Method for detecting p-xylene in honey and its application in identification of tung flower honey

Abstract

The application belongs to the field of food detection and identification, and particularly relates to a detection method of p-dimethoxybenzene in honey and application of the detection method in identification of tung flower honey, which adopts solid phase extraction for enrichment and purification and adopts gas chromatography-tandem mass spectrometer for detection. The internal standard method is adopted for quantification. The application detects p-dimethoxybenzene in tung flower honey for the first time, and the concentration range of p-dimethoxybenzene is 3.3-8.0 mu g / kg. Accordingly, when the content of the honey sample is in the above range, the honey sample can be determined as tung flower honey, otherwise, the honey sample can be determined as other honey or adulterated honey. The method for identifying tung flower honey has the advantages of high efficiency, convenience, high sensitivity and the like, and has important significance for detection and identification of tung flower honey, and has important practical significance for maintaining the order of the honey market and the legal rights and interests of consumers.

Description

Technical Field

[0001] This invention belongs to the field of food testing and identification, specifically a method for detecting p-phenylenedimethyl ether in honey and its application in the identification of tung honey. Background Technology

[0002] Honey is a natural sweet substance produced by bees from the nectar, secretions, or honeydew of plants, which they combine with their own secretions and then transform, dehydrate, and store in the honeycomb until mature. Paulownia, a nectar-producing plant, is widely cultivated in Henan and Anhui provinces of my country. Paulownia honey is a natural sweet substance produced by bees from the nectar of paulownia flowers, which they mix with their own secretions and then fully ferment. Paulownia flowers, as a traditional Chinese medicine, have antibacterial properties and are effective in relieving asthma, lowering blood pressure, detoxifying, and reducing swelling. Paulownia honey inherits these antibacterial, anti-swelling, asthma-relieving, and lung-moistening functions. Paulownia honey has a viscous texture; it is pale yellow before crystallization and becomes a light yellow, oily or granular substance after crystallization. Paulownia honey has the unique refreshing aroma of paulownia flowers, a sweet but not cloying taste, and a slightly acidic flavor.

[0003] Paulownia honey is mainly produced in the Yellow River and Yangtze River basins of my country. The paulownia flower season is short, generally lasting only ten days or so, and is highly susceptible to weather conditions, resulting in a relatively limited annual yield of tung honey, making it a relatively rare type of honey. Many unscrupulous merchants use adulteration and other methods to counterfeit tung honey for exorbitant profits. Therefore, there is an urgent need to develop a simple and effective method to provide technical support for the authenticity identification and quality evaluation of tung honey. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, at least one technical problem raised in the background art is solved.

[0005] The technical solution adopted by this invention to solve its technical problem is: the method for detecting p-phenylenediamine in honey according to this invention includes the following steps:

[0006] Step 1: Sample Processing

[0007] Weigh out a honey sample, dissolve it in deionized water and mix well;

[0008] Step 2: Solid-phase extraction

[0009] The sample solution after step one was subjected to solid-phase extraction to enrich, purify and elute p-phenylenedimethyl ether.

[0010] Step 3: Measurement

[0011] Gas chromatography-tandem mass spectrometry (GC-MS / MS) was used for qualitative and quantitative detection of p-phenylenedimethyl ether in honey samples, with quantification using the internal standard method. The GC-MS / MS detection conditions were as follows:

[0012] Gas chromatography conditions:

[0013] Chromatographic column: DB-5MS gas chromatographic column, specifications: 30 m × 250 μm × 0.25 μm;

[0014] Inlet temperature: 250 ℃;

[0015] The temperature program was set at 50 °C for 1 min, then increased to 250 °C at a rate of 30 °C / min and held for 3 min. The total running time was 10.67 min.

[0016] Column flow rate: 1.5 mL / min;

[0017] Carrier gas: High-purity helium with a purity of ≥99.999%;

[0018] Injection method: splitless injection;

[0019] Injection volume: 1 µL;

[0020] Mass spectrometry conditions:

[0021] Ion source: Electron impact ion source (EI);

[0022] Transmission line temperature: 280 ℃;

[0023] Ion source temperature: 280 ℃;

[0024] Detection method: Multiple reaction monitoring (MRM) scanning mode.

