Method for rapidly and accurately determining concentration of biogenic amine in fruit wine matrix

Abstract

The invention relates to a method for rapidly and accurately determining the concentration of biogenic amine in a fruit wine matrix, and belongs to the technical field of health inspection. The method comprises the following steps: firstly, drawing a biogenic amine detection standard curve through a mixed standard solution, then preparing a fruit wine matrix sample by taking a commercial molecular sieve as a dispersive solid-phase extraction reagent, detecting by adopting dispersive solid-phase extraction-liquid chromatography-tandem mass spectrometry, and finally determining the concentration of biogenic amine through an external standard quantification method. The method disclosed by the invention is simple, rapid, efficient and low in cost, can be further developed and applied to conventional analysis according to standards, and also has a good application prospect in other matrixes (such as drinking water, soy sauce and fish sauce).

Description

Technical Field

[0001] The invention relates to a method for quickly and accurately determining the concentration of biogenic amines in a fruit wine matrix, belonging to the technical field of sanitary inspection. Background Art

[0002] An efficient sample preparation process is the basis for accurate determination of complex samples. Liquid-liquid extraction and solid phase extraction are two common sample preparation methods. Their enrichment and concentration functions reduce matrix effects and improve detection efficiency (Ramos RM, Valente IM, Rodrigues JA, Analysis of biogenic amines in wines by salting-out assisted liquid–liquid extraction and high-performance liquid chromatography with fluorimetric detection, Talanta, 2014, 124, 146–151). However, the extensive use of liquid and solid reagents makes the sample preparation process complicated and time-consuming, and conflicts with the concept of green chemistry. Microextraction technology is an ideal alternative to liquid-liquid extraction and solid phase extraction because they are simple, rapid, green and efficient (Cao D, Xu X, Xue S, Feng X, Zhang L, An in situ derivatization combined with magnetic ionic liquid-based fast dispersive liquid-liquid microextraction for determination of biogenic amines in food samples, Talanta, 2019, 199, 212–219). Common microextraction techniques include dispersive liquid-liquid microextraction and dispersive solid phase microextraction. Dispersive solid phase microextraction is a new technology that uses a small amount of adsorbent to adsorb samples in solution, followed by desorption and detection. Newly emerging adsorbents include molecularly imprinted polymers, carbon-based adsorbents, metal organic frameworks, magnetic nanoparticles, molecular sieves, etc. ( G, RS,Ceylan B,Egeli D,Tekkeli EK, A, A review of the currently developed analytical methods for the determination of biogenic amines in food products, Food Chem., 2023, 398, 133919).

[0003] Molecular sieves are a class of inorganic porous materials composed of a silicon-oxygen skeleton. This type of material has many advantages, such as large specific surface area, uniform pore size distribution, and adjustable charge, which provides a basis for the physical adsorption of samples (Verboekend D, Nuttens N, Locus R, Van Aelst J, Verolme P, Groen JC, Pérez-Ramírez J, Sels BF, Synthesis, characterisation, and catalytic evaluation of hierarchical faujasite zeolites: milestones, challenges, and future directions, Chem. Soc. Rev., 2015, 45, 3331–3352). Niwa and his collaborators found that USY molecular sieves have a certain acidity, which is produced by the incorporation of aluminum ions into the silicon-oxygen skeleton (Niwa M, Suzuki K, Isamoto K, Katada N, Identification and Measurements of Strong Acid Site in Ultrastable Y(USY)Zeolite,J.Phys.Chem.B,2006,110,264–269). In this way, the hydrogen ions and oxygen ions distributed in the pores provide effective forces for the adsorption performance of the molecular sieve, such as electrostatic effects and hydrogen bonds. It can be imagined that the uniform pore structure and adjustable interactions make the molecular sieve a nearly perfect dispersed solid phase extraction material. However, the current research on molecular sieves is mainly focused on the exploration of gas adsorption and catalytic properties, but there are few reports on dispersed solid phase extraction (Li Y, Yu J, Emerging applications of zeolites in catalysis, separation and host–guest assembly, Nat.Rev.Mater.,2021,6,1156–1174).

[0004] Fruit wine is widely popular around the world due to its rich nutritional value (including various vitamins and amino acids) and good taste. Recently, biogenic amines in fruit wine have attracted people's attention. Studies have found that low concentrations of biogenic amines can assist in the secretion of gastric acid, participate in immune response and hormone synthesis, etc., so they are beneficial to human health; however, high concentrations of biogenic amines can cause symptoms such as headaches, allergic reactions, and abnormal blood pressure, and these symptoms will be enhanced in the presence of alcohol (Wójcik W, M, Puppel K, Biogenic amines: formation, action and toxicity–a review, J. Sci. Food Agr., 2020, 101, 2634–2640). Therefore, it is very important to quickly and accurately determine the biogenic amines in fruit wine.

