Method for detecting 2-hydroxynaringenin in plants

Abstract

The invention belongs to the technical field of analysis and detection, and particularly relates to a method for detecting 2-hydroxynaringenin in plants. The detection method comprises the following steps: extracting a plant sample to obtain a liquid to be detected; detecting the liquid to be detected by adopting high performance liquid chromatography-tandem mass spectrometry to obtain the peak area of the 2-hydroxynaringenin in the plant sample; and according to the peak area of the 2-hydroxynaringenin and a preset standard curve, calculating the content of the 2-hydroxynaringenin in the plant sample, the detection conditions of the ultra-high performance liquid chromatography are as follows: a mobile phase A is water; the mobile phase B is acetonitrile; the ion source temperature of the ultra-high performance liquid chromatography is 500 DEG C. The method has the advantages of favorable linear relationship (favorable linear relationship (r gt; 0.999)), favorable precision and favorable repeatability, and can accurately determine the content of 2-hydroxynaringenin in plants.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, specifically relating to a method for detecting 2-hydroxynaringenin in plants. Background Technology

[0002] Flavonoids are a class of natural polyphenolic compounds widely found in plants such as tea and fruits. They possess beneficial health effects, including antioxidant, anti-inflammatory, and cardiovascular protection properties. Almost all natural flavonoids exist in plants as O-glycosides or C-glycosides. 2-Hydroxynaringin (CAS No.: 58124-18-8) is a 2-hydroxyflavanone compound within the flavonoid family, with the molecular formula C2. 15 H 12 O6, with a molecular weight of 288.25, is generated from naringenin under the action of flavonoid-2-hydroxylase. 2-Hydroxynaringenin is a key precursor for the synthesis of vitexin (apigenin-8-C-glucoside, CAS No.: 3681-93-4) and isovitexin (apigenin-6-C-glucoside, CAS No.: 38953-85-4), and is an important substrate for the function of C-glycosyltransferase.

[0003] However, 2-hydroxynaringenin exhibits tautomerism in the reaction system (Formula I), and its chemical structure is unstable under acidic conditions. Its 2-hydroxyl group readily undergoes spontaneous removal and conversion to apigenin (CAS: 520-36-5), rendering it ineffective as a precursor for C-glycosylation reactions. Furthermore, its content in plants is typically extremely low and difficult to detect, and literature reports on its detection and quantitative analysis are scarce. Formula I. Summary of the Invention

[0004] In view of this, the present invention provides a method for detecting 2-hydroxynaringenin in plants. The method provided by the present invention can achieve qualitative and accurate quantification of 2-hydroxynaringenin.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for detecting 2-hydroxynaringenin in plants, comprising the following steps: Plant samples were extracted to obtain the test solution; High performance liquid chromatography-tandem mass spectrometry was used to detect the test solution and obtain the peak area of ​​2-hydroxynaringenin in the plant sample; The content of 2-hydroxynaringenin in the plant sample was calculated based on the peak area of ​​2-hydroxynaringenin and the predetermined standard curve. The detection conditions for the ultra-high performance liquid chromatography include: mobile phase A is water; mobile phase B is acetonitrile; The ion source temperature of the ultra-high performance liquid chromatography is 500℃.

[0006] Preferably, the flow rates of mobile phases A and B in the ultra-high performance liquid chromatography are 1 mL / min; the chromatographic column is an Atlantis Premier BEH Z-HILIC column; and the column temperature is 40℃.

[0007] Preferably, the mass spectrometry conditions for the ultra-high performance liquid chromatography include: simultaneous use of positive and negative ion modes, ion spray voltage of +5500V / -4500V, curtain gas of 35psi, ion source gas 1 of 50L / min, ion source gas 2 of 60L / min, collision gas of medium, scan type of multiple reaction monitoring, inlet voltage of ±10V, and collision cell outlet voltage of ±10V.

[0008] Preferably, the gradient elution procedure is as follows: 0~1min: The volume percentage of mobile phase A is 10%; 1~4 min: The volume percentage of the mobile phase A increases uniformly from 10% to 25%; 4~4.1 min: The volume percentage of the mobile phase A increases uniformly from 25% to 50%; 4.1~5.5 min: The volume percentage of mobile phase A is 50%; 5.5~5.6 min: The volume percentage of the mobile phase A decreases uniformly from 50% to 10%; 5.6~7.5 min: The volume percentage of the mobile phase A is 10%.

