Anemonin detection method based on high performance liquid chromatography-tandem mass spectrometry

Abstract

The invention discloses an anemonin detection method based on high performance liquid chromatography-tandem mass spectrometry, and belongs to the technical field of anemonin detection.The anemonin detection method comprises the steps that a sample to be detected is pretreated, and an extract solution is obtained; and performing high performance liquid chromatography tandem mass spectrometer detection on the extract solution, and performing qualitative and / or quantitative analysis on the anemonin according to a qualitative ion pair 193.0 / 97.0 and a quantitative ion pair 193.0 / 91.0. The method has the advantages of high sensitivity, low matrix effect, good precision and recovery rate and the like, can provide reliable guarantee for detection of low-content anemonin in the honey, and has important significance for quality supervision of the Chinese native honey.

Description

Technical Field

[0001] The invention belongs to the technical field of annularein detection, and specifically relates to annularein detection method based on high performance liquid chromatography tandem mass spectrometry, and is particularly suitable for the detection of annularein in honey. Background Art

[0002] Honey is a natural sweet substance that is fully brewed by bees after collecting nectar, secretions or honeydew from plants and mixing it with their own secretions. The National Food Safety Standard Honey stipulates that "the nectar, secretions or honeydew collected by bees should be safe and non-toxic, and must not come from poisonous nectar plants such as Tripterygium wilfordii, Macleania oleracea, and Stellera chamaejasme". However, China is vast and rich in resources, and the numerous poisonous nectar plants combined with the feeding habits of Chinese honeybees have increased the safety uncertainty of Chinese honey. At present, domestic standards and regulations on the safety of Chinese honeybee soil pay more attention to its pesticide and veterinary drug residues and microbial conditions, and pay relatively little attention to risk compounds from toxic nectar plants, and related detection technologies are limited. These risk compounds from toxic nectar plants can cause vomiting and diarrhea at the mildest, and even death at the worst. Therefore, in order to ensure the safety of Chinese honeybee soil and protect the health of consumers, it is necessary to strengthen the supervision of risk compounds from toxic nectar plants in honey and establish corresponding detection methods.

[0003] The phytotoxic components in honey can be mainly divided into two categories: alkaloids and terpenes. Alkaloid toxic components can be further divided into diterpenoid alkaloids, isoquinoline alkaloids, pyrrolizidine alkaloids and indole alkaloids. Terpenoid toxic components can be further divided into bicyclic sesquiterpenoid lactone compounds, diterpenoid alcohol (ester) components and diterpenoid components. Since these phytotoxic components usually have a certain toxic effect on bees, their content in Chinese bee honey is relatively low. At present, detection methods have been established at home and abroad for the detection of some alkaloids and terpenoid toxic components in honey. However, no research on anemonin in honey has been reported. Ananemonin is a dimer compound of proanemonin. Proanemonin and ananemonin are the main toxic compounds of many toxic nectar plants such as buttercup, nephrolepis, donkey hoof grass, scutellaria and clematis in the Ranunculaceae family. These plants first produce ranunculusin (the glycoside form of protoanemonin), which is first hydrolyzed by β-glucosidase to protoanemonin. Protoanemonin is extremely unstable and will quickly cyclodimerize to convert to protoanemonin (for the conversion process, see Figure 1 ). Studies have shown that analgesic acid is a strong irritant and can cause gastrointestinal diseases. In addition, due to the instability of ranunculus glycosides and protoanalgesic acid, they are not suitable for detection. Analgesic acid, which has more stable chemical properties, is suitable for detection. The above-mentioned Ranunculaceae plants are widely distributed in various regions of China, so potential honey poisoning incidents caused by analgesic acid in honey need to be taken seriously.

[0004] At present, there are few detection methods for anemonin, and the existing research targets anemonin in anemonin plant extracts, and there is no report on the detection of anemonin in honey. On the one hand, the content of anemonin in honey is much lower than that in plant extracts; on the other hand, the honey matrix has a great influence on the accurate detection of low-content anemonin. Therefore, there is an urgent need for a highly sensitive anemonin analysis method suitable for honey samples. Summary of the invention

[0005] In view of this, the present invention aims to establish a method for detecting low-content annulophantin by high performance liquid chromatography tandem mass spectrometry, so as to achieve accurate quantitative detection of the annulophantin content in honey, thereby achieving the purpose of ensuring the safety of native honey.

