A method for detecting zanhuang triploid jujube flower honey by ultra-high performance liquid chromatography fingerprinting

Abstract

The application discloses a kind of detection Zanhuang triploid jujube flower honey by ultra-high performance liquid chromatography fingerprint method, belong to analytical chemistry technical field.The application is extracted to the sample to be measured by trifluoroacetic acid, acetonitrile, after concentration and redissolving, the fingerprint of sample solution is determined by high performance liquid chromatography under 213nm wavelength, using traditional Chinese medicine fingerprint similarity software compares and calculates the similarity of sample and Zanhuang jujube flower honey control fingerprint, according to the similarity of sample is identified.Therefore, the detection method of the application can simultaneously analyze the overall composition and proportion relationship of multiple components, form the overall "fingerprint", improve the accuracy of identification.At the same time, the detection method of the application is more efficient, a large amount of chemical information can be obtained by instrument analysis once.Combined with automatic sample pretreatment equipment and intelligent data processing software, high-throughput screening can be realized, more suitable for market supervision and large-scale quality control.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical chemistry, in particular to a method for detecting Zanhuang triploid jujube flower honey by ultra performance liquid chromatography fingerprint. BACKGROUND

[0002] Zanhuang triploid jujube flower honey is a natural sweet substance produced by bees in Zanhuang County, Hebei Province, by collecting nectar from triploid jujube trees (Ziziphus jujuba Mill. Zizyphus jujuba Mill ) and mixing it with their own secretions, and then fully brewing it. As a major single-flower honey variety in China, jujube flower honey is deeply loved by people due to its unique health and beauty care effects. Due to the limited production and high production cost of jujube flower honey, the selling price of jujube flower honey is significantly higher than that of other common single-flower honeys. In order to pursue high profits, unscrupulous enterprises often use other cheap honeys to pass off as jujube flower honey, or adulterate other cheap honeys in jujube flower honey, and even more, adulterate high fructose syrup in jujube flower honey. At present, China has not yet formulated standards for jujube flower honey, and how to effectively evaluate and identify jujube flower honey has become one of the problems to be solved in the current bee product industry. Therefore, it is necessary to develop a rapid, efficient and accurate detection method for detecting Zanhuang triploid jujube flower honey. SUMMARY

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

[0004] The technical scheme for solving the above technical problems is as follows: a method for detecting Zanhuang triploid jujube flower honey by ultra performance liquid chromatography fingerprint is provided, comprising the following steps:

[0005] (1) preparing a reference solution;

[0006] (2) preparing a control sample solution;

[0007] (3) preparing a sample solution;

[0008] (4) determining the chromatographic conditions of UPLC:

[0009] ① mobile phase: A phase is 2wt% acetonitrile aqueous solution (containing 0.004wt% trifluoroacetic acid), and B phase is acetonitrile;

[0010] ② detection wavelength: 213nm;

[0011] ③Gradient 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%;

[0012] (5) UPLC detection: respectively take the reference solution of step (1), the control sample solution of step (2) and the sample solution of step (3), and perform UPLC detection, and record the chromatogram of 0 min-5 min;

[0013] (6) Establish fingerprint: integrate the characteristic chromatographic peaks of the reference solution, the control sample solution and the sample solution obtained in step (5), then establish the Zanhuang jujube flower honey control template by using Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System A version, and calculate the similarity of each sample by using Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System B version.

[0014] Further, the preparation of the reference solution in step (1) is specifically as follows: dissolve 4-hydroxyquinoline control in methanol, then dilute to 10 mL, mix well, and then take 1.0 mL of the solution, dilute to 25 mL with 2wt% acetonitrile aqueous solution, mix well to obtain the reference solution; wherein the 2wt% acetonitrile aqueous solution contains 0.004% trifluoroacetic acid.

[0015] Further, the preparation of the control sample solution in step (2) is specifically as follows: mix 3-20 Zanhuang triploid jujube flower honeys from different bee farms in Zanhuang County as the control sample.

[0016] Further, the preparation of the sample solution in step (3) is specifically as follows: add 2wt% acetonitrile aqueous solution to the unknown sample, mix well, then add acetonitrile, centrifuge, and then place at-20℃ for 40-80 min, collect the upper organic phase, add acetonitrile to the precipitate for secondary extraction, centrifuge, then combine the two acetonitrile extraction solutions, blow dry with nitrogen, then add 2wt% acetonitrile aqueous solution to dissolve and filter through a membrane; wherein the 2wt% acetonitrile aqueous solution contains 0.004wt% trifluoroacetic acid.

[0017] Further, the chromatographic column of UPLC in step (4) is C18, 2.1x50mm, 1.7µm; column temperature: 35℃; injection volume: 2µL; flow rate: 0.3mL / min.

[0018] Further, the result determination standard is that the fingerprint spectrum of the unknown sample should appear the peak No.2, and the chromatographic peaks No.1, No.2, No.3, No.4 and No.5 should be consistent with the retention time in the reference spectrum; the similarity of the five characteristic peaks is calculated by using the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, the similarity of the fingerprint spectrum of the unknown sample and the fingerprint spectrum of the Zanhuang triploid jujube flower honey control sample is greater than or equal to 0.90, and the unknown sample is determined as the Zanhuang triploid jujube flower honey when the above requirements are met.

