Method for detecting residual hydrogen peroxide in food by SPE-HPLC

Abstract

The present application relates to the field of food physics and chemistry detection technology, and in particular to a method for detecting residual hydrogen peroxide in food by SPE-HPLC. The present application uses phosphoric acid acetonitrile to extract the sample, adds sodium chloride after weighing, stirs the sample uniformly, extracts again, greatly enhances the dispersibility of the sample in the extraction liquid, significantly improves the removal capacity of the small column for polar impurities, greatly reduces the interference of pigment, oil and polysaccharide matrix, and makes the baseline more stable; effectively eliminates the impurity peak and baseline drift, and is more accurate in quantification. A mixed strong cation exchange reversed-phase solid-phase extraction column is used to remove the activation step, 3 mL of the to-be-purified liquid is directly passed through the column, the first 2 mL is discarded, and the third mL is collected; the operation process is simplified, the pretreatment time is shortened; the operation is simple and suitable for large-scale sample detection.

Description

Technical Field

[0001] This invention relates to the field of food physicochemical testing technology, specifically a method for determining the residual amount of hydrogen peroxide in various foods based on solid-phase extraction pretreatment followed by high-performance liquid chromatography. Background Technology

[0002] Existing national standards and routine literature employ HPLC detection methods involving pure water ultrasonic extraction, traditional HLB solid-phase extraction, and triphenylphosphine derivatization at room temperature for extended periods in the dark. These methods have the following significant drawbacks:

[0003] Conventional food matrices contain a large amount of pigments, polysaccharides, oils, and organic acids. Ordinary HLB purification has limited ability to remove interference, resulting in high baseline noise and numerous impurity peaks, which can easily lead to false positives and inaccurate quantification.

[0004] Traditional derivatization systems require a reaction time of more than 30 minutes in the dark, which is time-consuming and cumbersome, making them unsuitable for large-scale sample detection.

[0005] Hydrogen peroxide is extremely unstable and decomposes easily after prolonged pretreatment, resulting in low recovery rate and poor repeatability.

[0006] Conventional mobile phase systems have long elution times, high solvent consumption, and low detection efficiency.

[0007] Therefore, existing technologies generally suffer from problems such as strong matrix interference, time-consuming detection, poor stability, and high detection limits. There is an urgent need for an improved detection method with better purification effect, better stability, and higher accuracy. Summary of the Invention

[0008] To address the problems of strong matrix interference, time-consuming detection, poor stability, and high detection limits in the existing technologies, this invention provides a method for detecting hydrogen peroxide residues in food using SPE-HPLC.

[0009] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0010] A method for detecting hydrogen peroxide residue in food using SPE-HPLC includes the following steps:

[0011] (1) Take the sample to be tested, add sodium chloride and stir evenly, then add acetonitrile phosphate, vortex and centrifuge to obtain the supernatant;

[0012] (2) A mixed-type strong cation exchange reversed-phase solid-phase extraction column was used to purify the supernatant by solid-phase extraction to obtain a purified solution;

[0013] (3) The purified liquid was analyzed by high performance liquid chromatography using an acetonitrile-water mobile phase system, and the residual hydrogen peroxide content was quantified by external standard method.

[0014] Furthermore, in step (1), the mass ratio of the sample to be tested to sodium chloride is 1:4.

[0015] Further, in step (1), the ratio of the amount of the sample to be tested to acetonitrile phosphate is: 15~25mL of acetonitrile phosphate is added per gram of the sample to be tested.

[0016] Furthermore, in step (1), the acetonitrile phosphate is 0.5% acetonitrile phosphate, that is, an acetonitrile solution with a volume fraction of 0.5% phosphoric acid.

[0017] Furthermore, the centrifugation operation in step (1) is: centrifugation at 8000 rpm for 5 min.

[0018] Furthermore, the mixed-type strong cation exchange reversed-phase solid-phase extraction column in step (2) is: ProElut PXC 30 mg / 1 mL.