[0006] In order to achieve the above object, the present invention specifically adopts the following technical solutions: A method for detecting anemonin based on high performance liquid chromatography tandem mass spectrometry comprises the following steps: S1, pre-treating the sample to be tested to obtain an extract solution; S2. The extract solution is subjected to high performance liquid chromatography tandem mass spectrometry detection, and the anemonin in the sample to be tested is qualitatively and / or quantitatively analyzed according to the qualitative ion pair 193.0 / 97.0 and the quantitative ion pair 193.0 / 91.0.

[0007] Preferably, in the above-mentioned method for detecting anthracene, the declustering potential (DP) of the qualitative ion pair and the quantitative ion pair is 60 V, the collision energy (CE) of the qualitative ion pair is 16 eV, and the CE of the quantitative ion pair is 36 eV.

[0008] Preferably, in the above-mentioned anustonin detection method, the sample to be tested in step S1 is honey.

[0009] More preferably, when the sample to be tested is honey, the pre-treatment process of step S1 includes the following operations: S11, dissolving honey in ultrapure water, adjusting the pH to 2 with hydrochloric acid to obtain a honey solution; S12, loading the honey solution into an activated solid phase extraction column, and after all the honey solution flows through the solid phase extraction column, using a hydrochloric acid aqueous solution to remove substances not adsorbed by the filler; S13, eluting with methanol and collecting the eluate, drying the eluate to obtain a honey extract, and redissolving the honey extract to obtain a honey extract solution.

[0010] In the above pretreatment process, step S12 can be activated by using methanol and a hydrochloric acid aqueous solution with a pH value of 2 respectively, and step 13 can use a 50% formic acid methanol solution to redissolve the honey extract.

[0011] Preferably, in the above-mentioned method for detecting anustolianin, the chromatographic conditions in step S2 are: chromatographic column, C18 column (2.1mm×100 mm, 1.9 μm); mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is acetonitrile, gradient elution; injection volume is 3 μL, column temperature is 30°C, and flow rate is 0.3 mL / min. More preferably, the program of the above-mentioned gradient elution is: 0 min, 5% mobile phase B; 2 min, 10% mobile phase B; 6 min, 40% mobile phase B; 7 min, 5% mobile phase B; 10 min, 5% mobile phase B.

[0012] Preferably, in the above-mentioned angustifoliin detection method, the mass spectrometry conditions in step S2 are: electrospray ion source (ESI), positive ion mode scanning; multiple reaction monitoring (MRM) mode; electrospray voltage is 5500 V; ion source temperature is 500°C; curtain gas (CUR) pressure is 45 psi; nebulizer gas (GS1) pressure is 55 psi; auxiliary gas (GS2) pressure is 55 psi.

[0013] It is understood that in the above-mentioned method for detecting anemonin, anemonin can be quantitatively analyzed according to the peak area of ​​the quantitative ion pair in the sample to be tested and the standard curve. Preferably, the above-mentioned standard curve is drawn in the following manner: the anemonin standard is prepared into a series of standard working solutions of different concentrations with 80% methanol solution, the peak area of ​​the quantitative ion pair in each standard working solution is detected and obtained, and the standard curve is drawn according to the concentration and the peak area of ​​the quantitative ion pair.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention establishes a detection scheme for anemonin based on high performance liquid chromatography tandem mass spectrometry for the first time, and the investigation results of indicators such as the detection limit, quantitation limit, linear range, matrix effect, intra-day precision, inter-day precision and spiked recovery rate of the detection scheme of the present invention show that the detection scheme of the present invention is accurate and reliable.

[0015] Based on the high performance liquid chromatography tandem mass spectrometry analysis of anemonin, the present invention determines the qualitative ion pair and the quantitative ion pair, and optimizes the CE and DP parameters of the qualitative ion pair and the quantitative ion pair in the MRM mode, which ensures the high sensitivity of the detection scheme of the present invention, the good linear relationship within a certain range, and the good precision and recovery rate on the basis of accurate qualitative and quantitative determination.

[0016] The present invention successfully applies the anthracene detection scheme based on high performance liquid chromatography tandem mass spectrometry to the detection of anthracene in honey, filling the gap in the prior art for detecting anthracene in honey. Moreover, the detection limit of the detection scheme of the present invention is as low as 1.8 ng / mL, and the quantification limit is as low as 4.9 ng / mL. Therefore, a very small amount of anthracene in honey can be detected, and the method can be applied to the quality and safety supervision of native honey, and the reliability of the supervision is effectively guaranteed.

