Ultra-high performance liquid chromatography fingerprint method for detecting Zanhuang triploid jujube honey

Abstract

The invention discloses an ultra-high performance liquid chromatography fingerprint method for detecting Zanhuang triploid jujube honey, and belongs to the technical field of analytical chemistry. The method comprises the following steps: extracting a to-be-detected sample with trifluoroacetic acid and acetonitrile, concentrating and redissolving, determining the fingerprint spectrum of a sample solution under the wavelength of 213nm by using high performance liquid chromatography, comparing and calculating the similarity between the sample and a Zanhuang jujube flower honey control fingerprint spectrum by using traditional Chinese medicine fingerprint spectrum similarity software, and identifying the sample according to the similarity. Therefore, the detection method disclosed by the invention can be used for simultaneously analyzing the overall composition and proportional relation of various components to form an overall'fingerprint ', so that the identification accuracy is improved. Meanwhile, the detection method is higher in efficiency, and a large amount of chemical information can be obtained through one-time instrument analysis. By combining automatic sample pretreatment equipment and intelligent data processing software, high-throughput screening can be realized, and the method is more suitable for market supervision and large-scale quality control.

Description

Technical Field

[0001] The invention relates to the technical field of analytical chemistry, and in particular to an ultra-high performance liquid chromatography fingerprint method for detecting Zanhuang triploid jujube flower honey. Background Art

[0002] Zanhuang Triploid Jujube Flower Honey is collected by bees from triploid jujube trees in Zanhuang County, Hebei Province. Zizyphus jujuba Mill ) nectar, mixed with the plant's own secretions and then fully brewed into a natural sweetener. Jujube flower honey, as the main single-flower honey variety in my country, is deeply loved by people for its unique health and beauty benefits. Due to the limited production of jujube flower honey and the high production cost, the price of jujube flower honey is significantly higher than other common single-flower honeys. In pursuit of high profits, unscrupulous companies often use other cheap honeys to impersonate jujube flower honey, or blend jujube flower honey with other cheap honeys, or even worse, blend jujube flower honey with fructose syrup. At present, my country has not yet formulated standards for jujube flower honey. How to effectively evaluate and identify jujube flower honey has become one of the urgent problems that need to be solved in the current bee products industry. Therefore, it is very necessary to develop a rapid, efficient and accurate detection method for the detection of Zanhuang triploid jujube flower honey. Summary of the Invention

[0003] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an ultra-high performance liquid chromatography fingerprint method for detecting Zanhuang triploid jujube flower honey, so as to provide an efficient and accurate method for detecting Zanhuang triploid jujube flower honey.

[0004] The technical solution of the present invention to solve the above technical problems is as follows: providing an ultra-high performance liquid chromatography fingerprint method for detecting Zanhuang triploid jujube flower honey, comprising the following steps: (1) Prepare reference solution; (2) Prepare control sample solution; (3) Prepare sample solution; (4) Determine the chromatographic conditions for UPLC: ① Mobile phase: Phase A is 2 wt% acetonitrile aqueous solution (containing 0.004 wt% trifluoroacetic acid), and phase B is acetonitrile; ②Detection wavelength: 213nm; ③ Gradient elution program: 0→3 min→3.01 min→7 min→7.01 min→11 min; Mobile phase A: 95%→65%→5%→5%→95%→95%; Mobile phase B: 5%→35%→95%→95%→5%→5%; (5) Ultra-high performance liquid chromatography detection: aspirate the reference solution of step (1), the control sample solution of step (2), and the sample solution of step (3) respectively, perform ultra-high performance liquid chromatography detection, and record the chromatogram from 0 min to 5 min; (6) Establishing fingerprints: Integrate the characteristic chromatographic peaks of the reference solution, control sample solution, and test sample solution obtained in step (5), then use the Chinese medicine chromatographic fingerprint similarity evaluation system version A to establish a Zanhuang Jujube Flower Honey control template, and use the Chinese medicine chromatographic fingerprint similarity evaluation system version B to calculate the similarity of each sample.

[0005] Furthermore, the reference solution in step (1) is prepared by dissolving the 4-hydroxyquinoline reference substance in methanol, then diluting to 10 mL, mixing, absorbing 1.0 mL of the solution, and diluting to 25 mL with a 2 wt % acetonitrile aqueous solution, and mixing to obtain a reference solution; wherein the 2 wt % acetonitrile aqueous solution contains 0.004% trifluoroacetic acid.