[0009] Preferably, the extractant used for extraction is an aqueous methanol solution.

[0010] Preferably, the volume concentration of the methanol aqueous solution is 70%.

[0011] Preferably, the mass spectrometry data of the 2-hydroxynaringenin are those of the 2-hydroxynaringenin. m / z : 287.1, 93, retention time is 1.2 min, declustering voltage is -50 V, collision energy is -30 eV.

[0012] Preferably, the plant comprises the aboveground tissues of Arabidopsis thaliana and / or poplar.

[0013] This invention provides a method for detecting 2-hydroxynaringenin in plants, comprising the following steps: extracting plant samples to obtain a test solution; detecting the test solution using high performance liquid chromatography-tandem mass spectrometry to obtain the peak area of ​​2-hydroxynaringenin in the plant sample; calculating the content of 2-hydroxynaringenin in the plant sample based on the peak area of ​​2-hydroxynaringenin and a predetermined standard curve; the detection conditions of the ultra-high performance liquid chromatography include: mobile phase A is water; mobile phase B is acetonitrile; the ion source temperature of the ultra-high performance liquid chromatography is 500℃; the method has good linearity (good linearity (r>0.999)), good precision and repeatability, and can accurately determine the content of 2-hydroxynaringenin in plants. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0015] Figure 1 The standard curve for 2-hydroxynaringenin; Figure 2 The TIC chromatogram for 2-hydroxynaringenin standard is shown, where the blue peak represents 2-hydroxynaringenin. Detailed Implementation

[0016] This invention provides a method for detecting 2-hydroxynaringenin in plants, comprising the following steps: Plant samples were extracted to obtain the test solution; High performance liquid chromatography-tandem mass spectrometry was used to detect the test solution and obtain the peak area of ​​2-hydroxynaringenin in the plant sample; The content of 2-hydroxynaringenin in the plant sample was calculated based on the peak area of ​​2-hydroxynaringenin and the predetermined standard curve. The detection conditions for the ultra-high performance liquid chromatography (UHPLC) include: mobile phase A is water; mobile phase B is acetonitrile; the elution method is gradient elution; and the ion source temperature for the UHPLC is 500℃.

[0017] This invention involves extracting plant samples to obtain a test solution; In one embodiment of the present invention, the plant includes the aboveground tissues of Arabidopsis thaliana and / or poplar; the aboveground tissues include stems and leaves; the extraction process further includes freeze-drying the plant sample to obtain plant powder. In another embodiment of the present invention, the extractant used for extraction can be an aqueous methanol solution; the concentration of the aqueous methanol solution can be 70 vol%.

[0018] This invention uses high performance liquid chromatography to detect the test solution and obtain the peak area of ​​2-hydroxynaringenin in the plant sample; based on the peak area of ​​2-hydroxynaringenin and a predetermined standard curve, the content of 2-hydroxynaringenin in the plant sample is calculated.

[0019] In one embodiment of the present invention, the detection conditions of the ultra-high performance liquid chromatography include: mobile phase A is water; mobile phase B is acetonitrile; the flow rates of mobile phases A and B can be 1 mL / min; the chromatographic column can be an Atlantis PremierBEH Z-HILIC column with a size of 1.7 μm and a diameter of 2.1 μm. 100mm; column temperature can be 40℃; As one embodiment of the present invention, the mass spectrometry conditions of the ultra-high performance liquid chromatography include: ion source temperature of 500℃; simultaneous use of positive and negative ion modes (ESI+ / -); ion spray voltage of +5500V / -4500V; curtain gas of 35psi; ion source gas 1 of 50L / min; ion source gas 2 of 60L / min; collision gas of medium; scan type of multiple reaction monitoring (MRM); entrance potential of ±10V; and collision cell exit potential of ±10V.

[0020] In one embodiment of the present invention, the mass spectrometry data of 2-hydroxynaringenin can be [data missing]. m / z The values ​​are 287.1 and 93, the retention time is 1.2 min, the declustering voltage is -50 V, and the collision energy is -30 eV.

