A method for screening and quantitative determination of multiple active ingredients in health food

Abstract

The invention relates to a method for screening and quantitatively determining multiple active ingredients in health food, comprising the following steps: S1: preparing a standard reserve solution; S2: preparing a standard intermediate solution; S3: preparing a standard working solution; S4: sample processing; S5: liquid chromatography quadrupole-electrostatic field orbital trap high-resolution mass spectrometer analysis; S6: ultra-high performance liquid chromatography-quadrupole mass spectrometry analysis. The invention uses high-resolution technology to quickly screen the chemical components in the health food, and simultaneously uses ultra-high performance liquid chromatography-quadrupole mass spectrometry (UPLC-MS / MS) to quantitatively analyze three components of N-p-trans-coumaryltyramide, notoginseng alcohol and notoginseng saponin R2 (S type) in the health food.

Description

Technical Field

[0001] The present invention relates to the technical field of active ingredient detection of health food, and in particular to a method for screening and quantitatively determining multiple active ingredients in health food. Background Art

[0002] Health food is a food with specific health functions or for the purpose of supplementing vitamins and minerals, suitable for consumption by specific groups of people, with the function of regulating body functions, not for the purpose of treating diseases, and without any acute, subacute or chronic harm to the human body. The standard stipulates that health food should have effective ingredients corresponding to its functional effects and their minimum content.

[0003] As one of the functional ingredients in health foods, coumarin has a wide range of physiological functions. Studies have shown that coumarin compounds have anti-tumor activity, anti-inflammatory effects, antioxidant, anticoagulant, antibacterial, and antiviral effects. Trans-N-trans-p-coumaryltyramide has attracted much attention due to its anti-inflammatory, antiviral, antioxidant, and α-glucosidase inhibition activities, among which α-glucosidase inhibition activity is particularly significant; Qianghuo alcohol also has the effects of inducing cell apoptosis, clearing away heat and detoxification, analgesic, and anti-inflammatory. Studies have shown that Qianghuo alcohol has the potential to be used in acute myeloid leukemia (AML). Trisaponins play an important role in health foods. Studies have shown that notoginseng saponins can promote blood circulation and improve immunity. They also have the effects of lowering blood lipids, lowering blood pressure, anti-inflammatory, antioxidant, anti-tumor, anti-fibrosis, sedation and anticonvulsant, and improving microcirculation. Notoginseng saponin R2 (S type) is an active saponin isolated from Panax notoginseng. Current studies have shown that it has neuroprotective and anti-tumor effects.

[0004] At present, the general determination of whether health foods contain active ingredients such as N-p-trans-coumaryl tyramine, notoginseng alcohol, notoginseng saponin R2 (S type) and their quantification are based on establishing specific detection methods for specific active ingredients. At the same time, there are few studies on the determination of whether health foods contain multiple active ingredients such as N-p-trans-coumaryl tyramine, notoginseng alcohol, notoginseng saponin R2 (S type) and their quantification. Therefore, there is currently no method suitable for the screening and quantitative determination of multiple active ingredients. Summary of the invention

[0005] The purpose of the present invention is to design a method for screening and quantitatively determining multiple active ingredients in health foods, and to establish a method for simultaneously determining whether health foods contain active ingredients such as N-p-trans-coumaryltyramine, notoginseng alcohol, and notoginseng saponin R2 (S type), and a quantitative determination method, so as to solve the problems raised in the background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A method for screening multiple active ingredients in health food comprises the following steps:

[0008] S1: Prepare standard stock solutions of N-p-trans-coumaryltyramide, notoginseng alcohol, and notoginsenoside R2 (S type) at a concentration of 1 mg / mL respectively;

[0009] S2: prepare standard intermediate solutions of N-p-trans-coumaryltyramide, notoginseng alcohol, and notoginsenoside R2 (S type) at a concentration of 10 μg / mL respectively;

[0010] S3: Prepare standard working solutions of N-p-trans-coumaryltyramide, notoginseng alcohol, and notoginseng saponin R2 (S type) at a concentration of 1 μg / mL respectively;

[0011] S4: Weigh the sample into a centrifuge tube, add methanol and vortex to disperse the sample, then perform ultrasonic extraction at room temperature, centrifuge, transfer the supernatant to another centrifuge tube, repeat the extraction once for the residue, combine the extracts, take 1 mL of the extract liquid and blow it to near dryness with nitrogen, re-dissolve it with acetonitrile, filter it through a membrane, and obtain the solution to be analyzed;

[0012] S5: Use a liquid chromatography quadrupole-electrostatic field orbital trap high-resolution mass spectrometer to perform chromatography and mass spectrometry detection and analysis on the standard working solution in S3 and the solution to be analyzed in S4.