[0006] Furthermore, the control sample solution is prepared in step (2) by mixing Zanhuang triploid jujube flower honey from 3-20 different bee farms in Zanhuang County as a control sample.

[0007] Furthermore, the sample solution is prepared in step (3) as follows: adding 2 wt% acetonitrile aqueous solution to the unknown sample, mixing and then adding acetonitrile, centrifuging and placing at -20°C for 40-80 min, collecting the upper organic phase, adding acetonitrile to the precipitate for secondary extraction, centrifuging and combining the secondary acetonitrile extracts, drying with nitrogen, and then adding 2 wt% acetonitrile aqueous solution to dissolve and filter with a filter membrane; wherein the 2 wt% acetonitrile aqueous solution contains 0.004 wt% trifluoroacetic acid.

[0008] Furthermore, the UPLC column in step (4) was: C18, 2.1×50 mm, 1.7 μm; column temperature: 35°C; injection volume: 2 μL; flow rate: 0.3 mL / min.

[0009] Furthermore, the result judgment criteria are: peak No. 2 should appear in the fingerprint of the unknown sample, and the retention time of chromatographic peaks No. 1, 2, 3, 4 and 5 should be consistent with that in the reference material spectrum; the similarity of the five characteristic peaks is calculated using the Chinese medicine chromatographic fingerprint similarity evaluation system, and the similarity between the fingerprint of the unknown sample and the fingerprint of the Zanhuang triploid jujube flower honey control sample is ≥0.90. If the above requirements are met at the same time, the unknown sample is judged to be Zanhuang triploid jujube flower honey.

[0010] The present invention has the following beneficial effects: The detection principle of the present invention is as follows: the sample to be tested is extracted with trifluoroacetic acid and acetonitrile, concentrated and redissolved, and the fingerprint of the sample solution is measured by high-performance liquid chromatography at a wavelength of 213nm. The similarity between the sample and the reference fingerprint of Zanhuang jujube flower honey is compared and calculated using traditional Chinese medicine fingerprint similarity software, and the sample is identified based on the similarity. The detection method of the present invention has the following advantages: (1) Holistic and comprehensive: Traditional honey detection methods usually use a single indicator (a certain component content, such as sugar) for identification, but a single indicator can easily be circumvented by counterfeiters (such as adding a specific syrup to simulate the sugar indicator). The advantage of using fingerprint identification is that it can simultaneously analyze the overall composition and proportion relationship of multiple components to form an overall "fingerprint". It is almost impossible for counterfeiters to perfectly replicate the characteristic components and relative abundance of natural Zanhuang Jujube Flower Honey, which improves the accuracy of identification; (2) Objectivity and reliability: Traditional sensory evaluation is subjective and may lead to errors. The fingerprint method relies on ultra-high performance liquid chromatography to collect data, and the analysis results are presented in the form of digital maps. The identification process is scientific and objective. Through strict methodological validation (precision, repeatability, and stability), the reliability and comparability of the identification results can be guaranteed; (3) High efficiency and high throughput: Compared with testing multiple physical and chemical indicators or markers one by one, fingerprint analysis is more efficient and can obtain a large amount of chemical information in a single instrument analysis. Combined with automated sample pretreatment equipment and intelligent data processing software, high-throughput screening can be achieved, which is more suitable for market supervision and large-scale quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the reference spectrum of Zanhuang triploid jujube flower honey; peak 2 is 4-hydroxyquinoline, and peaks 1, 3-5 are unknown peaks; Figure 2 is the spectrum of 4-hydroxyquinoline; Figure 3 This is the map of Zanhuang triploid jujube flower honey; Figure 4 This is the detection chart of Zanhuang Hundred Flower Honey; Figure 5 This is the detection spectrum of Zanhuang acacia honey; Figure 6 For the detection map of other provinces' hundred-flower honey; Figure 7 For the detection map of Vitex honey in other provinces; Figure 8 For the detection of acacia honey in other provinces ; Figure 9 This is the detection spectrum of jujube honey from other provinces. DETAILED DESCRIPTION

