A method for detecting chemical components in Euonymus fortunei or compound Euonymus fortunei mixture based on UPLC-QTRAP-MS / MS

Abstract

This invention belongs to the field of detection and analysis technology, specifically relating to a method for detecting chemical components in Euonymus fortunei or compound Euonymus fortunei mixture based on UPLC-QTRAP-MS / MS. This invention employs the UPLC-QTRAP-MS / MS analytical method, which can simultaneously determine the content of 15 components in Euonymus fortunei herb or 34 components in compound Euonymus fortunei mixture, and has been applied to the content determination of multiple batches of compound Euonymus fortunei mixture. Systematic methodological validation has shown that this method has good precision, repeatability, and accuracy, meeting the requirements of multi-component quantitative analysis. The content determination results provide strong scientific evidence for establishing more scientific and reliable quality control methods and comprehensively and systematically evaluating the safety and rationality of its clinical use.

Description

Technical Field

[0001] This invention belongs to the field of detection and analysis technology, specifically relating to a method for detecting chemical components in Euonymus fortunei or compound Euonymus fortunei mixture based on UPLC-QTRAP-MS / MS. Background Technology

[0002] Fufangteng ( Euonymiherba (This refers to the creeping Euonymus, a plant belonging to the Celastraceae family.) Euonymusfortunei (Turcz.) Hand.Mazz, Holly Euonymus Euonymusjaponicus L. or baldless euonymus Euonymussubsessilis The dried aerial parts of *Sprague*. The *Compendium of Materia Medica* describes it as "treating all blood disorders, all qi disorders, and all cold disorders." *Sprague* is a traditional ethnic medicine of Guangxi, entering the liver, spleen, and kidney meridians. It has the effects of tonifying qi and blood, relaxing muscles and tendons, dispelling wind and dampness, and stopping bleeding. It is commonly used to treat qi and blood deficiency syndromes, lumbar muscle strain, rheumatic pain, fractures, traumatic bleeding, metrorrhagia, and metrorrhagia. Compound *Sprague* preparations, with *Sprague* as the principal ingredient and supplemented with red ginseng and astragalus, are widely used in clinical practice as a classic tonifying formula. This preparation follows the traditional Chinese medicine theory of tonification and has significant effects in tonifying qi and blood, strengthening the spleen and nourishing the heart. Modern pharmacological studies have confirmed that its active ingredients show definite efficacy in improving syndromes of heart and spleen deficiency, regulating cardiovascular homeostasis, and correcting immune imbalances.

[0003] The 2021 edition of the "Quality Standards for Zhuang Medicines of Guangxi Zhuang Autonomous Region" only describes the biological identification methods and thin-layer chromatography identification methods for *Euonymus fortunei*, lacking a binary quality control standard based on "component-content". Furthermore, the quality standards in Part I of the *Pharmacopoeia of the People's Republic of China* (2020 edition) regarding compound *Euonymus fortunei* preparations only include the determination of astragaloside A content in *Astragalus membranaceus* using thin-layer chromatography. Existing detection methods both domestically and internationally largely remain at the level of qualitative and quantitative analysis of single components, lacking multi-index component simultaneous detection schemes based on chromatography-mass spectrometry (GC-MS). This situation restricts the in-depth development and rational clinical application of compound *Euonymus fortunei* preparations. Summary of the Invention

[0004] The purpose of this invention is to provide a method for detecting chemical components in Euonymus fortunei or compound Euonymus fortunei mixture based on UPLC-QTRAP-MS / MS. The method provided by this invention can simultaneously detect multiple chemical components in Euonymus fortunei or compound Euonymus fortunei mixture.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for detecting chemical components in Euonymus fortunei or compound Euonymus fortunei preparations based on UPLC-QTRAP-MS / MS, comprising the following steps:

[0007] Extracting Euonymus fortunei or compound Euonymus fortunei mixture with methanol-water solution to obtain the test solution of Euonymus fortunei or compound Euonymus fortunei mixture;

[0008] The test solution of Euonymus fortunei or compound Euonymus fortunei mixture was analyzed by UPLC-QTRAP-MS / MS to obtain the peak area of ​​each chemical component to be tested in Euonymus fortunei or compound Euonymus fortunei mixture.

[0009] Based on the peak area of ​​each chemical component to be tested in Euonymus fortunei or Compound Euonymus fortunei Mixture and the corresponding predetermined standard curve of the chemical component, the content of each chemical component to be tested in Euonymus fortunei or Compound Euonymus fortunei Mixture is obtained; the predetermined standard curve of each chemical component to be tested is a linear relationship between the concentration and peak area of ​​each chemical component to be tested.

