Method for detecting content of new food raw material in food

Abstract

The present application relates to a kind of food new food material content detection method, the new food material is pyrroloquinolinequinone disodium salt.Method comprising the following steps: (1) preparation standard working solution;(2) preparation sample solution to be measured;(3) using high performance liquid chromatograph determination and analysis condition;(4) establish standard working curve;(5) result analysis.This method establishes the high performance liquid chromatography detection method of new food material pyrroloquinolinequinone disodium salt in beverage.The method can effectively reduce the interference of food matrix component, while can effectively guarantee the stability of target pyrroloquinolinequinone disodium salt in determination process, high sensitivity, good reproducibility, simple and fast operation, suitable for new food material pyrroloquinolinequinone disodium salt in beverage daily large batch rapid detection and regulatory requirements.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of food detection, in particular to a high performance liquid chromatography detection method for pyrroloquinoline quinone disodium salt, a new food raw material in beverages. BACKGROUND

[0002] Pyrroloquinoline quinone (PQQ) is a redox enzyme coenzyme with physiological functions similar to vitamins, and is widely present in various common foods such as fruits (such as kiwi fruit, kiwifruit), vegetables (such as parsley, green pepper), drinks (such as green tea, oolong tea), etc. The content is 3.65-61.0 ng / g; human breast milk contains 140-180 ng / mL of PQQ, which is a growth factor necessary for development. As a redox enzyme coenzyme, PQQ participates in the respiratory chain electron transfer, has multiple biological functions such as improving immunity, improving mitochondrial activity, promoting brain nerve factor growth, scavenging free radicals, delaying aging, preventing and treating liver damage, and outstanding antioxidant performance, and is called the king of immunity by the global biomedical community. According to relevant research, PQQ plays a crucial role in human nutrition. When PQQ is lacking in the diet of mammals, it will cause growth disorders, immune function damage, and abnormal reproductive function, etc. Like essential nutrients such as folic acid and biotin, the human immune system is extremely sensitive to PQQ levels. After PQQ is missing, the human body's immune function has multiple defects, such as decreased white blood cell function, reduced number, and inability to normally immune response, etc.

[0003] It is currently believed that the intestinal flora of animals and humans cannot synthesize PQQ or the amount of synthesis is far from meeting the needs of the body, and the PQQ in the body is mainly obtained exogenously through dietary means, and needs to be supplemented with exogenous PQQ. PQQ in daily diet is mainly derived from plants, but due to the limited intake of daily diet, PQQ-added products have emerged. Due to the unstable nature of PQQ itself, the form of PQQ usually added in products is the sodium salt derivative of PQQ, i.e. pyrroloquinoline quinone disodium salt (PQQ-2Na). Pyrroloquinoline quinone disodium salt is managed as a "generally recognized as safe substance (GRAS)" in the United States and can be used as a raw material for energy drinks, vitamin functional drinks, electrolyte drinks and other foods; as a dietary supplement or natural health food in the European Union and Canada. In March 2022, China approved pyrroloquinoline quinone disodium salt as a new food raw material, and the use range and maximum usage amount are limited to beverages (40 mg / kg, solid beverages are converted according to the mass of the liquid after brewing), and the recommended daily intake of adults (except infants, pregnant women or lactating women) is 20 mg. With the approval of pyrroloquinoline quinone disodium salt as a new food raw material that can be applied to beverage products, the market demand for PQQ is continuously released, and beverage products containing PQQ ingredients are gradually entering the market, such as the sunrise red ginseng turmeric drink of the brand black zero TM under Huaxi Biotechnology, PQQ-2Na solid beverage of Beijing Zhi Gan, PQQ-2Na solid beverage of Shandong Yusheng Kenzhiyi series, and solid beverage of bovine colostrum probiotic yeast beta glucan PQQ-2Na of Guangzhou Zhenpin Kang. The market for PQQ-2Na beverage products is increasing, and the quality of the products is uneven, and some businesses may use inferior products to sell or engage in fraudulent practices. Due to the lack of testing method standards, people cannot accurately determine the true content of PQQ-2Na in beverage products, and this also brings great difficulties to related supervision. Therefore, it is urgent to establish an accurate, reliable, sensitive and efficient detection method standard to meet the determination of pyrroloquinoline quinone disodium salt in food and the supervision needs of relevant departments.

[0004] Currently, there are few literature reports on the determination method of pyrroloquinoline quinone disodium salt in beverages. Liu et al. established a method for determining the content of new food raw material pyrroloquinoline quinone disodium salt in beverages by high performance liquid chromatography and mass spectrometry. This method has the following disadvantages: the pretreatment is complicated, the operability is not strong, the separation time is long, it is not suitable for large batch sample detection, the scope of application is narrow, it cannot cover all beverage matrices, the recovery rate of milk-containing beverages is poor, the sensitivity is poor, the detection limit is as high as 10 mg / kg, which is very close to the limit value, and thus it cannot effectively meet the determination requirements of various food matrices. SUMMARY

[0005] The purpose of the present application is to provide a high performance liquid chromatography detection method for the content of a new food raw material pyrroloquinoline quinone disodium salt in food, to make up for and solve the lack of the current detection method for the content of pyrroloquinoline quinone disodium salt in various beverage matrices specified in GB 2760 "National Food Safety Standard Food Additive Use Standard" and GB / T 10789-2015 "Beverage General", so that the method has strong applicability and practicality in the related field and can fully meet the limit requirements of relevant laws and regulations

[0006] The present method adds ethylenediaminetetraacetic acid disodium as a stabilizer to the beverage sample, dilutes with a diluent, and then purifies by centrifugation with an ultrafiltration centrifuge tube before determination by high performance liquid chromatography, to establish a high performance liquid chromatography determination method for the content of pyrroloquinoline quinone disodium salt in various types of beverages. The method selects 250nm as the detection wavelength, purifies with an ultrafiltration purification tube, effectively avoids the interference of various additives commonly found in food and coenzyme Q10 and nicotinamide mononucleotide that may exist simultaneously, and completes the detection of the project on an ordinary liquid chromatograph in 10min, with a quantitative detection limit of 0.5mg / kg, which is much lower than the limit value of 40mg / kg, and can realize the ultrafast and high-sensitivity quantitative detection of pyrroloquinoline quinone disodium salt in beverages. After adding ethylenediaminetetraacetic acid disodium solution as a stabilizer, the concentration of 0.050mg / L near the minimum point of the standard sample curve can ensure the stability of the concentration for more than three months. In comparison, the solution without adding ethylenediaminetetraacetic acid disodium solution as a stabilizer can only be stored for two months before the concentration begins to decay by more than half. At the same time, all actual samples can obtain stability for more than 24 hours, which is particularly important in the process of handling a large number of samples.

