Method for simultaneously detecting 3, 4, 5-trimethoxytoluene (TMT) and 2, 6-dimethoxy-4-methylphenol (DMMP) in honey and application of method

Abstract

The invention discloses a method for simultaneously detecting 3, 4, 5-trimethoxytoluene (TMT) and 2, 6-dimethoxy-4-methylphenol (DMMP) in honey and application of the method, solid-phase extraction is adopted for enrichment and purification, a gas chromatography-tandem mass spectrometer is used for detection, and TMT and DMMP are quantified by an external standard method. The detection results of 75 batches of honey samples show that the TMT and the DMMP are only detected in the Xinjiang safflower honey, and the content ranges of the TMT and the DMMP are respectively 0.59 mg / kg to 1.98 mg / kg and 23.13 mg / kg to 76.23 mg / kg. Therefore, when the honey sample is within the range, the honey sample is judged to be Xinjiang safflower honey, and otherwise, the honey sample is judged to be other honey or adulterated honey. The method for simultaneously determining the TMT and the DMMP in the honey through the gas chromatography-tandem mass spectrometry is established and applied to identification of the single-flower honey, has the advantages of being easy and convenient to operate, high in accuracy and good in sensitivity, and has important practical significance on authenticity evaluation and quality control of the Xinjiang safflower honey.

Description

Technical Field

[0001] The invention relates to the technical field of food detection and identification, and more specifically to a method for simultaneously detecting 3,4,5-trimethoxytoluene (TMT) and 2,6-dimethoxy-4-methylphenol (DMMP) in honey and an application thereof. Background Art

[0002] Xinjiang safflower (Carthamus tinctorius L.), an annual herb belonging to the genus Xinjiang safflower of the Asteraceae family, is rich in a variety of biological activities, has the effects of promoting blood circulation and removing blood stasis, reducing inflammation and swelling, and lowering blood lipids. It is an important medicinal nectar source plant in my country. The cultivation of Xinjiang safflower is mainly concentrated in Yumin County, Tacheng, Yili Prefecture, and Qitai County and Jimusar County in Changji Prefecture in the northwest. Xinjiang safflower honey is a substance that bees collect Xinjiang safflower nectar, add their own secretions for biotransformation, and finally store in the hive. Xinjiang safflower honey has been developed into a special honey because its nectar plant has high medicinal value. With the rise of the big health industry, the market demand for Xinjiang safflower honey has gradually increased, but due to its limited production, unscrupulous merchants often use other single flower honeys to impersonate Xinjiang safflower honey, or blend other cheap single flower honeys and fructose syrup in Xinjiang safflower honey. The problem of honey adulteration has seriously damaged the legitimate rights and interests of consumers and endangered the healthy development of the bee products industry. Therefore, it is urgent to develop a practical and effective method for authenticity identification of Xinjiang safflower honey.

[0003] Different single-flower honeys have their own characteristic ingredients, such as tricyanopyridine in jujube honey, hexanoic acid in rice honey, tiliatin in linden honey, etc. These unique compounds have been used as characteristic compounds to identify the authenticity of single-flower honey. Through experiments, it was found that TMT and DMMP can be used as characteristic ingredients to identify the authenticity of Xinjiang safflower honey. At present, there is no relevant detection method for detecting TMT and DMMP in honey. Summary of the invention

[0004] In view of this, the present invention provides a qualitative and quantitative detection method for TMT and DMMP in honey samples and applies it to honey detection, which is first enriched and purified and then detected by gas chromatography-tandem mass spectrometry, and is applied to honey identification.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] First, the present invention provides a method for simultaneously detecting 3,4,5-trimethoxytoluene TMT and 2,6-dimethoxy-4-methylphenol DMMP in honey, comprising the following steps:

[0007] Step 1: Sample processing

[0008] Weigh the honey sample, dissolve it with deionized water and mix well;

[0009] Step 2: Solid Phase Extraction

[0010] The sample solution treated in step 1 is subjected to solid phase extraction to enrich, purify and elute TMT and DMMP;

[0011] Step 3: Determination

[0012] Gas chromatography-tandem mass spectrometry was used to qualitatively and quantitatively detect TMT and DMMP in honey samples, and the external standard method was used for quantification;

[0013] Wherein, the gas chromatography-tandem mass spectrometry detection conditions are:

[0014] Gas chromatography conditions:

[0015] Chromatographic column: DB-5MS gas chromatography column, 30m×0.25mm×0.25μm;

[0016] Inlet temperature: 270°C;

[0017] The temperature was programmed to start at 60 °C, hold for 1 min, then increase to 270 °C at a rate of 20 °C / min, and hold for 3 min.