[0019] Further, in step (2), 2 mL of the liquid to be purified is added to a mixed strong cation exchange reversed-phase solid-phase extraction column, the effluent is discarded, 1 mL of the liquid to be purified is added, the effluent is collected, vortexed and mixed, and filtered through a 0.45 μm NY filter membrane.

[0020] Furthermore, the high-performance liquid chromatography column used in step (3) is a polar modified C18 reversed-phase column, and the flow rate gradient is set as follows: 0~8 min, flow rate 0.4 mL / min; 8~8.1 min, flow rate 0.4~1.0 mL / min; 8.1~23 min, flow rate 1.0 mL / min; 23~23.1 min, flow rate 1.0~0.4 mL / min; 23.1~25 min, flow rate 0.4 mL / min.

[0021] Furthermore, in step (3), the mobile phase acetonitrile:water = 5:95; v / v.

[0022] Furthermore, in step (3) of the external standard method, the solvent used for the hydrogen peroxide standard solution is 0.5% acetonitrile phosphate, and the spiking volume is no more than 1 mL.

[0023] The beneficial effects of this invention are:

[0024] 1. Using acetonitrile phosphate to extract samples, adding sodium chloride after weighing the sample, stirring it evenly before extraction, can greatly enhance the dispersibility of the sample in the extract, significantly improve the column's ability to remove polar impurities, greatly reduce interference from pigments, oils, and polysaccharide matrices, and make the baseline more stable; effectively eliminate impurity peaks and baseline drift, and make quantification more accurate.

[0025] 2. A mixed-type strong cation exchange reversed-phase solid-phase extraction column is used, eliminating the activation step. Take 3 mL of the solution to be purified and pass it directly through the column. Discard the first 2 mL and collect the 3 mL. This simplifies the operation process and shortens the pretreatment time. The operation is simple and suitable for large-scale sample detection.

[0026] 3. Select 0.5% acetonitrile phosphate as the solvent for preparing the standard intermediate solution and the sample extraction solvent. Hydrogen peroxide is more stable under these conditions. Perform a spiked recovery experiment by adding a certain concentration of hydrogen peroxide to the sample. The complex environment of some samples may cause the stability of hydrogen peroxide to deteriorate. Control the spiked volume to about 1 mL to ensure that the added hydrogen peroxide can exist stably in the sample and avoid loss of spiked recovery results.

[0027] 4. Liquid chromatography conditions: Using a constant flow rate to detect samples, the peak stability of impurities in the sample was poor. Changing the flow rate gradient setting effectively improved the stability of the sample impurity peaks. Employing a low-volume, high-elution-efficiency acetonitrile gradient elution, compared to traditional 5 mL large-volume elution, reduced solvent consumption by 60%, increased the target analyte recovery rate to 94.9%, and significantly improved stability.

[0028] 5. Improved acetonitrile-water mobile phase ratio and elution procedure, suitable for large-scale food screening; less organic solvent is used, green and low-consumption, resulting in lower detection costs. Attached Figure Description

[0029] Figure 1 Hydrogen peroxide standard curve;

[0030] Figure 2 Chromatogram of hydrogen peroxide standard solution (0.5 mg / mL);

[0031] Figure 3 Chromatogram of hydrogen peroxide standard solution (0.05 mg / mL);

[0032] Figure 4 Chromatogram of hydrogen peroxide standard solution (0.005 mg / mL);

[0033] Figure 5 Chromatogram of hydrogen peroxide detection in chicken feet with a concentration of 10 g / kg;

[0034] Figure 6 Chromatogram of hydrogen peroxide detection in chicken feet with a concentration of 1.0 g / kg;

[0035] Figure 7 Chromatogram of hydrogen peroxide detection in chicken feet with an added concentration of 0.1 g / kg;

[0036] Figure 8 Chromatogram of hydrogen peroxide (blank) detection in chicken feet;

[0037] Figure 9 Chromatogram of hydrogen peroxide detection in shrimp with an added concentration of 10 g / kg;

[0038] Figure 10 Chromatogram of hydrogen peroxide detection in shrimp with a concentration of 1.0 g / kg;