[0017] The present invention develops a honey sample pretreatment process suitable for high performance liquid chromatography tandem mass spectrometry detection, which greatly reduces the influence of matrix effect (ME); experimental data show that the matrix effect is 93.74%, which is in the range of 85% to 115%, indicating that the honey matrix has little effect on the quantification of anemonin. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the conversion process between ranunculusin, protoanemonin and ananemonin; Figure 2 The MRM chromatograms of the qualitative ion pair and the quantitative ion pair used in the present invention in the standard product of anemonin; wherein the upper figure is the MRM graph of the qualitative ion pair 193.0 / 97.0, and the lower figure is the MRM graph of the quantitative ion pair 193.0 / 91.0, and the ordinate represents the response value; Figure 3 It is the MRM chromatogram of the qualitative ion pair and the quantitative ion pair in the positive native honey sample (A) and the negative native honey sample (B) in Example 2 of the present invention; wherein, the upper figure in A is the MRM graph of the qualitative ion pair 193.0 / 97.0 of the positive sample, the lower figure in A is the MRM graph of the quantitative ion pair 193.0 / 91.0 of the positive sample, the upper figure in B is the MRM graph of the qualitative ion pair 193.0 / 97.0 of the negative sample, and the lower figure in B is the MRM graph of the quantitative ion pair 193.0 / 91.0 of the negative sample. DETAILED DESCRIPTION

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The term "comprises" and any variations thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusions.

[0020] In view of the lack of methods for detecting anolein in honey in the prior art, the present invention is the first to develop a targeted quantitative method for anolein using high performance liquid chromatography tandem mass spectrometry and conduct a methodological investigation, and ultimately provides an anolein detection method based on high performance liquid chromatography tandem mass spectrometry, which is successfully applied to the detection of anolein in honey.

[0021] The technical solution of the present invention will be described clearly and completely below in conjunction with specific embodiments. It should be understood that the embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0022] If no specific techniques or conditions are specified in the following examples, all of them are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions; if the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0023] Example 1 This example provides a method for detecting anemonin based on high performance liquid chromatography tandem mass spectrometry, as follows: (1) Sample pretreatment.

[0024] For honey samples, the pre-treatment operations are as follows: Honey was dissolved in ultrapure water and the pH was adjusted to 2 with a dilute hydrochloric acid solution to obtain a honey solution. The solid phase extraction column was activated with methanol and a hydrochloric acid aqueous solution with a pH of 2, respectively. The prepared honey solution was then loaded into the solid phase extraction column. After the honey solution had all flowed through the solid phase extraction column, a hydrochloric acid aqueous solution with a pH of 2 was flowed through the solid phase extraction column to remove the unadsorbed compounds. Methanol was then flowed through the solid phase extraction column and the eluent was collected. The eluent was dried to obtain a honey extract. Before use, the dried honey extract was reconstituted with a 50% formic acid methanol solution.

[0025] (2) Qualitative and quantitative ion pairs of anemonin.

[0026] The standard of anemonin was prepared into a 1 μg / mL solution with 80% methanol solution and analyzed by mass spectrometry using AB SCIEX 6500QTRAP equipment. In the positive ion mode, the qualitative ion pair was determined to be 193.0 / 97.0 and the quantitative ion pair was 193.0 / 91.0 (see MRM chromatogram Figure 2 ). The CE and DP parameters of the qualitative and quantitative ion pairs were optimized, and the optimization results are shown in Table 1.

[0027] Table 1 Ion pairs, collision energies and declustering voltages of anemonin

[0028] Note: * indicates quantitative ion pair.

[0029] (3) Detection conditions of high performance liquid chromatography-tandem mass spectrometry.

[0030] The liquid chromatography conditions were as follows: the chromatographic column was Thermo Hypersil Gold C18 (2.1 mm×100 mm, 1.9 μm); the mobile phase A was 0.1% formic acid in water, the mobile phase B was acetonitrile, and the gradient elution program was: 0 min, 5% mobile phase B; 2 min, 10% mobile phase B; 6 min, 40% mobile phase B; 7 min, 5% mobile phase B; 10 min, 5% mobile phase B; the injection volume was 3 μL, the column temperature was 30°C, and the flow rate was 0.3 mL / min.

