Method for simultaneously determining four flavonoid components in pine pollen based on ultra-high performance liquid chromatography-tandem mass spectrometry

Abstract

The invention relates to the technical field of detection, and provides a method for simultaneously determining four flavonoid components in pine pollen based on ultra-high performance liquid chromatography-tandem mass spectrometry, which comprises the following steps: preparing a sample to be detected and detecting the sample to be detected by using ultra-high performance liquid chromatography-tandem mass spectrometry. The method disclosed by the invention is simple and convenient to operate, small in detection sample dosage, high in analysis speed, good in stability, good in repeatability and high in accuracy, can be used for simultaneously determining the contents of four flavonoid components with relatively large pollen pini content difference, and provides a new direction for rapid refined quality detection of the pollen pini.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection, and particularly relates to a method for simultaneously determining 4 flavonoid components in pine pollen by ultra-high performance liquid chromatography tandem mass spectrometry. Background Art

[0002] Pine pollen is the dried pollen of Pinus massoniana Lamb. Pinus massoniana Lamb ( Pinus tabuliformis Carr ) or Pinus tabuliformis Carr. or the dried pollen of several plants of the same genus. Pine pollen is rich in various bioactive components such as flavonoids, volatile oils, sterols, nucleic acids, etc., and has various effects such as antioxidant, immune regulation, blood lipid regulation, blood sugar regulation, etc., and is widely used in health products, pharmaceuticals, cosmetics and other aspects.

[0003] In recent years, domestic and foreign research on pine pollen has mostly focused on pharmacological effects, health products, etc., while the research on the correlation between the material basis and biological effects of pine pollen has been reported less, and there is a lack of systematic research on the quality control of pine pollen. In addition, pine pollen is widely used in health foods, but only a few Chinese patent medicines use pine pollen as a raw material. Chinese invention patent CN102426156A discloses a method for determining the total flavonoid content in the supercritical extract of pine pollen, but this method can only detect the total flavonoid content. As the main flavonoid substances in pine pollen, taxifolin, rutin, naringenin, and kaempferol, there is currently no method for simultaneously and rapidly detecting taxifolin, rutin, naringenin, and kaempferol in pine pollen in the prior art.

[0004] Therefore, it is very necessary to develop a method for simultaneously determining 4 flavonoid components in pine pollen by ultra-high performance liquid chromatography tandem mass spectrometry. Summary of the Invention

[0005] To solve the above technical problems, the purpose of the present invention is to provide a method for simultaneously determining 4 flavonoid components in pine pollen by ultra-high performance liquid chromatography tandem mass spectrometry, and establish an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-Q-MS / MS) method for simultaneously determining the contents of 4 flavonoid active components, namely taxifolin, rutin, naringenin, and kaempferol, in pine pollen, in order to provide a direction for better developing pine pollen medicinal materials and scientifically and reasonably establishing the quality standard of pine pollen.

[0006] The purpose of the present invention is achieved as follows. The 4 flavonoid components are taxifolin, rutin, naringenin, and kaempferol, and the method includes: Preparation of the sample to be measured: Take the pine pollen sample, add petroleum ether for defatting, then add 60% ethanol as the extraction solvent for reflux extraction, and then filter, and take the subsequent filtrate as the sample to be measured; Detect the sample to be measured by ultra-high performance liquid chromatography tandem mass spectrometry, wherein: Chromatographic conditions: Chromatographic column: C18 chromatographic column; Mobile phase A: 0.1% formic acid aqueous solution; Gradient elution: From 0 min to 2.00 min, the volume ratio of mobile phase A to mobile phase B is maintained at 30:70; From 2.00 min to 3.00 min, the volume ratio of mobile phase A to mobile phase B is maintained at 55:45; From 3.00 min to 5.00 min, the volume ratio of mobile phase A to mobile phase B is maintained at 60:40; From 5.00 min to 6.00 min, the volume ratio of mobile phase A to mobile phase B is maintained at 30:70; Mass spectrometry conditions: Ion source: Electrospray ionization source; Detection mode: Negative ion scanning mode.

