Method for simultaneous detection of 3,4,5-trimethoxyl toluene (TMT) and 2,6-dimethoxyl-4-methyl phenol (DMMP) in honey and application thereof

Abstract

This invention discloses a method for the simultaneous detection of 3,4,5-trimethoxytoluene (TMT) and 2,6-dimethoxy-4-methylphenol (DMMP) in honey and its application. Solid-phase extraction is used for enrichment and purification, followed by gas chromatography-tandem mass spectrometry (GC-MS / MS) for detection. TMT and DMMP are quantified using the external standard method. Testing of 75 batches of honey samples showed that TMT and DMMP were detected only in Xinjiang safflower honey, with concentrations ranging from 0.59 to 1.98 mg / kg and 23.13 to 76.23 mg / kg, respectively. Therefore, honey samples falling within these ranges are identified as Xinjiang safflower honey; otherwise, they are identified as other types of honey or adulterated honey. This invention establishes a method for the simultaneous determination of TMT and DMMP in honey using gas chromatography-tandem mass spectrometry and applies it to the identification of single-flower honeys. This method is characterized by its simplicity, high accuracy, and good sensitivity, and has significant practical implications for the authenticity evaluation and quality control of Xinjiang safflower honey.

Description

Technical Field

[0001] This invention relates to the field of food detection and identification technology, and more specifically to a method for the simultaneous detection of 3,4,5-trimethoxytoluene (TMT) and 2,6-dimethoxy-4-methylphenol (DMMP) in honey, and its application. Background Technology

[0002] Xinjiang safflower (Carthamus tinctorius L.), belonging to the genus Carthamus in the Asteraceae family, is an annual herb rich in various bioactive substances. It possesses properties that promote blood circulation, reduce inflammation and swelling, and lower blood lipids, making it an important medicinal and nectar source plant in my country. The cultivation of Xinjiang safflower is mainly concentrated in Yumin County of Tacheng Prefecture, the Ili region, and Qitai County and Jimusar County of Changji Prefecture in northwestern China. Xinjiang safflower honey is produced by bees collecting nectar from Xinjiang safflower flowers, adding their own secretions for biotransformation, and finally storing it in the honeycomb. Due to the high medicinal value of its nectar source plant, Xinjiang safflower honey has been developed into a specialty honey. With the rise of the health industry, the market demand for Xinjiang safflower honey is gradually increasing. However, due to its limited production, unscrupulous merchants often use other monofloral honeys to impersonate Xinjiang safflower honey, or blend Xinjiang safflower honey with other cheap monofloral honeys and fructose syrup. The problem of honey adulteration has seriously damaged the legitimate rights and interests of consumers and harmed the healthy development of the bee product industry. Therefore, it is urgent to develop a practical and effective method for identifying the authenticity of Xinjiang safflower honey.

[0003] Different monofloral honeys have their characteristic components, such as cyanopyridine in jujube honey, hexanoic acid in rice honey, and linden resin in linden honey. These unique compounds have been used as characteristic compounds for the authenticity identification of monofloral honeys. Experiments have shown that TMT and DMMP can be used as characteristic components for identifying the authenticity of Xinjiang safflower honey. Currently, there are no known detection methods for 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. The method involves enrichment and purification followed by detection using gas chromatography-tandem mass spectrometry and is applied to honey identification.

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

[0006] First, this invention provides a method for the simultaneous detection of 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 out a honey sample, dissolve it in deionized water and mix well;

[0009] Step 2: Solid-phase extraction

[0010] The sample solution after step one was subjected to solid-phase extraction to enrich, purify and elute TMT and DMMP.

[0011] Step 3: Measurement

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

[0013] The detection conditions for gas chromatography-tandem mass spectrometry are as follows:

[0014] Gas chromatography conditions:

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

[0016] Inlet temperature: 270℃;

[0017] The temperature is programmed to rise from an initial temperature of 60°C for 1 minute, then rise to 270°C at a rate of 20°C / min and hold for 3 minutes.

[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); Transmission line temperature: 270℃; Ion source temperature: 250℃; Detection method: Multiple reaction monitoring (SRM) mode.

[0024]

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

[0026] Preferably, the honey dissolving and mixing process in step one is carried out by vortex mixing at 1000-2000 rpm.

[0027] Preferably, the solid-phase extraction step in step two is 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) Transfer the sample solution obtained in step one to the activated extraction column;

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

[0031] (2.4) Finally, use 3 mL of methanol to elute, blow the eluent dry with nitrogen at 40°C, then redissolve with 3 mL of acetone, vortex at 1000-2000 rpm to mix, filter the membrane and place it in a brown sample vial for testing.

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

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

[0034] In the preferred step three, the external standard method for quantification uses the following external standard curve equation:

[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 analyte in the sample, and Y is the peak area of ​​the target analyte in the sample.