[0019] The present application has the following advantages: the detection principle of the present application is as follows: the sample to be detected is extracted by trifluoroacetic acid and acetonitrile, concentrated and redissolved, and then the fingerprint spectrum of the sample solution is determined by high performance liquid chromatography at a wavelength of 213 nm, the similarity of the sample and the Zanhuang jujube flower honey control fingerprint spectrum is compared and calculated by using the traditional Chinese medicine fingerprint similarity software, and the sample is identified according to the similarity. The detection method of the present application has the following advantages:

[0020] (1) integrity and comprehensiveness: the traditional detection method of honey usually adopts the determination of a single index (the content of a certain component, such as sugar) for identification, but the single index can be easily evaded by the fraudsters (such as adding a specific sugar syrup to simulate the sugar index). The advantage of using fingerprint identification is that it can analyze the overall composition and proportion relationship of multiple components at the same time, forming a whole "fingerprint", and it is almost impossible for the fraudsters to perfectly copy the characteristic components and their relative abundance of the natural Zanhuang jujube flower honey, which improves the accuracy of identification;

[0021] (2) objectivity and reliability: the traditional sensory evaluation is subjective and may cause errors. The fingerprint method relies on the data collected by the ultra-high performance liquid chromatograph, and the analysis result is presented in the form of digital atlas, so the identification process is scientific and objective. Through strict methodological validation (precision, repeatability, stability), the reliability and comparability of the identification result can be guaranteed;

[0022] (3) high efficiency and high throughput: compared with detecting multiple physicochemical indexes or markers one by one, the fingerprint method is more efficient, and a large amount of chemical information can be obtained by one instrument analysis. Combined with automatic sample pretreatment equipment and intelligent data processing software, high-throughput screening can be realized, which is more suitable for market supervision and large-scale quality control. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is the control spectrum of Zanhuang triploid jujube flower honey; wherein, peak 2 is 4-hydroxyquinoline, and peaks 1, 3-5 are unknown peaks;

[0024] Figure 2 is the spectrum of 4-hydroxyquinoline;

[0025] Figure 3 is the spectrum of Zanhuang triploid jujube flower honey;

[0026] Figure 4 Detection atlas of Zanhuang honeydew honey;

[0027] Figure 5 Detection atlas of Zanhuang acacia honey;

[0028] Figure 6 Detection atlas of honey from other provinces;

[0029] Figure 7 Detection atlas of honey from other provinces;

[0030] Figure 8 Detection atlas of Zanhuang acacia honey;

[0031] Figure 9 Detection atlas of honey from other provinces; DETAILED DESCRIPTION

[0032] The following examples are intended to illustrate but not limit the present application. Unless otherwise indicated, conventional conditions or those manufacturer's recommendations were employed in the examples. Unless otherwise indicated, the reagents or instruments used were conventional products available commercially.

[0033] Example 1:

[0034] A method for detecting Zanhuang triploid jujube honey by ultra-high performance liquid chromatography fingerprinting includes the following steps:

[0035] (1) Preparation of reference solution: accurately weigh 10.0 mg of 4-hydroxyquinoline reference substance (calculated according to the concentration indicated on the certificate of standard substance), and place it in a 10 mL volumetric flask. Add appropriate amount of methanol to dissolve and dilute to the mark, and shake well. Precisely 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.

[0036] (2) Preparation of control sample solution: select 5 different apiaries in Zanhuang County, and mix 200 g of Zanhuang triploid jujube honey with a pollen rate of ≥60% to obtain a control sample.

[0037] (3) Preparation of sample solution: 2.0 g of unknown sample (unknown honey) was weighed into a 50 mL centrifuge tube, 4.0 mL of 2 wt% acetonitrile aqueous solution (containing 0.004 wt% trifluoroacetic acid) was added, vortexed to dissolve, then 2.0 mL of acetonitrile was added, vortexed to extract, centrifuged at 1500 rpm for 5 min, placed at -20°C for 60 min, collected the upper organic phase, then 2.0 mL of acetonitrile was added to the precipitate for secondary extraction, centrifuged at 1500 rpm for 5 min, the secondary acetonitrile extract was combined, blown to near dryness under nitrogen at 30°C, 0.5 mL of 2 wt% acetonitrile aqueous solution (containing 0.004 wt% trifluoroacetic acid) was added and ultrasonically dissolved, filtered through a 0.22 µm filter membrane, and then subjected to liquid chromatography.

[0038] (4) Chromatographic conditions:

[0039] Chromatographic column: C18, 2.1 x 50 mm, 1.7 µm, ACQUITY UPLC BEH C18 type recommended; ®

[0040] Mobile phase: A phase was 2 wt% acetonitrile aqueous solution (containing 0.004 wt% trifluoroacetic acid), B phase was acetonitrile, the gradient elution program is shown in Table 1.

