Method for detecting p-xylylene dimethyl ether in honey and application of method in identification of aegiceras corniculatum honey

Abstract

The invention belongs to the field of food detection and identification, and particularly relates to a method for detecting p-xylylene ether in honey and application of the method in identification of aegiceras corniculatum honey, solid-phase extraction is adopted for enrichment and purification, and a gas chromatography-tandem mass spectrometer is adopted for detection. And quantifying by adopting an internal standard method. According to the method, the p-xylylene ether is detected in the Aegiceras corniculatum honey for the first time, the concentration range of the p-xylylene ether is 3.3-8.0 g / kg, accordingly, when the content in the honey sample is within the range, the honey sample can be judged to be the Aegiceras corniculatum honey, otherwise, the honey sample is judged to be other honey or adulterated honey, the method for identifying the Aegiceras corniculatum honey has the advantages of high efficiency, simplicity, convenience, high sensitivity and the like, and the method is suitable for popularization and application. The method has important significance on detection and identification of the Aegiceras corniculatum honey, and also has important practical significance on maintaining the order of the honey market and legitimate rights and interests of consumers.

Description

TECHNICAL FIELD

[0001] 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 to identification of Paulownia honey. BACKGROUND

[0002] Honey is a natural sweet substance which is obtained by bees collecting nectar, secretion or honeydew, combining with their own secretion, transforming, dehydrating and storing in the nest, and then being matured in the nest. Paulownia is a honey plant which is widely planted in Henan and Anhui of China. Paulownia honey is a natural sweet substance which is obtained by bees collecting nectar of Paulownia flowers and mixing with their own secretion and then being fully brewed. It is reported that Paulownia flowers, as a traditional Chinese medicine, have the effects of inhibiting bacteria, relieving asthma, reducing pressure, detoxifying and reducing swelling. Paulownia honey inherits the functions of inhibiting bacteria, reducing swelling, relieving asthma and moistening the lung. Paulownia honey has a sticky texture, is light yellow when not crystallized, and is light yellow oil or fine granular after crystallization. Paulownia honey has a unique refreshing fragrance of Paulownia flowers, a sweet taste without greasiness, and a slightly sour taste.

[0003] Paulownia honey is mainly produced in the Yellow River Basin and the Yangtze River Basin of China. Paulownia flowers have a short flowering period, generally only more than ten days, and are easily affected by the climate, resulting in a limited annual yield of Paulownia honey. Paulownia honey is a relatively rare honey. Many unscrupulous traders use adulteration and other means to pretend to be Paulownia honey to make a profit. Therefore, it is urgent to develop a simple and effective method to provide technical support for the authenticity identification and quality evaluation of Paulownia honey. SUMMARY

[0004] In order to make up for the deficiencies of the prior art and solve at least one technical problem proposed in the background.

[0005] The technical scheme adopted by the application to solve the technical problem is that the detection method of p-dimethoxybenzene in honey comprises the following steps: Step 1: sample treatment Weigh the honey sample, dissolve it with deionized water and mix it evenly; Step 2: solid phase extraction Take the sample solution treated in step 1 to perform solid phase extraction to enrich, purify and elute p-dimethoxybenzene; Step 3: determination Qualitative and quantitative detection of p-dimethoxybenzene in the honey sample is performed by gas chromatography-tandem mass spectrometry, and the internal standard method is used for quantification. The detection conditions of the gas chromatography-tandem mass spectrometry are as follows: Gas chromatography conditions: Chromatographic column: DB-5MS gas chromatography column, specifications: 30 m x 250 μm x 0.25 μm; Inlet temperature: 250 DEG C; Programmed temperature: initial temperature 50 ℃, hold for 1 min, then increase to 250 ℃ at 30 ℃ / min, hold for 3 min, total running time is 10.67 min; Column flow rate: 1.5 mL / min; Carrier gas: high-purity helium with purity ≥ 99.999%; Injection mode: splitless injection; Injection volume: 1 µL; Mass spectrometry conditions: Ion source: electron impact ion source EI; Transfer line temperature: 280 ℃; Ion source temperature: 280 ℃; Detection mode: multiple reaction monitoring scan mode.

[0006] As a further technical solution of the application: in step one, the ratio of honey to deionized water is 1 g:3 mL.

[0007] As a further technical solution of the application: in step one, 20 µL of isotopic internal standard solution with a concentration of 1 mg / L is added to the honey sample solution, and vortexed on a vortex machine at a speed of 1000-2000 rpm.