[0005] A variety of methods have been developed to measure biogenic amines in fruit wine, including high performance liquid chromatography-ultraviolet detection, high performance liquid chromatography-fluorescence detection, high performance liquid chromatography-tandem mass spectrometry, and gas chromatography-mass spectrometry (Dong H, Xiao K, Modified QuEChERS combined with ultra high performance liquid chromatography tandem mass spectrometry to determine seven biogenic amines in Chinese traditional condiment soy sauce, Food Chem., 2017, 229, 502–508). It can be seen that most studies use liquid-liquid microextraction and solid phase microextraction techniques to extract biogenic amines, and commercial molecular sieves are rarely reported as dispersed solid phase extraction materials, although they are conducive to widespread promotion and application. Summary of the invention

[0006] The purpose of the present invention is to overcome the above-mentioned shortcomings and provide a method for quickly and accurately determining the concentration of biogenic amines in a fruit wine matrix. This method is simple, rapid, efficient, and low-cost, and can be widely used in routine analysis.

[0007] The technical solution of the present invention is a method for quickly and accurately determining the concentration of biogenic amines in a fruit wine matrix, and the steps are as follows:

[0008] (1) Drawing of standard curve of biogenic amines:

[0009] a. Confirmation of mixed standard solution parameters: Prepare mixed standard solutions of biogenic amines of different concentrations using a volumetric flask, transfer them to injection vials after preparation, and set aside; Use the mixed standard solution to determine the optimal experimental parameters of the liquid chromatography-tandem mass spectrometer;

[0010] b. Measure the mixed standard solution obtained in step a by using a liquid chromatography-tandem mass spectrometer; draw a standard curve of biogenic amines with the concentration of biogenic amines as the abscissa and the chromatographic peak area as the ordinate;

[0011] (2) Preparation of fruit wine samples: add fruit wine to USY molecular sieve, vortex, centrifuge to remove supernatant; continue to add desorption solvent, vortex centrifuge again, and collect supernatant; add desorption solvent for the third time, vortex centrifuge, and collect supernatant; measure two supernatants respectively, record the concentration as C1 and C2, and obtain juice sample extract;

[0012] (3) Measurement: The juice sample extract prepared in step (2) is measured using a liquid chromatography-tandem mass spectrometer to quantify the concentration of the fruit wine by using an external standard method combined with a standard curve.

[0013] Furthermore, the biogenic amine in step (1) a is specifically cadaverine, putrescine, tyramine, histamine or tryptamine;

[0014] Furthermore, when the biogenic amine is cadaverine, putrescine or tyramine, the concentrations of the prepared mixed standard solution are 10, 25, 50, 100, 200, 500 and 1000 μg / L respectively; when the biogenic amine is histamine or tryptamine, the concentrations of the prepared mixed standard solution are 1, 2.5, 5, 10, 20, 50 and 100 μg / L respectively.

[0015] Furthermore, the specific steps of step (2) are: adding 4-6 mg USY molecular sieve into a 15 mL centrifuge tube, then adding 4-6 mL fruit wine, vortexing for 1-3 minutes, and then centrifuging; after removing the supernatant, adding 1-2 mL of desorption solvent, vortexing again for 1-3 minutes, centrifuging, and collecting the supernatant; adding 1-2 mL of desorption solvent again, vortexing for 1-3 minutes, centrifuging, and collecting the supernatant; measuring the two supernatants separately, the concentrations are C1 and C2, respectively, in μg / L.

[0016] Furthermore, the model of the liquid chromatography-tandem mass spectrometer is SCIEX 5500LC-MS / MS;

[0017] Further, the gradient elution program of the liquid chromatography is: 0–2.0 min, 0% A; 2.0–5.5 min, 0%–60% A; 5.5–7.0 min, 60% A; 7.0–7.5 min, 60%–0% A; 7.5–12 min, 0% A; wherein A and B represent an aqueous solution containing 0.01 mol / L ammonium formate and 0.5% formic acid and an acetonitrile solution containing 0.01 mol / L ammonium formate and 0.5% formic acid, respectively;

[0018] The mass spectrometry conditions were as follows: curtain gas 40.0 psi; collision gas Medium; ionization voltage 5500 V; temperature 550° C.; nebulizer gas 55.0 psi; auxiliary heating gas 55.0 psi; and collision cell ejection voltage 13.0 V.