[0010] During UPLC-QTRAP-MS / MS detection, the chromatographic separation conditions included: mobile phase A was formic acid aqueous solution; mobile phase B was acetonitrile; and the chromatographic column was C. 18 The column; the flow rates of mobile phase A and mobile phase B are 0.3 mL / min; the elution method is gradient elution;

[0011] The conditions for mass spectrometry detection include: ion source: electrospray ion source; detection mode: multiple reaction ion detection.

[0012] Preferably, the conditions for the mass spectrometry detection include: curtain gas: 45 psi; jet gas: Low; ionization voltage: 5500 V; temperature: 450 ℃; spray gas: 50 psi; auxiliary heating gas: 20 psi.

[0013] Preferably, the chromatographic column is a CORTECS UPLC C18 column; the column temperature is 35 ℃; and the injection volume is 2 μL.

[0014] Preferably, when detecting Compound Euonymus fortunei Mixture, the gradient elution procedure is as follows:

[0015] 0-1 min: The volume percentage of mobile phase B increases from 5% to 14%;

[0016] 1-3 min: The volume percentage of mobile phase B increases from 14% to 40%;

[0017] 3-5 min: The volume percentage of mobile phase B is 40%;

[0018] In 5-6 minutes, the volume percentage of the dynamic phase B increased from 40% to 59%.

[0019] Over 6-8 minutes, the volume percentage of the dynamic phase B increased from 59% to 95%.

[0020] Preferably, when detecting Euonymus fortunei, the gradient elution procedure is as follows:

[0021] 0-1 min: The volume percentage of mobile phase B increases from 5% to 14%;

[0022] 1-4 min: The volume percentage of mobile phase B increases from 14% to 85%;

[0023] In 4-5 minutes, the volume percentage of the dynamic phase B increased from 85% to 95%.

[0024] Preferably, the chemical components to be tested in the compound Euonymus fortunei mixture are euonymus alcohol, pyrogallol, protocatechuic acid, p-hydroxybenzoic acid, gentian acid, syringic acid, mogroflavin, camelliaside B, rutin, verrucoside, kaempferol-3- O - At least two of the following: rutin, eugenol, ferulic acid, ginsenoside Re, ginsenoside Rg1, cyproterin B, styracin, salicylic acid, daidzein, quercetin, verrucoside isoflavone, ginsenoside Rb1, ginsenoside Rf, ginsenoside Rc, ginsenoside Ro, ginsenoside Rg2, ginsenoside Rh1, astragaloside A, ginsenoside Rd, styracin, ginsenoside Rg3, tripterygine, and ginsenoside Rk1.

[0025] Preferably, the chemical components to be tested in the Euonymus fortunei are at least two of the following: euonymus alcohol, rutin, kaempferol B, mogroflavin, syringaldehyde, tripterygine, cyprotinin B, p-hydroxybenzoic acid, salicylic acid, gentian acid, protocatechuic acid, ferulic acid, syringic acid, kaempferol, and quercetin.

[0026] Preferably, the volume concentration of the methanol aqueous solution is 70% when extracting Euonymus fortunei.

[0027] Preferably, when extracting the compound Euonymus fortunei mixture, the volume concentration of the methanol aqueous solution is 50%.

[0028] Preferably, the volume concentration of formic acid in the formic acid aqueous solution is 0.1%.

[0029] This invention employs a UPLC-QTRAP-MS / MS analytical method to simultaneously determine the content of 15 components in *Euonymus fortunei* herb or the content of 34 components in compound *Euonymus fortunei* compound preparations. This method has been applied to the content determination of multiple batches of compound *Euonymus fortunei* compound preparations. Systematic methodological validation has demonstrated that this method possesses good precision, repeatability, and accuracy, meeting the requirements for multi-component quantitative analysis. The content determination results provide strong scientific evidence for establishing more scientific and reliable quality control methods and for comprehensively and systematically evaluating the safety and rationality of its clinical use. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 A is the MRM chromatogram of the mixed reference standard, and B is the MRM chromatogram of the test solution; 1. Euonymus alcohol; 2. Pyrogallic acid; 3. Protocatechuic acid; 4. p-hydroxybenzoic acid; 5. Gentianic acid; 6. Syringic acid; 7. Mogroside; 8. Camellia glycoside B; 9. Rutin; 10. Verbena isoflavone glycoside; 11. Kaempferol; 12. Kaempferol-3- O - Rutin; 13. Syringaldehyde; 14. Ferulic acid; 15. Ginsenoside Re; 16. Ginsenoside Rg1; 17. Cynanchine B; 18. Arganin; 19. Salicylic acid; 20. Daidaidzein; 21. Quercetin; 22. Versicolor isoflavone; 23. Ginsenoside Rb1; 24. Ginsenoside Rf; 25. Ginsenoside Rc; 26. Ginsenoside Ro; 27. Ginsenoside Rg2; 28. Ginsenoside Rh1; 29. ​​Astragaloside A; 30. Ginsenoside Rd; 31. Arganin; 32. Tripterygium alkaloid; 33. Ginsenoside Rg3; 34. Ginsenoside Rk1;