[0007] The method is simple, sensitive, accurate and reliable, and is suitable for the determination of the content of pyrroloquinoline quinone disodium salt in various types of beverages.

[0008] The specific scheme is as follows:

[0009] A detection method for the content of a new food raw material in food, the new food raw material being pyrroloquinoline quinone disodium salt; wherein the detection method specifically comprises the following steps:

[0010] (1) Prepare a standard working solution by preparing a standard working solution of pyrroloquinoline quinone disodium salt with a diluent at a concentration of 0.020-10mg / L;

[0011] (2) Prepare a sample solution to be tested: weigh the sample to be tested in a container, add an aqueous solution of ethylenediaminetetraacetic acid disodium and diluent, ultrasonically treat, dilute with diluent, centrifuge, and obtain a sample solution to be tested for determination;

[0012] (3) The high performance liquid chromatograph is used for detection and analysis: the detector is a photodiode array detector; the chromatographic column is an RD-C18 column, the column temperature is 35°C; the mobile phase A is a mobile phase salt solution, the mobile phase B is acetonitrile; the flow rate is 1 mL / min; the injection volume is 20 μL; the gradient elution program is as follows: 0-3.5 min, 60% by volume of A; 3.5-3.6 min, 60% by volume of A-20% by volume of A; 3.6-6.0 min, 20% by volume of A; 6.0-6.1 min, 20% by volume of A-60% by volume of A; 6.1-10.0 min, 60% by volume of A;

[0013] (4) The standard curve is drawn: different concentrations of the standard working solution are injected into the high performance liquid chromatograph, gradient elution and detection are carried out under the chromatographic conditions of step (3), qualitative analysis is carried out according to the retention time, and the standard curve is drawn according to the corresponding relationship between the size of the peak area and the concentration; the standard curve is used for quantitative analysis of the target substance in the sample solution to be measured;

[0014] (5) The results are analyzed: the sample solution to be measured in step (2) is injected into the high performance liquid chromatograph, gradient elution and detection are carried out under the chromatographic conditions of step (3), the peak area of the target substance in the sample solution to be measured is measured, qualitative analysis is carried out according to the retention time, and quantitative analysis is carried out according to the standard curve prepared in (4), and the content of pyrroloquinoline quinone disodium salt in the sample to be measured is calculated.

[0015] Further, the food is a beverage;

[0016] Optionally, the beverage comprises at least one of a special use beverage, a protein beverage, a solid beverage, a flavored beverage, a tea beverage, a juice beverage, a carbonated beverage, a plant beverage or a coffee beverage.

[0017] Further, in step (1), the standard working solution is obtained by gradually diluting a standard stock solution and a standard intermediate solution;

[0018] Optionally, the diluent is a phosphate diluent formed by mixing a 12-hydrated disodium hydrogen phosphate aqueous solution and a 2-hydrated sodium dihydrogen phosphate aqueous solution.

[0019] Further, the standard stock solution is prepared as follows: pyrroloquinoline quinone disodium salt standard substance with a purity of 100% by mass is weighed in a volumetric flask, the diluent is used for dissolution, the volume of the volumetric flask to be prepared and the 10 g / L ethylenediaminetetraacetic acid disodium salt aqueous solution are added in a volume ratio of 25:1, and the 10 g / L ethylenediaminetetraacetic acid disodium salt aqueous solution is prepared to obtain the standard stock solution with a concentration of 1000 mg / L.

[0020] Further, the preparation of the standard intermediate solution: take the standard stock solution, dilute with the diluent, add the volume of the prepared capacity and the aqueous solution of disodium ethylenediaminetetraacetate according to the volume ratio of 25:1 to the aqueous solution of 10 g / L disodium ethylenediaminetetraacetate, and prepare the standard intermediate solution with a concentration of 100 mg / L.

[0021] Further, the preparation of the standard working solution is specifically as follows: take 0.020-10 mL of the standard intermediate solution in a volumetric flask, add the volume of the prepared capacity and the aqueous solution of disodium ethylenediaminetetraacetate according to the volume ratio of 25:1 to the aqueous solution of 10 g / L disodium ethylenediaminetetraacetate, and then dilute to the mark with the diluent, mix well, and prepare a series of standard working solutions with a mass concentration of 0.020-10 mg / L.

[0022] Further, in step (2), the specific steps for preparing the sample solution to be tested are as follows: weigh 2.0±0.001 g of the sample to be tested in a stoppered colorimetric tube, add the diluent with a volume ratio of 60% and the aqueous solution of 10 g / L disodium ethylenediaminetetraacetate with a volume ratio of 4%, ultrasonic extraction for 5-15 min, dilute and dilute with the diluent, mix well, obtain the sample solution, and take 0.8-2.0 mL of the sample solution in an ultrafiltration centrifuge tube, centrifuge at 4000-5000 r / min for 2-10 min, and obtain the sample solution to be tested for liquid chromatography determination;

[0023] Optionally, when the sample to be tested is a solid beverage, the solid beverage needs to be diluted according to the brewing ratio indicated on the product label, and then the sample solution to be tested is prepared.

[0024] Preferably, in step (3), the RD-C18 chromatographic column has a specification of 4.6 mm x 150 mm, and the particle size of the chromatographic column filler is 5 μm.

[0025] Optionally, the detection wavelength of the photodiode array detector is 190-800 nm.

[0026] Preferably, the detection wavelength of the photodiode array detector is 250 nm.

[0027] Further, in step (4), the standard curve coefficient is not less than 0.9999, and the quantitative detection limit of the method is 0.5 mg / kg.

[0028] Further, the sample stability RSD during 0-24 h is less than 3.3%, the sample precision RSD is less than 1.5%, the recovery rate of the detection method is in the range of 85.0%-109.4%, and the RSD is 0.3%-4.9%.

[0029] Advantages:

[0030] 1、The application can make up for and solve the deficiency of the current method for detecting the content of pyrroloquinoline quinone disodium salt in various beverage matrices specified in GB 2760 "National Food Safety Standard Food Additive Usage Standard" and GB / T 10789-2015 "Beverage General", the researched beverage matrices cover special use beverage, protein beverage, solid beverage, flavor beverage, tea beverage, fruit juice beverage, carbonated beverage, plant beverage, coffee beverage, and can completely meet the requirements of the existing laws and regulations of our country and the beverage matrix range required by food safety detection, and the applicability is wide. The existing technology is only for two food matrices of fruit juice beverage and protein beverage, and the technical limitation is obvious.