[0025] As a further technical solution of the present invention: In step one, the ratio of honey to deionized water is 1g:3mL.

[0026] As a further technical solution of the present invention: In step one, 20 µL of isotope internal standard solution with a concentration of 1 mg / L is added to the honey sample solution, and the solution is vortexed at a speed of 1000-2000 rpm on a vortex mixer.

[0027] As a further technical solution of the present invention: In step two, the solid phase extraction step includes:

[0028] The solid-phase extraction column was installed on a vacuum filtration device, and impurities were washed away with 3 mL of ethyl acetate, followed by activation with 3 mL of methanol and equilibration with 6 mL of deionized water.

[0029] Load the sample solution obtained in step one onto a solid phase extraction column;

[0030] Rinse with 3 mL of deionized water, then dry with a vacuum pump;

[0031] Finally, elute with 3 mL of ethyl acetate, blow down to 1 mL with nitrogen at 30 °C, vortex mix, and filter through a membrane into a 1 mL brown sample vial for testing.

[0032] As a further technical solution of the present invention: the flow rate of the sample solution loaded onto the solid phase extraction column is controlled at 1 mL / min.

[0033] As a further technical solution of the present invention: the extraction column adopts a 60 mg HLB solid phase extraction column.

[0034] As a further technical solution of the present invention: the filter membrane is a 0.22 µm nylon filter membrane.

[0035] As a further technical solution of the present invention: in the internal standard method quantification described in step three, the internal standard curve equation for p-phenylenedimethyl ether is:

[0036] Y = 0.06324X + 0.00635, linear correlation coefficient R 2 =0.9998;

[0037] Where X is the concentration of the target analyte in the sample, and Y is the ratio of the peak area of ​​the target analyte to that of the internal standard in the sample.

[0038] As a further technical solution of the present invention: the detection limit of the detection method is 0.3 µg / kg, the quantitation limit is 1.0 µg / kg, and the recovery rate of the detection method is higher than 80%.

[0039] The application of the above method in the identification of tung honey is characterized in that if the content of dimethyl ether in the honey sample is in the range of 3.3-8.0 µg / kg, then the honey sample is judged to be genuine tung honey; otherwise, the honey sample is judged to be other honey or adulterated tung honey.

[0040] The beneficial effects of this invention are as follows:

[0041] This invention identifies p-phenylenedimethyl ether as a characteristic marker of tung blossom honey for the first time, and proposes a detection method for p-phenylenedimethyl ether in honey using gas chromatography-tandem mass spectrometry. Based on its content, tung blossom honey can be effectively identified. The content of p-phenylenedimethyl ether in pure tung blossom honey is 3.3-8.0 µg / kg.

[0042] The identification method for tung blossom honey provided by this invention has high specificity and sensitivity, enabling effective separation and accurate quantification of p-phenylenediamine in honey. This method is simple to operate, highly efficient, and easy to promote and apply, and is of great significance for the authenticity identification and quality evaluation of tung blossom honey. Attached Figure Description

[0043] The invention will now be further described with reference to the accompanying drawings.

[0044] Figure 1 A linear curve defined by the internal standard;

[0045] Figure 2 The images show multiple reaction monitoring (SRM) chromatograms of p-phenylenedimethyl ether and its internal standard. (A) is the SRM chromatogram of the quantitative ion of p-phenylenedimethyl ether, (B), (C) and (D) are the SRM chromatograms of the auxiliary qualitative ion of p-phenylenedimethyl ether, (E) is the SRM chromatogram of the quantitative ion of the internal standard, and (F), (G) and (H) are the SRM chromatograms of the auxiliary qualitative ion of the internal standard.

[0046] Figure 3 The effect of solid phase extraction conditions on the extraction effect is shown in (A) for solid phase extraction column optimization, (B) for elution volume optimization, and (C) for elution volume optimization.