[0032] Figure 2 In Figure A, the MRM chromatogram of the reference standard is shown, and in Figure B, the MRM chromatogram of the Euonymus fortunei sample is shown. The components are: 1. Euonymus alcohol; 2. Protocatechuic acid; 3. p-hydroxybenzoic acid; 4. Gentian acid; 5. Syringic acid; 6. Mogroflavin; 7. Camellia glycoside B; 8. Rutin; 9. Kaempferol; 10. Syringaldehyde; 11. Ferulic acid; 12. Cynancoside B; 13. Salicylic acid; 14. Quercetin; 15. Tripterygium alkaloid. Detailed Implementation

[0033] This invention provides a method for detecting chemical components in Euonymus fortunei or compound Euonymus fortunei preparations based on UPLC-QTRAP-MS / MS, comprising the following steps:

[0034] Extracting Euonymus fortunei or compound Euonymus fortunei mixture with methanol-water solution to obtain the test solution of Euonymus fortunei or compound Euonymus fortunei mixture;

[0035] The test solution of Euonymus fortunei or compound Euonymus fortunei mixture was analyzed by UPLC-QTRAP-MS / MS to obtain the peak area of ​​each chemical component to be tested in Euonymus fortunei or compound Euonymus fortunei mixture.

[0036] Based on the peak area of ​​each chemical component to be tested in Euonymus fortunei or Compound Euonymus fortunei Mixture and the corresponding predetermined standard curve of the chemical component, the content of each chemical component to be tested in Euonymus fortunei or Compound Euonymus fortunei Mixture is obtained; the predetermined standard curve of each chemical component to be tested is a linear relationship between the concentration and peak area of ​​each chemical component to be tested.

[0037] This invention involves extracting Euonymus fortunei or compound Euonymus fortunei mixture with methanol-water solution to obtain a test solution of Euonymus fortunei or compound Euonymus fortunei mixture.

[0038] In one embodiment of the present invention, when extracting Euonymus fortunei, the volume concentration of the methanol aqueous solution can be 70%; when extracting compound Euonymus fortunei mixture, the volume concentration of the methanol aqueous solution can be 50%.

[0039] This invention uses UPLC-QTRAP-MS / MS to detect the test solution of Euonymus fortunei or compound Euonymus fortunei mixture, and obtains the peak area of ​​each chemical component to be tested in Euonymus fortunei or compound Euonymus fortunei mixture.

[0040] As one embodiment of the present invention, the chemical components to be tested in the compound Fufangteng mixture may be euonymus alcohol, pyrogallol, protocatechuic acid, p-hydroxybenzoic acid, gentian acid, syringic acid, mogroflavin, camelliaside B, rutin, verrucoside, kaempferol-3- O - At least two of the following: rutin, eugenol, ferulic acid, ginsenoside Re, ginsenoside Rg1, cyproterin B, styracin, salicylic acid, daidzein, quercetin, verrucoside isoflavone, ginsenoside Rb1, ginsenoside Rf, ginsenoside Rc, ginsenoside Ro, ginsenoside Rg2, ginsenoside Rh1, astragaloside A, ginsenoside Rd, styracin, ginsenoside Rg3, tripterygine, and ginsenoside Rk1.

[0041] As one embodiment of the present invention, when using UPLC-QTRAP-MS / MS to detect the test solution of Compound Fufangteng Mixture, the chromatographic separation conditions include: mobile phase A is an aqueous formic acid solution; the volume concentration of the aqueous formic acid solution can be 0.1%; mobile phase B is acetonitrile; the flow rates of mobile phase A and mobile phase B are 0.3 mL / min; and the chromatographic column is C10. 18 The column can be a CORTECS UPLC C18 column, the column temperature can be 35 ℃; the injection volume can be 2 μL, and the elution method is gradient elution.

