Liquid chromatography-tandem mass spectrometry determination method for six biogenic amines in food

Abstract

The invention discloses a liquid chromatography-tandem mass spectrometry determination method for six biogenic amines in food. The six biogenic amines are cadaverine, putrescine, tyramine, histamine, 2-phenylethylamine, 2-phenylethylamine and tryptamine; the food is frozen marine fish, marine shrimps, fresh oysters, mollusks, marine products, fermented meat products, pickled vegetables, fermented vegetable products, fermented bean products, infant supplementary food, fermented milk products and fermented wine. The infant supplementary food is one or more of infant canned supplementary food containing fish or shrimp, dried fish floss and fish sausage. The method provided by the invention adopts the high performance liquid chromatography-tandem mass spectrometry, and compared with the existing high performance liquid chromatography, the method reduces the use of organic reagents, simplifies the pretreatment process, improves the sensitivity, and has the advantages of wide detection range, high analysis speed, high sensitivity and the like when being used for detecting the content of biogenic amines in various food components.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis and relates to a liquid chromatography-tandem mass spectrometry method for determining six biogenic amines in food. Background Art

[0002] Biogenic amines (BAs) are a class of low-molecular-weight, nitrogen-containing organic compounds. Based on their source, they can be divided into endogenous and exogenous biogenic amines. Endogenous BAs are generally found in foods such as fruits, where they act as body regulators, playing a role in cell synthesis and maintaining cell membrane stability. Exogenous BAs are primarily found in fish and fish products, fermented foods (meat, dairy, vegetable products, and alcohol), and are formed from the α-carboxyl groups of free amino acids in foods through the enzymatic reaction of microbial amino acid decarboxylases. Among common biogenic amines, histamine and tyramine are the most abundant in foods and pose the greatest threat to human health. They are formed by the decarboxylation of histidine and tyrosine, respectively. Studies have shown that a certain amount of histamine entering the human circulatory system can cause a range of toxic symptoms, such as hypotension, edema, palpitations, urticaria, and increased blood viscosity. Large doses of histamine can even cause shock. Furthermore, biogenic amines exhibit additive and synergistic toxic effects (for example, the presence of diamines and polyamines inhibits the metabolism of monoamines, enhancing their toxicity). Some biogenic amines (such as cadaverine and putrescine) can react with nitrite to form heterocyclic carcinogens such as nitrosamines. Consequently, many countries have established limits on the amount of biogenic amines in food. Food poisoning incidents caused by excessive biogenic amine levels are common, necessitating an urgent need to monitor the biogenic amine content in food.

[0003] Currently, methods for detecting biogenic amines include GC-MS, ELISA, CE, LC, and LC-MS. However, the depth and scope of research on monitoring biogenic amine content in food are clearly insufficient. These methods suffer from insufficient sensitivity, complex pretreatment processes, and the need for numerous reagents and consumables, limiting their scope of application. These factors significantly hinder the monitoring and risk assessment of biogenic amines in food. Summary of the Invention

[0004] The present invention addresses these issues and provides a liquid chromatography-tandem mass spectrometry method for the simultaneous detection of six biogenic amines in food. This method omits derivatization during sample pretreatment, employing only a single extraction with 5% perchloric acid solution. Detection is then performed using LC-MS / MS. The detection limits for cadaverine, putrescine, 2-phenylethylamine, and tyramine are 1.0 mg / kg, and for histamine and tryptamine are 0.1 mg / kg. This significantly improves detection sensitivity and can be used for routine monitoring of biogenic amine content in food and risk assessment studies.

[0005] In order to achieve the above object, the technical solutions specifically adopted by the present invention are as follows:

[0006] In a first aspect, the present invention provides a method for the determination of six biogenic amines in food by liquid chromatography-tandem mass spectrometry, which can simultaneously detect six biogenic amines, namely, cadaverine, putrescine, tyramine, histamine, 2-phenylethylamine, and tryptamine, in food. The determination method comprises the following steps:

[0007] A. Sampling after pre-treatment of the food to be tested

[0008] The food is selected from frozen marine fish, marine shrimp, marine shellfish and crabs, mollusks, marine products, fermented meat products, pickled vegetables, fermented vegetable products, fermented soy products, infant supplementary food, fermented dairy products and fermented wine.

[0009] Furthermore, the fermented meat products include sausages, bacon, and ham; the fermented vegetable products include kimchi and pickled vegetables; and the infant supplementary food includes canned infant supplementary food containing fish and shrimp ingredients, fish floss, and fish sausage.

[0010] Further, the method for pre-processing food is as follows:

[0011] The pre-processing methods for frozen marine fish, marine shrimp, oysters, and mollusks are as follows: Marine fish: remove the scales and internal organs, and take the edible part including the muscle; Marine shrimp: remove the head, shell, and intestinal glands, and take the muscle part; Marine shellfish and crabs: remove the shells, take the edible part; then mince, homogenize and mix, and set aside;

[0012] The pretreatment method of seafood, fermented meat products, pickled vegetables, fermented vegetable products, fermented soy products, fermented dairy products and infant complementary foods is as follows: take the edible part, mince it, and mix it homogenously;

[0013] After sampling, solid samples should be stored below -18°C; liquid samples should be stored below 4°C.