[0039] Figure 11 Chromatogram of hydrogen peroxide detection in shrimp with an added concentration of 0.1 g / kg;

[0040] Figure 12 Chromatogram of hydrogen peroxide (blank) detection in shrimp;

[0041] Figure 13 Chromatogram of hydrogen peroxide detection in tripe at a concentration of 10 g / kg;

[0042] Figure 14 Chromatogram of hydrogen peroxide detection in tripe at a concentration of 1.0 g / kg;

[0043] Figure 15 Chromatogram of hydrogen peroxide detection in tripe at a concentration of 0.1 g / kg;

[0044] Figure 16 Chromatogram of hydrogen peroxide (blank) detection in beef tripe. Detailed Implementation

[0045] The present invention will be further described below with reference to specific embodiments.

[0046] A method for detecting hydrogen peroxide residue in food using SPE-HPLC includes the following steps:

[0047] (1) Take the sample to be tested, add sodium chloride and stir evenly. The mass ratio of the sample to sodium chloride is 1:4. Then add 0.5% acetonitrile phosphate, i.e. acetonitrile solution with a volume fraction of 0.5% phosphoric acid. The volume ratio of the sample to acetonitrile phosphate is: 15~25mL acetonitrile phosphate per gram of sample to be tested. Vortex for 5 min, centrifuge at 8000 rpm for 5 min, and obtain the supernatant.

[0048] (2) The supernatant was purified by solid-phase extraction using a mixed strong cation exchange reversed-phase solid-phase extraction column ProElut PXC 30 mg / 1 mL. 2 mL of the solution to be purified was added to the mixed strong cation exchange reversed-phase solid-phase extraction column, the effluent was discarded, and 1 mL of the solution to be purified was added. The effluent was collected, vortexed and mixed, and filtered through a 0.45 μm NY filter membrane to obtain the purified solution.

[0049] (3) The purified solution was analyzed by high performance liquid chromatography using an acetonitrile-water mobile phase system, and the residual hydrogen peroxide content was quantified by external standard method. The high performance liquid chromatography column used was a polar modified C18 reversed phase column, and the mobile phase was acetonitrile:water = 5:95, v / v. The flow rate gradient was set as follows: 0~8min, flow rate 0.4mL / min; 8~8.1min, flow rate 0.4~1.0mL / min; 8.1~23min, flow rate 1.0mL / min; 23~23.1min, flow rate 1.0~0.4mL / min; 23.1~25min, flow rate 0.4mL / min. The solvent used for the hydrogen peroxide standard solution in the external standard method was 0.5% acetonitrile phosphate, and the spiked volume was no more than 1 mL.

[0050] This method is applicable to the determination of hydrogen peroxide residue in samples such as chicken feet, shrimp, and beef tripe, with a limit of quantitation of 0.1 g / kg. The specific detection process is as follows.

[0051] I. Preparation of Experimental Instruments and Reagents

[0052] (I) Main Instruments

[0053] 1. High-performance liquid chromatograph: equipped with a diode array detector (PDA detector);

[0054] 2. Analytical balance: accuracy 0.0001 g;

[0055] 3. High-speed refrigerated centrifuge: maximum speed ≥10000 rpm;

[0056] 4. Multi-channel vortex oscillator;

[0057] 5. Solid-phase extraction column equipped with a vacuum negative pressure device;

[0058] 6.50 mL polypropylene centrifuge tubes, 10 mL volumetric flasks, pipettes (0.01–5 mL);

[0059] 7.0.45 μm Nylon (NY) organic phase filter tip, disposable syringe;

[0060] 8. ProElut PXC hybrid strong cation exchange reversed-phase solid-phase extraction column (30 mg / 1 mL, catalog number 68202).

[0061] 9. Liquid chromatography column: Spursil 5μm C18-EP polar modified reversed-phase column, 250 mm × 4.6 mm.