[0047] Figure 4 Multiple reaction monitoring (SRM) chromatograms and signal-to-noise ratios of terephthalic dimethyl ether ions at the limit of detection (LOD) and limit of quantitation (LOQ) levels are shown. (A) is the SRM chromatogram and signal-to-noise ratio of the ion at the LOD level, and (B) is the SRM chromatogram and signal-to-noise ratio of the ion at the LOQ level.

[0048] Figure 5 This is a graph showing the content of diphenyl ether in a single-flower honey sample. Detailed Implementation

[0049] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0050] The materials, reagents, and instruments involved in the following examples are as follows:

[0051] Gas chromatography-tandem mass spectrometry: Thermo TSQ 8000;

[0052] Chromatographic column: DB-5MS gas chromatographic column (30 m × 250 μm × 0.25 μm)

[0053] Vortex mixer: Vortex-Genie2 (Scientific Industries);

[0054] Electronic analytical balance: XP105 (Mettler Toledo);

[0055] Micro-adjustable pipettes: 20 µL, 100 µL, 200 µL, 1 mL, 5 mL (ThermoFisher).

[0056] p-phenylenedimethyl ether standard: 99% purity (Shandong Keyuan Biochemical Co., Ltd.);

[0057] Internal standard for terephthalic dimethyl ether isotopes: English name 1,4-Dimethoxybenzene-D6, purity 97.0% (Shanghai Zhenzhun Biotechnology Co., Ltd.);

[0058] Chromatographically pure methanol and ethyl acetate (Beijing Dicoma Technology Co., Ltd.);

[0059] Vacuum pump: DOA-P504-BN diaphragm vacuum pump (GAST Corporation, USA);

[0060] All water used in the experiments was ultrapure water.

[0061] Example 1

[0062] The method for detecting p-phenylenediamine in honey according to embodiments of the present invention includes the following steps:

[0063] Step 1: Plotting the Standard Curve

[0064] (1.1) Standard stock solution and preparation: Accurately weigh 100 mg of p-phenylenediamine standard and place it in a 10 mL volumetric flask. Dissolve it in pure methanol and dilute to volume to prepare a 10000 mg / L standard stock solution. Store it at 4 °C.

[0065] (1.2) Internal standard stock solution and preparation: Accurately weigh 10 mg of diphenyl ether internal standard and place it in a 10 mL volumetric flask. Dissolve it in pure methanol and dilute to volume to prepare a 1000 mg / L internal standard stock solution. Store it at 4 °C.

[0066] (1.3) Preparation of intermediate standard solutions and internal standard solutions: Accurately transfer 1 mL of the above standard stock solution into a 10 mL volumetric flask, dilute to volume with ethyl acetate, and mix well to prepare 1000 mg / L intermediate standard solution A; accurately transfer 1 mL of the above intermediate standard solution A and the internal standard stock solution into a 10 mL volumetric flask, dilute to volume with ethyl acetate, and mix well to prepare 100 mg / L intermediate standard solution B and intermediate internal standard solution B; then accurately transfer 100 µL of the above intermediate standard solution B and intermediate internal standard solution B into a 10 mL volumetric flask, dilute to volume with ethyl acetate, and prepare 1 mg / L intermediate standard solution C and intermediate internal standard solution C; all the above solutions are stored at 4 ℃.

[0067] (1.4) Preparation of a series of standard working solutions: Take 20 µL of internal standard intermediate solution C and an appropriate amount of p-phenylenediamine standard intermediate solution C, and dilute them with ethyl acetate to prepare a series of standard working solutions with target concentrations of 1.0 µg / L, 2.0 µg / L, 5.0 µg / L, 10.0 µg / L, 20.0 µg / L and 50.0 µg / L, respectively;

[0068] (1.5) A standard curve was plotted with the ratio of the peak area of ​​the quantitative ion of p-phenylenediamine to that of the quantitative ion of 20.0 µg / L internal standard as the Y-axis and the concentration of the target component as the X-axis. The linear curve is shown in Appendix. Figure 1 The equation for the standard curve of p-phenylenediamine and the linear correlation coefficient are shown in Table 1:

[0069] Table 1. Linear equations and correlation coefficients of the p-phenylenedimethyl ether standard curve

[0070]

[0071] Step Two: Sample Collection and Preparation

[0072] A total of 84 different types of honey were collected from different regions, including: tung blossom honey, jujube blossom honey, vitex honey, linden honey, rapeseed honey, acacia honey, and lychee honey.