[0019] Furthermore, the USY molecular sieve is an ultra-stable Y-type molecular sieve.

[0020] Furthermore, in step (2), the fruit wine needs to be placed at 4° C. for 20 minutes before being treated with USY molecular sieve.

[0021] Furthermore, in step (2), the centrifugal speed is 9000-11000 r / min, the centrifugal temperature is 4° C., and the centrifugal time is 4-6 minutes.

[0022] Furthermore, the desorption solvent in step (2) is a 40% methanol aqueous solution of perchloric acid with a volume concentration of 1%.

[0023] Furthermore, the concentration of the fruit wine in step (2) is calculated using the formula C=(C1+C2) / 5.

[0024] Furthermore, when the biogenic amine is cadaverine, putrescine or tyramine, the linear range of this method is 10-1000 μg / L, and the detection limit and quantification limit are 0.30 and 0.92 μg / L, respectively; when the biogenic amine is histamine or tryptamine, the linear range of this method is 1-100 μg / L, and the detection limit and quantification limit are 0.09 and 0.30 μg / L, respectively.

[0025] Furthermore, the relative standard deviations determined by this method are all within 5%.

[0026] Furthermore, in the fruit wine matrix, the intra-day precision and inter-day precision of this method were between 0.8%–9.9%, and the spike recovery was between 85%–101%.

[0027] Furthermore, the matrix effect of fruit wine determined by this method was between 91% and 110%, showing a negligible matrix effect.

[0028] Beneficial effects of the present invention: The present invention uses commercial molecular sieves as dispersed solid phase extraction reagents for the first time to achieve the dual goals of enrichment and removal of biogenic amines in food matrices. By optimizing the dispersed solid phase extraction conditions, the developed dispersed solid phase extraction-liquid chromatography-tandem mass spectrometry detection method shows nearly perfect method parameters in the detection of biogenic amines in fruit wine, including very low detection limits, negligible matrix effects, very good precision and recovery rate, so the external standard method can be used for quantification. Moreover, the external standard quantitative results were verified using the standard addition method, and there was no significant difference between the two. This method is not only simple, rapid, efficient and low-cost, but can also be further developed to promote the application of standards in routine analysis. It also has good application prospects in other matrices (such as drinking water, soy sauce and fish sauce). BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Characterization and selection of molecular sieves.

[0030] a. BJH pore size distribution of three molecular sieves: USY, TS-1, and MCM-41;

[0031] b, Experimental and simulated powder X-ray diffraction patterns of three molecular sieves: USY, TS-1, and MCM-41;

[0032] c. Morphology of USY molecular sieve;

[0033] d. Morphology of TS-1 molecular sieve;

[0034] e. Morphology of MCM-41 molecular sieve;

[0035] f. The extraction efficiency of biogenic amines by three molecular sieves, USY, TS-1 and MCM-41, as dispersed solid phase extraction reagents.

[0036] Figure 2 : Characterization of USY molecular sieve before and after extraction of biogenic amines and its adsorption mechanism.

[0037] a. Powder X-ray diffraction of USY molecular sieve before and after extraction of biogenic amines;

[0038] b, Morphology before extraction of biogenic amines by USY molecular sieve;

[0039] c. Morphology of biogenic amines extracted with USY molecular sieve;

[0040] d. ATR infrared spectra of biogenic amines before and after extraction with USY molecular sieve;

[0041] e, X-ray photoelectron spectra of Si 2p before and after extraction of biogenic amines by USY molecular sieve;

[0042] f. X-ray photoelectron spectrum of O1s before and after extraction of biogenic amines by USY molecular sieve.

[0043] Figure 3 : Optimization process of USY molecular sieve as dispersed solid phase extraction reagent for extracting biogenic amines from fruit wine.

[0044] a. Optimization of USY molecular sieve dosage; b. Optimization of extraction time; c. Optimization of desorption solvent; d. Optimization of desorption time; e. Optimization of desorption times; f. Recycling of USY molecular sieve.

[0045] Figure 4 : Chromatogram of standard solution and chromatogram of fruit wine extract.

[0046] a, chromatogram of cadaverine standard solution (200μg / L); b, chromatogram of putrescine standard solution (200μg / L); c, chromatogram of tyramine standard solution (200μg / L); d, chromatogram of histamine standard solution (20μg / L); e, chromatogram of tryptamine standard solution (20μg / L); f, chromatogram of cadaverine fruit wine extract; g, chromatogram of putrescine fruit wine extract; h, chromatogram of tyramine fruit wine extract; i, chromatogram of histamine fruit wine extract; j, chromatogram of tryptamine fruit wine extract. DETAILED DESCRIPTION

[0047] The following embodiments describe the present invention in more detail, but the embodiments do not limit the present invention.