[0013] Methanol is used as the extractant. Methanol has a strong extraction ability and can achieve the simultaneous extraction of multiple organic compounds. Methanol has a low boiling point and is easy to remove. Using methanol for extraction twice can ensure complete extraction. Acetonitrile is used as the re-solvent. Acetonitrile has a strong elution ability and the target peak on the chromatographic column has a sharp peak shape.

[0014] Preferably, 0.5 g of sample is placed in a centrifuge tube, 2.5 mL of methanol is added and vortexed to disperse the sample, then ultrasonicated at room temperature for 30 to 60 min, centrifuged, the supernatant is transferred to another centrifuge tube, the residue is extracted once again, the extracts are combined, 1 mL of the extract is blown to near dryness with nitrogen, and re-dissolved with 1 mL of acetonitrile.

[0015] Pass through the membrane to obtain the liquid to be analyzed.

[0016] Preferably, in the liquid chromatography quadrupole-electrostatic field orbital trap high-resolution mass spectrometer analysis, the chromatographic conditions are: chromatographic column: RRHD Ec li pse Plus C18, 3.0mm×150mm, 1.8μm; mobile phase A: 0.1% formic acid water, mobile phase B: acetonitrile; gradient elution conditions are: 0-4min, 40%A; 4-6min, 5%A; 6-8min, 5%A; 8-8.1min, 40%A; 8.1-10min, 40%A, flow rate: 0.2-0.4mL / min, column temperature: 30-40°C; injection volume: 1μL.

[0017] Preferably, in the liquid chromatography quadrupole-electrostatic field orbital trap high-resolution mass spectrometer analysis, the mass spectrometry conditions are: electrospray ion source (ESI), scanning mode: full scan-data dependent secondary scanning mode (Full MS / dd MS); sheath gas flow rate: 35arb, auxiliary gas flow rate: 10arb; spray voltage 3.5kV; ion transfer tube temperature 320°C, auxiliary gas heater temperature 320°C; primary scanning range: m / z 80~1000amu, primary scanning resolution: 70000, primary automatic gain control value: 2×105, injection time: 100ms; secondary scanning resolution: 17500, automatic gain control value: 2×105, injection time: 60ms; normalized collision energy: 20eV, 50eV, 80eV.

[0018] Preferably, before S5, a database and sample screening are established: a target database is established using Tracefinder software, and the data of the screened samples are matched with the targets in the database, thereby quickly determining whether the sample contains the target.

[0019] A method for quantifying multiple active ingredients in health food comprises the following steps:

[0020] S1: Prepare standard stock solutions of N-p-trans-coumaryltyramide, notoginseng alcohol, and notoginsenoside R2 (S type) at a concentration of 1 mg / mL respectively;

[0021] S2: prepare standard intermediate solutions I of N-p-trans-coumaryltyramide, notoginseng alcohol, and notoginseng saponin R2 (S type) at a concentration of 10 μg / mL respectively;

[0022] S3: prepare standard intermediate solutions II of N-p-trans-coumaryltyramide, notoginseng alcohol, and notoginseng saponin R2 (S type) at a concentration of 10 μg / mL respectively;

[0023] S4: Use S3 mixed standard intermediate solution II to prepare a series of standard working solutions, with the concentrations of N-p-trans-coumaryltyramide and notopterygium root alcohol being 1, 2, 5, 10, and 20 ng / mL, and the concentrations of notoginseng saponin R2 (S type) being 50, 100, 200, 500, and 1000 ng / mL, respectively.