[0031] 2、The application selects suitable detection wavelength, suitable type of RD-C18 column as stationary phase and suitable mobile phase system, continuously explores and optimizes the composition and proportion of various mobile phases, so that the chromatographic separation effect of ultra-high performance liquid instrument can be realized on a conventional ordinary liquid instrument, compared with 26min required by the existing technology, the complete separation of pyrroloquinoline quinone disodium salt in the beverage can be realized in 10min, the method is simple and fast, and better peak shape and sensitivity are obtained. This is particularly important when screening a large number of samples, which can greatly reduce solvent consumption, and the operator does not need to frequently prepare the mobile phase, does not need to run multiple instruments, and does not need to wait for the running of a large number of sample tasks, which greatly reduces the cost of consumables, time, instruments and labor.

[0032] 3、The ultrafiltration purification tube system originally applied in the field of biological medicine is innovatively introduced into the food detection system of the application. The ultrafiltration purification tube is originally used for concentrating protein, polypeptide and other substances in the biological medicine field, and the salts and solvents, small molecule substances in the sample solution are passed through, the application innovatively utilizes the principle in reverse to intercept protein, polypeptide and other interfering substances in food matrices such as protein beverage, solid beverage and coffee beverage, and obtains the sample solution and the target pyrroloquinoline quinone disodium salt therein, and it is found that the ultrafiltration centrifuge tube system can effectively remove the interfering substances such as pigments in the sample, so that the sample solution presents the characteristics of colorless, transparent and clear solution, and can be directly tested without further treatment. At the same time, the ultrafiltration centrifuge tube can eliminate impurity interference, and the target recovery rate can reach more than 85%, and the methodological results are satisfactory. The application of the ultrafiltration purification tube system can achieve more excellent effect than the existing technology, and obtain better methodological results.

[0033] Compared with the conventional purification system applied to the above food matrix, such as zinc acetate + potassium ferrocyanide purification system (2 mL of 200 g / L zinc acetate and 100 g / L potassium ferrocyanide are added to the sample, and diluted to 50 mL with diluent), 50% acetonitrile purification system (50% acetonitrile is added to the sample to make up to 50 mL), trichloroacetic acid purification system (10 g / L trichloroacetic acid is added to the sample to make up to 50 mL), sodium tungstate + sulfuric acid purification system (2 mL of 10% sodium tungstate solution and 10% sulfuric acid solution are added to the sample, and diluted to 50 mL with diluent), zinc sulfate + sodium hydroxide purification system (10 mL of 10% zinc sulfate solution is added to the sample, and the recovery rate is 0%, while the recovery rate of the ultrafiltration purification column system is less than 10%, the recovery rate of the 20% acetonitrile purification system is less than 85%, which is much lower than the ultrafiltration purification tube system used in the present application.

[0034] 4、The present application found in the experiment that part of the sample containing the target substance began to appear the phenomenon of attenuation after 6 hours. In order to ensure the stability of the target substance in the sample determination process, after comparing the water-soluble antioxidants such as disodium ethylenediaminetetraacetate (C 10 H 14 N2Na2O8), L(+)-ascorbic acid (C6H8O6), the stabilizer disodium ethylenediaminetetraacetate is innovatively added in the sample determination process, and the corresponding effective concentration is determined to be 0.4 g / L to ensure the stability of the target substance in the sample solution, while L(+)-ascorbic acid will make the target substance pyrroloquinoline quinone disodium salt attenuate by about 80%. The related experimental results show that the addition of disodium ethylenediaminetetraacetate (the addition level is 0.4 g / L) in the sample determination process can effectively ensure that the target substance in all food matrices such as special use beverage, protein beverage, solid beverage, flavor beverage, tea beverage, fruit juice beverage, carbonated beverage, plant beverage and coffee beverage remains stable for at least 24 hours, which is particularly important in large-scale detection. There is no stabilizing protection measure for the target substance pyrroloquinoline quinone disodium salt in the sample in the prior art. At the same time, the addition of disodium ethylenediaminetetraacetate as a stabilizer in the standard working solution can effectively prolong the effective period of the standard working solution, especially the lowest concentration of the standard curve. Experimental results show that after adding disodium ethylenediaminetetraacetate solution as a stabilizer, the lowest concentration 0.040 mg / L of the standard sample curve can ensure the concentration to be stable for more than three months, while the solution without adding disodium ethylenediaminetetraacetate solution as a stabilizer can only be placed for two months before the concentration begins to attenuate by more than half, thereby reducing the frequency of preparing the standard working solution by the experimental personnel and effectively improving the efficiency of the experimental personnel in the experimental process.

[0035] 5、The application puts forward a simple, fast and operable pretreatment method on the basis of careful investigation of a series of pretreatment conditions such as sample amount, extraction solvent (diluent and pure water), eight kinds of purification systems and two kinds of stabilizing systems. 10 g / L of ethylenediaminetetraacetic acid disodium solution is added to the sample to be tested, and then the sample is diluted in the purification tube and centrifuged to be loaded on the machine. However, the prior art needs to add liquid to the sample, vortex mixing, ultrasonic, repeated extraction of residues, mixing, supernatant transfer and other complex steps, which is extremely tedious, time-consuming and labor-consuming, and is not conducive to the rapid detection of large quantities of samples. At the same time, there is no residue in general beverage samples, and the pretreatment operation is not reasonable. After 20% acetonitrile extraction, the sample solution is still relatively turbid, which cannot meet the requirements of loading on the machine, and forcibly loading on the machine will greatly shorten the service life of the chromatographic column and the instrument.

[0036] 6、The quantitative detection limit of the application can reach 0.5 mg / kg, which is much lower than the limit value 40 mg / kg, and can realize high sensitivity quantitative detection of pyrroloquinoline quinone disodium salt in beverages. The quantitative detection limit of the prior art is as high as 10 mg / kg, which is very close to the limit value 40 mg / kg, and the sensitivity cannot effectively meet the determination requirements of the existing announcement.

[0037] 7、The application has good specificity. Within 10 minutes of running time, the target pyrroloquinoline quinone disodium salt can be fully separated from 13 kinds of food additives commonly used in beverages, such as benzoic acid, sorbic acid, saccharin, acesulfame, dehydroacetic acid, lemon yellow, new red, amaranth, carmine, sunset yellow, temptation red, bright blue and azo ruby, as well as common functional ingredients such as coenzyme Q10 and nicotinamide mononucleotide, which shows that the application has good specificity for determination of pyrroloquinoline quinone disodium salt in beverages, and is suitable for accurate determination of pyrroloquinoline quinone disodium salt content in beverages under complex matrix conditions.