[0041] Column temperature: 35°C;

[0042] Injection volume: 2 µL;

[0043] Flow rate: 0.3 mL / min;

[0044] Detection wavelength: 213 nm.

[0045] Table 1 Gradient elution program

[0046]

[0047] (5) Fingerprint determination: the reference solution of step (1), the control solution of step (2) and the sample solution of step (3) were respectively taken, detected according to the chromatographic conditions of step (4), the chromatogram from 0 min to 5 min was recorded, the characteristic chromatographic peaks were integrated, the Zanhuang triploid jujube flower honey control template was established by using Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System A (2012), and the similarity between the sample solutions was calculated by using Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System B (2012), the Zanhuang triploid jujube flower honey control spectrum is shown in Figure 1 , and the 4-hydroxyquinoline spectrum is shown in Figure 2 .

[0048] ​(6) Result determination: the fingerprint of the unknown sample should appear peak No. 2, and the retention time of peaks No. 1, No. 2, No. 3, No. 4 and No. 5 should be consistent with that of the reference spectrum; the similarity of the five characteristic peaks is calculated by using the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, the similarity of the fingerprint of the unknown sample and the control sample of Zanhuang triploid jujube flower honey is ≥0.90, and the unknown sample is determined to be Zanhuang triploid jujube flower honey when the above requirements are met.

[0049] (7) Method precision detection: the same sample is determined twice continuously under the repeated conditions, and the relative deviation is ≤0.5%.

[0050] Test example:

[0051] The ultra-high performance liquid chromatography fingerprint method of Example 1 is used to detect Zanhuang triploid jujube flower honey, Zanhuang multiflower honey, Zanhuang acacia honey, out-of-province jujube flower honey, out-of-province rattan honey, out-of-province acacia honey and out-of-province multiflower honey samples. The sample detection results are shown in Tables 2-4, and the chromatograms are shown in Figures 3-9 .

[0052] Table 2 Sample detection results (1)

[0053]

[0054] Table 3 Sample detection results (2)

[0055]

[0056] Table 4 Sample detection results (3)

[0057]

[0058] As shown in Tables 2-4, 4-hydroxyquinoline (>0.73 mg / kg) is detected in the five Zanhuang triploid jujube flower honey samples, and the maximum of 4-hydroxyquinoline in the non-Zanhuang jujube flower honey samples is only 0.82 mg / kg (out-of-province multiflower No. 3); the similarity of the Zanhuang triploid jujube flower honey samples is all >0.90, and the similarity of the non-Zanhuang jujube flower honey samples is all <0.90 (the highest is 0.895 for out-of-province multiflower No. 5).

[0059] The above only describes the preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for detecting Zanhuang triploid jujube flower honey by ultra-high performance liquid chromatography fingerprinting, characterized in that, The method comprises the following steps: (1) preparing a reference solution; (2) preparing a control sample solution; (3) preparing a sample solution: adding 2wt% acetonitrile aqueous solution to the unknown sample, mixing, then adding acetonitrile, centrifuging, and placing at-20℃ for 40-80min, collecting the upper organic phase, adding acetonitrile to the precipitate for secondary extraction, centrifuging, and then combining the secondary acetonitrile extract, blowing dry with nitrogen, then adding 2wt% acetonitrile aqueous solution to dissolve and filtering with a membrane; wherein the 2wt% acetonitrile aqueous solution contains 0.004wt% trifluoroacetic acid; (4) determining the chromatographic conditions of UPLC: ① mobile phase: A phase is 2wt% acetonitrile aqueous solution (containing 0.004wt% trifluoroacetic acid), and B phase is acetonitrile; ② detection wavelength: 213nm; ③ gradient elution program: 0→3min→3.01min→7min→7.01min→11min; mobile phase A: 95%→65%→5%→5%→95%→95%; mobile phase B: 5%→35%→95%→95%→5%→5%; the chromatographic column of the UPLC: C18, 2.1×50mm, 1.7µm; column temperature: 35℃; injection volume: 2µL; flow rate: 0.3mL / min; (5) UPLC detection: respectively taking the reference solution of step (1), the control sample solution of step (2) and the sample solution of step (3), and performing UPLC detection, and recording the chromatogram of 0min-5min; (6) establishing a fingerprint: integrating the characteristic chromatographic peaks of the reference solution, the control sample solution and the sample solution obtained in step (5), then establishing a Zanhuang jujube flower honey control template by using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System A version, and calculating the similarity of each sample by using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System B version.

2. The ultra-performance liquid chromatography fingerprinting method of claim 1, wherein, In step (1), the preparation of the reference solution is specifically as follows: dissolving 4-hydroxyquinoline control in methanol, then diluting to 10mL, mixing, then taking 1.0mL of the solution, diluting with 2wt% acetonitrile aqueous solution to 25mL, mixing, and then preparing the reference solution; wherein the 2wt% acetonitrile aqueous solution contains 0.004% trifluoroacetic acid.

3. The UPLC fingerprint method according to claim 1, characterized in that, In step (2), the preparation of the control sample solution is specifically as follows: mixing 3-20 Zanhuang triploid jujube flower honeys from different apiaries in Zanhuang County to obtain a control sample.

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