[0007] Among them, detection by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS / MS) means separating the sample by high performance liquid chromatography, using a triple quadrupole mass spectrometer as the detector. Because the mass-to-charge ratios of taxifolin, rutin, naringenin, and kaempferol are different, after mass spectrometry separation, they pass through the detector in a certain order to obtain a chromatogram, and the standard curve method is used for quantitative analysis.

[0008] Preferably, for the preparation of the sample to be tested, 1 g of pine pollen sample is taken, defatted with 50 times the amount (mass multiple) of petroleum ether, 100 ml of 60% ethanol is used, the reflux extraction time is 1 h, cooled to room temperature and made up to the original weight, filtered through a 0.22 µm microporous filter membrane, and the subsequent filtrate is taken to obtain the sample.

[0009] Preferably, in the chromatographic conditions, the specifications of the chromatographic column are: 1.7 µm, 2.1 mm × 100 mm; Flow rate: 0.3 mL / min; Column temperature: 40 °C; Injection volume: 1 µL.

[0010] Preferably, in the mass spectrometry conditions, the ion source temperature is 500 °C; the ion source voltage is -4.5 kV; the spray gas Gas1 is 55 Psi; the auxiliary heating gas Gas2 is 55 Psi; the curtain gas is 7 Psi; the monitoring mode is multiple reaction monitoring MRM.

[0011] Preferably, for the mass spectrometry conditions of taxifolin, rutin, naringenin, and kaempferol, the mass spectrometry parameters are shown in Table 1. In Table 1, * represents the quantitative ion; Table 1 Mass spectrometry parameters of each component .

[0012] Preferably, the regression equations and correlation coefficients r of the 4 flavonoid components are shown in Table 2; Table 2 Regression equations and correlation coefficients r of each component , In the table, x is the mass concentration of each component, and y is the peak area of the quantitative ion pair.

[0013] Advantages of the present invention: The method of the present invention is simple to operate, requires less sample for detection, has a fast analysis speed, good stability, good repeatability, and high accuracy. It can simultaneously determine the contents of 4 flavonoid components with large content differences in pine pollen, providing a new direction for the rapid and refined quality detection of pine pollen. Description of the Drawings

[0014] Figure 1 It is the TIC chromatogram of the reference substance for Example 1 (1 is taxifolin, 2 is rutin, 3 is naringenin, 4 is kaempferol); Figure 2 It is the liquid chromatogram of 4 flavonoid components in the sample solution of Example 1 (A is taxifolin, B is rutin, C is naringenin, D is kaempferol). Detailed Embodiments

[0015] The present invention will be further described below in conjunction with the embodiments and the drawings, but it is not limited to the present invention in any way. Any transformation or substitution based on the teachings of the present invention belongs to the protection scope of the present invention.