[0042] The gradient elution procedure can be as follows:

[0043] 0-1 min: The volume percentage of mobile phase B increases from 5% to 14%;

[0044] 1-3 min: The volume percentage of mobile phase B increases from 14% to 40%;

[0045] 3-5 min: The volume percentage of mobile phase B is 40%;

[0046] In 5-6 minutes, the volume percentage of the dynamic phase B increased from 40% to 59%.

[0047] Over 6-8 minutes, the volume percentage of the dynamic phase B increased from 59% to 95%.

[0048] The conditions for mass spectrometry detection include: Ion source: electrospray ionization source; Detection mode: multiple reaction ion detection (MRM); Curtain gas (CUR): 45 psi; Collision gas (CAD): Low; Ionization voltage (IS): 5500 V; Temperature (TEM): 450 ℃; Spray gas (GS1): 50 psi; Auxiliary heating gas (GS2): 20 psi.

[0049] As one embodiment of the present invention, the chemical components to be tested in the Euonymus fortunei can be at least two of the following: euonymus alcohol, rutin, kaempferol B, mogroflavin, syringaldehyde, tripterygine, cyprotinin B, p-hydroxybenzoic acid, salicylic acid, gentian acid, protocatechuic acid, ferulic acid, syringic acid, kaempferol and quercetin.

[0050] As one embodiment of the present invention, when detecting the test solution of Euonymus fortunei by UPLC-QTRAP-MS / MS, the gradient elution program can be as follows: 0-1 min: the volume percentage of mobile phase B increases from 5% to 14%; 1-4 min: the volume percentage of mobile phase B increases from 14% to 85%; 4-5 min: the volume percentage of mobile phase B increases from 85% to 95%. Other chromatographic separation conditions and mass spectrometry detection conditions are preferably the same as those used when detecting the test solution of compound Euonymus fortunei mixture, and will not be described in detail here.

[0051] Table 1. Mass spectrometry parameters of compound Euonymus fortunei mixture or chemical components in Euonymus fortunei.

[0052]

[0053]

[0054] To further illustrate the present invention, the following detailed description of the invention's solutions, in conjunction with the accompanying drawings and embodiments, is provided, but should not be construed as limiting the scope of protection of the present invention.

[0055] Instruments and reagents involved in the examples

[0056] ① Instruments

[0057] A60 / 220.R2 type 1 / 100,000 balance (Radwag); 5424R type high-speed centrifuge (Eppendorf); SCIEX QTRAP6500+ liquid chromatography-mass spectrometry system (SCIEX); Milli-Q7005 ultrapure water preparation system (Millipore); Analyst analysis software (SCIEX).

[0058] ② Drug testing

[0059] Reference standards: rutin (batch number: DST210520-017), ginsenoside Rg3 (batch number: DST191107-011(S)), argentin (batch number: MUST-17041101), camelliaside B (batch number: DSTDS008001), mogroside (batch number: DSTDL003001), ginsenoside Rf (batch number: MUST-13101010), eugenol (batch number: DSTDD030901), tripterygine (batch number: DST220812-078) Ginsenoside Rg1 (batch number: DST190828-009), cypronoside B (batch number: DST240424-078), ginsenoside Re (batch number: MUST-13041201), p-hydroxybenzoic acid (batch number: DSTDD011401), ginsenoside Rk1 (batch number: DST200409-034), salicylic acid (batch number: DSTDS012401), gentic acid (batch number: DSTDL001301), protocatechuic acid (batch number: DSTDY008101), ginseng Ginsenoside Rg2 (batch number: DST190517-010), euonymol (batch number: DSTDW002701), ferulic acid (batch number: DST191112-001), syringic acid (batch number: DST200927-057), kaempferol (batch number: DST230914-003), ginsenoside Rd (batch number: DSTDRO01502), quercetin (batch number: DSTDH002802), ginsenoside Rh1 (batch number: DST170405-035(S)), daidzein ( Batch No.: DST180128-020), Ginsenoside Ro (Batch No.: DST221230-031), Astragaloside A (Batch No.: DSTDM001102), Ginsenoside Rb1 (Batch No.: DST191212-006), Vernix isoflavone glycoside (Batch No.: DSTDM001301), Astragaloside A (Batch No.: DSTDH001503), Pyrogallic acid (Batch No.: DJ0172-0020), Vernix isoflavone (Batch No.: DST220226-012), Kaempferol-3- ORutin (batch number: DST18110-075) and ginsenoside Rc (batch number: MUST-13081902) were purchased from Chengdu Dester Biotechnology Co., Ltd. and Chengdu Mansite Biotechnology Co., Ltd.; acetonitrile and methanol were of chromatographic grade and purchased from Fisher; ultrapure water was prepared by an ultrapure water system.