[0038] 8、The analysis instrument used in the application is a common high performance liquid chromatograph, and common brand chromatographic columns instead of well-known brand chromatographic columns are used, which can effectively reduce the detection cost. Compared with expensive instruments such as liquid chromatograph-mass spectrometer and ultra-high performance liquid chromatograph, the application is more easy to popularize and apply due to its low cost. BRIEF DESCRIPTION OF DRAWINGS

[0039] In order to more clearly illustrate the technical solutions of the application, the following will briefly introduce the drawings. Obviously, the drawings described in the following description only relate to some embodiments of the application, and not limit the application.

[0040] Figure 1 is the high performance liquid chromatogram of the pyrroloquinoline quinone disodium salt standard working solution (10.0 mg / L) provided by the embodiment 2 of the application.

[0041] Figure 2It is the DAD spectrum of the pyrroloquinolinequinone disodium salt standard working solution provided in Embodiment 2 of the present application.

[0042] Figure 3A It is the chromatogram of 13 food additive standard solutions provided in Embodiment 3 of the present application: 1. benzoic acid; 2. sorbic acid; 3. saccharin; 4. acesulfame potassium; 5. dehydroacetic acid; 6. lemon yellow; 7. new red; 8. amaranth; 9. cochineal; 10. sunset yellow; 11. allura red; 12. brilliant blue; 13. azorubin.

[0043] Figure 3B It is the chromatogram of the pyrroloquinolinequinone disodium salt standard working solution (1.0 mg / L) provided in Embodiment 3 of the present application.

[0044] Figure 4A It is the chromatogram of the pyrroloquinolinequinone disodium salt standard working solution (5.0 mg / L) provided in Embodiment 3 of the present application.

[0045] Figure 4B It is the chromatogram of the coenzyme Q10 standard working solution (5.0 mg / L) provided in Embodiment 3 of the present application.

[0046] Figure 4C It is the chromatogram of the nicotinamide mononucleotide standard working solution (5.0 mg / L) provided in Embodiment 3 of the present application.

[0047] Figure 5 It is the effect comparison chart before and after purification of the protein beverage provided in Embodiment 3 of the present application: the protein beverage before purification (left one); the protein beverage after purification by the ultrafiltration centrifugal tube (left two); the protein beverage after purification by the ultrafiltration purification column (middle); the protein beverage after purification by the 20% acetonitrile purification system (right two); the protein beverage after purification by the 50% acetonitrile purification system (right one).

[0048] Figure 6 It is the effect comparison chart before and after purification of the solid beverage provided in Embodiment 3 of the present application: the solid beverage before purification (left one); the solid beverage after purification by the ultrafiltration centrifugal tube (left two); the solid beverage after purification by the ultrafiltration purification column (middle); the solid beverage after purification by the 20% acetonitrile purification system (right two); the solid beverage after purification by the 50% acetonitrile purification system (right one).

[0049] Figure 7 It is the effect comparison chart before and after purification of the coffee beverage provided in Embodiment 3 of the present application: the coffee beverage before purification (left one); the coffee beverage after purification by the ultrafiltration centrifugal tube (left two); the coffee beverage after purification by the ultrafiltration purification column (middle); the coffee beverage after purification by the 20% acetonitrile purification system (right two); the coffee beverage after purification by the 50% acetonitrile purification system (right one).

[0050] Figure 8is a stability test result graph of different samples to be tested within 24 h provided by embodiment 3 of the present application.

[0051] Figure 9A is a chromatogram of disodium ethylenediaminetetraacetate standard working solution (0.4 g / L) provided by embodiment 3 of the present application.

[0052] Figure 9B is a chromatogram of L(+)-ascorbic acid standard working solution (0.4 g / L).

[0053] Figure 9C is a chromatogram of pyrroloquinoline quinone disodium salt standard working solution (6.0 mg / L) provided by embodiment 3 of the present application.

[0054] Figure 10A is a chromatogram of target pyrroloquinoline quinone disodium salt standard working solution (1.6 mg / L) provided by embodiment 3 of the present application, which is measured after 0.4 g / L disodium ethylenediaminetetraacetate is added.

[0055] Figure 10B is a chromatogram of target pyrroloquinoline quinone disodium salt standard working solution (1.6 mg / L) provided by embodiment 3 of the present application, which is measured after 0.4 g / L L(+)-ascorbic acid is added.

[0056] Figure 10C is a chromatogram of target pyrroloquinoline quinone disodium salt standard working solution (1.6 mg / L) provided by embodiment 3 of the present application. DETAILED DESCRIPTION

[0057] The preferred embodiments of the present application will be described in more detail below. Although the preferred embodiments of the present application are described below, it is understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. If no specific technique or condition is specified in the examples, the technique or condition described in the literature in the art or according to the product manual is used. If no manufacturer of the reagent or instrument is specified, it is a conventional product that can be commercially available. In the following examples, unless otherwise specified, "%" means volume percent, and "parts" means weight parts.

[0058] Example 1

[0059] 1. Reagents and materials

[0060] Unless otherwise specified, all reagents are of analytical purity, and water is first-grade water specified in GB / T 6682.

[0061] 1.1 Acetonitrile: chromatographically pure.

[0062] 1.2 Disodium hydrogen phosphate dodecahydrate.

[0063] 1.3 Sodium dihydrogen phosphate dihydrate.

[0064] 1.4 Tetrabutylammonium bromide.

[0065] 1.5 Potassium dihydrogen phosphate dihydrate.

[0066] 1.6 Zinc acetate.

[0067] 1.7 Potassium ferrocyanide.

[0068] 1.8 Trichloroacetic acid.

[0069] 1.9 Sodium tungstate.

[0070] 1.10 Sulfuric acid.

[0071] 1.11 Zinc sulfate.

[0072] 1.12 Sodium hydroxide.

[0073] 1.13 Disodium ethylenediaminetetraacetate (C 10 H 14 N2Na2O8).

[0074] 1.14 L(+)-ascorbic acid (C6H8O6).

[0075] 1.15 Standard: Pynroloquinoline quinone disodium salt (CAS No. 122628-50-6): purity ≥ 95%, or certified standard material published by the state.

[0076] 1.16 Diluent: weigh 2.26 g of disodium hydrogen phosphate dodecahydrate (1.2) and 1.98 g of sodium dihydrogen phosphate dihydrate (1.3) and dissolve in 1000 mL of water.

[0077] 1.17 Mobile phase salt solution: weigh 6.45 g of tetrabutylammonium bromide (1.4) and 2.72 g of potassium dihydrogen phosphate dihydrate (1.5) and dissolve in 1000 mL of water.

[0078] 1.18 Disodium ethylenediaminetetraacetate solution (10 g / L): weigh 10 g of disodium ethylenediaminetetraacetate (1.13) and dissolve in 1000 mL of diluent (1.16).

[0079] 1.19 L(+)-ascorbic acid solution (10 g / L): weigh 10 g of L(+)-ascorbic acid (1.14) and dissolve in 1000 mL of diluent (1.16).