[0016] Example 1 This example is based on ultra-high performance liquid chromatography tandem mass spectrometry for the simultaneous determination of 4 flavonoid components in pine pollen. The specific method is as follows: The first step is solution preparation: Reference substance solution: Taxifolin reference substance (batch number 111816 - 202403), rutin reference substance (batch number 100080 - 202012), purchased from the National Institutes for Food and Drug Control; Kaempferol reference substance (batch number B21126, purity 98%), naringenin reference substance (batch number B21596, purity 98%); Take appropriate amounts of taxifolin, rutin, naringenin, and kaempferol reference substances, accurately weigh them, and place them in 10 mL volumetric flasks respectively. Add methanol to dissolve and make up to the mark, shake well, and obtain reference substance solutions with taxifolin, rutin, naringenin, and kaempferol mass concentrations of 0.1036, 0.1002, 0.1008, and 0.1018 mg / mL respectively; Accurately measure appropriate amounts of the above reference substance solutions respectively, place them in the same 10 mL volumetric flask, add 50% methanol to make up to the mark, shake well, and obtain a mixed reference substance solution with taxifolin, rutin, naringenin, and kaempferol mass concentrations of 1.0360, 0.2505, 1.7150, and 25.20 μg / mL respectively; Test solution: Pinus yunnanensis pollen samples (batch numbers 20240106, 20240615, 20241213), purchased from Kunming, Yunnan Province; weigh 1.0 g of the pine pollen sample accurately, transfer it to a 250 mL conical flask, add petroleum ether for defatting at 50 times the amount (mass multiple), then add 100 mL of 60% ethanol and reflux for 1 h. Let it cool and make up the weight, filter through a 0.22 µm microporous membrane, and take the subsequent filtrate to obtain the test solution; Second step, methodological investigation: (1) Specificity test: Take the reference solution and test solution prepared in the first step, and inject them into an ultra-high performance liquid chromatography-tandem mass spectrometry for injection and determination. Ultra-high performance liquid chromatograph: LC-40 type from Shimadzu Corporation, Japan; mass spectrometer: 5500+QTRAP type from AB SCIEX Corporation; Chromatographic conditions: Chromatographic column: ACQUITY UPLC BEH C18 chromatographic column, specifications: 1.7 µm, 2.1 mm × 100 mm; Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: chromatographic grade methanol; Gradient elution: From 0 min to 2.00 min, the volume ratio of mobile phase A to mobile phase B is maintained at 30:70; From 2.00 min to 3.00 min (excluding the 2.00 min point value here), the volume ratio of mobile phase A to mobile phase B is maintained at 55:45; From 3.00 min to 5.00 min (excluding the 3.00 min point value here), the volume ratio of mobile phase A to mobile phase B is maintained at 60:40; From 5.00 min to 6.00 min (excluding the 5.00 min point value here), the volume ratio of mobile phase A to mobile phase B is maintained at 30:70; Flow rate: 0.3 mL / min; Column temperature: 40 °C; Injection volume: 1 µL; Mass spectrometry conditions: Ion source: electrospray ionization source; Detection mode: negative ion scanning mode; Ion source temperature: 500 °C; Ion source voltage: -4.5 kV; Spray gas Gas1: 55 Psi; Auxiliary heating gas Gas2: 55 Psi; Curtain gas: 7 Psi; Monitoring mode: multiple reaction monitoring MRM; The mass spectrometry parameters of taxifolin, rutin, naringenin, and kaempferol are shown in Table 1. In Table 1, * is the quantitative ion; Table 1 Mass spectrometry parameters of each component ; Monitoring the target ions in MRM mode can effectively exclude matrix interference; in the chromatograms of the test solution and the reference solution, the retention times of each component peak are consistent, the peak shapes are good, and there is no interference. The chromatograms are shown in Figure 1 ; (2)Investigation of linear relationship: Accurately pipette an appropriate amount of the mixed reference substance solution in the first step, and successively dilute it with 50% methanol to obtain a series of mass concentration of the mixed reference substance solution of taxifolin (0.2072 μg / mL, 0.4144 μg / mL, 0.6216 μg / mL, 0.8288 μg / mL, 1.036 μg / mL); rutin (0.02004 μg / mL, 0.04008 