[0048] Example 1 Detection method of cadaverine, putrescine, tyramine, histamine and tryptamine in fruit wine

[0049] (1) Drawing of standard curve of biogenic amines:

[0050] a. Preparation of mixed standard solutions: Use volumetric flasks to prepare seven mixed standard solutions, in which the concentrations of cadaverine, putrescine and tyramine are 10, 25, 50, 100, 200, 500 and 1000 μg L –1 The concentrations of histamine and tryptamine were 1, 2.5, 5, 10, 20, 50, and 100 μg L –1 ;

[0051] b. Call the set SCIEX 5500 LC-MS / MS instrument method. The liquid chromatography gradient elution program is: 0–2.0 min, 0% A; 2.0–5.5 min, 0%–60% A; 5.5–7.0 min, 60% A; 7.0–7.5 min, 60%–0% A; 7.5–12 min, 0% A. A and B represent an aqueous solution containing 0.01 mol / L ammonium formate and 0.5% formic acid and an acetonitrile solution containing 0.01 mol / L ammonium formate and 0.5% formic acid, respectively. The mass spectrometry conditions are: curtain gas 40.0 psi; collision gas Medium; ionization voltage 5500 V; temperature 550 ° C; nebulizer gas 55.0 psi; auxiliary heating gas 55.0 psi; collision chamber ejection voltage 13.0 V. For other liquid chromatography-mass spectrometry instruments, refer to the above instrument parameter settings;

[0052] c. Test the standard solution with an injection volume of 1 μL; plot a standard curve with the biogenic amine concentration as the horizontal axis and the chromatographic peak area as the vertical axis.

[0053] (2) Preparation of fruit wine samples: USY molecular sieve was used as a dispersed solid phase extraction reagent to extract biogenic amines from fruit wine. The fruit wine sample was placed in a 4°C refrigerator for 20 minutes. 5 mg of USY molecular sieve was added to a 15 mL centrifuge tube, and then 5 mL of fruit wine was added, vortexed for 2 minutes, and then centrifuged at 4°C for 5 minutes at a speed of 10,000 r / min. After removing the supernatant, 1 mL of 40% methanol aqueous solution containing 1% perchloric acid was added, vortexed for 2 minutes, centrifuged, and the supernatant was collected. 1 mL of 40% methanol aqueous solution containing 1% perchloric acid was added again, vortexed for 2 minutes, and centrifuged at 4°C for 5 minutes at a speed of 10,000 r / min, and the supernatant was collected. The two supernatants were measured separately, and the concentrations were C1 and C2, respectively, in μg / L.

[0054] (3) Measurement: The parameters described in step (1) b are used to detect and analyze the fruit wine extract; based on the standard curve, the concentration of the fruit wine sample is quantified using the external standard method C = (C1 + C2) / 5.

[0055] Example 2 Specific parameter optimization

[0056] Various conditions were optimized during the development process in Example 1, including the selection of molecular sieves, the adsorption mechanism of USY molecular sieves, the optimization process of the method of extracting biogenic amines from fruit wine using USY molecular sieves as dispersed solid phase extraction reagents, instrument parameters, parent ion and daughter ion information, various parameters of the method (including linear range, detection limit, quantitative limit, precision, accuracy and matrix effect), the test results of 6 fruit wine samples and verification using the standard addition method, chromatograms of biogenic amine standard solutions and fruit wine extracts, and comparisons with the latest methods reported in the literature. The specific results are as follows:

[0057] 1. Characterization and selection experimental results of molecular sieves are as follows Figure 1 shown.

[0058] in Figure 1 -a: BJH pore size distribution of three molecular sieves: USY, TS-1, and MCM-41;

[0059] Figure 1 -b: Experimental and simulated powder X-ray diffraction patterns of three molecular sieves, USY, TS-1, and MCM-41.

[0060] Figure 1 -c: Morphology of USY molecular sieve.

[0061] Figure 1 -d: morphology of TS-1 molecular sieve.

[0062] Figure 1 -e: morphology of MCM-41 molecular sieve.

[0063] Figure 1 -f: Extraction efficiency of biogenic amines using three molecular sieves, USY, TS-1 and MCM-41, as dispersed solid phase extraction reagents.