[0018] Column flow rate: 1.5 mL / min;

[0019] Carrier gas: high purity helium;

[0020] Injection method: splitless injection;

[0021] Injection volume: 1 μL;

[0022] Mass spectrometry conditions:

[0023] Ion source: electron impact ion source EI; transfer line temperature: 270°C; ion source temperature: 250°C; detection method: multiple reaction monitoring scanning mode SRM, in which,

[0024]

[0025] Preferably, in step 1, the mass volume ratio of honey to water is 0.2 g / mL.

[0026] Preferably, the honey dissolving and mixing process described in step 1 adopts vortex mixing at 1000-2000 rpm.

[0027] Preferably, the solid phase extraction step in step 2 is specifically as follows:

[0028] (2.1) Install the extraction column on a vacuum filtration device, activate it with 3 mL of methanol, and then balance it with 6 mL of deionized water;

[0029] (2.2) transferring the sample solution obtained in step 1 to the activated extraction column;

[0030] (2.3) Rinse with 3 mL of deionized water and then dry with a vacuum pump;

[0031] (2.4) Finally, use 3 mL of methanol for elution. Blow the eluent to dryness with nitrogen at 40°C, then reconstitute with 3 mL of acetone, vortex mix at 1000-2000 rpm, filter the membrane and place it in a brown injection bottle for testing.

[0032] Furthermore, the extraction column adopts an HLB solid phase extraction column.

[0033] Furthermore, the filter membrane is a 0.22 μm nylon filter membrane.

[0034] In the external standard method quantification described in the preferred step 3, the external standard curve equation is:

[0035] TMT: Y = 2.302e4X-3.352e5, linear correlation coefficient R 2 =0.9992;

[0036] DMMP: Y = 2.603e3X + 1.273e4, linear correlation coefficient R 2 =0.9994;

[0037] Where X is the concentration of the target in the sample, and Y is the peak area of ​​the target in the sample.

[0038] Preferably, in the method, the detection limits of TMT and DMMP are both 0.02 mg / kg, the quantification limits are both 0.06 mg / kg, and the recovery rates are both >80%.

[0039] The present invention also provides an application of the method described in the above technical solution in the identification of Xinjiang safflower honey.

[0040] Furthermore, Xinjiang safflower honey contains TMT and DMMP. Based on the weight of honey, the contents of TMT and DMMP in Xinjiang safflower honey range from 0.59-1.98 mg / kg and 23.13-76.23 mg / kg, respectively.

[0041] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a method for simultaneously detecting 3,4,5-trimethoxytoluene TMT and 2,6-dimethoxy-4-methylphenol DMMP in honey and its application, which has the following beneficial effects:

[0042] The invention determines that TMT and DMMP can be used as characteristic markers of Xinjiang safflower honey, and proposes a detection method for simultaneously detecting TMT and DMMP in honey by gas chromatography-tandem mass spectrometry. Xinjiang safflower honey can be effectively identified according to their contents. The content range of TMT in pure Xinjiang safflower honey is 0.59-1.98 mg / kg, and the content range of DMMP is 23.13-76.23 mg / kg.

[0043] The method for detecting TMT and DMMP in honey provided by the present invention has the advantages of simple and quick operation, strong practicality, high sensitivity, good accuracy and precision, and can achieve effective separation and accurate quantification of TMT and DMMP in honey. The method is of great significance for the authenticity identification and quality evaluation of honey, and at the same time provides a new indicator for protecting the rights and interests of honey consumers and maintaining the healthy and orderly development of the honey consumption market. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0045] Figure 1 is the linear curve of TMT and DMMP;

[0046] Figure 2 is the multiple reaction monitoring (SRM) chromatogram of TMT and DMMP;

[0047] Figure 3 The effect of elution volume on extraction effect;

[0048] Figure 4 The multiple reaction monitoring chromatograms and signal-to-noise ratios of TMT and DMMP quantification ions at the limit of quantification (LOQ) level;

[0049] Figure 5 This is a graph showing the TMT and DMMP contents in single flower honey samples. DETAILED DESCRIPTION

[0050] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] The instruments, reagents and materials in the following examples are as follows:

[0052] Gas chromatography-tandem mass spectrometer: TSQ 8000 (Thermo Fisher Scientific, USA);

[0053] MPS multifunctional sample pretreatment platform (Gerstel, Germany);

[0054] Chromatographic column: Agilent DB-5MS capillary column (30m×0.25mm×0.25μm);

[0055] Vortex mixer: Vortex-Genie2 (Scientific Industries);

[0056] Electronic analytical balance: XP105 (Mettler Toledo);

[0057] Ultrapure water generator: Mili-Q (Millipore);

[0058] Micro-adjustable pipette: range 20μL, 100μL, 200μL, 1mL, 5mL (ThermoFisher);

[0059] TMT standard: purity 98% (McLean, Shanghai McLean Biochemical Technology Co., Ltd.);

[0060] DMMP standard: purity 97% (McLean, Shandong Keyuan Biochemical Co., Ltd.);

[0061] Volumetric flask: 10mL (Tianbo Glass Instrument Co., Ltd.)