[0062] (II) Reagent Preparation

[0063] 1.0.5% Phosphate Acetonitrile Extract: Accurately measure 5 mL of chromatographic grade phosphoric acid using a graduated cylinder, slowly add it to 1000 mL of chromatographic grade acetonitrile, shake well, store in a sealed container at room temperature away from light, and prepare and use immediately. Avoid strong light exposure throughout the preparation process to prevent phosphoric acid oxidation and acetonitrile volatilization.

[0064] 2. Hydrogen peroxide stock solution (30 mg / mL): Purchase commercially available 3% (W / V) hydrogen peroxide standard solution directly, store in the dark at 4°C, and keep it on the shelf for 7 days after opening. Shake well before each use and handle in the dark throughout the process to reduce the decomposition and loss of hydrogen peroxide.

[0065] 3. Preparation of a series of standard intermediate solutions: (1) 10 mg / mL standard intermediate solution: Accurately transfer 4 mL of 30 mg / mL hydrogen peroxide stock solution, add 8 mL of 0.5% acetonitrile phosphate, vortex mix, and store in the dark for later use; (2) 1.0 mg / mL standard intermediate solution: Accurately transfer 0.4 mL of 30 mg / mL stock solution, add 11.6 mL of 0.5% acetonitrile phosphate, and mix; (3) 0.1 mg / mL standard intermediate solution: Accurately transfer 0.04 mL of 30 mg / mL stock solution, add 11.96 mL of 0.5% acetonitrile phosphate, and mix; After all intermediate solutions are prepared, store in the dark and use within 24 hours to avoid long-term storage that could lead to degradation of the target substance.

[0066] II. Sample Extraction Steps

[0067] This extraction method is suitable for three typical highly interfering food matrices: chicken feet, shrimp, and beef tripe. The entire operation is performed in the dark, with a short settling time to inhibit hydrogen peroxide decomposition. The steps are as follows:

[0068] 1. Sample homogenization and weighing: Take fresh edible parts to be tested, remove bones, shells, fascia and other non-edible parts, and use a high-speed homogenizer to fully pulverize into a fine homogenate; accurately weigh 1.00 g of sample homogenate and transfer it to a 50 mL polypropylene centrifuge tube.

[0069] 2. Salting-out dispersion pretreatment: Add 4.00 g of sodium chloride (sample to NaCl mass ratio 1:4) to the centrifuge tube and stir thoroughly with a clean glass rod for 3–4 min until the sodium chloride and sample matrix are completely mixed. Sodium chloride can destroy the structure of hydrated proteins and polysaccharides in the sample, reduce the solubility of pigments and oils, and at the same time improve the stability of hydrogen peroxide in organic extracts.

[0070] 3. Organic phase extraction: Add 20 mL of 0.5% acetonitrile phosphate to the mixed system (15~25 mL per gram of sample is applicable; for batch testing, 20 mL is used for simplified operation), tighten the centrifuge tube cap, and place it on a vortex mixer to shake at high speed for 5 min to ensure that the sample and the extract are in full contact and to fully dissolve the hydrogen peroxide residue in the sample.

[0071] 4. Centrifugation to obtain supernatant: After shaking, place the centrifuge tube in a high-speed centrifuge, set the speed to 8000 rpm, and centrifuge for 5 min. After centrifugation, let stand for 1 min to separate the layers. The clear organic phase on the upper layer is the extraction supernatant. Carefully transfer 3 mL of the supernatant to a clean centrifuge tube for solid-phase extraction purification. During the transfer process, avoid aspirating the lower layer of protein and lipid precipitates to prevent the introduction of impurities that may interfere with subsequent detection.

[0072] III. ProElut PXC Solid Phase Extraction Purification

[0073] This invention eliminates the traditional solid-phase extraction column activation and rinsing steps, significantly shortening the pretreatment time, making it suitable for large-scale sample screening. No organic solvent pre-rinsing is required throughout the process. Operation steps:

[0074] 1. Assemble the solid-phase extraction negative pressure device, fix the ProElut PXC 30 mg / 1 mL column to the support, and place the collection tube below;

[0075] 2. First loading: Slowly add 2 mL of the supernatant to be purified into the column, turn on the negative pressure, and discard all the eluent after it has completely flowed out; this step removes most of the macromolecular pigments, organic acids, and positively charged protein impurities from the sample.