[0064] Dimond software shows that the molecular sizes of cadaverine, putrescine, tyramine, histamine and tryptamine are 0.43, 0.36, 0.33, 0.47 and 0.35 nanometers, respectively. Therefore, it is particularly important to find suitable molecular sieves as dispersed solid phase extraction reagents to adsorb these biogenic amines. Studies have found that the sizes of commercial molecular sieves USY, TS-1 and MCM-41 are 0.71, 0.56 and 2.66 nanometers, respectively (as can be clearly seen from the BJH pore size distribution), which are relatively matched with the pore size of biogenic amines. Powder X-ray diffraction shows that USY and TS-1 are highly crystalline molecular sieves (as can be seen from consistent experimental data and crystal simulation data), while MCM-41 is an amorphous structure. Highly crystalline USY and TS-1 have uniform sizes, which are conducive to the efficient adsorption of biogenic amines. Scanning electron microscopy imaging showed that USY and MCM-41 displayed brick-shaped and irregular flake structures with a particle size of about 1 μm, while TS-1 displayed a spherical structure with a particle size between 5–20 μm.

[0065] When USY, TS-1, and MCM-41 molecular sieves were used as dispersed solid phase extraction reagents to extract biogenic amines, USY had the highest extraction efficiency, both close to 100%, while TS-1 only showed extremely high extraction efficiency for cadaverine, putrescine, and histamine, and MCM-41 had the worst extraction efficiency. Based on these results, we believe that the extraction efficiency of molecular sieves depends on their pore size distribution, crystallinity, dispersibility, and the molecular size of biogenic amines. USY had the highest extraction efficiency and was therefore selected as the dispersed solid phase extraction reagent.

[0066] 2. Characterization of USY molecular sieve before and after extraction of biogenic amines and its adsorption mechanism Figure 2 shown.

[0067] Figure 2 -a: Powder X-ray diffraction of USY molecular sieve before and after extraction of biogenic amines.

[0068] Figure 2 -b: Morphology before extraction of biogenic amines by USY molecular sieve.

[0069] Figure 2 -c: Morphology after extraction of biogenic amines by USY molecular sieve.

[0070] Figure 2 -d: ATR infrared spectra of biogenic amines before and after extraction with USY molecular sieve.

[0071] Figure 2 -e: X-ray photoelectron spectra of Si 2p before and after extraction of biogenic amines by USY molecular sieve.

[0072] Figure 2 -f: X-ray photoelectron spectrum of O1s before and after extraction of biogenic amines by USY molecular sieve.

[0073] The study found that the powder X-ray diffraction pattern, morphology, ATR infrared spectrum and X-ray photoelectron spectrum of USY molecular sieve did not change before and after the adsorption of biogenic amines, indicating that the adsorption of USY on biogenic amines is physical adsorption, which may be mixed with negligible chemical adsorption. USY is an inorganic silicon-aluminum-oxygen crystalline material. Compared with the silicon-oxygen skeleton, the incorporation of aluminum ions requires the recruitment of hydrogen ions to balance the charge. Therefore, the hydrogen ions and oxygen ions in the skeleton may form strong electrostatic interactions and hydrogen bonds with the biogenic amines. These interactions stabilize the physical adsorption, thus leading to the best adsorption effect.

[0074] 3. Optimization process of USY molecular sieve as dispersed solid phase extraction reagent for extracting biogenic amines from fruit wine Figure 3 shown.

[0075] Figure 3 -a: Optimization of USY molecular sieve dosage.

[0076] Figure 3 -b: Optimize extraction time.

[0077] Figure 3 -c: Optimization of desorption solvent.

[0078] Figure 3 -d: Optimization of desorption time.

[0079] Figure 3 -e: Optimization of desorption times.

[0080] Figure 3 -f: Recycling of USY molecular sieve.

[0081] From the characterization results, it can be seen that the USY molecular sieve has the best extraction effect on the five biogenic amines when the USY dosage is 5 mg, the extraction time is 2 min, the desorption solvent is 40% methanol aqueous solution containing 1% perchloric acid, the desorption time is 2 min, and the desorption number is 2 times. Moreover, the USY molecular sieve is recycled three times, and the adsorption effect is still very good, indicating that the USY molecular sieve can be reused. It is worth mentioning that the molecular sieve is baked in an oven at 110°C for 2 hours after use and then used again.

[0082] 4. The specific results of the optimization of the instrument parameters of the liquid chromatography-mass spectrometry instrument and the parent ion and product ion information are shown in Table 1-2.