[0062] Acetone: chromatographically pure (purity 99.9%, Dima Company);

[0063] Methanol: chromatographic grade (purity 99.9%, Dima Company);

[0064] Vacuum pump: DOA-P504-BN diaphragm vacuum pump (GAST, USA);

[0065] Ultrapure water was used in all tests.

[0066] The specific measurement steps are as follows:

[0067] Step 1: Draw the standard curve

[0068] (1.1) Preparation of standard stock solution: Accurately weigh 0.01 g of TMT and DMMP standards and place them in 10 mL volumetric flasks, respectively. Dissolve them in pure acetone and dilute to the mark to prepare 1000 mg / L standard stock solutions. Store at 4 °C.

[0069] (1.2) Preparation of standard working solution: Accurately pipette 1 mL of each of the above standard stock solutions into a 10 mL volumetric flask, dilute to the mark with acetone, and prepare a 100 mg / L mixed standard working solution; Accurately pipette 0.1 mL of the above standard stock solutions into a 10 mL volumetric flask, dilute to the mark with acetone, and prepare a 10 mg / L mixed standard working solution, and store at 4°C;

[0070] (1.3) Preparation of a series of standard solutions: Take an appropriate amount of the mixed standard working solution and dilute it with acetone to form a series of standard solutions with concentrations of 0.02 mg / L, 0.05 mg / L, 0.10 mg / L, 0.20 mg / L, 0.50 mg / L and 1.00 mg / L respectively;

[0071] (1.4) With the peak area of ​​TMT and DMMP quantitative ions as the Y-axis and the concentration of the target component as the X-axis, a standard curve is drawn. The linear curve is shown in the attached figure. Figure 1 , TMT and DMMP standard curve equations and linear correlation coefficients are shown in Table 1:

[0072] Table 1 Linear equations and correlation coefficients of TMT and DMMP standard curves

[0073]

[0074] Step 2: Sample processing and measurement

[0075] (2.1) Accurately weigh 1 g (accurate to 0.01 g) of honey sample into a 50 mL centrifuge tube, add 5 mL of deionized water, and vortex mix at 1000-2000 rpm for later use;

[0076] (2.2) Install the HLB solid phase extraction column on a solid phase extraction vacuum filtration device, activate it with 3 mL of methanol, and equilibrate the extraction column with 6 mL of deionized water;

[0077] (2.3) Transfer the honey sample solution in (2.1) above to the activated HLB solid phase extraction column and drip it at a rate of about 1 mL / min. After the loading is completed, rinse it with 3 mL of deionized water and finally dry it with a vacuum pump;

[0078] (2.4) Elute with 3 mL of methanol solution at a flow rate of about 1 mL / min, blow dry with nitrogen at 40°C, and finally reconstitute with 3 mL of acetone, vortex mix at 1000-2000 rpm for later use;

[0079] (2.5) The above solution was filtered through a 0.22 μm nylon filter membrane and transferred to a brown injection vial for gas chromatography-tandem mass spectrometry detection. The detection conditions were as follows:

[0080] Gas chromatography conditions:

[0081] Chromatographic column: DB-5MS gas chromatography column (30m×0.25mm×0.25μm);

[0082] Inlet temperature: 270°C;

[0083] The temperature was programmed to start at 60°C, hold for 1 min, then increase to 270°C at 20°C / min, and hold for 3 min.

[0084] Column flow rate: 1.5mL / min; Carrier gas: high-purity helium; Injection method: splitless injection; Injection volume: 1μL;

[0085] Mass spectrometry conditions:

[0086] Ion source: electron impact ion source EI; transfer line temperature: 270°C; ion source temperature: 250°C; detection method: multiple reaction monitoring scanning mode SRM; among them,

[0087]

[0088] See the attached picture for the multiple reaction monitoring (SRM) chromatograms of TMT and DMMP. Figure 2 .