[0060] In the example, 10 batches of Compound Fufangteng Mixture were provided by Guangxi Zhongyi Bainianle Pharmaceutical Co., Ltd., which were numbered S1-S10 respectively. The batch numbers of S1-S10 were 20230905, 20230301, 20230302, 20230303, 20230304, 20230504, 20230505, 20231011, 20231001, and 20240304.

[0061] The medicinal material of Euonymus fortunei is produced in Yulin City, Guangxi Zhuang Autonomous Region, and its batch number is 20230204.

[0062] Example 1

[0063] 1. Solution preparation

[0064] 1.1. Preparation of reference solution

[0065] Accurately weigh the following reference standards: rutin, ginsenoside Rg3, argentin, kaempferol B, mogroside, ginsenoside Rf, syringaldehyde, tripterygine, ginsenoside Rg1, cyprotinin B, ginsenoside Re, p-hydroxybenzoic acid, ginsenoside Rk1, salicylic acid, gentic acid, protocatechuic acid, ginsenoside Rg2, ferulic acid, syringic acid, kaempferol, ginsenoside Rd, quercetin, ginsenoside Rh1, daidzein, ginsenoside Ro, argentin, ginsenoside Rb1, verrucoside, astragaloside A, pyrogallol, verrucoside, and kaempferol-3- O - Take appropriate amounts of rutin and ginsenoside Rc, dissolve them in methanol, and prepare a 1 mg / mL reference solution; accurately weigh the reference standard euonymus alcohol, dissolve it in ultrapure water, and prepare a 1 mg / mL reference solution.

[0066] 1.2 Preparation of the test solution

[0067] Take 1 mL of Compound Euonymus fortunei Mixture and place it in a 50 mL volumetric flask. Add 50% methanol to the mark and sonicate for 30 min (300 W, 40 kHz). Cool and replenish the liquid level to the mark. Filter the solution through a 0.22 μm microporous membrane to obtain the test solution. Take 1 mL of the filtrate and place it in a 10 mL volumetric flask. Add 50% methanol to the mark to obtain the diluted test solution.

[0068] 1.3 Detection conditions

[0069] The chromatographic separation conditions included: mobile phase A was an aqueous formic acid solution; mobile phase B was acetonitrile; the chromatographic column was a CORTECSUPLC C18 column (2.1 × 100 mm, 1.6 μm); the flow rates of mobile phases A and B were 0.3 mL / min; the column temperature was 35℃; the injection volume was 2 μL; the volume concentration of formic acid in the aqueous formic acid solution was 0.1%; the elution method was gradient elution; the gradient elution program was as follows:

[0070] 0-1 min: The volume percentage of mobile phase B increases from 5% to 14%;

[0071] 1-3 min: The volume percentage of mobile phase B increases from 14% to 40%;

[0072] 3-5 min: The volume percentage of mobile phase B is 40%;

[0073] In 5-6 minutes, the volume percentage of the dynamic phase B increased from 40% to 59%.

[0074] Over 6-8 minutes, the volume percentage of the dynamic phase B increased from 59% to 95%.

[0075] The conditions for mass spectrometry detection include: Ion source: electrospray ionization source; Detection mode: multiple reaction ionization; Curtain gas (CUR): 45 psi; Collision gas (CAD): Low; Ionization voltage (IS): 5500 V; Temperature (TEM): 450 ℃; Spray gas (GS1): 50 psi; Auxiliary heating gas (GS2): 20 psi.