[0014] B. Sample Pretreatment

[0015] A certain mass of sample was weighed using an electronic scale with an accuracy of 0.01 g and placed in a centrifuge tube. 5% perchloric acid solution was added at a weight-to-volume ratio of 1:10. The sample was then vortexed, sonicated on ice, and centrifuged at 5000 rpm. The supernatant was transferred to a graduated test tube of the same volume as the centrifuge tube. 400 g / L sodium hydroxide solution was added to the supernatant to adjust the pH to 2-3, and the volume was fixed with 0.5% perchloric acid solution. A certain volume of the solution was transferred to a centrifuge tube, and an equal volume of 0.5% perchloric acid solution was added. The mixture was vortexed and mixed thoroughly. 0.25 mL of the solution was transferred to a glass tube, 0.75 mL of acetonitrile was added, and the mixture was passed through a 0.22 μm filter membrane for analysis by liquid chromatography-tandem mass spectrometry. The sample was diluted 400-fold.

[0016] When testing in the laboratory, the preferred operating steps are as follows: take 2 g as the sample, place it in a 50 mL centrifuge tube, add 20 mL of 5% perchloric acid solution, vortex for 5 minutes, sonicate in an ice bath for 20 minutes, and centrifuge at 5000 r / min for 10 minutes. Transfer the supernatant to another 50 mL graduated test tube, add 400 g / L sodium hydroxide solution to the above supernatant to adjust the pH to 2-3, and make up to 50 mL with 0.5% perchloric acid solution; take 2 mL of the above solution to a centrifuge tube, add 2 mL of 0.5% perchloric acid solution, and shake to mix. Pipette 0.25 mL of the above solution into a glass tube, add 0.75 mL of acetonitrile, mix, pass through a 0.22 μm filter membrane, and provide for liquid chromatography-tandem mass spectrometry determination.

[0017] C. Liquid chromatography detection

[0018] Chromatographic column: Waters XBridge HILIC with dimensions of 150 mm × 2.1 mm and a filter pore size of 3.5 μm;

[0019] Flow rate: 0.3 mL / min;

[0020] Column temperature: 35°C;

[0021] Injection volume: 2 μL;

[0022] Elution: Mobile phase A is an aqueous solution containing 10 mmol / L ammonium formate and 0.5% formic acid, and mobile phase B is an acetonitrile solution containing 10 mmol / L ammonium formate and 0.5% formic acid. The specific gradient elution program is as follows:

[0023]

[0024] D. Mass spectrometry

[0025] Positive ion scanning, a spray voltage of 5500 V, an ion transfer capillary temperature of 550°C, a curtain gas pressure of 40 psi, a nebulizer gas pressure of 55 psi, an auxiliary heating gas pressure of 55 psi, and a collision gas pressure of 5 psi were used. Mass spectrometry was performed on six biogenic amines using different collision energies. The MRM parent ions, product ions, and collision energies are shown in the table below.

[0026]

[0027] E. Concentration determination

[0028] A standard series of solutions of six biogenic amines were prepared in advance, and a standard curve was prepared using the liquid chromatography determination conditions set in step C and the mass spectrometry determination conditions set in step D, and qualitative and quantitative determinations of the six biogenic amines in food were performed.

[0029] Specifically, in step E, cadaverine, putrescine, 2-phenylethylamine, and tyramine are combined according to a first concentration gradient to prepare a standard series of solutions, and histamine and tryptamine are combined according to a second concentration gradient to prepare a standard series of solutions, all of which are prepared freshly for use;

[0030] The first concentration gradient combination was 10 μg / L, 25 μg / L, 50 μg / L, 100 μg / L, 200 μg / L, 500 μg / L, and 1000 μg / L; the second concentration gradient combination was 1.0 μg / L, 2.5 μg / L, 5.0 μg / L, 10 μg / L, 20 μg / L, 50 μg / L, and 100 μg / L.

[0031] Preferably, the preparation method of the standard series solutions includes preparing a standard stock solution, a standard intermediate solution, a biogenic amine mixed standard working solution, and a standard series solution. The standard stock solution has a shelf life of one year, the standard intermediate solution has a shelf life of one month, and the biogenic amine mixed standard working solution has a shelf life of two weeks. The standard series solutions are prepared and used immediately.

[0032] 1) Preparation of standard stock solutions of six biogenic amines: Weigh cadaverine, putrescine hydrochloride, tyramine hydrochloride, histamine hydrochloride, 2-phenylethylamine, and tryptamine, dissolve in water in a 10 mL volumetric flask, bring to volume, and mix thoroughly to prepare a mixed standard stock solution of 5.0 g / L of cadaverine, putrescine, and tyramine-2-phenylethylamine and 1.0 g / L of histamine and tryptamine. Store at 4°C.

[0033] When preparing standard solutions, the concentrations of the six biogenic amine standards are calculated based on the biogenic amine monomers. The hydrochloride salts should be converted when weighing the standards. All standard solutions should be stored in brown containers and protected from light.

[0034] 2) Prepare the standard intermediate solution, which includes the following steps:

[0035] S1: Mixed standard intermediate solution of cadaverine, putrescine, 2-phenylethylamine, and tyramine: Pipette 0.4 mL of each standard stock solution of cadaverine, putrescine, and tyramine into a 10 mL volumetric flask, dilute with water and bring to volume, mix well, and store at 4°C in the dark.