[0024] S5: Weigh the sample into a centrifuge tube, add methanol and vortex to disperse the sample, then ultrasonicate at room temperature for 30 to 60 minutes, centrifuge, transfer the supernatant to another centrifuge tube, repeat the extraction of the residue, combine the extracts, blow the extracts with liquid nitrogen until nearly dry, re-dissolve with acetonitrile, pass through a membrane, and obtain the solution to be analyzed.

[0025] S6: Liquid chromatography-mass spectrometry is used to perform chromatography and mass spectrometry detection and analysis on the series of standard working solutions in S4 and the solution to be analyzed in S5.

[0026] Preferably, in the liquid chromatography-mass spectrometry analysis, ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry is used to quantitatively analyze the target effective ingredients.

[0027] Preferably, in S5, 0.5 g of sample is weighed into a centrifuge tube, 2.5 mL of methanol is added and vortexed to disperse the sample, then ultrasonicated at room temperature for 30 to 60 min, centrifuged, the supernatant is transferred to another centrifuge tube, the residue is extracted once again, the extracts are combined, 1 mL of the extract is blown to near dryness with nitrogen, re-dissolved with 1 mL of acetonitrile, and filtered to obtain the solution to be analyzed.

[0028] Preferably, in the liquid chromatography-mass spectrometry analysis, the chromatographic conditions are: chromatographic column: EVOO C18, 100×2.1mm, 2.6μm; mobile phase A: 0.1% formic acid water, mobile phase B: acetonitrile; gradient elution conditions: 0-2min, 40%A; 2-4min, 5%A; 4-5min, 5%A; 5-5.1min, 40%A; 5.1-6.5min, 40%A, flow rate: 0.2-0.4mL / min; column temperature: 30-40℃; injection volume: 1μL.

[0029] Preferably, in the liquid chromatography-mass spectrometry analysis, the mass spectrometry conditions are: electrospray ion source (ESI); positive / negative ions; scanning mode: multiple reaction secondary scanning (PRM); curtain gas: 25psi; collision gas: 9psi; temperature: 550°C; ionization voltage: 5500V; spray gas: 55psi; auxiliary heating gas: 55psi.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The present invention uses high-resolution technology to quickly screen the chemical components in health foods, and uses ultra-performance liquid chromatography-quadrupole mass spectrometry (UPLC-MS / MS) to quantitatively analyze the three active ingredients in health foods, namely N-p-trans-coumaryltyramine, notoginseng alcohol, and notoginseng saponin R2 (S type).

[0032] 2. The present invention adopts ultra-high performance liquid chromatography and utilizes gradient elution mode to achieve simultaneous determination of the content of active ingredients such as N-p-trans-coumaryl tyramine, qianghuo alcohol, and notoginseng saponin R2 (S type). The test method established by the present invention can better separate and determine the content of N-p-trans-coumaryl tyramine, qianghuo alcohol, and notoginseng saponin R2 (S type) in health food, and the chromatographic peak has a good peak shape and a high degree of separation. Under the chromatographic conditions, the concentration and peak area of ​​N-p-trans-coumaryl tyramine, qianghuo alcohol, and notoginseng saponin R2 (S type) show a good linear relationship, and the recovery rate of various components is above 80%. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 It is the selected ion chromatogram of N-p-trans-coumaryltyramide;

[0035] Figure 2 This is the selected ion chromatogram of notopterygium alcohol;

[0036] Figure 3 This is the selected ion chromatogram of notoginsenoside R2 (S type); DETAILED DESCRIPTION

[0037] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0038] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0039] When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs.

[0040] The present invention does not limit the source of raw materials. Unless otherwise specified, the raw materials used are common commercial products.

[0041] Health foods are commercially available;

[0042] The water used in the experiment is first-class water;

[0043] N-p-trans-coumaryltyramine, notoginseng alcohol, and notoginseng saponin R2 (S type) are standard substances certified by the state and awarded with standard substance certificates, and the purity of the standard substances is above 99.4%;

[0044] Methanol and acetonitrile were commercially available chromatographic grade.