[0076] 2. Methodological Examination

[0077] 2.1. Standard curve, detection line, limit of quantitation

[0078] Accurately measure an appropriate amount of the reference stock solution and prepare it with methanol to contain eurygol and ginsenoside Rb1 at a concentration of 2000 ng / mL, kaempferol at a concentration of 1600 ng / mL, and kaempferol-3- O- A mixed reference solution with the following concentrations: rutin (1500 ng / mL), ginsenosides Rg1, Rc, Ro, and Rg3 (1000 ng / mL), sennaoside, ferulic acid, protocatechuic acid, gentianic acid, pyrogallic acid, mogroside, camelliaside B, ginsenoside Rf, Rh1, Rk1, verrucoside, ginsenoside Rg2, Re, syringic acid, p-hydroxybenzoic acid, and salicylic acid (800 ng / mL), verrucoside and astragaloside A (600 ng / mL), syringaldehyde and cytosine B (400 ng / mL), and sennaoside, daidzein, rutin, quercetin, tripterygine, and ginsenoside Rd (200 ng / mL). The mixed reference solution was successively diluted with methanol by 2, 2, 2, 2.5, 2, 2.5, 2, and 2 times to obtain nine concentrations of mixed reference solution, which were then injected for analysis. The regression equation and correlation coefficient were calculated with the peak area (Y) of the analyte as the ordinate and the concentration (X) of the analyte as the abscissa. The mixed reference solution was then progressively diluted, and the limit of detection and limit of quantitation were defined as the reference concentrations corresponding to a signal-to-noise ratio (S / N) ≥ 3 and the lowest point of the linear range, respectively. The results are shown in Table 2. Figure 1 In the image, A is the MRM chromatogram of the mixed reference standard, and B is the MRM chromatogram of the test solution. Figure 1 It can be seen that the target compounds do not interfere with each other and have good peak shapes.

[0079] Table 2. Standard curve regression equations, limits of detection, and limits of quantitation for 34 chemical components of Compound Fufangteng Mixture.

[0080]

[0081]

[0082] 2.2. Precision Experiment

[0083] Intra-day precision: 1 mL of the compound Fufangteng mixture sample (S1) was taken and the test solution was prepared according to the method described in section "1.2 Preparation of Test Solution". The sample was injected six times consecutively under chromatographic conditions, and the peak areas of the compounds were recorded. The RSD values ​​of the peak areas of the 34 compounds were calculated. The results are shown in Table 3. The RSD values ​​were all less than 9.94%, indicating good intra-day precision of the instrument.

[0084] Table 3. Intra-day precision of 34 components of Compound Euonymus fortunei Mixture (n=6)

[0085]

[0086]

[0087] 2.3. Daytime precision

[0088] Take 1 mL of the compound Euonymus fortunei mixture sample (S1) and prepare the test solution according to the method in section "1.2 Preparation of Test Solution". Inject the sample continuously for 3 days under chromatographic conditions, repeating the injection twice a day, and record the peak area of ​​each compound. Calculate the RSD values ​​of the peak areas of 34 compounds, and the results are shown in Table 4. The RSD values ​​are all less than 9.21%, indicating good daytime precision of the instrument.

[0089] Table 4. Day-to-day precision of 34 components in Compound Euonymus fortunei Mixture (n=6)

[0090]

[0091]

[0092] 2.4. Repeatability Experiment

[0093] Take 1 mL of the compound Fufangteng mixture sample (S1), and prepare 6 parallel test solutions using the method described in section "1.2 Preparation of Test Solution". Inject each solution into the solution and calculate the content of the compounds. Calculate the RSD values ​​of the contents of 34 compounds, and the results are shown in Table 5. As shown in Table 5, the RSD values ​​are all less than 8.99%, indicating that the method has good repeatability.

[0094] Table 5. Repeatability of 34 components of Compound Euonymus fortunei Mixture (μg / mL, n=6)

[0095]

[0096]

[0097] 2.5. Stability Test

[0098] Take 1 mL of the compound Euonymus fortunei mixture sample (S1) and prepare the test solution according to the method in section "1.2 Preparation of Test Solution". Inject the sample at 0, 2, 4, 8, 12 and 24 h and record the peak area of ​​the compounds. Calculate the RSD values ​​of the peak areas of 34 compounds. The results are shown in Table 6. As can be seen from Table 6, the RSD values ​​are all less than 9.13%, indicating that the test solution has good stability within 24 h.

[0099] Table 6. Stability of 34 components in Compound Euonymus fortunei Mixture (n=6)

[0100]

[0101]

[0102] 2.6. Recovery Experiment

[0103] Six samples (S1) of Compound Euonymus fortunei Mixture were taken, each 0.5 mL. A certain amount of mixed reference solution was added to each sample. Six test solutions were prepared in parallel using the method described in section "1.2 Preparation of Test Solution". The solutions were then injected and analyzed separately, and the recovery rates were calculated. The results are shown in Table 7. As can be seen from Table 7, the average recovery rates of each component ranged from 81.71% to 119.68%.