[0036] S2: Mixed standard intermediate solution of histamine and tryptamine: Pipette 0.2 mL of the standard stock solution of histamine and tryptamine respectively into a 10 mL volumetric flask, dilute with water and make up to the mark, mix well to obtain a histamine and tryptamine concentration of 20 mg / L, and store at 4°C in the dark.

[0037] 3) Prepare a mixed standard working solution of biogenic amines using the standard intermediate solution obtained in step 2), specifically comprising the following steps: Accurately pipette 0.1 mL of the mixed standard intermediate solution of cadaverine, putrescine, 2-phenylethylamine, and tyramine prepared in step S1, and 0.1 mL of the mixed standard intermediate solution of histamine and tryptamine prepared in step S2 into a 10 mL volumetric flask, dilute to the mark with 0.5% perchloric acid solution-acetonitrile solution, mix thoroughly, and obtain a concentration of 2 mg / L for cadaverine, putrescine, 2-phenylethylamine, and tyramine, and a concentration of 0.2 mg / L for histamine and tryptamine. Store at 4°C in the dark.

[0038] The preparation method of 0.5% perchloric acid solution-acetonitrile solution is:

[0039] A1: Accurately measure 5 mL of perchloric acid, dilute to 1000 mL with water, mix thoroughly, and adjust the pH with 400 g / L sodium hydroxide solution to obtain a 0.5% perchloric acid solution with a pH of 2-3.

[0040] A2: Accurately measure 25 mL of the 0.5% perchloric acid solution obtained in step A1, dilute to 1000 mL with acetonitrile, and mix thoroughly to obtain a 0.5% perchloric acid solution-acetonitrile solution.

[0041] 4) preparing a series of standard solutions using the biogenic amine mixed standard working solution obtained in step 3), specifically comprising the following steps: accurately pipetting 0.05 mL, 0.125 mL, 0.25 mL, 0.5 mL, 1.0 mL, 2.5 mL, and 5.0 mL of the biogenic amine mixed standard working solution obtained in step 3), respectively, into 10 mL volumetric flasks, diluting and calibrating to volume with 0.5% perchloric acid solution-acetonitrile solution, wherein the concentrations of cadaverine, putrescine, 2-phenylethylamine, and tyramine are all in the first concentration gradient combination; the concentrations of histamine and tryptamine are both in the second concentration gradient combination; preparing and using immediately;

[0042] The first concentration gradient combination was 10 μg / L, 25 μg / L, 50 μg / L, 100 μg / L, 200 μg / L, 500 μg / L, and 1000 μg / L; the second concentration gradient combination was 1.0 μg / L, 2.5 μg / L, 5.0 μg / L, 10 μg / L, 20 μg / L, 50 μg / L, and 100 μg / L.

[0043] When exploring detection conditions in this method, the method first tests the sample using a standard solution, followed by testing actual samples. The limits of detection (LOD) and quantification (LOQ) of this method are: for a 2g sample, a 50mL extract volume, and a dilution factor of 400, the limits of detection and quantification are 2.0 mg / kg and 5.0 mg / kg for cadaverine, putrescine, 2-phenylethylamine, and tyramine, respectively; and 0.2 mg / kg and 0.5 mg / kg for histamine and tryptamine, respectively.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] 1. Sample pretreatment omits the derivatization process, requiring only a single extraction with 5% perchloric acid solution. This method is simple and time-saving, while significantly reducing the use of organic reagents, making it an efficient and environmentally friendly method. The present invention, through simple sample pretreatment and instrumental analysis, not only expands the scope of detection to include a variety of food matrices, including frozen marine fish, marine shrimp, oysters, mollusks, seafood, fermented meat products, pickled vegetables, fermented vegetable products, fermented soy products, infant supplementary foods, fermented dairy products, and fermented alcoholic beverages, but also utilizes a simple, rapid method with minimal reagent usage, reducing experimental costs.

[0046] 2. LC-MS / MS demonstrated excellent separation, specificity, sensitivity, and reproducibility. Across different samples and spike levels, recoveries of the six biogenic amines ranged from 70% to 120%, with RSDs no greater than 15%, demonstrating the method's high accuracy and stability.

[0047] 3. In the method of the present invention, the detection limit of cadaverine, putrescine, 2-phenylethylamine and tyramine is 1.0 mg / kg, and the detection limit of histamine and tryptamine is 0.1 mg / kg, which greatly improves the detection sensitivity and can be used for routine monitoring of biogenic amine content in food and risk assessment research.

[0048] Therefore, the present invention not only expands the detection sample range to a variety of food matrices such as frozen marine fish, marine shrimp, oysters, mollusks, seafood products, fermented meat products, pickled vegetables, fermented vegetable products, fermented soy products, infant supplementary food, fermented dairy products and fermented alcoholic beverages through a simple sample pretreatment method and instrumental analysis method, but also the method is simple, fast, highly accurate, and uses a small amount of organic reagents, making it an efficient and environmentally friendly method. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The quantitative ion chromatogram of the standard solution of cadaverine, putrescine, tyramine, histamine, 2-phenylethylamine and tryptamine detected by the method provided by the present invention is shown. DETAILED DESCRIPTION

[0050] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] " Scope " disclosed herein is in the form of lower limit and upper limit. Can be respectively one or more lower limits, and one or more upper limits. A given range is limited by selecting a lower limit and an upper limit. The selected lower limit and upper limit define the boundary of a particular range. All ranges that can be limited in this way are inclusive and combinable, i.e. any lower limit can be combined with any upper limit to form a range. For example, for a particular parameter, a range of 100 ~ 140 and 500 ~ 900 is listed, and it is understood that a range of 100 ~ 140 and 500 ~ 900 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range 3,4 and 5 are listed, then the following ranges can all be expected: 1 ~ 2, 1 ~ 4, 1 ~ 5, 2 ~ 3, 2 ~ 4 and 2 ~ 5.