[0031] The mass spectrometry conditions were as follows: electrospray ion source, positive ion mode scanning; multiple reaction monitoring mode; electrospray voltage was 5500 V; ion source temperature was 500°C; curtain gas pressure was 45 psi; nebulizer gas pressure was 55 psi; and auxiliary gas pressure was 55 psi.

[0032] (4) Validation of the anemonin detection method.

[0033] The standard of anemonin was dissolved in 80% methanol, and the methodological validation was carried out by determining the detection limit, quantification limit, linear range, matrix effect, intra-day precision, inter-day precision and spike recovery according to the "Guidelines for Validation of Analytical Methods in Chinese Pharmacopoeia 9101", as follows: ① Determination of detection limit and quantification limit.

[0034] The signal-to-noise ratio (S / N) method was used, and the concentrations of the anemonin solution corresponding to S / N = 3 and S / N = 10 were used as the detection limit and quantification limit. Specifically, the liquid phase diagram of 80% methanol was used as a blank control, a low concentration of anemonin solution was prepared and adjusted according to the signal-to-noise ratio. The concentrations corresponding to S / N of 3 and 10 were the detection limit and quantification limit.

[0035] ②Linear range and linear equation.

[0036] The standard solution of anemonin was prepared with 80% methanol solution into standard working solutions with a series of mass concentrations of 25, 50, 75, 100, 250, and 500 ng / mL, and the solvent standard curve was drawn according to the concentration and the peak area of ​​the quantitative ion pair.

[0037] ③Matrix effect (ME).

[0038] A series of standard working solutions of anemonin at mass concentrations of 25, 50, 75, 100, 250, and 500 ng / mL were prepared using a blank honey matrix solution (i.e., an extract solution of a honey sample determined to be free of anemonin, the preparation method of which is described in step (1)). A matrix-matched standard curve was drawn based on the concentration and peak area of ​​the quantitative ion pair. ME was calculated using the following formula: ME = matrix-matched calibration curve slope / solvent standard curve slope.

[0039] ④Precision.

[0040] Intra-day precision was determined using 75, 100, and 250 ng / mL of anemonin solution, and the results were expressed as relative standard deviation (RSD%). Inter-day precision was also determined using 75, 100, and 250 ng / mL of anemonin solution, monitored for three consecutive days, and the results were expressed as RSD%.

[0041] ⑤ Spike recovery rate.

[0042] The concentration of annelidone solution was determined by adding 75, 100, and 250 ng / mL to blank honey, and the results were expressed as recovery rate (%).

[0043] The above test results are shown in Table 2. From the test results, it can be seen that the detection limit is 1.8 ng / mL, the quantification limit is 4.9 ng / mL, indicating that the method of the present invention has high sensitivity; the linear range is 25~500 ng / mL, the linear equation is y=103.54x-9.75, R 2 The RSD of intra-day precision was 2.41-8.11%, the RSD of inter-day precision was 5.84-12.09%, and the recovery rate of spiked samples was 97.52-99.91%, showing good precision and recovery, which met the requirements of the Guiding Principles for Validation of Analytical Methods of Chinese Pharmacopoeia 9101.

[0044] Table 2 Verification results of the method of the present invention

[0045] Example 2 According to the detection method in Example 1, this example quantitatively analyzed the anemonin in 42 collected native honey samples, and the specific operation was as follows: 42 samples of wild honey were weighed 5 g each and dissolved in 20 mL of ultrapure water. The samples were stirred to completely dissolve and the pH value was adjusted to 2 with hydrochloric acid to obtain wild honey solution. A Waters Oasis HLB solid phase extraction column was used and pre-activated with 5 mL of methanol and 5 mL of pH=2 hydrochloric acid aqueous solution. The wild honey solution was loaded into the activated solid phase extraction column and the honey solution was dripped at a rate of 1 drop / s (solid phase extraction instrument can be used for assistance). After all the honey solution flowed through the solid phase extraction column, 5 mL of pH=2 hydrochloric acid aqueous solution was added to the solid phase extraction column to remove the compounds not adsorbed by the filler. Subsequently, 5 mL of methanol was added to elute the adsorbed compounds and the eluate was collected. The eluate was blown to dryness with a nitrogen blower and stored to prepare the honey extract. Before use, the dried honey extract needed to be reconstituted with 50% formic acid methanol solution.