[0080] 1.20 Standard stock solution preparation: Weigh 0.25 g (0.0001 g) of pyrroloquinoline quinone disodium salt standard substance, which is converted to 100% mass, dissolve in a 25 mL brown volumetric flask, add 1 mL of 10 g / L disodium ethylenediaminetetraacetate solution (1.18), dissolve with diluent and dilute to 25 mL, prepare a standard stock solution with a concentration of 1000 mg / L. Store in a 4°C refrigerator for standby use, valid for 6 months.

[0081] 1.21 Standard intermediate solution configuration (100 mg / L): Accurately pipette 10.0 mL of pyrroloquinoline quinoline disodium salt standard stock solution (1000 mg / L) into a 100 mL volumetric flask, add 4 mL of 10 g / L disodium ethylenediaminetetraacetate solution (1.18), dilute to the mark with diluent (1.16), mix well, prepare a standard intermediate solution with a mass concentration of 100 mg / L, store in a 4°C refrigerator for standby use, valid for 3 months.

[0082] 1.22 Standard working solution preparation: Pipette 0.020 mL, 0.050 mL, 0.10 mL, 0.50 mL, 1.0 mL, 5.0 mL and 10 mL of pyrroloquinoline quinoline disodium salt standard intermediate solution (100 mg / L) into a 100 mL volumetric flask, respectively, add 4 mL of 10 g / L disodium ethylenediaminetetraacetate solution (1.18), dilute to the mark with diluent (1.16), mix well. The mass concentration of the pyrroloquinoline quinoline disodium salt standard series working solution is 0.020 mg / L, 0.050 mg / L, 0.10 mg / L, 0.50 mg / L, 1.0 mg / L, 5.0 mg / L, 10 mg / L. Store in a 4°C refrigerator for standby use, valid for 1 month.

[0083] 2 Instruments and equipment

[0084] 2.1 High performance liquid chromatograph: equipped with photodiode array detector.

[0085] 2.2 Analytical balance: sensitivity of 0.0001 g and 0.001 g.

[0086] 2.3 High speed centrifuge: speed not less than 4500 r / min.

[0087] 2.4 Cuvette with stopper: 50 mL.

[0088] 2.5 Microporous filter membrane: pore size 0.45 μm, water phase.

[0089] 2.6 Anavo PES ultrafiltration centrifuge tube, 15 mL, 10 kDa, or equivalent.

[0090] 2.7 Anavo Que ultrafiltration purification column, 180 mg / 2.5 ml SPE, or equivalent.

[0091] 2.8 Homogenizer.

[0092] 2.9 Ultrasonic cleaner: operating frequency 40kHz, power 800W, temperature control range room temperature to 80℃, or equivalent.

[0093] 3 methods

[0094] 3.1 High Performance Liquid Chromatography Conditions

[0095] a) Chromatographic column: RD-C 18 Column (4.6×150mm, 5.0μm);

[0096] b) Mobile phase: Mobile phase A is a mobile phase salt solution, and mobile phase B is acetonitrile; gradient elution program: 0–3.5 min, 60% A; 3.5–3.6 min, 60% A–20% A; 3.6–6.0 min, 20% A; 6.0–6.1 min, 20% A–60% A; 6.1–10.0 min, 60% A.

[0097] c) Flow rate: 1.0 mL / min;

[0098] d) Column temperature: 35℃;

[0099] e) Injection volume: 20 μL;

[0100] f) Detection conditions for photodiode array detector: detection wavelength 250nm.

[0101] 3.2 Plotting the Standard Curve

[0102] Take standard working solutions (1.22) and perform chromatographic determination according to the chromatographic conditions in 3.1. Plot a standard curve by performing linear regression on the peak area (Y) of each analyte against the corresponding mass concentration (X, mg / L) to obtain the linear regression equation.

[0103] 4 Sample Testing Procedures

[0104] 4.1 Sample Pretreatment

[0105] Weigh 2g (accurate to 0.001g) of sample (solid beverages should be diluted according to the mixing ratio indicated on the label before testing) into a 50mL stoppered colorimetric tube (2.4), add 30mL of diluent (1.16) and 2mL of disodium EDTA solution (1.18), place in an ultrasonic cleaner (2.9) for ultrasonic extraction for 10min, dilute with diluent (1.16) and bring to a final volume of 50mL, mix well, take 1.0mL of sample solution into an ultrafiltration centrifuge tube (2.6), centrifuge at 4500r / min for 5min, and the sample solution to be tested is used for liquid chromatography determination.

[0106] 4.2 Blank test

[0107] The same determination procedure was taken for parallel operation except that no sample was added.

[0108] 4.3 Preparation of standard working curve

[0109] The standard working solution was injected into the high performance liquid chromatograph, and the corresponding peak area was determined. The mass concentration of pyrroloquinoline quinone disodium salt in the standard working solution was taken as the abscissa, and the peak area was taken as the ordinate to draw the standard curve. The high performance liquid chromatogram of the pyrroloquinoline quinone disodium salt standard working solution is shown in the accompanying drawings of the specification Figure 1 The DAD spectrum of the pyrroloquinoline quinone disodium salt standard working solution is shown in the accompanying drawings of the specification Figure 2 .

[0110] 4.4 Determination of sample solution (to-be-tested sample solution)

[0111] The sample solution was injected into the liquid chromatograph to obtain the peak area, and the mass concentration of pyrroloquinoline quinone disodium salt in the sample solution was obtained according to the standard curve. The response values of pyrroloquinoline quinone disodium salt in the standard working solution and the sample solution should be within the linear response range of the instrument. If the content exceeds the linear range of the standard curve, the sample amount or the constant volume needs to be adjusted for re-detection.

[0112] 5 Expression of analysis results

[0113] The content of pyrroloquinoline quinone disodium salt in the sample was calculated according to formula (1):

[0114]

[0115] In the formula:

[0116] X1 — the content of pyrroloquinoline quinone disodium salt in the sample, in milligrams per kilogram (mg / kg);

[0117] C — the mass concentration of pyrroloquinoline quinone disodium salt in the sample solution, in milligrams per liter (mg / L); C0 — the mass concentration of pyrroloquinoline quinone disodium salt in the sample blank solution, in milligrams per liter (mg / L);

[0118] C0 — the mass concentration of pyrroloquinoline quinone disodium salt in the sample blank solution, in milligrams per liter (mg / L);

[0119] V — the constant volume of the sample, in milliliters (mL);

[0120] m — the sample amount, in grams (g);

[0121] 1000 — unit conversion coefficient.

[0122] The calculation result was expressed as the arithmetic mean of two independent determination results obtained under repeatability conditions, and the result was rounded to three significant digits.