[0038] Preferably, the detection limits for TMT and DMMP in the method are both 0.02 mg / kg, the quantitation limits are both 0.06 mg / kg, and the recoveries are both >80%.

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

[0040] Furthermore, Xinjiang safflower honey contains TMT and DMMP. By weight, the content of TMT and DMMP in Xinjiang safflower honey ranges from 0.59 to 1.98 mg / kg and 23.13 to 76.23 mg / kg, respectively.

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

[0042] This invention identifies TMT and DMMP as characteristic markers of Xinjiang safflower honey and proposes a method for simultaneously detecting TMT and DMMP in honey using gas chromatography-tandem mass spectrometry. Based on their content, Xinjiang safflower honey can be effectively identified. 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 detection method for TMT and DMMP in honey provided by this invention has the advantages of being simple and quick to operate, highly practical, highly sensitive, and accurate and precise. It can effectively separate and accurately quantify TMT and DMMP in honey. This method is of great significance for the identification of the authenticity and quality evaluation of honey, and at the same time provides new indicators for protecting the rights and interests of honey consumers and maintaining the healthy and orderly development of the honey consumption market. Attached Figure Description

[0044] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0045] Figure 1 Linear curves for TMT and DMMP;

[0046] Figure 2 Chromatograms of TMT and DMMP using multiple reaction monitoring (SRM).

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

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

[0049] Figure 5 This is a graph showing the TMT and DMMP content in a single-flower honey sample. Detailed Implementation

[0050] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

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

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

[0053] MPS Multifunctional Sample Pretreatment Platform (Gerstel GmbH, 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 pipettes: 20μL, 100μL, 200μL, 1mL, 5mL (ThermoFisher);

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

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

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

[0062] Acetone: chromatographic grade (99.9% purity, Dima Company);

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

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

[0065] All water used in the experiments was ultrapure water.

[0066] The specific measurement steps are as follows:

[0067] Step 1: Plotting 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 a 1000 mg / L standard stock solution. Store at 4 °C.

[0069] (1.2) Preparation of standard working solutions: Accurately transfer 1 mL of each of the above standard stock solutions into a 10 mL volumetric flask, and dilute to the mark with acetone to prepare a 100 mg / L mixed standard working solution; accurately transfer 0.1 mL of the above standard stock solutions into a 10 mL volumetric flask, and dilute to the mark with acetone to 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 mixed standard working solution and dilute it with acetone to prepare 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) Plot a standard curve with the peak areas of TMT and DMMP quantitative ions as the Y-axis and the concentration of the target component as the X-axis. The linear curve is shown in the appendix. Figure 1 The standard curve equations and linear correlation coefficients for TMT and DMMP are shown in Table 1:

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

[0073]

[0074] Step 2: Sample processing and determination

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

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

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

[0078] (2.4) Elute with 3 mL of methanol solution, controlling the flow rate to about 1 mL / min, then blow dry with nitrogen at 40 °C, and finally redissolve with 3 mL of acetone, vortex at 1000-2000 rpm for later use.

[0079] (2.5) Pass the above solution through a 0.22 μm nylon filter membrane and transfer it to a brown vial for gas chromatography-tandem mass spectrometry detection. The detection conditions are as follows:

[0080] Gas chromatography conditions:

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

[0082] Inlet temperature: 270℃;

[0083] The temperature is increased by 60°C initially and held for 1 minute, then increased to 270°C at a rate of 20°C / min and held for 3 minutes.

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

[0085] Mass spectrometry conditions:

[0086] Ion source: Electron impact ion source (EI); Transmission line temperature: 270℃; Ion source temperature: 250℃; Detection method: Multiple reaction monitoring (SRM) scan mode; where,

[0087]

[0088] The chromatograms of TMT and DMMP multiple reaction monitoring (SRM) are attached. 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 different eluent volumes on the experimental results was investigated (see Appendix). Figure 3 The results showed that different rinsing volumes had little difference in the removal effect on the content of the target substance and impurities such as sugars, and the final rinsing solution was 3 mL of deionized water.

[0092] Elution buffer volume optimization: When the eluent volume was 1 mL, the content of TMT and DMMP in the effluent was the highest. When the eluent volume was 3 mL, no target substances were detected in the effluent. Therefore, the final eluent volume was selected as 3 mL.

[0093] Limits of detection, limits of quantitation, recovery, and precision of the method:

[0094] The detection limits for TMT and DMMP at a signal-to-noise ratio (S / N ≥ 3) of 0.02 mg / kg were obtained; the method limits of quantification at a signal-to-noise ratio (S / N ≥ 10) of 0.06 mg / kg were obtained. The extracted ion chromatograms for the corresponding quantitative ions of the two substances are attached. Figure 4 .