[0052] In this disclosure, unless otherwise specified, the numerical range "a to b" is an abbreviation for any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0 to 5" indicates that all real numbers between "0" and "5" are listed herein, and "0 to 5" is merely an abbreviation for these numerical combinations.

[0053] In the present invention, unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined with each other to form a new technical solution.

[0054] 1. Determination of testing conditions

[0055] The present invention provides a liquid chromatography-tandem mass spectrometry method for determining six biogenic amines in food, using a standard solution method to determine the detection conditions; the six biogenic amines are cadaverine, putrescine, tyramine, histamine, 2-phenylethylamine, and tryptamine. The method comprises the following steps:

[0056] 1) Preparation of standard stock solutions of six biogenic amines: Weigh cadaverine, putrescine hydrochloride, tyramine hydrochloride, histamine hydrochloride, 2-phenylethylamine, and tryptamine, dissolve in water in a 10 mL volumetric flask, bring to volume, and mix thoroughly to prepare a mixed standard stock solution of 5.0 g / L of cadaverine, putrescine, and tyramine-2-phenylethylamine and 1.0 g / L of histamine and tryptamine. Store at 4°C.

[0057] The concentrations of the six biogenic amine standards were calculated based on the monomers of each biogenic amine. The hydrochloride salts were converted when weighing the standards. All standard solutions were stored in brown containers and protected from light.

[0058] 2) Prepare the standard intermediate solution, which includes the following steps:

[0059] S1: Mixed standard intermediate solution of cadaverine, putrescine, 2-phenylethylamine, and tyramine: Pipette 0.4 mL of each standard stock solution of cadaverine, putrescine, and tyramine into a 10 mL volumetric flask, dilute with water and bring to volume, mix well, and store at 4°C in the dark.

[0060] S2: Mixed standard intermediate solution of histamine and tryptamine: Pipette 0.2 mL of the standard stock solution of histamine and tryptamine respectively into a 10 mL volumetric flask, dilute with water and make up to the mark, mix well to obtain a histamine and tryptamine concentration of 20 mg / L, and store at 4°C in the dark.

[0061] 3) Using the standard intermediate solution obtained in step 2), a mixed standard working solution of biogenic amines was prepared, specifically comprising the following steps: accurately pipetting 0.1 mL of the mixed standard intermediate solution of cadaverine, putrescine, 2-phenylethylamine, and tyramine prepared in step S1 and 0.1 mL of the mixed standard intermediate solution of histamine and tryptamine prepared in step S2 into a 10 mL volumetric flask, diluting the solution to the mark with 0.5% perchloric acid solution-acetonitrile solution, and mixing the solution until the concentrations of cadaverine, putrescine, 2-phenylethylamine, and tyramine were 2 mg / L, and the concentrations of histamine and tryptamine were 0.2 mg / L. The solution was stored at 4°C in the dark.

[0062] The preparation method of 0.5% perchloric acid solution-acetonitrile solution is:

[0063] A1: Accurately measure 5 mL of perchloric acid, dilute to 1000 mL with water, mix thoroughly, and adjust the pH with 400 g / L sodium hydroxide solution to obtain a 0.5% perchloric acid solution with a pH of 2-3.

[0064] A2: Accurately measure 25 mL of the 0.5% perchloric acid solution obtained in step A1, dilute to 1000 mL with acetonitrile, and mix thoroughly to obtain a 0.5% perchloric acid solution-acetonitrile solution.

[0065] 4) preparing a series of standard solutions using the mixed standard working solution of biogenic amines obtained in step 3), specifically comprising the following steps: accurately pipetting 0.05 mL, 0.125 mL, 0.25 mL, 0.5 mL, 1.0 mL, 2.5 mL, and 5.0 mL of the mixed standard working solution of biogenic amines obtained in step 3) into 10 mL volumetric flasks, respectively, diluting and calibrating to volume with 0.5% perchloric acid solution-acetonitrile solution, wherein the concentrations of cadaverine, putrescine, 2-phenylethylamine, and tyramine are all in the first concentration gradient combination; the concentrations of histamine and tryptamine are both in the second concentration gradient combination; preparing and using immediately;

[0066] The first concentration gradient combination was 10 μg / L, 25 μg / L, 50 μg / L, 100 μg / L, 200 μg / L, 500 μg / L, and 1000 μg / L; the second concentration gradient combination was 1.0 μg / L, 2.5 μg / L, 5.0 μg / L, 10 μg / L, 20 μg / L, 50 μg / L, and 100 μg / L;

[0067] 5) HPLC analysis using a 150 mm × 2.1 mm Waters XBridge HILIC column with a 3.5 μm pore size at a flow rate of 0.3 mL / min for the sample and standard solutions, a column temperature of 35°C, and an injection volume of 2 μL in mobile phases A and B. The specific gradient elution program is shown in Table 1.