[0045] The embodiment of the present invention provides a method for screening multiple active ingredients in health food, comprising the following steps:

[0046] S1: Preparation of standard stock solution: accurately add 5 mg of N-p-trans-coumaryl tyramine, notoginseng alcohol, and notoginseng saponin R2 (S type) respectively into a 5 mL volumetric flask, dilute with methanol and make up to volume to obtain a 1 mg / mL standard stock solution of N-p-trans-coumaryl tyramine, notoginseng alcohol, and notoginseng saponin R2 (S type), and store in a frozen state;

[0047] S2: Preparation of mixed standard solution: Accurately pipette 0.1 mL of N-p-trans-coumaryl tyramine, notoginseng alcohol, and notoginseng saponin R2 (S type) standard stock solutions into 10 mL volumetric flasks, dilute to scale with methanol, and obtain standard intermediate solutions of N-p-trans-coumaryl tyramine, notoginseng alcohol, and notoginseng saponin R2 (S type) with a concentration of 10 μg / mL;

[0048] S3: Accurately pipette 0.1 mL of the mixed standard intermediate solution prepared in S2, dilute it with acetonitrile and make it up to 10 mL to obtain a standard working solution of N-p-trans-coumaryltyramide, notoginseng alcohol and notoginseng saponin R2 (S type) with a concentration of 1 μg / mL;

[0049] S4: Weigh 0.5 g of sample into a centrifuge tube, add 2.5 mL of methanol and vortex to disperse the sample, then ultrasonicate at room temperature for 30 min, centrifuge at 8500 r / min for 2 min, transfer the supernatant to another centrifuge tube, repeat the extraction of the residue, combine the extracts, take 1 mL of the extract liquid, blow it to near dryness with nitrogen, re-dissolve it with 1 mL of acetonitrile, pass through a membrane, and obtain the solution to be analyzed.

[0050] S5: In the liquid chromatography quadrupole-electrostatic field orbital trap high-resolution mass spectrometer analysis, the mixed standard working solution in S3 and the solution to be analyzed in S4 are subjected to chromatography and mass spectrometry detection and analysis.

[0051] Wherein, in the analysis of liquid chromatography quadrupole-electrostatic field orbital trap high-resolution mass spectrometer, the chromatographic conditions are: chromatographic column: RRHD Ec lipse Plus C18, 3.0mm×150mm, 1.8μm; mobile phase A: 0.1% formic acid water, mobile phase B: acetonitrile; gradient elution conditions are: 0-4min, 40%A; 4-6min, 5%A; 6-8min, 5%A; 8-8.1min, 40%A;

[0052] 8.1~10min, 40% A, flow rate: 0.2mL / min; column temperature: 40℃; injection volume: 1μL. Mass spectrometry conditions were: electrospray ion source (ESI), scan mode: full scan-data dependent secondary scan mode (Full MS / dd MS); sheath gas flow: 35arb, auxiliary gas flow: 10arb; spray voltage 3.5kV; ion transfer tube temperature 320℃, auxiliary gas heater temperature 320℃; primary scan range: m / z 80~1000amu, primary scan resolution: 70000, primary automatic gain control value: 2×10 5 , injection time: 100ms; secondary scanning resolution: 17500, automatic gain control value: 2×10 5 , injection time: 60ms; normalized collision energy: 20, 50, 80eV.

[0053] The selected ion chromatogram of N-p-trans-coumaryltyramide is shown in Figure 1 As shown, the selected ion chromatogram of Qianghuo alcohol is as follows Figure 2 As shown, the selected ion chromatogram of notoginseng saponin R2 (S type) is as follows Figure 3 Under the experimental instrument conditions, the accurate mass numbers of the detection ions of the three active ingredients are shown in Table 1, and four active ingredient data comparison libraries were established for rapid screening of target substances in health foods.

[0054] The target database is established by using Tracefinder software, and the data of the screening samples are matched with the targets in the database to quickly determine whether the samples contain the target.