[0095] 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 (low, medium, and high) of standard working solutions (see Table 2) were added to a blank honey sample (excluding TMT and DMMP). The sample was analyzed and the recovery rate was calculated according to the above detection method. The results are shown in Table 2.

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

[0097]

[0098] Table 2 shows that the recoveries and relative standard deviations of TMT and DMMP 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.

[0099] Example 1

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

[0101] Sample source: The honey samples used in this study were 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°C before testing.

[0102] 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: 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 above honey samples were detected, compared, and analyzed. The results showed that TMT and DMMP were only found in Xinjiang safflower honey (see attached image). Figure 5 Therefore, it was ultimately determined that TMT and DMMP can be used to differentiate and identify Xinjiang safflower honey and other honey varieties.

[0104] Based on the test results of the above experimental samples, the following criteria can be used to identify the plant source of Xinjiang safflower honey:

[0105] A) If TMT and DMMP are detected, and their contents are 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 if the content of the detected target substance is not within the range described in A, then the sample is not pure Xinjiang safflower honey (adulterated or fake) or not Xinjiang safflower honey (other types of honey or non-honey).

[0107] To further verify this, five batches of honey samples were purchased from online e-commerce platforms. The TMT and DMMP tests were performed using the method of this invention, and the authenticity of each honey sample was determined using the aforementioned criteria. The results are shown in Table 3.

[0108] Table 3. Determination results of TMT and DMMP in commercially available honey samples and their authenticity identification.

[0109] 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 Authentic Xinjiang safflower honey; b Not detected; c This is not genuine Xinjiang safflower honey.

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

[0112] The various embodiments described in this specification are presented in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for simultaneous detection of 3,4,5-trimethoxytoluene (TMT) and 2,6-dimethoxy-4-methylphenol (DMMP) 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 TMT and DMMP. The solid-phase extraction steps are as follows: (2.1) Install the HLB solid-phase extraction column on a vacuum filtration device, activate it with 3 mL of methanol and then equilibrate it with 6 mL of deionized water; (2.2) Transfer the sample solution obtained in step one to the activated extraction column; (2.3) Rinse with 3 mL of deionized water, then dry with a vacuum pump; (2.4) Finally, 3 mL of methanol was used for elution. The eluent was dried under nitrogen at 40 °C, then reconstituted with 3 mL of acetone, vortexed at 1000~2000 rpm, filtered through the membrane, and placed in a brown sample vial for testing. Step 3: Measurement Gas chromatography-tandem mass spectrometry was used to qualitatively and quantitatively detect TMT and DMMP in honey samples, and external standard method was used for quantification. The detection conditions for gas chromatography-tandem mass spectrometry are as follows: Gas chromatography conditions: Chromatographic column: DB-5MS gas chromatographic column, 30 m × 0.25 mm × 0.25 μm; Inlet temperature: 270 ℃; The temperature was programmed to rise initially to 60 °C, held for 1 min, and then increased to 270 °C at a rate of 20 °C / min, held for 3 min. Column flow rate: 1.5 mL / min; Carrier gas: High-purity helium; Injection method: splitless injection; Injection volume: 1 mL; Mass spectrometry conditions: Ion source: Electron impact ion source (EI); Transmission line temperature: 270 ℃; Ion source temperature: 250 ℃; Detection method: Multiple reaction monitoring (SRM) mode. 。 2. The method for simultaneous detection of 3,4,5-trimethoxytoluene (TMT) and 2,6-dimethoxy-4-methylphenol (DMMP) in honey according to claim 1, characterized in that, In step one, the mass-to-volume ratio of honey to water is 0.2 g / mL.

3. The method for simultaneous detection of 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 one is carried out by vortex mixing at 1000~2000 rpm.

4. The method for simultaneous detection of 3,4,5-trimethoxytoluene (TMT) and 2,6-dimethoxy-4-methylphenol (DMMP) in honey according to claim 1, characterized in that, The filter membrane is a 0.22 μm nylon filter membrane.

5. The method for simultaneous detection of 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 three, the equation of the external standard curve 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 analyte in the sample, and Y is the peak area of ​​the target analyte in the sample.

6. 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 1-5, characterized in that, In the method described, the limits of detection for TMT and DMMP were both 0.02 mg / kg, the limits of quantitation were both 0.06 mg / kg, and the recoveries were both >80%.

7. The application of the method according to any one of claims 1-6 in the identification of Xinjiang safflower honey.

8. The application according to claim 7, characterized in that, Xinjiang safflower honey contains TMT and DMMP. By weight, the content of TMT and DMMP in Xinjiang safflower honey ranges from 0.59 to 1.98 mg / kg and 23.13 to 76.23 mg / kg, respectively.

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