[0076] 3. Second elution and collection: Add the remaining 1 mL of supernatant to be purified to the same PXC column, slowly pass the column under negative pressure, and collect the elution after all the liquid has flowed out.

[0077] 4. Pretreatment before membrane filtration: Vortex the collected purified solution for 30 seconds, draw up the solution with a disposable syringe, filter it through a 0.45 μm nylon organic filter, transfer the filtrate to a liquid chromatography vial, and immediately analyze it on the instrument; if it cannot be analyzed immediately, it should be sealed and stored in the dark for no more than 2 hours to prevent hydrogen peroxide degradation and low recovery rate.

[0078] IV. Detection Conditions and Gradient Programs for High Performance Liquid Chromatography

[0079] (a) Basic constant chromatographic parameters

[0080] Chromatographic column: Spursil 5μm C18-EP, 250×4.6 mm polar modified C18 column;

[0081] Mobile phase ratio: Acetonitrile: Ultrapure water = 5:95 (V / V), the mobile phase is used after ultrasonic degassing for 20 min;

[0082] Detection wavelength: 220 nm for PDA detector;

[0083] Column oven temperature: constant at 30 ℃;

[0084] Injection volume: 20 μL;

[0085] Total testing time: 25 min / single needle sample.

[0086] (ii) Segmented velocity gradient program

[0087] The flow velocity gradient settings are shown in Table 1.

[0088] Table 1 Gradient elution program

[0089]

[0090] Gradient Function Description

[0091] 0~8min: Slowly elute weakly polar impurities at low flow rates, separate matrix impurity peaks, and avoid overlap between impurities and target peaks.

[0092] 8~8.1 min: Uniformly increase to 1.0 flow rate and switch briefly to quickly wash away moderately polar interfering substances.

[0093] 8.1~23 min: High flow rate for rapid rinsing of the column to strongly adsorb oil and pigment residues, shortening the overall detection cycle and reducing solvent consumption.

[0094] 23~23.1 min: The flow rate is gradually reduced to 0.4 mmol / L to balance the column pressure and prevent drastic baseline fluctuations.

[0095] 23.1~25 min; Low flow rate to equilibrate the column, ensuring stable peak shape for the next sample injection.

[0096] (III) On-machine testing

[0097] 1. After powering on, equilibrate the column with the mobile phase at 0.4 mL / min for 30 min. Inject the sample only after the baseline has stabilized and there is no drift.

[0098] 2. After confirming the absence of interfering peaks with impurities using the initial blank solvent (0.5% acetonitrile phosphate), proceed sequentially with the standard working solution, sample purification solution, and matrix-spikened sample.

[0099] 3. Insert one intermediate concentration standard solution for every 10 samples tested to calibrate, correct chromatographic response drift, and ensure quantitative accuracy.

[0100] V. Preparation of Standard Series Working Solutions and Plotting of Standard Curves

[0101] 1. Preparation of standard series: Accurately transfer 0.025 mL, 0.05 mL, 0.5 mL, 1.0 mL, 2.5 mL, and 5.0 mL of 1.0 mg / mL hydrogen peroxide standard intermediate solution into six 10 mL volumetric flasks, respectively. Dilute to the mark with 0.5% acetonitrile phosphate, and shake well to obtain a gradient of standard working solution concentrations: 0.0025 mg / mL, 0.005 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.25 mg / mL, and 0.5 mg / mL.

[0102] 2. Curve acquisition: Six gradient standard solutions were injected sequentially into the liquid chromatograph, and the peak area of ​​hydrogen peroxide characteristic peaks corresponding to each concentration was recorded. With the standard solution concentration as the x-axis and the chromatographic peak area response value as the y-axis, the instrument automatically fitted a linear standard curve. The linear correlation coefficient R² ≥ 0.999 is required to meet the quantitative requirements.