[0012] The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, the experiments were performed under conventional conditions or those recommended by the manufacturer. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0013] Example 1: An ultra-high performance liquid chromatography fingerprint method for detecting Zanhuang triploid jujube flower honey comprises the following steps: (1) Preparation of reference solution: Accurately weigh 10.0 mg of 4-hydroxyquinoline reference substance (converted to the concentration indicated on the standard substance certificate) and place it in a 10 mL volumetric flask. Add an appropriate amount of methanol to dissolve and dilute to the mark, then shake well. Accurately absorb 1.0 mL and place it in a 25 mL volumetric flask. Dilute to the mark with 2 wt% acetonitrile aqueous solution (containing 0.004 wt% trifluoroacetic acid), shake well, and prepare a 40 mg / L reference solution.

[0014] (2) Preparation of control sample solution: 5 different bee farms in Zanhuang County were selected, and 200 g of Zanhuang triploid jujube flower honey with a pollen rate ≥ 60% was taken from each bee farm and mixed evenly as the control sample.

[0015] (3) Preparation of sample solution: Weigh 2.0 g of unknown sample (unknown honey) into a 50 mL centrifuge tube, add 4.0 mL of 2 wt% acetonitrile aqueous solution (containing 0.004 wt% trifluoroacetic acid), vortex to dissolve, then add 2.0 mL of acetonitrile, vortex to mix and extract, centrifuge at 1500 rpm for 5 min, place at -20 ° C for 60 min, collect the upper organic phase, add 2.0 mL of acetonitrile to the precipitate for secondary extraction, centrifuge at 1500 rpm for 5 min, combine the secondary acetonitrile extracts, blow to dryness with nitrogen at 30 ° C, add 0.5 mL of 2 wt% acetonitrile aqueous solution (containing 0.004 wt% trifluoroacetic acid), ultrasonically dissolve, filter through 0.22 µm filter membrane, and wait for liquid chromatography determination.

[0016] (4) Chromatographic conditions: Chromatographic column: C18, 2.1×50mm, 1.7µm, ACQUITY UPLC is recommended ® BEH C18 model; Mobile phase: Phase A is 2 wt% acetonitrile aqueous solution (containing 0.004 wt% trifluoroacetic acid), phase B is acetonitrile, and the gradient elution program is shown in Table 1.

[0017] Column temperature: 35°C; Injection volume: 2 µL; Flow rate: 0.3 mL / min; Detection wavelength: 213nm.

[0018] Table 1 Gradient elution program

[0019] (5) Fingerprint determination: the reference solution of step (1), the reference solution of step (2) and the sample solution of step (3) were respectively aspirated, and the chromatographic conditions of step (4) were used to detect the chromatogram from 0 min to 5 min. The characteristic chromatographic peaks were integrated. The Chinese medicine chromatographic fingerprint similarity evaluation system version A (2012) was used to establish the Zanhuang triploid jujube flower honey reference template. The Chinese medicine chromatographic fingerprint similarity evaluation system version B (2012) was used to calculate the similarity between the sample solutions. The Zanhuang triploid jujube flower honey reference template is shown in Figure 1 , 4-hydroxyquinoline spectrum see Figure 2 .

[0020] (6) Result determination: The fingerprint of the unknown sample should have peak 2, and the retention times of chromatographic peaks 1, 2, 3, 4 and 5 should be consistent with those in the reference material spectrum; the similarity of the five characteristic peaks is calculated using the Chinese medicine chromatographic fingerprint similarity evaluation system. The similarity between the fingerprint of the unknown sample and the fingerprint of the Zanhuang triploid jujube flower honey control sample is ≥0.90. If the above requirements are met at the same time, the unknown sample is determined to be Zanhuang triploid jujube flower honey.

[0021] (7) Method precision test: The same sample was measured twice in succession under repeated conditions, and the relative deviation was ≤0.5%.

[0022] Test example: The ultra-high performance liquid chromatography fingerprint method of Example 1 was used to detect Zanhuang triploid jujube honey, Zanhuang multi-flower honey, Zanhuang acacia honey, other provinces jujube honey, other provinces vitex honey, other provinces acacia honey and other provinces multi-flower honey samples. The sample test results are shown in Tables 2-4 and the spectra are shown in Figure 3-9 .