[0068] Table 1 Gradient elution program

[0069]

[0070] 6) Using positive ion scanning (ESI + ), 5500 V spray voltage, 550°C ion transfer capillary temperature, 40 psi curtain gas pressure CUR, 55 psi nebulizer gas pressure GS1, 55 psi auxiliary heating gas pressure GS2, 5 psi collision gas CAD, and different collision energies were used for mass spectrometry determination of the six biogenic amines. The multi-reaction monitoring (MRM) parent ions, product ions, and collision energies are shown in Table 2;

[0071] Table 2 Summary of the qualitative ions, quantitative ions and collision energies of six biogenic amines

[0072]

[0073] 7) The standard series solutions obtained in step 4) are used to prepare a standard curve using the liquid chromatography conditions set in step 5) and the mass spectrometry conditions set in step 6) and to perform qualitative and quantitative determination of the six biogenic amines in the food.

[0074] The validity period of the standard stock solution of the present invention is 1 year, the validity period of the standard intermediate solution is 1 month, and the validity period of the biogenic amine mixed standard working solution is 2 weeks.

[0075] The liquid chromatography-tandem mass spectrometry method for determining six biogenic amines provided by the present invention is applicable to the determination of six biogenic amines in frozen marine fish, marine shrimp, oysters, mollusks, seafood products, fermented meat products, pickled vegetables, fermented vegetable products, fermented soy products, infant supplementary foods (canned infant supplementary foods containing fish and shrimp ingredients, fish floss, fish sausage, etc.), fermented dairy products and fermented alcoholic beverages.

[0076] The limits of detection (LOD) and quantification (LOQ) of this method are as follows: when the solid sample weight is 2 g (liquid sample 2 mL), the fixed volume of the extract is 50 mL, and the dilution factor is 400, the limits of detection and quantification of cadaverine, putrescine, 2-phenylethylamine and tyramine are 2.0 mg / kg and 5.0 mg / kg, respectively; and the limits of detection and quantification of histamine and tryptamine are 0.2 mg / kg and 0.5 mg / kg, respectively.

[0077] The principle of the liquid chromatography-tandem mass spectrometry method for determining six biogenic amines provided by the present invention is that the sample is subjected to 5% perchloric acid, ultrasonic extraction in an ice bath, centrifugation, and the supernatant is collected. The pH is adjusted and the supernatant is diluted to a constant volume, and then the supernatant is determined by liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode (positive ion mode) and quantified by external standard method.

[0078] The reagents used in the method provided by the present invention and their preparation methods are as follows:

[0079] Unless otherwise specified, all reagents used were of analytical grade, and the experimental water was grade 1 water as specified in GB / T 6682; perchloric acid (HClO4, 70%-72%); sodium hydroxide (NaOH); formic acid (CH3COOH): chromatographic grade; acetonitrile (CH3CN): chromatographic grade; ammonium formate (HCOONH4): chromatographic grade; 5% perchloric acid solution: accurately weigh 50 mL of perchloric acid, dilute to 1000 mL with water, and mix thoroughly; 400 g / L sodium hydroxide solution: accurately weigh 200 g of sodium hydroxide, dissolve in water, dilute to 500 mL, and mix thoroughly; 0.5% perchloric acid solution (pH 2-3): accurately weigh 5 mL of perchloric acid, dilute to 1000 mL with water, and mix thoroughly; 0.5% perchloric acid solution-acetonitrile solution: accurately weigh 25 mL of 0.5% perchloric acid solution, dilute to 100 mL with acetonitrile, and mix thoroughly;

[0080] Mobile phase A (containing 10 mmol / L ammonium formate and 0.5% formic acid in water): Accurately weigh 630 mg of ammonium formate and 5.0 mL of formic acid, dilute to 1000 mL with water, and mix thoroughly.

[0081] Mobile phase B (containing 10 mmol / L ammonium formate and 0.5% formic acid in acetonitrile): Accurately weigh 630 mg of ammonium formate and dissolve it in 100 mL of water. Accurately weigh 5.0 mL of formic acid, dilute to 1000 mL with acetonitrile, and mix thoroughly.

[0082] The six biogenic amine standards used in the method provided by the present invention are as follows:

[0083] Cadaverine (1,5-Diaminopentane, C5H 14 N2, CAS No.: 462-94-2) standard (purity> 98%);

[0084] Putrescinedihydrochloride (C4H 12 N2·2HCl, CAS No.: 333-93-7) standard (purity> 99%);

[0085] Tyramine hydrochloride (C8H 11 NO·HCl, CAS No.: 60-19-5) standard (purity > 98%);

[0086] Histamine dihydrochloride (C5H9N3·2HCl, CAS No.: 56-92-8) standard (purity>99%);

[0087] 2-phenylethylamine (CAS No.: 64–04-0, C8H 11 N, 98%),);

[0088] Tryptamine (C 10 H 12 N2, CAS No.: 61-54-1) standard (purity> 98%).

[0089] The method for drawing the standard curve of the method provided by the present invention is as follows:

[0090] Inject the standard solution series into a liquid chromatography-tandem mass spectrometer and plot a standard working curve using the peak areas of the six biogenic amine quantification ions as the ordinate and the corresponding biogenic amine concentrations as the abscissa. The response values ​​for the target compounds in both the standard and sample solutions should be within the instrument's linear range.