[0104] Table 7. Recovery rates of 34 components in Compound Euonymus fortunei Mixture (n=6)

[0105]

[0106]

[0107] 2.7. Dilution Effect Experiment

[0108] Accurately measure the stock solution of the reference standard and prepare a mixed reference solution containing euonymus alcohol and ginsenoside Rb1 at a concentration of 2000 ng / mL, kaempferol at a concentration of 1600 ng / mL, ginsenoside Rk1 at a concentration of 800 ng / mL, and verrucoside and astragaloside A at a concentration of 600 ng / mL. Dilute the above solution 5, 10, and 20 times, repeating the dilution method 6 times, and inject the samples for analysis. Record the concentration of each compound and compare it with the prepared concentration to determine the accuracy. The results are shown in Table 8. The accuracy of each component ranged from -3.44% to 6.36%.

[0109] Table 8. Accuracy of dilution effect of the six components of Compound Euonymus fortunei Mixture (n=6)

[0110]

[0111] 2.8. Content Determination

[0112] Take 1 mL of each of 10 batches of Compound Euonymus fortunei Mixture and prepare the test solution according to the method under "1.2 Preparation of Test Solution". Measure the concentration of 34 compounds using "1.3 Detection Conditions" and record the concentrations. Calculate the content of each compound based on the standard curve. The results are shown in Table 9.

[0113] Table 9. Content of 34 chemical components (μg / mL, n=3) in Compound Euonymus fortunei Mixture (Batches S1-S10)

[0114]

[0115]

[0116] Note: C1. Celastrol; C2. Pyrogallic acid; C3. Protocatechuic acid; C4. p-Hydroxybenzoic acid; C5. Gentianic acid; C6. Syringic acid; C7. Mogroflavin; C8. Camellia glycoside B; C9. Rutin; C10. Verbena isoflavone glycoside; C11. Kaempferol; C12. Kaempferol-3- O - Rutin; C13. Syringaldehyde; C14. Ferulic acid; C15. Ginsenoside Re; C16. Ginsenoside Rg1; C17. Cynanchin B; C18. Arganin; C19. Salicylic acid; C20. Daidzein; C21. Quercetin; C22. Verrucoside isoflavone; C23. Ginsenoside Rb1; C24. Ginsenoside Rf; C25. Ginsenoside Rc; C26. Ginsenoside Ro; C27. Ginsenoside Rg2; C28. Ginsenoside Rh1; C29. Astragaloside A; C30. Ginsenoside Rd; C31. Arganin; C32. Tripterygium alkaloid; C33. Ginsenoside Rg3; C34. Ginsenoside Rk1.

[0117] Example 2

[0118] 3. Solution preparation

[0119] 3.1. Preparation of reference solution

[0120] Accurately weigh appropriate amounts of the reference standards rutin, kaempferol B, mogroflavin, syringaldehyde, tripterygine, cyprotinin B, p-hydroxybenzoic acid, salicylic acid, gentian acid, protocatechuic acid, ferulic acid, syringic acid, kaempferol, and quercetin, dissolve them in methanol, and prepare a 1 mg / mL reference solution; accurately weigh the reference standard euonymus alcohol, dissolve it in ultrapure water, and prepare a 1 mg / mL reference solution.

[0121] 3.2. Preparation of the test solution

[0122] Weigh 500.0 mg of Euonymus fortunei powder and place it in a 50 mL volumetric flask. Add 70% methanol to the mark and sonicate for 40 min (300 W, 40 kHz). Cool and replenish the liquid level to the mark. Take the supernatant and filter it through a 0.22 μm microporous membrane to obtain the test solution. Take 1 mL of the filtrate and place it in a 10 mL volumetric flask. Add 70% methanol to the mark to obtain the diluted test solution.

[0123] 3.3. Testing Conditions

[0124] The chromatographic separation conditions included: mobile phase A was a 0.1% (v / v) formic acid aqueous solution; mobile phase B was acetonitrile; the chromatographic column was a CORTECS UPLC C18 column; the flow rates of mobile phases A and B were 0.3 mL / min; the column temperature was 35 ℃; the injection volume was 2 μL; and the elution method was gradient elution.

[0125] The gradient elution procedure is as follows:

[0126] 0-1 min: The volume percentage of mobile phase B increases from 5% to 14%;

[0127] 1-4 min: The volume percentage of mobile phase B increases from 14% to 85%;

[0128] In 4-5 minutes, the volume percentage of the mobile phase B increased from 85% to 95%.