[0073] Step 3: Sample pretreatment:

[0074] (3.1) Accurately weigh 5 g (accurate to 0.01 g) of honey sample into a 50 mL centrifuge tube, add 15 mL of deionized water, add 20 µL of 1.0 mg / L internal standard solution, and vortex at 1000-2000 rpm for later use.

[0075] (3.2) Install the HLB solid phase extraction column on the vacuum filtration device for solid phase extraction, wash the impurities in the extraction column with 3 mL of ethyl acetate, then activate it with 3 mL of methanol, and balance the extraction column with 6 mL of deionized water.

[0076] (3.3) Load the honey sample solution from (3.1) above onto the HLB solid phase extraction column at a rate of about 1 mL / min. After loading, rinse with 3 mL of deionized water and finally dry with a vacuum pump.

[0077] (3.4) Elute with 3 mL of ethyl acetate at a flow rate of about 1 mL / min, then purge with nitrogen at 30 °C to 1 mL, vortex to mix and set aside;

[0078] (3.5) The sample was transferred through a 0.22 µm filter membrane to a brown vial, and then detected by gas chromatography-tandem mass spectrometry under the following conditions:

[0079] Gas chromatography conditions:

[0080] Chromatographic column: DB-5MS gas chromatographic column (30 m × 250 μm × 0.25 μm);

[0081] Inlet temperature: 250 ℃;

[0082] The temperature program is as follows: initial temperature 50 ℃, hold for 1 min, then increase to 250 ℃ at 30 ℃ / min and hold for 3 min;

[0083] Carrier gas: High-purity helium, purity ≥ 99.999%;

[0084] Column flow rate: 1.5 mL / min;

[0085] Injection method: splitless injection;

[0086] Injection volume: 1 μL;

[0087] Mass spectrometry conditions:

[0088] Ion source: Electron impact ion source (EI);

[0089] Ion source temperature: 280 ℃;

[0090] Transmission line temperature: 280 ℃;

[0091] Detection method: Multiple reaction monitoring (SRM) mode, SRM parameter settings are as follows:

[0092]

[0093] The multiple reaction monitoring (SRM) chromatogram is attached. Figure 2 .

[0094] Optimization and screening of experimental conditions:

[0095] The experimental conditions were optimized by selecting tung blossom honey with a high content of tung blossom flowers.

[0096] Solid-phase extraction column optimization: The extraction effects of four solid-phase extraction columns (60 mg C18, 1000 mg C18, 60 mg HLB, and 200 mg HLB) on the target components were investigated. (See Appendix) Figure 3 (A) Finally, a 60 mg HLB solid-phase extraction column is preferred as the pretreatment solid-phase extraction column;

[0097] Eluent optimization: The effect of different eluent volumes on the extraction efficiency of the sample solution on the experimental results was investigated (see Appendix). Figure 3(B) indicates that different rinsing volumes have little difference in the enrichment effect of the target analyte and the removal effect of impurities such as sugars. Therefore, 3 mL of deionized water was finally selected as the rinsing solution.

[0098] Elution volume optimization: The effect of different elution volumes on the extraction efficiency of the sample solution on the experimental results was investigated (see Appendix). Figure 3 (C) When the eluent volume is 1 mL, the content of p-phenylenediamine in the effluent is the highest. When the eluent volume is 3 mL, no p-phenylenediamine is detected in the effluent. Therefore, the final eluent volume is 3 mL.