[0046] By external standard method, the anemonin in the above honey samples was quantified under the detection conditions of Example 1. The results are shown in Table 3. Among the 42 honey samples, only one sample was detected with anemonin, and its content was 100.19 μg / kg, with a positive rate of 2.38%. The MRM chromatograms of the qualitative ion pairs and quantitative ion pairs of the positive honey samples and the negative honey samples are shown in Table 3. Figure 3 Although anulmonin was detected in only a few honeys, the harm and potential risks of anulmonin to the human body cannot be ignored. It can be seen that the method of the present invention is suitable for the detection of anulmonin in honey samples and can be used as a reliable means of monitoring the quality and safety of native honey.

[0047] Table 3 Content of anemonin in honey samples

[0048] In summary, the present invention provides a new detection scheme for the detection of annulophantin, and it has the advantages of high sensitivity, low matrix effect, good precision and recovery rate, etc. It can provide reliable guarantee for the detection of low-content annulophantin in honey, and is of great significance to the quality supervision of Chinese native honey.

[0049] It should be noted that the above embodiments are only part of the embodiments of the present invention rather than all the embodiments, and are only used to illustrate the technical solution of the present invention rather than to limit it. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. A method for detecting anemonin based on high performance liquid chromatography tandem mass spectrometry, characterized in that: The following steps are involved: S1, pre-treating the sample to be tested to obtain an extract solution; S2. The extract solution is subjected to high performance liquid chromatography tandem mass spectrometry detection, and the anemonin is qualitatively and / or quantitatively analyzed according to the qualitative ion pair 193.0 / 97.0 and the quantitative ion pair 193.0 / 91.

0.

2. The method for detecting anuranol according to claim 1, characterized in that: The declustering voltages of the qualitative ion pair and the quantitative ion pair are both 60 V, the collision energy of the qualitative ion pair is 16 eV, and the collision energy of the quantitative ion pair is 36 eV.

3. The method for detecting anuranol according to claim 1, characterized in that: The sample to be tested is honey.

4. The method for detecting anuranol according to claim 3, characterized in that: Step S1 includes: S11, dissolving honey in ultrapure water, adjusting the pH to 2 with hydrochloric acid to obtain a honey solution; S12, loading the honey solution into an activated solid phase extraction column, and after all the honey solution flows through the solid phase extraction column, using a hydrochloric acid aqueous solution to remove substances not adsorbed by the filler; S13, eluting with methanol and collecting the eluate, drying the eluate to obtain a honey extract, and redissolving the honey extract to obtain a honey extract solution.

5. The method for detecting anuranol according to claim 4, characterized in that: The solid phase extraction column is activated by using methanol and a hydrochloric acid aqueous solution with a pH value of 2 for activation.

6. The method for detecting anuranol according to claim 4, characterized in that: The honey extract is re-dissolved using a 50% formic acid methanol solution.

7. The method for detecting anuranol according to claim 1, characterized in that: The chromatographic conditions in step S2 are: chromatographic column, C18 column 2.1 mm×100 mm, 1.9 μm; mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is acetonitrile, gradient elution; injection volume is 3 μL, column temperature is 30°C, and flow rate is 0.3 mL / min.

8. The method for detecting anuranol according to claim 7, characterized in that: The gradient elution procedure was: 0 min, 5% mobile phase B; 2 min, 10% mobile phase B; 6 min, 40% mobile phase B; 7 min, 5% mobile phase B; 10 min, 5% mobile phase B.

9. The method for detecting anuranol according to claim 1, characterized in that: The mass spectrometry conditions in step S2 are: electrospray ion source, positive ion mode scanning; multiple reaction monitoring mode; electrospray voltage is 5500 V; ion source temperature is 500°C; curtain gas pressure is 45 psi; nebulizer gas pressure is 55 psi; Auxiliary gas pressure is 55 psi.

10. The method for detecting anuranol according to claim 1, characterized in that: In step S2, quantitative analysis is performed based on the peak area of ​​the quantitative ion pair in the sample to be tested and the standard curve, wherein the standard curve is drawn as follows: the anemonin standard is prepared into a series of standard working solutions of different concentrations using 80% methanol solution, the peak area of ​​the quantitative ion pair in each standard working solution is detected and obtained, and the standard curve is drawn based on the concentration and the peak area of ​​the quantitative ion pair.

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