention was between 0.013-0.089 mg / L, and the detection limit of the national standard method was between 0.026-0.159 mg / L.

[0055] Table 4 Results of determination of biogenic amines by the method of the present invention and the national standard method

[0056]

[0057] Soy sauce was selected as the matrix, and 5.0, 10.0, and 20.0 mg / L of mixed standard working solutions of biogenic amines were added respectively, and the samples were injected continuously for 6 times after derivatization. As shown in the following table, the average recovery rate of the method of the present invention was between 93.3% and 99.73%, and the RSD was between 1.15% and 4.27%. The average recovery rate of the national standard method was between 88.29% and 100.30%, and the RSD was between 1.21% and 5.31%. The average recovery rate of all biogenic amines was greater than 90%, and RSD < 5%, indicating that the present method had good precision and could meet the requirements for quantitative and qualitative analysis of biogenic amines in soy sauce.

[0058] Table 5 Recovery of biogenic amines in soy sauce detected by the patented method of the present invention (n=6)

[0059]

[0060] Table 6 Recovery of biogenic amines in soy sauce detected by national standard method (n=6)

[0061]

[0062] It can be seen that the method of the present invention has the characteristics of simple operation, small reagent consumption, fast detection speed, high detection accuracy and good reproducibility.

[0063] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for simultaneously detecting multiple biogenic amines in food using high performance liquid chromatography, characterized in that The following steps are involved: Take 1 mL of the biogenic amine standard curve solution and the food sample or its biogenic amine extract, and add 100 μL of the internal standard working solution, 100 μL of the sodium hydroxide solution, 200 μL of the saturated sodium bicarbonate solution, 1 mL of the FNBT derivatization reagent, and 50 μL of N,N-diisopropylethylamine respectively before the derivatization reaction, mix well, and perform the derivatization reaction at 60°C in the dark for 30 minutes; take out and let stand, cool to room temperature, centrifuge, take the bottom droplets and dissolve them in methanol, filter and perform high performance liquid chromatography detection, record the chromatogram, and calculate according to the internal standard method; The biogenic amines include putrescine, cadaverine, spermine, spermidine, tryptamine, β-phenylethylamine, histamine, tyramine and octopamine; The biogenic amine standard curve solution is prepared by the following steps: Prepare a 1000 mg / L stock solution of each biogenic amine standard with 0.1 mol / L hydrochloric acid solution, then take the same volume of each biogenic amine stock solution, mix and dilute to obtain a mixed standard solution with a concentration of 100 mg / L; take 0.10, 0.25, 0.50, 1.00, 1.50, 2.50 and 5.00 mL of the 100 mg / L biogenic amine mixed standard solution, dilute to 10 mL with 0.10 mol / L hydrochloric acid, and prepare biogenic amine standard curve solutions with concentrations of 1.00, 2.50, 5.00, 10.00, 15.00, 25.00 and 50.00 mg / L; When the food is alcohol or condiment, there is no need to extract biogenic amines, and the sample can be directly measured after dilution; When the food is aquatic products, meat, dairy products and fermented food, extraction and purification are required to obtain a food sample biogenic amine extract; the concentration of the sodium hydroxide solution is 2 mol / L; the FNBT derivatization reagent is a 4% v / v 4-fluoro-3-nitrotrifluorotoluene methanol solution; The centrifugation is 8000 r / min for 10 minutes; The filtration is performed using a filter membrane with a pore size of 0.22-0.45 μm; the HPLC detection is performed using an octadecylsilane bonded silica gel column, the column temperature is 30°C, an ultraviolet detector or a diode array detector is used, and the ultraviolet detection wavelength is 254 nm; the mobile phase A is ultrapure water, the mobile phase B is acetonitrile, the flow rate is 1.0 mL / min, and the gradient elution procedure is as follows: 。

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