[0021] As one embodiment of the present invention, obtaining the predetermined standard curve includes the following steps: A series of 2-hydroxynaringenin standard solutions were prepared, and then ultra-high performance liquid chromatography-tandem mass spectrometry was used for detection. The peak area of ​​2-hydroxynaringenin at each concentration was used as the ordinate and the concentration was used as the abscissa for linear fitting to obtain the predetermined standard curve.

[0022] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0023] Instruments: Ultra Performance Liquid Chromatography (UPLC) (ExionLC™ AD) and Tandem Mass Spectrometry (MS / MS) (QTRAP® 6500+), Eppendorf 5424R centrifuge, RADWAG AS 60 / 220.R2 electronic balance, Shanghai Jingxin MIX-200, multi-tube vortex mixer.

[0024] Reagents: Merck chromatographic grade methanol, CNW chromatographic grade acetonitrile, Sigma-Aldrich chromatographic grade formic acid, standard 2-hydroxynaringenin (Acmec, H64940-5mg, HPLC ≥98%), and ultrapure water prepared by a Milli-Q7005 ultrapure water preparation system. The plant material was Arabidopsis thaliana (…). Arabidopsis thaliana ) or poplar ( Populus alba × Populus glandulosa The above-ground tissues (including all stems and leaves).

[0025] Example 1 (1) High-performance liquid chromatography-tandem mass spectrometry detection conditions: mobile phase A was water; mobile phase B was acetonitrile; flow rate was 1 mL / min; chromatographic column was an Atlantis Premier BEH Z-HILIC column (1.7 μm, 2.1 μm). The column temperature was 40℃ (100 mm). The gradient elution program was as follows: 0~1 min: the volume percentage of mobile phase A was 10%; 1~4 min: the volume percentage of mobile phase A increased uniformly from 10% to 25%; 4~4.1 min: the volume percentage of mobile phase A increased uniformly from 25% to 50%; 4.1~5.5 min: the volume percentage of mobile phase A was 50%; 5.5~5.6 min: the volume percentage of mobile phase A decreased uniformly from 50% to 10%; 5.6~7.5 min: the volume percentage of mobile phase A was 10%. The ion source temperature was 500℃, using both positive and negative ion modes. The ion spray voltage was +5500V / -4500V, the curtain gas was 35psi, ion source gas 1 was 50L / min, ion source gas 2 was 60L / min, the collision gas was medium, the scan type was multiple reaction monitoring, the inlet voltage was ±10V, and the collision cell outlet voltage was ±10V. The mass spectrometry data of 2-hydroxynaringenin were... m / z : 287.1, 93, retention time is 1.2 min, declustering voltage is -50 V, collision energy is -30 eV; (2) Standard curve: The standard of 2-hydroxynaringenin (HPLC ≥ 98%) was dissolved in dimethyl sulfoxide (DMSO) to prepare standard solutions of 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL and 10 ng / mL. The mass spectrometry peak intensity data of the corresponding quantitative signals of the standard at each concentration were obtained. The standard curve was obtained by plotting the concentration as the abscissa and the peak area as the ordinate. See Figure 1 ,Depend on Figure 1 We know that the linear equation is y = 7.35231e 4 x + 766.49858 (r = 0.99978), the detection limit is 0.05 μg / L.

[0026] (3) Plant sample preparation: Fresh plant samples were freeze-dried under vacuum for 3 days, and then ground into powder using a ball mill (30 Hz, 1.5 min). 50 mg of the powder sample was weighed into a 2 mL centrifuge tube, and 500 μL of 70% methanol aqueous solution was added. The mixture was vortexed for 5 min. Then, the mixture was centrifuged at 12000 rpm / min for 10 min at 4 °C. 300 μL of the supernatant was transferred into a 1.5 mL centrifuge tube and allowed to stand at 20 °C for 30 min. Then, the mixture was centrifuged again at 12000 rpm / min for 10 min at 4 °C. 100 μL of the supernatant was used for subsequent liquid chromatography-mass spectrometry detection.

[0027] Example 2 Instrument precision test: A standard solution with a concentration of 660 μg / L was prepared, and then injected continuously 6 times under the ultra-high performance liquid chromatography-mass spectrometry conditions described above. The RSD of the peak area of ​​2-hydroxynaringenin was calculated. The RSD value was 3.21%, indicating that the instrument precision was good.