[0083] According to the experiment, the gradient elution program of liquid chromatography is 0–2.0min, 0% A; 2.0–5.5min, 0%-60% A; 5.5-7.0min, 60% A; 7.0-7.5min, 60%-0% A; 7.5-12min, 0% A. A and B represent an aqueous solution containing 0.01mol / L ammonium formate and 0.5% formic acid and an acetonitrile solution containing 0.01mol / L ammonium formate and 0.5% formic acid, respectively. The mass spectrometry conditions are: curtain gas 40.0psi; collision gas Medium; ionization voltage 5500V; temperature 550℃; nebulizer gas 55.0psi; auxiliary heating gas 55.0psi; collision chamber ejection voltage 13.0V. Other liquid chromatography-mass spectrometry instruments can refer to the above instrument parameter settings.

[0084] Table 1: Gradient elution program for liquid chromatography and conditions for tandem mass spectrometry

[0085] Mass spectrometry parameters Optimization value Curtain gas (psi) 40.0 Collision gas Medium Ionization voltage (V) 5500 Temperature(℃) 550 Atomizing gas (psi) 55.0 Auxiliary heating gas (psi) 55.0 Collision cell ejection voltage (V) 13.0

[0086] The parent ion and daughter ion information of the five biogenic amines are as follows: cadaverine: parent ion 103.0, daughter ion 86.1 (quantitative) and 69.0; putrescine: parent ion 89.1, daughter ion 72.0 (quantitative) and 30.0; tyramine: parent ion 138.2, daughter ion 121.0 (quantitative) and 77.0; histamine: parent ion 112.2, daughter ion 95.1 (quantitative) and 68.0; tryptamine: parent ion 161.2, daughter ion 144.1 (quantitative) and 117.0.

[0087] Table 2: Precursor and daughter ion information of five biogenic amines

[0088]

[0089]

[0090] The product ions marked with an asterisk are used as quantification ions, and the other product ions are used as qualification ions.

[0091] Example 3 Test results

[0092] 1. The linear range, relative standard deviation (RSD), detection limit and quantification limit of the biogenic amine content in the fruit wine matrix detected by the method of Example 1-2 were tested. The specific results are shown in Table 3.

[0093] Table 3 Linear range, relative standard deviation (RSD), detection limit and quantification limit of the method in standard solutions.

[0094]

[0095] a Cadaverine, putrescine and tyramine were measured at 100 μg L –1 Histamine and tryptamine were measured at 10 μg L –1 Measurable.

[0096] b The detection limit and quantification limit were determined by three-fold and ten-fold signal-to-noise ratios, respectively (n=10).

[0097] As can be seen from Table 3, the linear range of cadaverine, putrescine and tyramine is 10–1000 μg / L, and the linear range of histamine and tryptamine is 1–100 μg / L. Each sample was tested in parallel 6 times, and the relative standard deviations were all within 5%. The detection limits and quantification limits of cadaverine, putrescine and tyramine were 0.30 and 0.92 μg / L, respectively, and the detection limits and quantification limits of histamine and tryptamine were 0.09 and 0.30 μg / L, respectively.

[0098] 2. The intra-day precision, inter-day precision, recovery rate and matrix effect obtained by three spiking concentrations of low, medium and high in fruit wine samples were tested. The test results are shown in Table 4.

[0099] Table 4 Intra-day precision, inter-day precision, recovery and matrix effect obtained by three spiking concentrations of low, medium and high in fruit wine samples.

[0100]

[0101] a n = 6; b n = 3; c n=6

[0102] d Matrix effect = (slope of the standard curve obtained in the fruit wine sample / slope of the standard curve obtained in the standard solution) × 100

[0103] It can be seen from the results that the three spike concentrations of low, medium and high are 15.0, 100.0, 200.0 μg / L (cadaverine, putrescine and tyramine) and 1.5, 10.0, 20.0 μg / L (histamine and tryptamine). At these three spike concentrations, the intra-day precision was measured 6 times, the inter-day precision was measured 3 times, and the recovery was measured 6 times. The matrix effect was calculated by the ratio of the slope of the standard curve drawn in the fruit wine matrix to the slope of the standard curve drawn in the standard solution. It can be seen that the intra-day precision and inter-day precision are both between 0.8%–9.9%, the spike recovery is between 85%–101%, and the matrix effect is between 91%–110%. This high recovery and negligible matrix effect indicate that the molecular sieve not only has perfect adsorption function, but also has excellent impurity removal function.

[0104] 3. The six fruit wine samples were tested using the dispersed solid phase microextraction-liquid chromatography-tandem mass spectrometry method. Each sample was measured three times. The specific results are shown in Table 5.