[0055] Table 1 Parent ion and fragment ion information of N-p-coumaroyltyramide, notoginseng alcohol, and notoginseng saponin R2 (S type)

[0056]

[0057] The embodiment of the present invention provides a method for quantifying multiple active ingredients in health food, comprising the following steps:

[0058] S1: Prepare standard stock solutions of N-p-trans-coumaryltyramide, notoginseng alcohol, and notoginsenoside R2 (S type) at a concentration of 1 mg / mL respectively;

[0059] S2: prepare standard intermediate solution I of N-p-trans-coumaryltyramide, notoginseng alcohol, and notoginseng saponin R2 (S type) at a concentration of 10 μg / mL;

[0060] S3: prepare standard intermediate solution II with concentrations of 0.1 μg / mL N-p-trans-coumaryltyramide, notoginseng alcohol and 1 μg / mL notoginseng saponin R2 (S type) respectively;

[0061] S4: Use S3 mixed standard intermediate solution II to prepare a series of standard working solutions, the concentrations of N-p-trans-coumaryltyramide and notopterygium root alcohol are 1, 2, 5, 10, 20 ng / mL, and the concentration of notoginseng saponin R2 (S type) is 50, 100, 200, 500, 1000 ng / mL;

[0062] S4: Weigh 0.5 g of sample into a centrifuge tube, add 2.5 mL of methanol and vortex to disperse the sample, then ultrasonicate at room temperature for 30 to 60 min, centrifuge, transfer the supernatant to another centrifuge tube, extract the residue repeatedly, combine the extracts, take 1 mL of the extract liquid and blow it to near dryness with nitrogen, re-dissolve it with 1 mL of acetonitrile, filter it through a membrane, and obtain the solution to be analyzed;

[0063] S5: Ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry analysis, performing chromatography and mass spectrometry detection and analysis on the series of standard working solutions in S3 and the solution to be analyzed in S4.

[0064] Among them, in ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry analysis, the chromatographic conditions are: chromatographic column: EVOO C18, 100×2.1mm, 2.6μm; mobile phase A: 0.1% formic acid water, mobile phase B: acetonitrile; gradient elution conditions: 0-2min, 40%A; 2-4min, 5%A; 4-5min, 5%A; 5-5.1min, 40%A; 5.1-6.5min, 40%A, flow rate: 0.2mL / min; column temperature: 40℃; injection volume: 1μL.

[0065] The mass spectrometry conditions were as follows: electrospray ion source (ESI); positive / negative ions; scanning mode: multiple reaction secondary scanning (PRM); curtain gas: 25 pSi; collision gas: 9 pSi; temperature: 550°C; ionization voltage: 5500 V; spray gas: 55 pSi; auxiliary heating gas: 55 pSi.

[0066] The mass spectrometry parameters of N-p-trans-coumaryltyramide, notoginseng alcohol, notoginseng saponin R2 (S type), and notoginseng saponin Fc are shown in Table 2.

[0067] Table 2 Mass spectrometry parameters of N-p-coumaroyltyramide, notoginseng alcohol, and notoginseng saponin R2 (S type)

[0068]

[0069] Note: * indicates quantitative ion

[0070] (1) Linear equation, correlation coefficient and limit of quantification

[0071] The mixed standard curve working solution was prepared according to S3, and the samples were injected and measured according to the chromatographic conditions. The linear range was investigated, and the standard curve was drawn with the injection volume (X, μg) as the horizontal axis and the corresponding peak area (Y) as the vertical axis. The linear equation, linear range, correlation coefficient, detection limit and quantification limit were calculated. The detection limit (RSN=3) and quantification limit (RSN=10) of the target were determined according to the signal-to-noise ratio. The results are shown in Table 3.

[0072] Table 3 Linear range, correlation coefficient, detection limit and quantification limit of three active ingredients in health foods

[0073]

[0074] (2) Method repeatability

[0075] Weigh 0.5g of blank sample, 6 portions in total, add the mixed target and mix well, extract according to the extraction method under S4, and analyze under the conditions under S5. The average contents of N-p-trans-coumaryltyramine, notoginseng alcohol, and notoginseng saponin R2 (S type) were measured to be 0.0572mg / kg, 0.0574mg / kg, and 2.62mg / kg, and their RSDs were 7.67%, 7.23%, and 0.99%, respectively. This shows that the test method of the present invention has good repeatability.

[0076] (3) Instrument stability

[0077] The instrument was run 6 times at the punctuation point under S3, and the change in the peak area of ​​the target substance was observed. As shown in Table 4, the RSDs of the peak areas of the target substance were 6.51%, 6.25%, and 0.99%, respectively.