[0123] Example 2

[0124] In this example, the raw materials of special purpose beverages, vitamin functional beverages, electrolyte beverages, protein beverages, solid beverages, energy flavor beverages, tea beverages, fruit juice beverages, carbonated beverages, plant beverages, coffee beverages, etc. were pretreated according to the 4-sample testing procedure in Example 1, and then determined by the determination method in Example 1. The specific determination procedure is as follows: 6 methodology investigation, including linearity, detection limit, quantification limit, recovery rate, precision, repeatability, and stability

[0125] 6.1 Linearity, detection limit, and quantification limit

[0126] A series of standard working solutions of pyrroloquinoline quinone disodium salt with a mass concentration of 0.020-10 mg / L were prepared, and linear regression was performed on the peak area versus the mass concentration (unit: mg / L). The linear equation, correlation coefficient, linear range, detection limit, and quantification limit are shown in Table 1. The results show that the pyrroloquinoline quinone disodium salt component has a good linear relationship in the linear range, and the correlation coefficient r = 0.9999. The method detection limit and quantification limit are obtained at a signal-to-noise ratio (S / N) = 3 and (S / N) = 10. The detection limit of the pyrroloquinoline quinone disodium salt component is 0.15 mg / kg, and the quantification limit is 0.50 mg / kg. The method quantification limit can fully meet the limit requirements of pyrroloquinoline quinone disodium salt in foods in China.

[0127] Table 1 Linear equation, correlation coefficient, linear range, detection limit, and quantification limit of pyrroloquinoline quinone disodium salt

[0128]

[0129] 6.2 Recovery rate and precision

[0130] Under the optimized test conditions, the blank matrix of vitamin functional beverages (special purpose beverages), electrolyte beverages (special purpose beverages), protein beverages, solid beverages, energy flavor beverages (flavor beverages), tea beverages, fruit flavor beverages (flavor beverages), fruit juice beverages, carbonated beverages, plant beverages, and coffee beverages were tested for the recovery rate with a spiked level of 0.5, 2.5, 40.0, and 250.0 mg / kg, respectively. Each level was analyzed in duplicate for 6 times, and the results are shown in Table 2. As can be seen from Table 2, the recovery rate range of this method is 85.0%-109.4%, and the RSDs are 0.3%-4.9%. The results show that this method has good accuracy and precision, and is suitable for the routine analysis and detection of pyrroloquinoline quinone disodium salt in beverages.

[0131] Table 2 Recovery rate and relative standard deviation (n = 6) of pyrroloquinoline quinone disodium salt in beverages

[0132]

[0133]

[0134] 6.3 Precision experiment:

[0135] Under the optimized test conditions, the vitamin functional beverage (special purpose beverage), electrolyte beverage (special purpose beverage), protein beverage, solid beverage, energy flavor beverage (flavor beverage), tea beverage, fruit flavor beverage (flavor beverage), fruit juice beverage, carbonated beverage, plant beverage, coffee beverage blank matrix were added with the pyrroloquinoline quinone disodium salt standard intermediate solution, and the labeled lactobacillus solid beverage added with pyrroloquinoline quinone disodium salt actual sample, and the content was measured by repeating the sample injection for 6 times, and the relative standard deviation (RSD) was calculated, and the results were shown in Table 3. As shown in Table 3, the method had good precision, and could meet the determination requirements of the standard method.

[0136] Table 3 Precision test results of samples

[0137]

[0138]

[0139] 6.4 Reproducibility experiment:

[0140] Under the optimized test conditions, the vitamin functional beverage (special purpose beverage), electrolyte beverage (special purpose beverage), protein beverage, solid beverage, energy flavor beverage (flavor beverage), tea beverage, fruit flavor beverage (flavor beverage), fruit juice beverage, carbonated beverage, plant beverage, coffee beverage blank matrix were added with the pyrroloquinoline quinone disodium salt standard intermediate solution, and the labeled lactobacillus solid beverage added with pyrroloquinoline quinone disodium salt actual sample, and the content was measured by repeating the sample injection for 6 times, and the relative standard deviation (RSD) was calculated, and the results were shown in Table 3. As shown in Table 3, the method had good precision, and could meet the determination requirements of the standard method.

[0141] Table 4 Reproducibility test results of samples

[0142]

[0143]

[0144] 6.5 Stability of standard working solution:

[0145] Take 0.05, 1.0, 10, 100, 1000 mg / L of pyrroloquinoline quinone disodium salt standard working solution, standard intermediate solution and standard stock solution, respectively, at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 3 days, 30 days, 60 days, 90 days, HPLC analysis, measure its pyrroloquinoline quinone disodium salt concentration, calculate its relative standard deviation (RSD). As shown in Table 5, the stability of pyrroloquinoline quinone disodium salt standard working solution, standard intermediate solution and standard stock solution is good.

[0146] Table 5 Stability test results of solutions with different pyrroloquinoline quinone disodium salt concentrations

[0147]

[0148] 6.6 Stability:

[0149] Take the vitamin functional beverage (special purpose beverage), electrolyte beverage (special purpose beverage), protein beverage, solid beverage, energy flavor beverage (flavor beverage), fruit flavor beverage (flavor beverage), coffee beverage, fruit juice beverage, carbonated beverage, plant beverage, tea beverage blank matrix sample, add pyrroloquinoline quinone disodium salt standard intermediate solution, the addition level is 40 mg / kg, add 10 g / L disodium ethylenediaminetetraacetate solution, the addition level is 0.4 g / L, and the lactic acid bacteria solid beverage sample marked with the addition of pyrroloquinoline quinone disodium salt, respectively, at 0, 2, 4, 6, 8, 10, 12, 24 h, HPLC analysis, measure its pyrroloquinoline quinone disodium salt content, calculate its relative standard deviation (RSD), the results are shown in Table 6. As shown in Table 6, when the concentration of EDTA in the sample solution is 0.4 g / L, the stability of all types of spiked beverage matrix is good, which can meet the test requirements.

[0150] In summary, the beverage matrix studied in the present application covers special purpose beverage, protein beverage, solid beverage, tea beverage, flavor beverage, fruit juice beverage, carbonated beverage, plant beverage, coffee beverage and other beverage matrices, which can completely meet the requirements of the existing laws and regulations of our country and the scope and limitation requirements of the "Announcement on 32 kinds of 'three new foods' such as Kanzan cherry" for beverage matrix. The method can effectively reduce the interference of food matrix components, while effectively ensuring the stability of the target pyrroloquinoline quinone disodium salt during the determination process, has high sensitivity, good reproducibility, simple and rapid operation, and is suitable for daily large-scale rapid detection and supervision requirements of pyrroloquinoline quinone disodium salt as a new food material in beverages.