[0105] Table 5 Test results of 6 fruit wine samples and verification using standard addition method.

[0106] Analytes <![CDATA[Sample 1 a > <![CDATA[Sample 2 a > <![CDATA[Sample 3 a > <![CDATA[Sample 4 a > <![CDATA[Sample 5 a > <![CDATA[Sample 6 a > <![CDATA[Sample 6 b > <![CDATA[p-value c > Cadaverine 9.6±0.4 nd nd 15.2±0.3 9.0±0.4 9.1±0.3 9.2±0.4 0.184 Putrescine 84.4±1.1 50.5±0.9 nd 100.2±1.4 80.4±2.1 85.4±1.3 85.2±1.5 0.225 Tyramine 10.9±0.3 3.5±0.2 7.3±0.2 17.3±0.7 11.2±0.3 11.8±0.8 12.2±0.5 0.147 histamine 34.8±0.5 21.8±0.8 6.8±0.2 15.3±0.3 26.4±0.7 37.8±1.1 38.2±0.9 0.074 Tryptamine nd nd nd nd nd nd nd sum 139.7 75.8 14.1 148 127 144.1 144.8

[0107] nd: Not detected

[0108] a Quantification was performed using an external standard method.

[0109] b Quantification was performed using the method of standard addition.

[0110] c The t-test was used to compare whether there was a significant difference between the external standard method and the standard addition method.

[0111] The results showed that putrescine had the highest detection amount, and tryptamine could not be detected. Moreover, there was no significant difference between the external standard quantitative method and the standard addition method at p>0.05, indicating that the external standard quantitative method was reliable.

[0112] 4. Perform chromatographic detection on the standard solution and the fruit wine extract. The specific chromatogram is as follows: Figure 4 shown.

[0113] Figure 4 -a: Chromatogram of cadaverine standard solution (200 μg / L).

[0114] Figure 4 -b: Chromatogram of putrescine standard solution (200 μg / L).

[0115] Figure 4 -c: Chromatogram of tyramine standard solution (200 μg / L).

[0116] Figure 4 -d: Chromatogram of histamine standard solution (20 μg / L).

[0117] Figure 4 -e: Chromatogram of tryptamine standard solution (20μg / L).

[0118] Figure 4 -f: Chromatogram of cadaverine fruit wine extract.

[0119] Figure 4 -g: Chromatogram of putrescine fruit wine extract.

[0120] Figure 4 -h: Chromatogram of tyramine wine extract.

[0121] Figure 4 -i: Chromatogram of histamine wine extract.

[0122] Figure 4 -j: Chromatogram of tryptamine fruit wine extract.

[0123] from Figure 4 As can be seen from aj, in the standard solution and the wine extract, the chromatograms of cadaverine, putrescine, tyramine, histamine, and tryptamine Figure 1 The results were consistent, showing negligible matrix effect, which once again demonstrated that the external standard quantification method was reliable.

[0124] Compared with the prior art, the overall method of the present invention is not only simple, fast and efficient, but can also be further promoted and applied to the routine analysis of biogenic amines. The problems caused by derivatization treatment include complex and time-consuming sample preparation, and high detection limits and quantification limits. Therefore, many researchers have reported an integrated method of derivatization and dispersed liquid-liquid microextraction / dispersed solid phase microextraction, which can reduce the detection limit, quantification limit and matrix effect of the analyte. It is conceivable that the integrated strategy of non-derivatization and dispersed solid phase microextraction can produce a simpler, faster and more efficient sample processing method, but such examples have not been reported in the detection of biogenic amines. The method described in the present invention has achieved this goal for the first time. It is not only simple, fast and efficient, but also has a low detection limit and a high recovery rate. What is even more gratifying is that USY molecular sieve is a commercial reagent with a low price, which provides a good opportunity for the promotion and application of biogenic amine detection.