[0008] As a further technical solution of the application: in step two, the solid phase extraction includes: Install the solid phase extraction column on the vacuum filtration device, wash off impurities with 3 mL of ethyl acetate, then activate with 3 mL of methanol, and 6 mL of deionized water; Load the sample solution obtained in step one onto the solid phase extraction column; Select 3 mL of deionized water for elution, then dry with a vacuum pump; Finally, elute with 3 mL of ethyl acetate, nitrogen blow at 30 ℃ to 1 ml, vortex well, then filter membrane filter to 1 mL brown sample bottle for testing.

[0009] As a further technical solution of the application: the flow rate of sample solution loading onto the solid phase extraction column is controlled at 1 mL / min.

[0010] As a further technical solution of the application: the extraction column uses a 60 mg HLB solid phase extraction column.

[0011] As a further technical solution of the application: the filter membrane is a 0.22 µm nylon filter membrane.

[0012] As a further technical solution of the application: in the internal standard method quantification described in step three, the internal standard curve equation for p-dimethyl anisole is: Y=0.06324X+0.00635, linear correlation coefficient R 2 =0.9998. Wherein, X is the concentration of the target in the sample, Y is the ratio of the peak area of the target and the internal standard in the sample.

[0013] As a further technical solution of the application: the detection limit of the detection method is 0.3 µg / kg, and the quantification limit is 1.0 µg / kg; the recovery rate of the detection method is higher than 80%.

[0014] The application of the above method in the identification of tung flower honey is characterized in that the content of p-dimethyl anisole in the honey sample is in the range of 3.3-8.0 µg / kg, and then the honey sample is judged to be real tung flower honey, otherwise, the honey sample is judged to be other honey or tung flower honey.

[0015] The beneficial effects of the application are as follows: The application first determines p-dimethyl anisole as a characteristic marker of tung flower honey, and proposes a detection method for detecting p-dimethyl anisole in honey by using a gas chromatograph-tandem mass spectrometer, so that the tung flower honey can be effectively identified according to the content of p-dimethyl anisole, and the content of p-dimethyl anisole in pure tung flower honey is 3.3-8.0 µg / kg.

[0016] The identification method of tung flower honey provided by the application has high specificity and sensitivity, can effectively separate and accurately quantify p-dimethyl anisole in honey, has the characteristics of simple operation and high efficiency, is convenient for popularization and application, and has important significance for the authenticity identification and quality evaluation of tung flower honey. BRIEF DESCRIPTION OF DRAWINGS

[0017] The application will be further described below with reference to the drawings.

[0018] Figure 1 It is a linear curve for internal standard quantitative; Figure 2 It is a multiple reaction monitoring (SRM) chromatogram of p-dimethyl anisole and its internal standard, wherein (A) is an SRM chromatogram of a quantitative ion of p-dimethyl anisole, (B), (C) and (D) are all SRM chromatograms of auxiliary qualitative ions of p-dimethyl anisole, (E) is an SRM chromatogram of a quantitative ion of the internal standard, and (F), (G) and (H) are all SRM chromatograms of auxiliary qualitative ions of the internal standard; Figure 3 It is an influence of solid phase extraction conditions on extraction effect, wherein (A) is solid phase extraction column optimization, (B) is elution volume optimization, and (C) is elution volume optimization; Figure 4The benzene dimethyl ether standard solution was prepared as follows: 100 mg of benzene dimethyl ether standard substance was accurately weighed and placed in a 10 mL volumetric flask, dissolved with pure methanol and diluted to the mark, and prepared into a 10000 mg / L standard stock solution, which was stored at 4 DEG C. Figure 5 The benzene dimethyl ether standard solution was prepared as follows: 100 mg of benzene dimethyl ether standard substance was accurately weighed and placed in a 10 mL volumetric flask, dissolved with pure methanol and diluted to the mark, and prepared into a 10000 mg / L standard stock solution, which was stored at 4 DEG C. DETAILED DESCRIPTION

[0019] In order to make the technical means, creative features, purposes and effects realized by the present application easy to understand, the present application will be further described below in combination with specific embodiments.