[0103] 3. Chromatograms of standard hydrogen peroxide solutions at different concentrations are shown below. Figures 2-4 As shown, the standard curve is as follows Figure 1 As shown, the peaks are symmetrical, without tails or extraneous peaks, and the baseline is stable.

[0104] VI. Matrix Spike Recovery Test

[0105] Three types of highly interfering food blank matrices—chicken feet, shrimp, and beef tripe—were selected. Three spiking levels were set: 0.1 g / kg, 1.0 g / kg, and 10 g / kg, respectively. The spiking volume was strictly controlled to ≤1 mL to ensure the stability of hydrogen peroxide.

[0106] 1. Blank matrix pretreatment: Blank samples were pre-tested to confirm the background hydrogen peroxide content (chicken feet blank 0.0075 g / kg, shrimp blank 0.0065 g / kg, beef tripe blank 0.0087 g / kg). Blank chromatograms are shown in Figures 8, 12, and 16, with no interference from the characteristic peaks of the target analyte.

[0107] 2. Three-gradient spiking operations: (1) 10 g / kg high concentration spiking: Weigh 1 g blank matrix, add 1 mL of 10 mg / mL standard intermediate solution, stir and mix well, let stand for 10 min, then add 4 g sodium chloride, and process according to the extraction and purification process of this invention; (2) 1.0 g / kg medium concentration spiking: Weigh 1 g blank matrix, add 1 mL of 1.0 mg / mL standard intermediate solution, and the rest of the operation is the same as above; (3) 0.1 g / kg low concentration spiking (method limit of quantitation): Weigh 1 g blank matrix, add 1 mL of 0.1 mg / mL standard intermediate solution, and the rest of the operation is the same as above; All spiking operations are carried out in the dark, and the spiking volume is uniformly controlled at 1 mL to avoid large volume aqueous solution from damaging the acidic stability of the acetonitrile phosphate system and reduce the loss of hydrogen peroxide decomposition.

[0108] 3. Chromatograms of hydrogen peroxide recovery from different concentrations in chicken feet, shrimp, tripe, etc. are shown below. Figures 5-16 As shown in Table 2, the recovery results are as follows.

[0109] Table 2 Recovery rate of hydrogen peroxide in chicken feet, shrimp, tripe, etc.

[0110]

[0111] Recovery rate statistics: Samples spiked at three levels for each matrix were analyzed using the instrument. The measured content was calculated based on the standard curve, and the spiked recovery rate was calculated after subtracting the blank background.

[0112] Chicken feet: 86.3%~93.8%;

[0113] Shrimp: 81.2%~93.2%;

[0114] Beef tripe: 90.1%~94.9%; The low, medium and high concentration recoveries of the three types of matrices all meet the requirements of the food physicochemical testing standards. The limit of quantitation for low concentration is 0.1 g / kg, which can meet the screening requirements for hydrogen peroxide residue limits in food. The chromatograms of the spiked samples are shown in Figures 5~7, 9~11, and 13~15. The target peaks are well separated and there are no false positive peaks.

[0115] Hydrogen peroxide is relatively stable under acidic conditions, so 0.5% acetonitrile phosphate was chosen as the solvent for preparing the standard intermediate solution and for sample extraction. In the spiked recovery experiment, a certain concentration of hydrogen peroxide was added to the sample. However, the complex environment of some samples can degrade the stability of hydrogen peroxide; therefore, the spiked volume needs to be controlled to approximately 1 mL to ensure that the added hydrogen peroxide remains relatively stable in the sample and to avoid loss of spiked recovery results.

[0116] The sample was extracted using 0.5% acetonitrile phosphate. The sample was poorly dispersed in the extract, so sodium chloride was added after weighing the sample and stirred until homogeneous before extraction.

[0117] Using a 30 mg / 1 mL PXC column, skip the activation step, directly pass 3 mL of the solution to be purified through the column, discard the first 2 mL, and collect the 3 mL. This significantly improves detection efficiency, avoids the decomposition of hydrogen peroxide over long periods, and results in higher recovery and better stability. It simplifies the operation process, shortens pretreatment time, and is easy to use, making it suitable for large-scale sample detection.