Claims

1. A method for rapidly and accurately determining the concentration of biogenic amines in a fruit wine matrix, characterized in that Here are the steps: (1) Drawing of standard curve of biogenic amines: a. Confirmation of mixed standard solution parameters: Prepare mixed standard solutions of biogenic amines of different concentrations using a volumetric flask, transfer them to injection vials after preparation, and set aside; Use the mixed standard solution to determine the optimal experimental parameters of the liquid chromatography-tandem mass spectrometer; b. Measure the mixed standard solution obtained in step a by using a liquid chromatography-tandem mass spectrometer; draw a standard curve of biogenic amines with the concentration of biogenic amines as the abscissa and the chromatographic peak area as the ordinate; (2) Preparation of fruit wine samples: add fruit wine to USY molecular sieve, vortex, and centrifuge to remove the supernatant; Continue to add desorption solvent, vortex centrifuge again, and collect the supernatant; add desorption solvent for the third time, vortex centrifuge, and collect the supernatant; measure the two supernatants respectively, record the concentrations as C1 and C2, and obtain the juice sample extract; (3) Measurement: The juice sample extract prepared in step (2) is measured using a liquid chromatography-tandem mass spectrometer to quantify the concentration of the fruit wine by using an external standard method combined with a standard curve.

2. The method for quickly and accurately determining the concentration of biogenic amines in a fruit wine matrix as claimed in claim 1, characterized in that: The biogenic amine in step (1) a is specifically cadaverine, putrescine, tyramine, histamine or tryptamine; Furthermore, when the biogenic amine is cadaverine, putrescine or tyramine, the concentrations of the prepared mixed standard solution are 10, 25, 50, 100, 200, 500 and 1000 μg / L respectively; when the biogenic amine is histamine or tryptamine, the concentrations of the prepared mixed standard solution are 1, 2.5, 5, 10, 20, 50 and 100 μg / L respectively.

3. The method for quickly and accurately determining the concentration of biogenic amines in a fruit wine matrix as claimed in claim 1, characterized in that step( 2) The specific steps are: add 4-6 mg USY molecular sieve into a 15 mL centrifuge tube, then add 4-6 mL fruit wine, vortex for 1-3 minutes, and then centrifuge; after removing the supernatant, add 1-2 mL desorption solvent, vortex again for 1-3 minutes, centrifuge, and collect the supernatant; add 1-2 mL desorption solvent again, vortex for 1-3 minutes, centrifuge, and collect the supernatant; measure the two supernatants separately, and the concentrations are C1 and C2, respectively, in μg / L.

4. The method for rapidly and accurately determining the concentration of biogenic amines in a fruit wine matrix according to claim 1, characterized in that: The model of the liquid chromatography-tandem mass spectrometer is SCIEX 5500LC-MS / MS; The gradient elution program of liquid chromatography was: 0–2.0 min, 0% A; 2.0–5.5 min, 0%–60% A; 5.5–7.0 min, 60% A; 7.0–7.5 min, 60%–0% A; 7.5–12 min, 0% A; wherein A and B represent an aqueous solution containing 0.01 mol / L ammonium formate and 0.5% formic acid and an acetonitrile solution containing 0.01 mol / L ammonium formate and 0.5% formic acid, respectively; The mass spectrometry conditions were as follows: curtain gas 40.0 psi; collision gas Medium; ionization voltage 5500 V; temperature 550° C.; nebulizer gas 55.0 psi; auxiliary heating gas 55.0 psi; and collision cell ejection voltage 13.0 V.

5. The method for rapidly and accurately determining the concentration of biogenic amines in a fruit wine matrix according to claim 1, characterized in that: The USY molecular sieve is an ultrastable Y-type molecular sieve.

6. The method for rapidly and accurately determining the concentration of biogenic amines in a fruit wine matrix according to claim 3, characterized in that: In step (2), the fruit wine needs to be placed at 4° C. for 20 minutes before being treated with USY molecular sieve.

7. The method for rapidly and accurately determining the concentration of biogenic amines in a fruit wine matrix according to claim 3, characterized in that: The centrifugal speed in step (2) is 9000-11000 r / min, the centrifugal temperature is 4° C., and the centrifugal time is 4-6 minutes.

8. The method for rapidly and accurately determining the concentration of biogenic amines in a fruit wine matrix according to claim 3, characterized in that: The desorption solvent in step (2) is a 40% methanol aqueous solution of perchloric acid with a volume concentration of 1%.

9. The method for rapidly and accurately determining the concentration of biogenic amines in a fruit wine matrix according to claim 3, characterized in that: The concentration of the fruit wine in step (2) is calculated using the formula C=(C1+C2) / 5.

10. The method for rapidly and accurately determining the concentration of biogenic amines in a fruit wine matrix according to claim 1, characterized in that: When the biogenic amine is cadaverine, putrescine or tyramine, the linear range of this method is 10-1000 μg / L, and the detection limit and quantification limit are 0.30 and 0.92 μg / L, respectively; when the biogenic amine is histamine or tryptamine, the linear range of this method is 1-100 μg / L, and the detection limit and quantification limit are 0.09 and 0.30 μg / L, respectively.

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