[0023] Table 2 Sample test results (1)

[0024] Table 3 Sample test results (2)

[0025] Table 4 Sample test results (3)

[0026] As shown in Table 2-4, 4-hydroxyquinoline (>0.73 mg / kg) was detected in all five Zanhuang triploid jujube flower honey samples, while the highest 4-hydroxyquinoline content in non-Zanhuang jujube flower honey samples was only 0.82 mg / kg (Baihua No. 3 from another province). The similarities of the Zanhuang triploid jujube flower honey samples were all >0.90, while the similarities of the non-Zanhuang jujube flower honey samples were all <0.90 (the highest was 0.895 for Baihua No. 5 from another province).

[0027] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An ultra-high performance liquid chromatography fingerprint method for detecting Zanhuang triploid jujube flower honey, characterized in that: The following steps are involved: (1) Prepare reference solution; (2) Prepare control sample solution; (3) Prepare sample solution; (4) Determine the chromatographic conditions for UPLC: ① Mobile phase: Phase A is 2 wt% acetonitrile aqueous solution (containing 0.004 wt% trifluoroacetic acid), and phase B is acetonitrile; ②Detection wavelength: 213nm; ③ Gradient elution program: 0→3 min→3.01 min→7 min→7.01 min→11 min; Mobile phase A: 95%→65%→5%→5%→95%→95%; Mobile phase B: 5%→35%→95%→95%→5%→5%; (5) Ultra-high performance liquid chromatography detection: aspirate the reference solution of step (1), the control sample solution of step (2), and the sample solution of step (3) respectively, perform ultra-high performance liquid chromatography detection, and record the chromatogram from 0 min to 5 min; (6) Establishing fingerprints: Integrate the characteristic chromatographic peaks of the reference solution, control sample solution, and test sample solution obtained in step (5), then use the Chinese medicine chromatographic fingerprint similarity evaluation system version A to establish a Zanhuang Jujube Flower Honey control template, and use the Chinese medicine chromatographic fingerprint similarity evaluation system version B to calculate the similarity of each sample.

2. The ultra-high performance liquid chromatography fingerprint method according to claim 1, characterized in that The reference solution prepared in step (1) is specifically prepared by dissolving the 4-hydroxyquinoline reference substance in methanol, then diluting to 10 mL, mixing, absorbing 1.0 mL of the solution and diluting to 25 mL with a 2 wt % acetonitrile aqueous solution, and mixing to obtain a reference solution; wherein the 2 wt % acetonitrile aqueous solution contains 0.004% trifluoroacetic acid.

3. The ultra-high performance liquid chromatography fingerprint method according to claim 1, characterized in that The control sample solution prepared in step (2) is specifically prepared by mixing Zanhuang triploid jujube flower honey from 3-20 different bee farms in Zanhuang County as a control sample.

4. The ultra-high performance liquid chromatography fingerprint method according to claim 1, characterized in that The sample solution prepared in step (3) is specifically as follows: adding a 2 wt % acetonitrile aqueous solution to the unknown sample, mixing and then adding acetonitrile, centrifuging and placing at -20°C for 40-80 minutes, collecting the upper organic phase, adding acetonitrile to the precipitate for secondary extraction, centrifuging and combining the secondary acetonitrile extracts, drying with nitrogen, and then adding a 2 wt % acetonitrile aqueous solution to dissolve and filter; wherein the 2 wt % acetonitrile aqueous solution contains 0.004 wt % trifluoroacetic acid.

5. The ultra-high performance liquid chromatography fingerprint method according to claim 1, characterized in that UPLC column in step (4): C18, 2.1 × 50 mm, 1.7 µm; Column temperature: 35°C; injection volume: 2µL; flow rate: 0.3mL / min.

6. The ultra-high performance liquid chromatography fingerprint method according to claim 1, characterized in that The result judgment criteria are: the fingerprint of the unknown sample should have peak 2, and the retention times of peaks 1, 2, 3, 4 and 5 should be consistent with those in the reference material spectrum; The similarity of the five characteristic peaks was calculated using the Chinese medicine chromatographic fingerprint similarity evaluation system. The similarity between the fingerprint of the unknown sample and the fingerprint of the Zanhuang triploid jujube flower honey control sample was ≥0.

90. If the above requirements were met at the same time, the unknown sample was judged to be Zanhuang triploid jujube flower honey.

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