[0118] Under liquid chromatography conditions, using a constant flow rate to detect samples reveals poor peak stability of impurities. Changing the flow rate gradient significantly reduces interference from complex food matrices, improving the stability of impurity peaks, effectively eliminating unwanted peaks and baseline drift, resulting in more accurate quantification. Employing a low-volume, high-efficiency acetonitrile gradient elution method reduces solvent usage by 60% compared to traditional 5 mL bulk elution, while increasing the target analyte recovery to 94.9% and demonstrating significant stability.

[0119] The improved acetonitrile-water mobile phase ratio and elution procedure are suitable for large-scale food screening; less organic solvent is used, making it green and low-cost, and the detection cost is lower.

Claims

1. A method for detecting hydrogen peroxide residue in food using SPE-HPLC, characterized in that, Includes the following steps: (1) Take the sample to be tested, add sodium chloride and stir evenly, then add acetonitrile phosphate, vortex and centrifuge to obtain the supernatant; (2) A mixed-type strong cation exchange reversed-phase solid-phase extraction column was used to purify the supernatant by solid-phase extraction to obtain a purified solution; (3) The purified liquid was analyzed by high performance liquid chromatography using an acetonitrile-water mobile phase system, and the residual hydrogen peroxide content was quantified by external standard method.

2. The method for detecting residual hydrogen peroxide in food using SPE-HPLC as described in claim 1, characterized in that: In step (1), the mass ratio of the sample to be tested to sodium chloride is 1:

4.

3. The method for detecting residual hydrogen peroxide in food using SPE-HPLC as described in claim 1, characterized in that: In step (1), the ratio of the sample to acetonitrile phosphate is: 15-25 mL of acetonitrile phosphate is added per gram of sample to be tested.

4. The method for detecting residual hydrogen peroxide in food using SPE-HPLC as described in any one of claims 1 to 3, characterized in that: In step (1), the acetonitrile phosphate is 0.5% acetonitrile phosphate, that is, an acetonitrile solution with a volume fraction of 0.5% phosphoric acid.

5. The method for detecting residual hydrogen peroxide in food using SPE-HPLC as described in claim 1, characterized in that: The centrifugation operation in step (1) is: centrifuge at 8000 rpm for 5 min.

6. The method for detecting residual hydrogen peroxide in food using SPE-HPLC as described in claim 1, characterized in that: The mixed-type strong cation exchange reversed-phase solid-phase extraction column used in step (2) is ProElut PXC 30 mg / 1 mL.

7. The method for detecting residual hydrogen peroxide in food using SPE-HPLC as described in any one of claims 1 to 6, characterized in that: In step (2), 2 mL of the solution to be purified is added to a mixed strong cation exchange reversed-phase solid-phase extraction column, the effluent is discarded, and then 1 mL of the solution to be purified is added. The effluent is collected, vortexed and mixed, and then filtered through a 0.45 μm NY filter membrane.

8. The method for detecting residual hydrogen peroxide in food using SPE-HPLC as described in any one of claims 1 to 7, characterized in that: The high-performance liquid chromatography column used in step (3) is a polar modified C18 reversed-phase column, and the flow rate gradient is set as follows: 0~8 min, flow rate 0.4 mL / min; 8~8.1 min, flow rate 0.4~1.0 mL / min; 8.1~23 min, flow rate 1.0 mL / min; 23~23.1 min, flow rate 1.0~0.4 mL / min; 23.1~25 min, flow rate 0.4 mL / min.

9. The method for detecting residual hydrogen peroxide in food using SPE-HPLC as described in claim 1, characterized in that: In step (3), the mobile phase acetonitrile:water = 5:95; v / v.

10. The method for detecting residual hydrogen peroxide in food using SPE-HPLC as described in claim 1, characterized in that: In step (3) of the external standard method, the solvent used for the hydrogen peroxide standard solution is 0.5% acetonitrile phosphate, and the spiking volume is no more than 1 mL.

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