[0151] Table 6 Stability test results of samples

[0152]

[0153]

[0154] Example 3 was explored according to the test steps and conditions provided in Example 1 and Example 2, specifically including the following:

[0155] 6.7 Specificity experiment

[0156] (1) The separation of the target pyrroloquinoline quinone disodium salt and 13 kinds of food additives commonly used in beverages under the chromatographic conditions of the present study was investigated, and the results are shown in Figure 3 (A and B). The results show that within 10 minutes of running time, the target pyrroloquinoline quinone disodium salt can be fully separated from the 13 kinds of food additives commonly used in beverages, indicating that the method provided by the present application has good specificity for the determination of pyrroloquinoline quinone disodium salt in beverages, and is suitable for accurate determination of the content of pyrroloquinoline quinone disodium salt in beverages under complex matrix conditions.

[0157] (2) The separation of the target pyrroloquinoline quinone disodium salt and coenzyme Q10, nicotinamide mononucleotide under the chromatographic conditions of the present study was investigated, and the results are shown in Figure 4 (A, B and C).

[0158] The results show that under the chromatographic conditions of the present study, the coenzyme Q10 standard working solution does not peak ( Figure 4B ), the nicotinamide mononucleotide standard chromatographic peak is eluted out within 2 min ( Figure 4C ), the target pyrroloquinoline quinone disodium salt can be fully separated from nicotinamide mononucleotide without being affected by coenzyme Q10, indicating that the method has good specificity for the determination of pyrroloquinoline quinone disodium salt in beverages, and is suitable for accurate determination of the content of pyrroloquinoline quinone disodium salt in beverages under complex matrix conditions.

[0159] 6.8 Visual comparison of purification effect of sample purification system

[0160] The actual purification effect of protein beverage, solid beverage and coffee beverage samples was compared, and the results are shown in Figure 5 , Figure 6 and Figure 7 . As can be seen from Figures 5-7 , the protein beverage, solid beverage and coffee beverage samples under the purification system of the ultrafiltration purification column, the 20% acetonitrile filter purification tube, the ultrafiltration purification column, the 20% acetonitrile purification system and the 50% acetonitrile purification system have not been significantly improved compared to before purification.

[0161] While the protein beverage, solid beverage and coffee beverage samples purified by the ultrafiltration centrifuge tube purification system have significantly removed impurities such as pigments, proteins and polypeptides, and the sample solution is clear and transparent, which can be directly tested on the machine, indicating that the ultrafiltration centrifuge tube purification system can meet the purification requirements of the present application.

[0162] 6.9 Optimization of the sample stability system

[0163] The vitamin functional beverage, electrolyte beverage, protein beverage, solid beverage, energy flavor beverage, fruit flavor beverage, coffee beverage, fruit juice beverage, carbonated beverage, plant beverage, tea beverage blank matrix sample was added with the pyrroloquinoline quinone disodium salt standard intermediate solution, the addition level was 40 mg / kg, and the lactic acid bacteria solid beverage sample with added pyrroloquinoline quinoline disodium salt was added with the pyrroloquinoline quinone disodium salt standard intermediate solution, and HPLC analysis was performed at 0, 2, 4, 6, 8, 10, 12, 24 h, the content of pyrroloquinoline quinone disodium salt was measured, and the relative standard deviation (RSD) was calculated, and the results are shown in Table 7. As can be seen from Table 7, the content of the target peak in the vitamin functional beverage (special purpose beverage), electrolyte beverage (special purpose beverage), protein beverage, solid beverage, energy flavor beverage (flavor beverage), fruit flavor beverage (flavor beverage), and coffee beverage added sample changes within 24 h, and the RSDs range from 1.3% to 3.1%, and the stability is good, while in the fruit juice beverage, tea beverage, carbonated beverage, plant beverage added sample, the target peak pyrroloquinoline quinone disodium salt decays after 6 hours, which is caused by the problem of the matrix, and the RSD is large, but the experiment is repeated twice, and the pyrroloquinoline quinone disodium salt standard working solution is in an extremely stable state within 24 h, and the reproducibility is good.

[0164] Table 7 Sample stability experiment results

[0165]

[0166]

[0167] From Figure 8 It can be seen that the recovery rate of the target pyrroloquinoline quinone disodium salt in the fruit juice beverage, tea beverage, carbonated beverage, plant beverage added sample presents a significant downward trend with time, and the target peak pyrroloquinoline quinoline disodium salt begins to decay from about 6 hours. Current studies have shown that PQQ has very high reactivity, is easy to react with substances containing amino groups and divalent metal ions, and is converted into imidazo pyrroloquinoline, so in some beverage matrices, there are substances that can react with pyrroloquinoline quinone disodium salt, which causes the pyrroloquinoline quinone disodium salt in some matrices to be in an unstable state.

[0168] In order to solve the instability of pyrroloquinoline quinone disodium salt in sample matrixes such as juice beverage, tea beverage, carbonated beverage, plant beverage and the like, we attempt to add water-soluble antioxidants to ensure the stability of the target substance in the above sample matrixes. Disodium ethylenediaminetetraacetate and L(+)-ascorbic acid are common water-soluble antioxidants. We first investigate the separation of disodium ethylenediaminetetraacetate, L(+)-ascorbic acid and the target peak pyrroloquinoline quinone disodium salt under the chromatographic conditions of the present application, and the results are shown in Fig. 9. The results show that disodium ethylenediaminetetraacetate does not peak under the chromatographic conditions of the present application ( Figure 9A ), L(+)-ascorbic acid is eluted out by the mobile phase within 2 min ( Figure 9B ), the target substance pyrroloquinoline quinone disodium salt can be fully separated from L(+)-ascorbic acid and is not affected by disodium ethylenediaminetetraacetate ( Figure 9C ).

[0169] In addition, we further investigate the concentration change of the target substance pyrroloquinoline quinone disodium salt before and after adding disodium ethylenediaminetetraacetate and L(+)-ascorbic acid under the chromatographic conditions of the present application, and the results are shown in Fig. 10. The results show that the addition of L(+)-ascorbic acid causes the concentration of the target substance pyrroloquinoline quinone disodium salt to decrease significantly ( Figure 10B ), and the content is only 21.6% of that without the addition ( Figure 10C ). However, the concentration of the target substance pyrroloquinoline quinone disodium salt does not change when the concentration of disodium ethylenediaminetetraacetate in the standard sample is 0.4 g / L ( Figure 10A ). Therefore, the concentration of disodium ethylenediaminetetraacetate in the sample solution is selected to be 0.4 g / L to further investigate the stability of the target substance pyrroloquinoline quinoline disodium salt in nine types of beverage matrixes within 24 h. The results show that when the concentration of disodium ethylenediaminetetraacetate in the sample solution is 0.4 g / L, the stability of all types of spiked beverage matrixes is good, which can meet the test requirements.