[0020] The materials, reagents and instruments involved in the following examples are as follows: Gas chromatography-tandem mass spectrometer: Thermo TSQ 8000 type; Chromatographic column: DB-5MS gas chromatographic column (30 m x 250 μm x 0.25 μm) Vortex mixer: Vortex-Genie2 (Scientific Industries); Electronic analytical balance: XP105 (Mettler Toledo); Micro adjustable pipette: range 20 μL, 100 μL, 200 μL, 1 mL, 5 mL (Thermo Fisher); Benzene dimethyl ether standard substance: purity 99% (Shandong Keyuan Biochemical Co., Ltd.); Benzene dimethyl ether isotopic internal standard substance: English name 1,4-Dimethoxybenzene-D6, purity 97.0% (Shanghai Zhenzhun Biological Technology Co., Ltd.); Chromatographic pure methanol, ethyl acetate (Beijing Dikema Technology Co., Ltd.); Vacuum pump: DOA-P504-BN diaphragm vacuum pump (GAST Company, USA); The test water is ultrapure water.

[0021] Example 1 The detection method of benzene dimethyl ether in honey according to the embodiment of the present application comprises the following steps: Step 1: Standard curve drawing (1.1) Standard stock solution and preparation: accurately weigh 100 mg of benzene dimethyl ether standard substance and place it in a 10 mL volumetric flask, dissolve and dilute to the mark with pure methanol, and prepare into a 10000 mg / L standard stock solution, which is stored at 4 DEG C. (1.2) Preparation of internal standard stock solution: 10 mg of p-xylylene was accurately weighed into a 10 mL volumetric flask, dissolved with pure methanol and diluted to the mark, and prepared into a 1000 mg / L internal standard stock solution, which was stored at 4 ℃; (1.3) Preparation of standard intermediate solution and internal standard intermediate solution: 1 mL of the above standard stock solution was accurately transferred into a 10 mL volumetric flask, diluted with ethyl acetate to the mark, mixed and prepared into a 1000 mg / L standard intermediate solution A; 1 mL of the above standard intermediate solution A and internal standard stock solution were accurately transferred into a 10 mL volumetric flask, diluted with ethyl acetate to the mark, mixed and prepared into a 100 mg / L standard intermediate solution B and internal standard intermediate solution B; 100 μL of the above standard intermediate solution B and internal standard intermediate solution B were accurately transferred into a 10 mL volumetric flask, respectively, diluted with ethyl acetate to the mark, and prepared into a 1 mg / L standard intermediate solution C and internal standard intermediate solution C; the above solutions were stored at 4 ℃; (1.4) Preparation of series of standard working solutions: 20 μL of the internal standard intermediate solution C and a proper amount of p-xylylene standard intermediate solution C were taken, diluted with ethyl acetate to the mark 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; (1.5) The ratio of the peak area of the p-xylylene quantitative ion to the 20.0 μg / L internal standard quantitative ion was taken as the Y axis, and the concentration of the target component was taken as the X axis to draw a standard curve, and the linear curve is shown in FIG. 1. Figure 1 The standard curve equation and linear correlation coefficient of p-xylylene are shown in Table 1: Table 1 Linear equation and correlation coefficient of p-xylylene standard curve Step two: sample collection and preparation: A total of 84 different kinds of honey samples were collected from different regions, including the following sample types: tung tree honey, jujube flower honey, wattle honey, linden tree honey, rape honey, acacia honey, and litchi honey; Step three: sample pretreatment: (3.1) 5 g (accurate to 0.01 g) of honey sample was accurately weighed into a 50 mL centrifuge tube, 15 mL of deionized water was added, 20 μL of 1.0 mg / L internal standard solution was added, and vortex mixing was performed on a vortex mixer at 1000-2000 rpm for mixing, ready for use; (3.2) The HLB solid phase extraction column was installed on the vacuum filtration device for solid phase extraction, 3 mL of ethyl acetate was used to wash the impurities in the extraction column, then 3 mL of methanol was used for activation, and 6 mL of deionized water was used to balance the extraction column; (3.3) The honey sample solution in (3.1) above is loaded onto the HLB solid phase extraction column at a speed of about 1 mL / min, after loading is completed, 3 mL of deionized water is used for elution, and finally a vacuum pump is used to dry; (3.4) 3 mL of ethyl acetate is used for elution, the flow rate is controlled at about 1 mL / min, then nitrogen blowing is carried out at 30°C to 1 mL, vortex mixing is carried out after vortex mixing, and then it is ready for use; (3.5) Transferred to a brown sample injection vial through a 0.22 µm filter membrane, and then detected by gas chromatography-tandem mass spectrometry, wherein the detection conditions are as follows: Gas chromatography conditions: Chromatographic column: DB-5MS gas chromatography column (30 m x 250 µm x 0.25 µm); Inlet temperature: 250°C; Programmed temperature: initial temperature 50°C, hold for 1 min, then increase to 250°C at 30°C / min, hold for 3 min; Carrier gas: high-purity helium, purity ≥ 99.999%; Column flow rate: 1.5 mL / min; Injection mode: splitless injection; Injection volume: 1 µL; Mass spectrometry conditions: Ion source: electron impact ion source EI; Ion source temperature: 280°C; Transfer line temperature: 280°C; Detection mode: multiple reaction monitoring scan mode (SRM), SRM parameter settings are as follows: The multiple reaction monitoring (SRM) chromatogram is shown in the accompanying Figure 2 .