μg / mL, 0.06012 μg / mL, 0.08016 μg / mL, 0.1002 μg / mL); naringenin (5.0400 μg / mL, 10.0800 μg / mL, 15.1200 μg / mL, 20.1600 μg / mL, 25.20 μg / mL), kaempferol (0.3430 μg / mL, 0.6860 μg / mL, 1.0290 μg / mL, 1.3720 μg / mL, 1.7150 μg / mL). Determine according to the chromatographic conditions and mass spectrometry conditions in the first step. Perform linear regression with the mass concentration (x, ng / mL) of each component as the abscissa and the peak area of the quantitative ion pair (y) as the ordinate. The results are shown in Table 2; Table 2 Results of the investigation of the linear relationship of each component (n = 6, n represents the sample size) ; Taxifolin, rutin, naringenin, and kaempferol showed good linear relationships in the ranges of 0.2072 - 1.0360 μg / mL, 0.0501 - 0.2505 μg / mL, 5.04 - 25.20 μg / mL, and 0.01715 - 1.7150 μg / mL (r = 0.99994, 0.9995, 0.99995, 0.9998); (3)Determination of the limit of quantitation and limit of detection: Accurately pipette the mixed reference substance solution in the first step, and successively dilute it with 50% methanol. Determine by ultra-high performance liquid chromatography-tandem mass spectrometry according to the same chromatographic conditions and mass spectrometry conditions in (1); Take the mass concentrations at signal-to-noise ratios (S / N) of 10 and 3 as the limit of quantitation and limit of detection, respectively; The limits of quantitation of taxifolin, rutin, naringenin, and kaempferol were 3.9632, 15.045, 0.1081, and 6.4556 ng / mL, respectively, and the limits of detection were 1.1889, 4.5135, 0.0324, and 1.9366 ng / mL, respectively; (4)Precision test: Take the mixed reference substance solution with the mass concentrations of taxifolin, rutin, naringenin, and kaempferol being 207.20, 20.04, 5040.00, and 343.00 ng / mL respectively under the investigation of linear relationship. According to the same chromatographic conditions and mass spectrometry conditions in (1), use ultra-high performance liquid chromatography-tandem mass spectrometry to inject samples continuously for 6 times; the RSDs of the peak areas of taxifolin, rutin, naringenin, and kaempferol are 0.013%, 0.016%, 0.0097%, and 0.0095% (n = 6) respectively, indicating that the precision of the instrument is good; (5)Stability test: Take the sample (batch number 20240106), prepare the test solution according to the method in the first step, and at 0, 2, 4, 6, 8, 12, and 24 h at room temperature, inject samples using ultra-high performance liquid chromatography-tandem mass spectrometry according to the same chromatographic conditions and mass spectrometry conditions in (1); the RSDs of the peak areas of taxifolin, rutin, naringenin, and kaempferol are 2.22%, 3.11%, 1.83%, and 4.07% (n = 7) respectively, indicating that the test solution has good stability when placed at room temperature for 24 h; (6)Repeatability test: Take 6 portions of the sample (batch number 20240106), each portion being 1.0 g, prepare the test solution according to the method in the first step, inject samples using ultra-high performance liquid chromatography-tandem mass spectrometry according to the same chromatographic conditions and mass spectrometry conditions in (1), and calculate the content of the components to be measured; the RSDs of the contents of taxifolin, rutin, naringenin, and kaempferol are 2.22%, 3.22%, 1.25%, and 0.062% (n = 6) respectively, indicating that the method has good repeatability; (7)Spiked recovery test: Take 6 portions of the sample (batch number 20240106), each portion being 0.5 g, accurately weigh, add the mixed reference substance solution in the first step according to the mass ratio of 1:1, prepare the test solution according to the method in the first step, inject samples using ultra-high performance liquid chromatography-tandem mass spectrometry according to the same chromatographic conditions and mass spectrometry conditions in (1), and calculate the spiked recovery rate; Table 3 shows that the RSDs (peak areas) of the contents of taxifolin, rutin, naringenin, and kaempferol are 3.10%, 4.81%, 4.23%, and 6.78% (n = 6) respectively, indicating that the method of the present invention has high accuracy. In the table refers to the average percentage content; Table 3 Results of spiked recovery test (n = 6, n represents the sample size) .