[0091] The qualitative determination method provided by the present invention is as follows:

[0092] Qualitative analysis of each target compound is performed based on retention time and the relative abundance of two pairs of ions (characteristic ion pair / quantitative ion pair) in the chromatographic peaks. The relative deviation in retention time between the target compound in the test sample solution and the standard solution must be no greater than 5%. Furthermore, the relative abundance ratio of the two daughter ions of the target compound in the test sample must be consistent with that of the standard solution of equivalent concentration, and the relative abundance deviation must not exceed the requirements in Table 3. The presence of biogenic amines in the sample is confirmed.

[0093] Table 3 Permissible deviation of relative ion abundance for qualitative confirmation

[0094]

[0095] The quantitative determination method provided by the present invention is as follows:

[0096] Take the test sample solution and a series of standard solutions and perform the analysis. The peak area of ​​the characteristic ion mass chromatographic peak of the target compound in the sample solution and the standard solution should be within the linear range of the instrument. Calculate the concentration using the external standard method based on the standard curve.

[0097] like Figure 1 Shown is the quantitative ion chromatogram of cadaverine, putrescine, tyramine, histamine, 2-phenylethylamine, and tryptamine standard solutions, wherein the concentrations of cadaverine, tyramine, 2-phenylethylamine, and putrescine are all 100 μg / L, and the concentrations of histamine and tryptamine are 10 μg / L.

[0098] The blank experiment of the present invention is performed using the same determination steps except that no sample is added.

[0099] The instruments and consumables used in the present invention are as follows:

[0100] Liquid chromatography-tandem mass spectrometer: equipped with electrospray ionization source (ESI);

[0101] Analytical balance: sensitivity 0.0 0001 g and 0.01 g;

[0102] Homogenizer;

[0103] Multi-tube vortex mixer;

[0104] Ultrasonic extractor;

[0105] Centrifuge: speed ≥6500 r / min;

[0106] Polytetrafluoroethylene hydrophobic filter membrane (Jinlong): 0.22 μm.

[0107] The contents of the six biogenic amines in the sample of the present invention are calculated according to the following formula, and the blank value needs to be deducted from the calculation result.

[0108]

[0109] C is the concentration of biogenic amines in the sample solution obtained from the standard working curve, in micrograms per liter (μg / L);

[0110] V——final volume of sample solution, in milliliters (mL);

[0111] m——sample weight, in grams (g);

[0112] f ——dilution factor.

[0113] Note: The result is expressed as the arithmetic mean of two independent determinations obtained under repeatability conditions, with three significant figures retained. The corresponding biogenic amine content in the sample is expressed in milligrams per kilogram (mg / kg).

[0114] Before analyzing actual samples, the laboratory must achieve the required precision and accuracy. Verify the reliability of the analytical method by analyzing spiked samples. Prepare at least three blank samples and spike each with the standard solution. Analyze the prepared spiked samples according to the above method. The recovery of each target compound should be within 70–120%, with a precision (RSD) no greater than 15%. If a standard reference material with a similar matrix to the sample is available, it can be used in place of the spiked sample for precision and accuracy testing.

[0115] When preparing standard stock solutions, especially tryptamine, ultrasonic dissolution can be used to ensure complete dissolution.

[0116] Some biogenic amine standards are in the form of biogenic amine hydrochloride. When preparing standard solutions, the concentration should be calculated based on the monomers of each biogenic amine, and the hydrochloride should be converted when weighing the standard.

[0117] Ultrasonic extraction was performed using ice bath ultrasound to ensure extraction efficiency and method repeatability.

[0118] When adjusting the pH of the solution, it is not advisable to add too much sodium hydroxide solution. The alkalinity of the solution is too strong, which will affect the life and efficiency of the chromatographic column.

[0119] Different brands of filter membranes have a significant impact on the detection results of biogenic amines. It is recommended to investigate the impact of the filter membrane before use.

[0120] If the content of biogenic amines in the sample is too high, such as fermented bean curd, broad bean paste, etc., the dilution factor can be appropriately increased so that the response value is within the range of the standard curve.

[0121] A method blank test is required for each batch of samples.

[0122] It is recommended that a quality control sample be included with each batch of samples. This can be a standard reference material or a spiked sample of known concentration. The measured value of the target compound should be within 70% to 120% of the standard value, depending on the spiked concentration.

[0123] Use calibration fluid to promptly calibrate the mass spectrometer to ensure that the mass number of the monitored ions does not change.

[0124] In order to ensure the accuracy of the analysis results, it is required that 10% of the parallel samples be measured when analyzing each batch of samples, and the relative deviation of the parallel sample results should not exceed 15%.

[0125] During the measurement process, it is recommended to intersperse the measurement of the same standard solution with the sample measurement to check the stability of the instrument.

[0126] Application Examples

[0127] In this embodiment, the method for determining six biogenic amines in food by liquid chromatography-tandem mass spectrometry comprises the following steps:

[0128] B1: Pre-process various foods including frozen seafood fish, seafood shrimp, oysters, mollusks, seafood products, fermented meat products, pickled vegetables, fermented vegetable products, fermented soy products, infant supplementary foods (canned infant supplementary foods containing fish and shrimp ingredients, fish floss, fish sausage, etc.), fermented dairy products and fermented alcoholic beverages. Specifically:

[0129] For frozen seafood, shrimp, oysters, and mollusks, fish: remove the scales and internal organs, and take out the edible parts such as the muscle; shrimp: remove the head, shell, and intestines, and take out the muscle; shellfish: remove the shells and take out the edible parts; crab: remove the shells and take out the edible parts; then mince, mix well, and set aside.