[0099] Limits of detection, limits of quantitation, recovery and precision of the method

[0100] The method detection limit was 0.3 µg / kg at a sample concentration corresponding to a signal-to-noise ratio of at least 3 times (S / N≥3), and the method quantitation limit was 1.0 µg / kg at a sample concentration corresponding to a signal-to-noise ratio of at least 10 times (S / N≥10). The multiple reaction monitoring (SRM) chromatograms of the corresponding quantitative ions are attached. Figure 4 .

[0101] The recovery rate and precision of this invention were verified by spiked recovery of blank samples. The specific operation was as follows: three concentration levels of standard solutions (low, medium, and high) were added to a blank honey sample (excluding p-phenylenediamine). The sample was analyzed and detected according to the above detection method, and the recovery rate was calculated. The results are shown in Table 2.

[0102] Table 2 Recovery and precision results at different spiking levels ( n =6)

[0103]

[0104] Table 2 shows that the recovery rate and relative standard deviation of diphenyl ether met the analytical requirements at the three concentrations, indicating that the method has high accuracy and precision, and the determination results are true and reliable.

[0105] Example 2

[0106] The application of p-phenylenedimethyl ether in honey as a characteristic marker of tung blossom honey, as described in this embodiment of the invention, includes the following steps:

[0107] Sample source: The honey samples used in this invention are all natural mature honeys collected during the flowering and nectar-producing seasons of various nectar-producing plants. All samples were purchased directly from beekeepers and stored at 4 ℃ before testing.

[0108] The analytical method established by this invention was used to test honey samples from seven different nectar-producing plants. Specifically, the honey sample types included were: 24 batches of tung blossom honey, 10 batches of jujube blossom honey, 10 batches of rapeseed honey, 10 batches of acacia honey, 10 batches of vitex honey, 10 batches of lychee honey, and 10 batches of linden honey.

[0109] The content of p-phenylenediamine in all the honey samples was detected, compared, and analyzed. The results showed that p-phenylenediamine was detected only in tung blossom honey, and not in the other honey samples. (See attached image) Figure 5 Therefore, p-phenylenedimethyl ether was ultimately determined to be a characteristic marker of tung blossom honey, with a content range of 3.3-8.0 μg / kg in the honey.

[0110] Based on the test results of the above experimental samples, the criteria for identifying tung flower honey can be determined as follows:

[0111] A) If phenyl dimethyl ether is detected and the content is in the range of 3.3-8.0 μg / kg, then the sample is pure tung flower honey;

[0112] B) If diphenyl ether is not detected, or if the content of the target substance is less than 3.3 μg / kg, the sample is not pure tung honey (adulterated or fake) or not tung honey (other types of honey or non-honey).

[0113] To further verify this, seven batches of labeled tung blossom honey samples were purchased from online e-commerce platforms. The method of this invention was used to detect phenylenediamine, and the authenticity of each honey sample was determined using the aforementioned criteria. The results are shown in Table 3.

[0114] Table 3. Results of the determination of p-phenylenediamine in commercially available honey samples and identification of the authenticity of tung blossom honey.

[0115]

[0116] Note: ND not detected; Y is pure tung flower honey; N is not pure tung flower honey or is not tung flower honey.

[0117] The results showed that three batches of samples met the criteria for identification in this embodiment and were identified as genuine tung flower honey. In the other two batches of samples, the content of diphenyl ether was less than 3.3 μg / kg, and diphenyl ether was not detected in the two batches of samples. They were identified as non-pure tung flower honey or non-tung flower honey. The results show that the method of the present invention has good practicality and can be used for the identification of the authenticity of tung flower honey.