[0017] Example 2 This example is based on the simultaneous determination of 4 flavonoid components in pine pollen by ultra-high performance liquid chromatography-tandem mass spectrometry, and is carried out according to the method of Example 1. Specifically, for 3 batches of pine pollen samples (batch numbers are 20240106, 20240615, and 20241213), the test solution is prepared according to the method of Example 1. 1.0 mL of the sample is taken, and according to the chromatographic conditions and mass spectrometry conditions of Example 1, ultra-high performance liquid chromatography-tandem mass spectrometry is used for injection and determination 3 times. The content of the component to be measured is calculated by substituting into the regression equation, and the results are shown in Table 4; Table 4 Results of the determination of the content of each component (ug / g, n = 2, n represents the sample size) 。

[0018] Comparative Experiment 1 When preparing the sample to be measured, the extraction solvents are selected as pure 20%, 40%, 60%, 80%, and 100% ethanol, and the rest of the process is carried out according to the present invention; the influence on the sample yield is compared, and the results are shown in Table 5. Considering comprehensively, it is found that when the ethanol concentration of the present invention is 60%, the response of each compound is the highest; Table 5 Results of the comparative experiment of the extraction solvent 。

[0019] Comparative Experiment 2 When preparing the sample to be measured, the extraction methods are maceration, ultrasonic method, and reflux extraction method, and the rest of the process is carried out according to the present invention; the results are shown in Table 6, and the results show that the response of each component is the highest by the reflux extraction method; Table 6 Results of the comparative experiment of the extraction method 。

[0020] Comparative Experiment 3 When preparing the sample to be measured, the reflux extraction time is set to 20 min, 30 min, 40 min, 60 min, and 90 min, and the rest of the process is carried out according to the present invention; the influence on the extraction effect is compared, and the results are shown in Table 7. When the reflux extraction time is 60 min, the extraction is basically complete. Considering comprehensively, the extraction time is selected as 60 min; Table 7 Results of the comparative experiment of the extraction time 。

Claims

1. A method for simultaneously determining four flavonoid components in pine pollen based on ultra-performance liquid chromatography-tandem mass spectrometry, characterized in that The four flavonoid components are taxifolin, rutin, naringenin, and kaempferol, and the method comprises: Preparation of the sample to be tested: Take a pine pollen sample, add petroleum ether to defatted, then add 60% ethanol as the extraction solvent for reflux extraction, then filter, and take the filtrate as the sample to be tested; The sample to be tested is detected by ultra-high performance liquid chromatography tandem mass spectrometry, wherein: Chromatographic conditions: Chromatographic column: ACQUITY UPLC BEH C18 Column, specification: 1.7um, 2.1mm×100mm; Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: Chromatographic grade methanol; Gradient elution: From 0 min to 2.00 min, the volume ratio of mobile phase A to mobile phase B was maintained at 30:70; From 2.00 min to 3.00 min, the volume ratio of mobile phase A to mobile phase B was maintained at 55:45; From 3.00 min to 5.00 min, the volume ratio of mobile phase A to mobile phase B was maintained at 60:40; From 5.00 min to 6.00 min, the volume ratio of mobile phase A to mobile phase B was maintained at 30:70; Flow rate: 0.3mL / min; column temperature: 40℃; injection volume: 1µL; Mass spectrometry conditions: ion source: electrospray ion source; detection method: negative ion scanning mode.

2. The method for simultaneously determining four flavonoid components in pine pollen based on ultra-performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that The sample preparation is to take 1g of pine pollen sample, add 50 times the amount of petroleum ether to defatting, use 100ml of 60% ethanol, reflux extraction time 1h, cool to make up the weight, filter using a 0.22uL microporous filter membrane, and take the filtrate.

3. The method for simultaneously determining four flavonoid components in pine pollen based on ultra-performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that The mass spectrometry conditions are as follows: ion source temperature: 500° C.; ion source voltage: −4.5 kV; spray gas Gas1: 55 Psi; auxiliary heating gas Gas2: 55 Psi; curtain gas: 7 Psi; and monitoring mode: multiple reaction monitoring MRM.

4. The method for simultaneously determining four flavonoid components in pine pollen based on ultra-performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that Among the mass spectrometry conditions, the mass spectrometry parameters of taxifolin, rutin, naringenin, and kaempferol are shown in Table 1 , in which * represents a quantitative ion; Table 1 Mass spectrometry parameters of each component 。 5. The method for simultaneously determining four flavonoid components in pine pollen based on ultra-performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that The regression equations and correlation coefficients r of the four flavonoid components are shown in Table 2 ; Table 2 Regression equations and correlation coefficients of each component ， In the table, x is the mass concentration of each component, and y is the peak area of ​​the quantitative ion pair.

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