[0130] Seafood, fermented meat products (sausage, bacon, ham, etc.), pickled vegetables, fermented vegetable products (kimchi, pickles, etc.), fermented soy products, fermented dairy products, and infant complementary foods (canned infant complementary foods containing fish or shrimp, fish floss, fish sausage, etc.): Take the edible part, mince, and mix homogenously;

[0131] B2: Solid samples should be stored below -18°C; liquid samples should be stored below 4°C;

[0132] B3: Weigh 2 g (accurate to 0.01 g) of the sample obtained in step B2 as a test sample into a 50 mL centrifuge tube. Add 20 mL of 5% perchloric acid solution, vortex for 5 minutes, sonicate in an ice bath for 20 minutes, and centrifuge at 5000 rpm for 10 minutes. Transfer the supernatant to another 50 mL graduated test tube. Add 400 g / L sodium hydroxide solution to the supernatant to adjust the pH to 2-3, and then dilute to 50 mL with 0.5% perchloric acid solution. Transfer 2 mL of this solution to a centrifuge tube, add 2 mL of 0.5% perchloric acid solution, and vortex to mix. Pipette 0.25 mL of this solution into a glass tube, add 0.75 mL of acetonitrile, mix, and filter through a 0.22 μm filter. The results of the six biogenic amines in different foods are shown in Table 4 below:

[0133] Table 4 Contents of six biogenic amines in different foods

[0134]

[0135]

[0136] “nd” means not detected.

[0137] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A method for determining six biogenic amines in food by liquid chromatography-tandem mass spectrometry, characterized in that: The six biogenic amines are cadaverine, putrescine, tyramine, histamine, 2-phenylethylamine and tryptamine, and the determination method comprises the following steps: A. Sample the food to be tested after pre-treatment; B. Weigh a certain mass of sample using an electronic scale with an accuracy of 0.01 g and place it in a centrifuge tube. Add 5% perchloric acid solution at a weight-to-volume ratio of 1:10, then perform vortexing, ice-bath sonication, and centrifugation at 5000 rpm. Transfer the supernatant to another graduated test tube of the same volume as the centrifuge tube. Add 400 g / L sodium hydroxide solution to the supernatant to adjust the pH to 2-3, and make up to volume with 0.5% perchloric acid solution. Transfer a certain volume of the solution to a centrifuge tube, add an equal volume of 0.5% perchloric acid solution, and vortex to mix. Pipette a certain volume of the solution into a glass tube, add 3 volumes of acetonitrile, mix, and filter through a 0.22 μm filter membrane for determination by liquid chromatography-tandem mass spectrometry. The sample is diluted 400 times. C. Liquid chromatography detection Chromatographic column: Waters XBridge HILIC, 150 mm × 2.1 mm, 3.5 μm pore size; Flow rate: 0.3 mL / min; Column temperature: 35°C; Injection volume: 2 μL; Elution: Mobile phase A is an aqueous solution containing 10 mmol / L ammonium formate and 0.5% formic acid, and mobile phase B is an acetonitrile solution containing 10 mmol / L ammonium formate and 0.5% formic acid. The specific gradient elution program is as follows: ； D. Mass spectrometry Positive ion scanning, 5500V spray voltage, 550°C ion transfer capillary temperature, 40psi curtain gas pressure CUR, 55psi nebulizer gas pressure GS1, 55psi auxiliary heating gas pressure GS2, 5psi collision gas CAD, and different collision energies were used for mass spectrometry determination of six biogenic amines. The multi-reaction monitoring (MRM) parent ions, product ions, and collision energies are shown in the table below: ； E. Concentration determination A standard series of solutions of six biogenic amines were prepared in advance, and a standard curve was prepared using the liquid chromatography determination conditions set in step C and the mass spectrometry determination conditions set in step D, and qualitative and quantitative determinations of the six biogenic amines in food were performed.

2. The assay method according to claim 1, wherein: in, In step A, the food is selected from frozen marine fish, marine shrimp, marine shellfish and crabs, mollusks, marine products, fermented meat products, pickled vegetables, fermented vegetable products, fermented soy products, infant supplementary food, fermented dairy products and fermented alcoholic beverages. The fermented meat products include sausages, bacon, and ham; the fermented vegetable products include kimchi and pickled vegetables; and the infant supplementary food includes canned infant supplementary food containing fish and shrimp ingredients, fish floss, and fish sausage.

3. The measuring method according to claim 2, wherein: in, The pre-processing method for frozen marine fish, marine shrimp, oysters, and mollusks is as follows: Marine fish: remove the scales and internal organs, and take the edible part including the muscle; Marine shrimp: remove the head, shell, and intestinal glands, and take the muscle part; Marine shellfish and crabs: remove the shells, take the edible part; then mince, homogenize and mix, and set aside; The pretreatment method of seafood, fermented meat products, pickled vegetables, fermented vegetable products, fermented soy products, fermented dairy products and infant complementary foods is as follows: take the edible part, mince it, and mix it homogenously; After sampling, solid samples should be stored below -18°C; liquid samples should be stored below 4°C.