[0118] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting p-phenylenediamine in honey, characterized in that: Includes the following steps: Step 1: Sample Processing Weigh out a honey sample, dissolve it in deionized water and mix well; Step 2: Solid-phase extraction The sample solution after step one was subjected to solid-phase extraction to enrich, purify and elute p-phenylenedimethyl ether. Step 3: Measurement Gas chromatography-tandem mass spectrometry (GC-MS / MS) was used for qualitative and quantitative detection of p-phenylenedimethyl ether in honey samples, with quantification using the internal standard method. The GC-MS / MS detection conditions were as follows: Gas chromatography conditions: Chromatographic column: DB-5MS gas chromatographic column, specifications: 30 m × 250 μm × 0.25 μm; Inlet temperature: 250 ℃; The temperature program was set at 50 °C for 1 min, then increased to 250 °C at a rate of 30 °C / min and held for 3 min. The total running time was 10.67 min. Column flow rate: 1.5 mL / min; Carrier gas: High-purity helium with a purity of ≥99.999%; Injection method: splitless injection; Injection volume: 1 µL; Mass spectrometry conditions: Ion source: Electron impact ion source (EI); Transmission line temperature: 280 ℃; Ion source temperature: 280 ℃; Detection method: Multiple reaction monitoring (SRM) mode, SRM parameter settings are as follows: Optimization and screening of experimental conditions: The experimental conditions were optimized by selecting tung blossom honey with a high content of tung blossom flowers. Solid-phase extraction column optimization: A 60 mg HLB solid-phase extraction column was used as the pretreatment solid-phase extraction column; Optimization of rinsing solution: Select 3 mL of deionized water as the rinsing solution; Elution volume optimization: When the eluent volume is 1 mL, the content of p-phenylenediamine in the effluent is the highest. When the eluent volume is 3 mL, no p-phenylenediamine is detected in the effluent. Therefore, the final eluent volume is 3 mL. Limits of detection, limits of quantitation, recovery and precision of the method The method detection limit is 0.3 µg / kg with a signal-to-noise ratio of not less than 3 times (S / N≥3) corresponding to the sample concentration, and the method quantitation limit is 1.0 µg / kg with a signal-to-noise ratio of not less than 10 times (S / N≥10) corresponding to the sample concentration, corresponding to the multiple reaction monitoring (SRM) chromatogram of the quantitative ions; In the internal standard method quantification described in step three, the internal standard curve equation for p-phenylenediamine is as follows: Y = 0.06324X + 0.00635, linear correlation coefficient R 2 =0.9998; Where X is the concentration of the target analyte in the sample, and Y is the ratio of the peak area of ​​the target analyte to that of the internal standard in the sample; The detection limit of this method is 0.3 µg / kg, and the quantitation limit is 1.0 µg / kg; the recovery rate of this method is higher than 80%. If the content of diphenyl ether in a honey sample is in the range of 3.3-8.0 µg / kg, the honey sample is determined to be genuine tung blossom honey; otherwise, the honey sample is determined to be other types of honey or adulterated tung blossom honey.

2. The method for detecting p-phenylenediamine in honey according to claim 1, characterized in that: In step one, the ratio of honey to deionized water is 1g:3mL.

3. The method for detecting p-phenylenediamine in honey according to claim 2, characterized in that: In step one, 20 µL of an isotope internal standard solution with a concentration of 1 mg / L is added to the honey sample solution, and the mixture is vortexed at a speed of 1000-2000 rpm on a vortex mixer.

4. The method for detecting p-phenylenediamine in honey according to claim 3, characterized in that: Step two, the solid-phase extraction steps include: The solid-phase extraction column was installed on a vacuum filtration device, and impurities were washed away with 3 mL of ethyl acetate, followed by activation with 3 mL of methanol and equilibration with 6 mL of deionized water. Load the sample solution obtained in step one onto a solid phase extraction column; Rinse with 3 mL of deionized water, then dry with a vacuum pump; Finally, elute with 3 mL of ethyl acetate, blow down to 1 mL with nitrogen at 30 °C, vortex mix, and filter through a membrane into a 1 mL brown sample vial for testing.

5. The method for detecting p-phenylenediamine in honey according to claim 4, characterized in that: The flow rate of the sample solution loaded onto the solid-phase extraction column was controlled at 1 mL / min.

6. The method for detecting p-phenylenediamine in honey according to claim 5, characterized in that: The extraction column used was a 60mg HLB solid-phase extraction column.

7. The method for detecting p-phenylenediamine in honey according to claim 6, characterized in that: The filter membrane is a 0.22 µm nylon filter membrane.

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