[0022] Optimization of experimental conditions: The experimental conditions are optimized by selecting high-content tung flower honey: Solid phase extraction column optimization: the extraction effects of four solid phase extraction columns with fillers of 60 mg C18, 1000 mg C18, 60 mg HLB and 200 mg HLB on target components are investigated, see the accompanying Figure 3 (A), finally, 60 mg of HLB solid phase extraction column is selected as the solid phase extraction column for pretreatment; Elution liquid optimization: the effects of extraction effects of sample solution under different elution solvent volumes on experimental results are investigated, see the accompanying Figure 3(B), it is shown that different elution volumes have little difference on the enrichment effect of target and the removal effect of impurities such as sugars, and finally the elution liquid is selected as 3 mL of deionized water; Elution volume optimization: the influence of the extraction effect of the sample solution under different elution volumes on the experimental results was investigated, and the results are shown in the following (C). Figure 3 (C), when the elution volume is 1 mL, the content of p-xylylene ether in the effluent is the highest, and when the elution volume is 3 mL, no p-xylylene ether is detected in the effluent, so the volume of the elution liquid is finally selected as 3 mL.

[0023] Limit of detection, limit of quantification, recovery rate and precision of the method The limit of detection of the method is 0.3 µg / kg corresponding to the sample concentration of not less than 3 times the signal-to-noise ratio (S / N≥3), and the limit of quantification of the method is 1.0 µg / kg corresponding to the sample concentration of not less than 10 times the signal-to-noise ratio (S / N≥10), and the multiple reaction monitoring (SRM) chromatogram corresponding to the quantification ion is shown in the following (D). Figure 4 .

[0024] The recovery rate and precision of the application are verified by adding standard solutions of low, medium and high concentrations to blank samples, and the specific operation is as follows: standard solutions of low, medium and high concentrations are added to blank honey samples (not containing p-xylylene ether), and the recovery rate is calculated by analyzing and detecting according to the above detection method, and the results are shown in Table 2. Table 2 Recovery rate and precision results of different addition levels n =6) Table 2 shows that under three concentration additions, the recovery rate and relative standard deviation of p-xylylene ether meet the analysis requirements, indicating that the method has high accuracy and precision, and the determination results are true and reliable.

[0025] Example 2 The application of p-xylylene ether in honey as a characteristic marker of tung honey according to the application embodiment includes the following steps: Sample source: the honey samples used in the application are all natural mature honey collected during the flowering and honey flow season of each honey plant, and all samples are directly purchased from bee farmers and stored at 4 ℃ before detection.

[0026] The honey samples of 7 different honey plants collected are detected by the analysis and detection method established by the application, and the specific types of honey samples include 24 batches of tung honey, 10 batches of jujube honey, 10 batches of rape honey, 10 batches of acacia honey, 10 batches of willow honey, 10 batches of litchi honey and 10 batches of linden honey.

[0027] The content of p-dianisole in all the above honey samples was detected, compared and analyzed, and it was found that p-dianisole was detected in tung flower honey only, and was not detected in other honey samples, see Appendix Figure 5 Therefore, it is finally determined that p-dianisole can be used as a characteristic marker of tung flower honey, and the content thereof in tung flower honey is 3.3-8.0 μg / kg.

[0028] According to the detection results of the above experimental samples, it can be determined that the basis for discriminating tung flower honey is: A) p-dianisole is detected, and the content thereof is in the range of 3.3-8.0 μg / kg, then the sample is pure tung flower honey; B) if p-dianisole is not detected, or the content of the target substance is lower than 3.3 μg / kg, then the sample is non-pure tung flower honey (doped or adulterated) or non-tung flower honey (other honey or non-honey).