[0028] Example 3 Repeatability test: Six portions of fresh poplar leaves were prepared in parallel according to step (3) of Example 1, and then detected under the conditions of ultra-high performance liquid chromatography-mass spectrometry described above. The RSD value was 4.71%, which proved that the method had good repeatability.

[0029] Example 4 Stability test: Fresh poplar leaves were prepared according to step (3) of Example 1. The resulting test solution was injected at 0, 2, 4, 8, 12 and 24 h respectively. The RSD value of the peak area was calculated to be 2.32%, indicating good stability.

[0030] Example 5 Fresh poplar leaves were freeze-dried under vacuum for 3 days, then ground into powder using a ball mill (30 Hz, 1.5 min). 50 mg of the poplar leaf powder sample was weighed into a 2 mL centrifuge tube, and 500 μL of 70% methanol-water solution was added. The mixture was vortexed for 5 min. Subsequently, it was centrifuged at 12000 rpm / min for 10 min at 4 °C, and 300 μL of the supernatant was transferred to a 1.5 mL centrifuge tube and allowed to stand at 20 °C for 30 min. Then, it was centrifuged again at 12000 rpm / min for 10 min at 4 °C, and 100 μL of the supernatant was used for subsequent liquid chromatography-mass spectrometry (LC-MS) analysis.

[0031] The supernatant was analyzed under the ultra-high performance liquid chromatography-mass spectrometry conditions described above, and the resulting peak area was y=7.35231e. 4 x + 766.49858 (r = 0.99978) yielded a 2-hydroxynaringenin content of 0.00074832 μg / mg.

[0032] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for detecting 2-hydroxynaringenin in plants, characterized in that, Includes the following steps: Plant samples were extracted to obtain the test solution; The peak area of ​​2-hydroxynaringenin in the plant sample was obtained by detecting the test solution using ultra-high performance liquid chromatography-tandem mass spectrometry. The content of 2-hydroxynaringenin in the plant sample was calculated based on the peak area of ​​2-hydroxynaringenin and the predetermined standard curve. The detection conditions for the ultra-high performance liquid chromatography include: mobile phase A is water; Mobile phase B is acetonitrile; The ion source temperature of the ultra-high performance liquid chromatography is 500℃.

2. The detection method according to claim 1, characterized in that, The flow rates of mobile phases A and B in the ultra-high performance liquid chromatography were 1 mL / min; the chromatographic column was an Atlantis Premier BEH Z-HILIC column; and the column temperature was 40℃.

3. The detection method according to claim 1, characterized in that, The mass spectrometry conditions for the ultra-high performance liquid chromatography (UHPLC) include: simultaneous use of positive and negative ion modes, ion spray voltage of +5500V / -4500V, curtain gas of 35 psi, ion source gas 1 of 50 L / min, ion source gas 2 of 60 L / min, collision gas of medium, scan type of multiple reaction monitoring, inlet voltage of ±10V, and collision cell outlet voltage of ±10V.

4. The detection method according to claim 1, characterized in that, The gradient elution procedure is as follows: 0~1min: The volume percentage of mobile phase A is 10%; 1~4 min: The volume percentage of the mobile phase A increases uniformly from 10% to 25%; 4~4.1 min: The volume percentage of the mobile phase A increases uniformly from 25% to 50%; 4.1~5.5 min: The volume percentage of mobile phase A is 50%; 5.5~5.6 min: The volume percentage of the mobile phase A decreases uniformly from 50% to 10%; 5.6~7.5 min: The volume percentage of the mobile phase A is 10%.

5. The detection method according to claim 1, characterized in that, The extractant used for extraction is an aqueous methanol solution.

6. The detection method according to claim 5, characterized in that, The volume concentration of the methanol aqueous solution is 70%.

7. The detection method according to claim 1, characterized in that, The mass spectrometry data of the 2-hydroxynaringenin are as follows: m / z : 287.1, 93, retention time is 1.2 min, declustering voltage is -50 V, collision energy is -30 eV.

8. The detection method according to claim 1, characterized in that, The plants include the aboveground tissues of Arabidopsis thaliana and / or poplar.

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