[0078] Table 4 Instrument stability data (n=6)

[0079]

[0080]

[0081] (4) Recovery and precision of the method

[0082] According to item S4, the spike recovery experiment was carried out on the low background samples, 6 samples were weighed independently for spike determination, and then the spike recovery rate and relative standard deviation were calculated. The results are shown in Table 5. The average spike recovery rate was 81.51% to 102.88%, and the relative standard deviation was 1.50% to 3.44%.

[0083] Table 5: Results of spike recovery test of the method (n=6)

[0084]

[0085] (5) UPLC-MS / MS determination of three active ingredients in health foods

[0086] The contents of N-p-trans-coumaryltyramine and notoginsenoside R2 (S type) in health food samples were 0.231 mg / kg and 3.19 mg / kg respectively, and notoginseng alcohol was not detected. This method provides a technical reference for the detection of active ingredients in health foods, and has positive significance for exploring the functions of health foods, meeting consumer needs, and promoting the development of the health food industry.

[0087] Table 6 Contents of three active ingredients in health foods

[0088] Compound Content: mg / kg RSD% N-p-trans-coumaryl tyramide 0.231 3.99 Notopterygium 0 - <![CDATA[Notoginsenoside R2 (S form)]]> 3.19 7.97

[0089] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for screening multiple active ingredients in health food, characterized in that: The steps include: S1: Prepare standard stock solutions of N-p-trans-coumaryltyramide, notoginseng alcohol, and notoginsenoside R2 (S type) at a concentration of 1 mg / mL respectively; S2: prepare standard intermediate solutions of N-p-trans-coumaryltyramide, notoginseng alcohol, and notoginsenoside R2 (S type) at a concentration of 10 μg / mL respectively; S3: Prepare standard working solutions of N-p-trans-coumaryltyramide, notoginseng alcohol, and notoginseng saponin R2 (S type) at a concentration of 1 μg / mL respectively; S4: Weigh the sample into a centrifuge tube, add methanol and vortex to disperse the sample, then perform ultrasonic extraction at room temperature, centrifuge, transfer the supernatant to another centrifuge tube, repeat the extraction once for the residue, combine the extracts, blow the extract liquid with nitrogen until it is nearly dry, then re-dissolve it with acetonitrile, filter it through a membrane, and obtain the solution to be analyzed; S5: Liquid chromatography quadrupole-electrostatic field orbital trap high-resolution mass spectrometer performs chromatography and mass spectrometry detection and analysis on the standard working solution in S3 and the solution to be analyzed in S4.

2. The method for screening multiple active ingredients in health food according to claim 1, characterized in that: In S4, 0.5 g of sample was weighed into a centrifuge tube, 2.5 mL of methanol was added and vortexed to disperse the sample, then ultrasonicated at room temperature for 30 to 60 min, centrifuged, and the supernatant was transferred to another centrifuge tube. The residue was extracted again, the extracts were combined, 1 mL of the extract was blown to near dryness with nitrogen, re-dissolved with 1 mL of acetonitrile, and filtered to obtain the solution to be analyzed.

3. The method for screening multiple active ingredients in health food according to claim 1, characterized in that: In the liquid chromatography quadrupole-electrostatic field orbital trap high-resolution mass spectrometer analysis, the chromatographic conditions are: chromatographic column: RRHD Eclipse Plus C18, 3.0 mm×150 mm, 1.8 μm; mobile phase A: 0.1% formic acid water, mobile phase B: acetonitrile; gradient elution conditions are: 0-4 min, 40% A; 4-6 min, 5% A; 6-8 min, 5% A; 8-8.1 min, 40% A; 8.1~10min, 40%A; flow rate 0.2~0.4mL / min; column temperature 30~40℃; injection volume: 1μL.