4. The measuring method according to claim 1, wherein: in, In step B, take 2 g as a sample and place it in a 50 mL centrifuge tube. Add 20 mL of 5% perchloric acid solution, vortex for 5 minutes, sonicate in an ice bath for 20 minutes, and centrifuge at 5000 r / min for 10 minutes. Transfer the supernatant to another 50 mL graduated test tube, add 400 g / L sodium hydroxide solution to the supernatant to adjust the pH to 2-3, and make up to 50 mL with 0.5% perchloric acid solution; take 2 mL of the above solution to a centrifuge tube, add 2 mL of 0.5% perchloric acid solution, and shake to mix. Pipette 0.25 mL of the above solution into a glass tube, add 0.75 mL of acetonitrile, mix, pass through a 0.22 μm filter membrane, and provide for liquid chromatography-tandem mass spectrometry determination.

5. The measuring method according to claim 1, wherein: in, In step E, cadaverine, putrescine, 2-phenylethylamine and tyramine are combined according to a first concentration gradient to prepare a standard series of solutions, and histamine and tryptamine are combined according to a second concentration gradient to prepare a standard series of solutions, all of which are prepared freshly for use; The first concentration gradient combination was 10 μg / L, 25 μg / L, 50 μg / L, 100 μg / L, 200 μg / L, 500 μg / L, and 1000 μg / L; the second concentration gradient combination was 1.0 μg / L, 2.5 μg / L, 5.0 μg / L, 10 μg / L, 20 μg / L, 50 μg / L, and 100 μg / L.

6. The measuring method according to claim 5, wherein: in, The preparation method of the standard series solution includes preparing a standard stock solution, preparing a standard intermediate solution, preparing a biogenic amine mixed standard working solution, and preparing a standard series solution. The validity period of the standard stock solution is 1 year, the validity period of the standard intermediate solution is 1 month, and the validity period of the biogenic amine mixed standard working solution is 2 weeks. The standard series solutions are prepared and used immediately.

7. The assay method according to claim 6, wherein: in, Prepare standard stock solutions as follows: Weigh cadaverine, putrescine hydrochloride, tyramine hydrochloride, histamine hydrochloride, 2-phenylethylamine, and tryptamine separately, dissolve in a 10-mL volumetric flask, dilute to the mark, and mix thoroughly. Prepare standard stock solutions of cadaverine, putrescine, tyramine, and 2-phenylethylamine at 5.0 g / L, and histamine and tryptamine at 1.0 g / L, respectively. Store at 4°C in the dark. The steps for preparing the standard intermediate solution are as follows: S1: Mixed standard intermediate solution of cadaverine, putrescine, 2-phenylethylamine, and tyramine: Pipette 0.4 mL of each standard stock solution of cadaverine, putrescine, 2-phenylethylamine, and tyramine into a 10 mL volumetric flask, dilute to the mark with water, and mix thoroughly. The concentration of cadaverine, putrescine, 2-phenylethylamine, and tyramine is 200 mg / L. Store at 4°C in the dark. S2: Mixed standard intermediate solution of histamine and tryptamine: Pipette 0.2 mL of the standard stock solution of histamine and tryptamine respectively into a 10 mL volumetric flask. Dilute with water and make up to the mark in the volumetric flask. Mix well to obtain a histamine and tryptamine concentration of 20 mg / L. Store at 4°C in the dark. Prepare the mixed standard working solution of biogenic amines as follows: Accurately pipette 0.1 mL of the mixed standard intermediate solution of cadaverine, putrescine, 2-phenylethylamine, and tyramine prepared in step S1 and 0.1 mL of the mixed standard intermediate solution of histamine and tryptamine prepared in step S2 into a 10 mL volumetric flask. Dilute to volume with 0.5% perchloric acid solution-acetonitrile solution and mix thoroughly. The concentrations of cadaverine, putrescine, 2-phenylethylamine, and tyramine are 2 mg / L, and the concentrations of histamine and tryptamine are 0.2 mg / L. Store at 4°C in the dark. The method for preparing the standard series solutions is as follows: accurately pipette 0.05 mL, 0.125 mL, 0.25 mL, 0.5 mL, 1.0 mL, 2.5 mL, and 5.0 mL of the mixed standard working solution of biogenic amines into 10 mL volumetric flasks, respectively, dilute and adjust the volume with 0.5% perchloric acid solution-acetonitrile solution, where the concentrations of cadaverine, putrescine, 2-phenylethylamine, and tyramine are all in the first concentration gradient combination; the concentrations of histamine and tryptamine are both in the second concentration gradient combination.

8. The assay method according to claim 7, wherein: in, When preparing the standard stock solutions, the concentrations of the six biogenic amine standards are calculated based on the monomers of each biogenic amine. When weighing the standards, the hydrochloride salts should be converted. The preparation method of 0.5% perchloric acid solution-acetonitrile solution is: (1) Accurately measure 5 mL of perchloric acid, dilute it to 1000 mL with water, mix well, and adjust the pH with 400 g / L sodium hydroxide solution to obtain a 0.5% perchloric acid solution with a pH of 2-3; (2) Accurately measure 25 mL of the 0.5% perchloric acid solution obtained in step (1), dilute it to 1000 mL with acetonitrile, and mix well to obtain a 0.5% perchloric acid solution-acetonitrile solution.

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