[0029] To further verify, 7 batches of labeled tung flower honey samples were purchased from an online e-commerce platform, p-dianisole was detected by the method of the application, and the authenticity of each honey sample was discriminated by the above discrimination basis, and the results are shown in Table 3: Table 3 Determination results of p-dianisole in commercially available honey samples and authenticity identification of tung flower honey Note: ND not detected; Y pure tung flower honey, N non-pure tung flower honey or non-tung flower honey.

[0030] It was found that 3 batches of samples met the discrimination basis of the embodiment, and were determined to be real tung flower honey, and the content of p-dianisole in the other 2 batches of samples was lower than 3.3 μg / kg, p-dianisole was not detected in 2 batches of samples, and was identified as non-pure tung flower honey or non-tung flower honey. The results show that the method of the application has good practicability and can be used for authenticity identification of tung flower honey.

[0031] The basic principles, main features and advantages of the application are shown and described above. Those skilled in the art should understand that the application is not limited by the above embodiments, and the above embodiments and descriptions in the specification are only to illustrate the principles of the application. Without departing from the spirit and scope of the application, various changes and improvements can be made to the application, and these changes and improvements all fall within the scope of the claimed application. The scope of protection of the application is defined by the appended claims and their equivalents.

Claims

1. A method for the detection of p-xylylene oxide in honey, characterized in that: Comprise the following steps: Step one: sample processing Weigh the honey sample, dissolve it with deionized water and mix well; Step two: solid phase extraction Take the sample solution treated in step one for solid phase extraction, and enrich, purify and elute p-xylylene ether; Step three: determination Use gas chromatography-tandem mass spectrometry to qualitatively and quantitatively detect p-xylylene ether in the honey sample, and use the internal standard method for quantification, wherein the gas chromatography-tandem mass spectrometry detection conditions are as follows: Gas chromatography conditions: Chromatographic column: DB-5MS gas chromatography column, specifications: 30 m x 250 μm x 0.25 μm; Inlet temperature: 250 ℃; Programmed temperature: initial temperature 50 ℃, hold for 1 min, then increase to 250 ℃ at 30 ℃ / min, hold for 3 min, total running time 10.67 min; Column flow rate: 1.5 mL / min; Carrier gas: high-purity helium with purity ≥99.999%; Injection mode: splitless injection; Injection volume: 1 μL; Mass spectrometry conditions: Ion source: electron impact ion source EI; Transfer line temperature: 280 ℃; Ion source temperature: 280 ℃; Detection mode: multiple reaction monitoring scan mode.

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

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

4. The method for detecting p-xylylene oxide in honey according to claim 3, characterized by: In step two, the steps of solid phase extraction include: Install the solid phase extraction column on the vacuum filtration device, and use 3 mL of ethyl acetate to wash away impurities, then use 3 mL of methanol to activate, and 6 mL of deionized water to balance; Load the sample solution obtained in step one into the solid phase extraction column; Select 3 mL of deionized water for leaching, then dry with a vacuum pump; Finally, use 3 mL of ethyl acetate for elution, nitrogen blow at 30 ℃ to 1 ml, vortex mixing, then filter membrane filtration to 1 mL brown sample bottle for testing.

5. The method for detecting p-xylylene oxide in honey according to claim 4, characterized by the fact that: The flow rate of sample solution loading into the solid phase extraction column is controlled at 1 mL / min.

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

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

8. The method for detecting p-xylylene oxide in honey according to claim 7, characterized by: In the internal standard method quantification in step three, the internal standard curve equation of p-xylylene ether is: Y = 0.06324X + 0.00635, linear correlation coefficient R = 0.9998 2 = 0.9998; Wherein, X is the concentration of the target substance in the sample, and Y is the ratio of the peak area of the target substance to the internal standard substance in the sample.

9. The method for detecting p-xylylene oxide in honey according to claim 8, characterized by: The detection limit of this detection method is 0.3 μg / kg, and the quantification limit is 1.0 μg / kg; the recovery rate of this detection method is higher than 80%.

10. Use of the method according to any one of claims 1 to 9 for the identification of tung flower honey, characterized in that, If the content of p-xylylene ether in the honey sample is in the range of 3.3-8.0 μg / kg, the honey sample is judged to be real tung honey, otherwise, the honey sample is judged to be other honey or adulterated tung honey.

Images