4. The method for screening multiple active ingredients in health food according to claim 1, characterized in that: In the liquid chromatography quadrupole-electrostatic field orbital trap high-resolution mass spectrometer analysis, the mass spectrometry conditions are: electrospray ion source (ESI), scanning mode: full scan-data dependent secondary scanning mode (Full MS / dd MS); sheath gas flow rate: 35 arb, auxiliary gas flow rate: 10 arb; spray voltage 3.5 kV; ion transfer tube temperature 320°C, auxiliary gas heater temperature 320°C; primary scanning range: m / z 80-1000 amu, primary scanning resolution: 70000, primary automatic gain control value: 2×10 5 , injection time: 100ms; secondary scanning resolution: 17500, automatic gain control value: 2×10 5 , injection time: 60ms; normalized collision energy: 20eV, 50eV, 80eV.

5. The method for screening multiple active ingredients in health food according to claim 1, characterized in that: Before S5, establish a database and sample screening: use Tracefinder software to establish a target database, and match the data of the screening samples with the database targets to quickly determine whether the samples contain the target.

6. A method for quantifying multiple active ingredients in health food, characterized in that: The following steps are involved: S1: Prepare standard stock solutions of N-p-trans-coumaryltyramide, notoginseng alcohol, and notoginsenoside R2 (S type) at a concentration of 1 mg / mL respectively; S2: prepare standard intermediate solutions I of N-p-trans-coumaryltyramide, notoginseng alcohol, and notoginseng saponin R2 (S type) at a concentration of 10 μg / mL respectively; S3: prepare standard intermediate solution II with concentrations of 0.1 μg / mL N-p-trans-coumaryltyramide, notoginseng alcohol and 1 μg / mL notoginseng saponin R2 (S type) respectively; S4: Use S3 mixed standard intermediate solution II to prepare a series of standard working solutions. The concentrations of N-p-trans-coumaryltyramide and notopterygium root alcohol are 1, 2, 5, 10, and 20 ng / mL, respectively, and the concentration of notoginseng saponin R2 (S type) is 50, 100, 200, 500, and 1000 ng / mL. S5: Weigh the sample into a centrifuge tube, add methanol and vortex to disperse the sample, then perform ultrasonic extraction at room temperature, centrifuge, transfer the supernatant to another centrifuge tube, repeat the extraction once for the residue, combine the extracts, blow the extracts to near dryness with liquid nitrogen, re-dissolve with acetonitrile, filter through a membrane, and obtain the solution to be analyzed; S6: In the liquid chromatography-mass spectrometry analysis, the standard working solution in S4 and the solution to be analyzed in S5 are subjected to chromatography and mass spectrometry detection and analysis.

7. The method for quantifying multiple active ingredients in health food according to claim 6, characterized in that: Ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry was used for quantitative analysis of the target active ingredients.

8. The method for quantifying multiple active ingredients in health food according to claim 6, characterized in that: In S5, 0.5 g of sample was weighed into a centrifuge tube, 2.5 mL of methanol was added and vortexed to disperse the sample, then ultrasonicated at room temperature for 30 to 60 min, centrifuged, and the supernatant was transferred to another centrifuge tube. The residue was extracted again, the extracts were combined, 1 mL of the extract was blown to near dryness with nitrogen, re-dissolved with 1 mL of acetonitrile, and filtered to obtain the solution to be analyzed.

9. The method for quantifying multiple active ingredients in health food according to claim 6, characterized in that: In the liquid chromatography-mass spectrometry analysis, the chromatographic conditions are: chromatographic column: EVOO C18, 100×2.1mm, 2.6μm; mobile phase A: 0.1% formic acid water, mobile phase B: acetonitrile; gradient elution conditions: 0-2min, 40%A; 2-4min, 5%A; 4-5min, 5%A; 5-5.1min, 40%A; 5.1-6.5min, 40%A; flow rate: 0.2-0.4mL / min; column temperature: 30-40℃; injection volume: 1μL.

10. The method for quantifying multiple active ingredients in health food according to claim 6, characterized in that: In the liquid chromatography-mass spectrometry analysis, the mass spectrometry conditions are: electrospray ionization source (ESI); positive / negative ion; scanning mode: multiple reaction secondary scanning (PRM); Curtain gas: 25psi; Collision gas: 9psi; Temperature: 550°C; Ionization voltage: 5500V; Spray gas: 55psi; Auxiliary heating gas: 55psi.

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