[0089] Optimization and screening of experimental conditions:

[0090] The experimental conditions were optimized by selecting high-content Xinjiang safflower honey:

[0091] Eluent optimization: The effect of sample solution on the experimental results at different eluent volumes was investigated. Figure 3 , indicating that different elution volumes have little difference in the removal effect on the content of the target and impurities such as sugars, and the final elution solution is 3mL of deionized water;

[0092] Optimization of eluent volume: When the eluent volume was 1 mL, the contents of TMT and DMMP in the effluent were the highest. When the eluent volume reached 3 mL, no target was detected in the effluent, so the final eluent volume was 3 mL.

[0093] Detection limit, quantification limit, recovery and precision of the method:

[0094] The detection limit of TMT and DMMP was 0.02 mg / kg when the sample concentration was not less than 3 times the signal-to-noise ratio (S / N≥3); the quantification limit of TMT and DMMP was 0.06 mg / kg when the sample concentration was not less than 10 times the signal-to-noise ratio (S / N≥10). The extracted ion chromatograms of the quantitative ions of the two substances are shown in the attached Figure 4 .

[0095] The recovery rate and precision of the present invention were verified by using a blank sample spike recovery method. The specific operation is as follows: standard working solutions of low, medium and high concentration levels (see Table 2) were added to blank honey samples (without TMT and DMMP), and the analysis and detection were performed according to the above detection method, and the recovery rate was calculated. The results are shown in Table 2:

[0096] Table 2 Recovery and precision results at different addition levels (n=6)

[0097]

[0098] Table 2 shows that at three concentrations, the recoveries and relative standard deviations of TMT and DMMP met the analytical requirements, indicating that this method has high accuracy and precision, and the determination results are true and reliable.

[0099] Example 1

[0100] Establishment and application of TMT and DMMP as honey characteristic markers:

[0101] Sample source: The honey samples used in this study were all natural mature honeys collected from the flowering and nectar flow seasons of various nectar-producing plants. All samples were purchased directly from beekeepers and stored at 4°C before testing.

[0102] By using the analytical detection method established by the present invention, honey samples collected from seven different nectar plants were detected, specifically including: 15 batches of Xinjiang safflower honey, 10 batches of acacia honey, 10 batches of vitex honey, 10 batches of linden honey, 10 batches of jujube honey, 10 batches of rapeseed honey, and 10 batches of fennel honey.

[0103] The contents of TMT and DMMP in all the honey samples were tested, compared and analyzed. The results showed that TMT and DMMP were only found in Xinjiang safflower honey. Figure 5 Therefore, it was finally determined that TMT and DMMP can be used to distinguish and identify Xinjiang safflower honey and other honey types.

[0104] According to the test results of the above experimental samples, the identification basis of the following Xinjiang safflower honey plant source can be determined as follows:

[0105] A) TMT and DMMP are detected, and the content is in the range of 0.59-1.98 mg / kg and 23.13-76.23 mg / kg respectively, then the sample is pure Xinjiang safflower honey;

[0106] B) If neither TMT nor DMMP is detected, or the detected target content is not within the range described in A, the sample is not pure Xinjiang safflower honey (adulterated or counterfeit) or non-Xinjiang safflower honey (other types of honey or non-honey).

[0107] For further verification, 5 batches of honey samples were purchased from an online e-commerce platform, and the method of the present invention was used to detect TMT and DMMP. The authenticity of each honey sample was judged using the above-mentioned discrimination basis. The results are shown in Table 3:

[0108] Table 3 Determination results of TMT and DMMP in commercial honey samples and authenticity identification

[0109] serial number TMT(mg / kg) DMMP(mg / kg) Authenticity determination A1 0.81 24.42 <![CDATA[Y a ]]> A2 1.515 45.57 Y A3 <![CDATA[ND b ]]> ND <![CDATA[N c ]]> A4 0.95 34.41 Y A5 ND ND N

[0110] Note: a Real Xinjiang safflower honey; b Not detected; c Not authentic Xinjiang safflower honey.

[0111] The results showed that 3 of the 5 batches of Xinjiang safflower honey samples met the identification criteria of this embodiment and were determined to be authentic Xinjiang safflower honey, and the other 2 batches of samples were identified as non-authentic Xinjiang safflower honey. This result shows that the method of the present invention has good practicality and can be used for authenticity identification of Xinjiang safflower honey.

[0112] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments, and the same and similar parts between the embodiments can be referred to each other. The above description of the disclosed embodiments enables professionals and technicians in this field to implement or use the present invention. Various modifications to these embodiments will be obvious to professionals and technicians in this field, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown in this article, but will comply with the widest range consistent with the principles and novel features disclosed herein.