[0170] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details of the above embodiments. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

[0171] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present application will not further describe various possible combinations.

[0172] In addition, various different embodiments of the present application can also be combined in any manner, as long as they do not deviate from the idea of the present application, and they should also be considered as disclosed by the present application.

Claims

1. A method for detecting the content of a new food material in a food product, characterized by: The food is a beverage, and the new food raw material is pyrroloquinoline quinone disodium salt; wherein the detection method specifically comprises the following steps: (1) preparing a standard working solution, pyrroloquinoline quinone disodium salt is prepared into a standard working solution with a concentration of 0.020 ~10 mg / L by using a diluent; the diluent is a phosphate diluent formed by mixing a twelve-hydrated disodium hydrogen phosphate aqueous solution and a dihydrated sodium dihydrogen phosphate aqueous solution; (2) preparing a sample solution to be detected: weighing the sample to be detected in a container, adding the diluent and an ethylenediaminetetraacetic acid disodium aqueous solution, ultrasonic treatment, and then using the diluent to make up the volume, and then performing ultrafiltration centrifugal treatment to obtain the sample solution to be detected, and then performing detection; (3) using a high performance liquid chromatograph for detection and analysis: the detector is a photodiode array detector; the chromatographic column is an RD-C18 column, and the column temperature is 35℃; the mobile phase A is a mobile phase salt solution, which is prepared by weighing 6.45 g of tetrabutylammonium bromide and 2.72 g of dihydrogen potassium phosphate dihydrate into 1000 mL of water; the mobile phase B is acetonitrile; the flow rate is 1 mL / min; the injection volume is 20 μL; the gradient elution program is: 0 ~ 3.5 min, 60% A; 3.5 ~ 3.6 min, 60% A ~ 20% A; 3.6 ~ 6.0 min, 20% A; 6.0 ~ 6.1 min, 20% A ~ 60% A; 6.1 ~ 10.0 min, 60% A; (4) drawing a standard curve: injecting different concentrations of the standard working solution into the high performance liquid chromatograph, performing gradient elution and detection under the chromatographic conditions of step (3), and qualitatively determining the retention time, and drawing a standard curve according to the corresponding relationship between the size of the peak area and the concentration, and using the standard curve to quantitatively determine the target substance in the sample solution to be detected; (5) result analysis: injecting the sample solution to be detected in step (2) into the high performance liquid chromatograph, performing gradient elution and detection under the chromatographic conditions of step (3), measuring the peak area of the target substance in the sample solution to be detected, qualitatively determining the retention time, quantitatively determining according to the standard curve prepared in (4), and calculating the content of pyrroloquinoline quinone disodium salt in the sample to be detected.

2. The method for detecting the content of new food raw materials in food according to claim 1, characterized in that: The beverage includes at least one of a special use beverage, a protein beverage, a solid beverage, a flavor beverage, a tea beverage, a fruit juice beverage, a carbonated beverage, a plant beverage, or a coffee beverage.

3. The method for detecting the content of new food raw materials in food according to claim 1, characterized in that: In step (1), the standard working solution is obtained by gradually diluting a standard stock solution and a standard intermediate solution.

4. The method for detecting the content of new food materials in food according to claim 3, characterized in that: The standard stock solution is prepared by weighing pyrroloquinoline quinone disodium salt standard with a purity of 100% into a volumetric flask, dissolving with the diluent, and adding the required volume and 10 g / L ethylenediaminetetraacetic acid disodium aqueous solution according to a volume ratio of 25:1 to prepare a standard stock solution with a concentration of 1000 mg / L.

5. The method for detecting the content of new food materials in food according to claim 3 or 4, characterized in that: The preparation of the standard intermediate solution: pipette the standard stock solution, dilute with the diluent, add the volume of the prepared solution and the aqueous solution of disodium ethylenediaminetetraacetate in a volume ratio of 25:1 to the aqueous solution of 10 g / L disodium ethylenediaminetetraacetate to obtain the standard intermediate solution with a concentration of 100 mg / L.

6. The method for detecting the content of new food materials in food according to claim 1 or 4, characterized in that, The specific steps for preparing the standard working solution are as follows: pipette 0.020-10 mL of the standard intermediate solution into a volumetric flask, add the volume of the prepared solution and the aqueous solution of disodium ethylenediaminetetraacetate in a volume ratio of 25:1 to the aqueous solution of 10 g / L disodium ethylenediaminetetraacetate, and then dilute to the mark with the diluent, mix well, and prepare a series of standard working solutions with a mass concentration of 0.020-10 mg / L.

7. The method for detecting the content of new food materials in food according to claim 1, characterized in that: In step (2), the specific steps for preparing the sample solution are as follows: weigh 2.0±0.001 g of the sample to be tested into a stoppered colorimetric tube, add the diluent with a volume ratio of 60% and the aqueous solution of 10 g / L disodium ethylenediaminetetraacetate with a volume ratio of 4%, ultrasonically extract for 5-15 min, dilute with the diluent and dilute to the mark, mix well, obtain the sample solution, and take 0.8-2.0 mL of the sample solution into an ultrafiltration centrifuge tube, centrifuge at 4000-5000 r / min for 2-10 min to obtain the sample solution for liquid chromatography.

8. The method for detecting the content of new food raw materials in food according to claim 7, characterized in that: When the sample to be tested is a solid beverage, the solid beverage needs to be diluted according to the dilution ratio indicated on the product label before the sample solution is prepared.

9. The method for detecting the content of new food ingredients in food according to claim 1, characterized in that, In step (3), the RD-C18 chromatographic column has a specification of 4.6 mm×150 mm, and the particle size of the chromatographic column filler is 5 μm.

10. The method for detecting the content of new food raw materials in food according to claim 9, characterized in that: The detection wavelength of the photodiode array detector is 190-800 nm.

11. The method for detecting the content of new food raw materials in food according to claim 10, characterized in that: The detection wavelength of the photodiode array detector is 250 nm.

12. The method according to claim 1, wherein the food material is a food additive. In step (4), the standard curve coefficient is not less than 0.9999; and the quantitative detection limit of the method is 0.5 mg / kg. ​ 13. The method for detecting the content of new food ingredients in food according to claim 1, characterized in that, The RSD of the sample stability determination within 0-24 h is less than 3.3%; the sample precision determination RSDs are less than 1.5%; the recovery rate of the detection method is in the range of 85.0% to 109.4%, and the RSDs are in the range of 0.3% to 4.9%.

Images