Claims

1. A method for simultaneously detecting 3,4,5-trimethoxytoluene (TMT) and 2,6-dimethoxy-4-methylphenol (DMMP) in honey, characterized in that: The following steps are involved: Step 1: Sample processing Weigh the honey sample, dissolve it with deionized water and mix it well; Step 2: Solid Phase Extraction The sample solution treated in step 1 is subjected to solid phase extraction to enrich, purify and elute TMT and DMMP; Step 3: Determination Gas chromatography-tandem mass spectrometry was used to qualitatively and quantitatively detect TMT and DMMP in honey samples, and the external standard method was used for quantification; Wherein, the gas chromatography-tandem mass spectrometry detection conditions are: Gas chromatography conditions: Chromatographic column: DB-5MS gas chromatography column, 30m×0.25mm×0.25μm; Inlet temperature: 270°C; The temperature was programmed to be 60 °C initially, maintained for 1 min, then raised to 270 °C at a rate of 20 °C / min, and maintained for 3 min. Column flow rate: 1.5 mL / min; Carrier gas: high purity helium; Injection method: splitless injection; Injection volume: 1 μL; Mass spectrometry conditions: Ion source: electron impact ion source EI; transfer line temperature: 270°C; ion source temperature: 250°C; detection method: multiple reaction monitoring scanning mode SRM, in which, 2. The method for simultaneously detecting 3,4,5-trimethoxytoluene (TMT) and 2,6-dimethoxy-4-methylphenol (DMMP) in honey according to claim 1, characterized in that: The mass volume ratio of honey and water in step 1 is 0.2 g / mL.

3. The method for simultaneously detecting 3,4,5-trimethoxytoluene (TMT) and 2,6-dimethoxy-4-methylphenol (DMMP) in honey according to claim 1, characterized in that: The honey dissolving and mixing process described in step 1 adopts vortex mixing at 1000-2000 rpm.

4. The method for simultaneously detecting 3,4,5-trimethoxytoluene (TMT) and 2,6-dimethoxy-4-methylphenol (DMMP) in honey according to claim 1, characterized in that: The solid phase extraction step described in step 2 is specifically as follows: (2.1) Install the extraction column on a vacuum filtration device, activate it with 3 mL of methanol, and then balance it with 6 mL of deionized water; (2.2) transferring the sample solution obtained in step 1 to the activated extraction column; (2.3) Rinse with 3 mL of deionized water and then dry with a vacuum pump; (2.4) Finally, use 3 mL of methanol for elution. Blow the eluent to dryness with nitrogen at 40°C, then reconstitute with 3 mL of acetone, vortex mix at 1000-2000 rpm, filter the membrane and place it in a brown injection bottle for testing.

5. The method for simultaneously detecting 3,4,5-trimethoxytoluene (TMT) and 2,6-dimethoxy-4-methylphenol (DMMP) in honey according to claim 4, characterized in that: The extraction column adopts an HLB solid phase extraction column.

6. The method for simultaneously detecting 3,4,5-trimethoxytoluene (TMT) and 2,6-dimethoxy-4-methylphenol (DMMP) in honey according to claim 4, characterized in that: The filter membrane is a 0.22 μm nylon filter membrane.

7. The method for simultaneously detecting 3,4,5-trimethoxytoluene (TMT) and 2,6-dimethoxy-4-methylphenol (DMMP) in honey according to claim 1, characterized in that: In the external standard method quantification described in step 3, the external standard curve equation is: TMT: Y = 2.302e4X-3.352e5, linear correlation coefficient R 2 =0.9992; DMMP: Y = 2.603e3X + 1.273e4, linear correlation coefficient R 2 =0.9994; Where X is the concentration of the target in the sample, and Y is the peak area of ​​the target in the sample.

8. The method for simultaneous detection of 3,4,5-trimethoxytoluene (TMT) and 2,6-dimethoxy-4-methylphenol (DMMP) in honey according to any one of claims 1 to 7, characterized in that: In the method, the detection limits of TMT and DMMP are both 0.02 mg / kg, the quantification limits are both 0.06 mg / kg, and the recovery rates are both >80%.

9. Use of the method according to any one of claims 1 to 8 in identifying Xinjiang safflower honey.

10. The use according to claim 9, characterized in that: Xinjiang safflower honey contains TMT and DMMP. Calculated by honey weight, the contents of TMT and DMMP in Xinjiang safflower honey range from 0.59-1.98 mg / kg and 23.13-76.23 mg / kg, respectively.

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