[0129] The conditions for mass spectrometry detection include: Ion source: electrospray ionization source; Detection mode: multiple reaction ionization; Curtain gas (CUR): 45 psi; Collision gas (CAD): Low; Ionization voltage (IS): 5500 V; Temperature (TEM): 450 ℃; Spray gas (GS1): 50 psi; Auxiliary heating gas (GS2): 20 psi.

[0130] 4. Methodological Examination

[0131] 4.1. Standard curve, detection line, limit of quantitation

[0132] Accurately measure an appropriate amount of the reference solution and prepare a mixed reference solution with methanol containing 6000 ng / mL of euonymol, 1600 ng / mL of kaempferol, 1000 ng / mL of mogroside, 800 ng / mL of protocatechuic acid, gentianic acid, camelliaside B, rutin, ferulic acid, syringic acid, p-hydroxybenzoic acid, and salicylic acid, 400 ng / mL of syringaldehyde and cyprodin B, and 200 ng / mL of quercetin and tripterygium alkaloid. Dilute this solution sequentially with methanol by 2, 2, 2, 2.5, 2, 2.5, 2, and 2 times to obtain nine mixed reference solutions of different concentrations, and inject them for analysis. Calculate the regression equation and correlation coefficient with the peak area (Y) of the analyte as the ordinate and the concentration (X) of the analyte as the abscissa. The mixed reference solution was gradually diluted, and the concentrations corresponding to a signal-to-noise ratio (S / N) ≥ 3 and the lowest point of the linear range were used as the detection limit and limit of quantitation, respectively. The results are shown in Table 10. The compounds do not interfere with each other; the MRM chromatogram is shown below. Figure 2 , Figure 2 In the image, A is the MRM chromatogram of the reference standard, and B is the MRM chromatogram of the Euonymus fortunei sample. Figure 2 It can be seen that the target compounds do not interfere with each other and have good peak shapes.

[0133] Table 10 Standard curve regression equations, limits of detection, and limits of quantitation for 15 chemical components of Euonymus fortunei medicinal material.

[0134]

[0135] 4.2. Precision Experiment

[0136] Intra-day precision: 500.0 mg of Euonymus fortunei powder was weighed and a test solution was prepared using the method described in section 3.2, "Preparation of Test Solution". The solution was injected six times consecutively under chromatographic conditions, and the peak areas of the compounds were recorded. The RSD values ​​of the peak areas of the 15 compounds were calculated. The results are shown in Table 11. All RSD values ​​were less than 5.54%, indicating good intra-day precision of the instrument.

[0137] Table 11 Intra-day precision of 15 components of Euonymus fortunei (n=6)

[0138]

[0139] 4.3. Daytime precision

[0140] Weigh 500.0 mg of Euonymus fortunei powder and prepare the test solution using the method described in section "3.2 Preparation of Test Solution". Inject the sample continuously for 3 days under chromatographic conditions, repeating the injection twice daily, and record the peak areas of the compounds. Calculate the RSD values ​​of the peak areas of 15 compounds, and the results are shown in Table 12. The RSD values ​​are all less than 5.86%, indicating good daytime precision of the instrument.

[0141] Table 12. Day-to-day precision of 15 components of Euonymus fortunei (n=6)

[0142]

[0143] 4.4. Repeatability Experiment

[0144] Weigh 500.0 mg of Euonymus fortunei powder and prepare six parallel test solutions using the method described in section "3.2 Preparation of Test Solution". Inject each solution into the solution and calculate the content of the compounds. Calculate the RSD values ​​of the contents of each of the 15 compounds. The results are shown in Table 13. The RSD values ​​are all less than 8.05%, indicating that the method has good repeatability.

[0145] Table 13. Repeatability of 15 components of Euonymus fortunei (μg / g, n=6)

[0146]

[0147] 4.5. Stability Test

[0148] Weigh 500.0 mg of Euonymus fortunei powder and prepare the test solution using the method described in section 3.2, "Preparation of Test Solution". Inject the solution at 0, 2, 4, 8, 12, and 24 h, and record the peak areas of the compounds. Calculate the RSD values ​​of the peak areas of the 15 compounds, and the results are shown in Table 14. The RSD values ​​are all less than 5.05%, indicating that the test solution has good stability within 24 h.

[0149] Table 14. Stability of 15 components in Euonymus fortunei (n=6)

[0150]

[0151] 4.6. Recovery Experiment

[0152] Six portions of Euonymus fortunei powder, each 250 mg, were weighed and added to a certain amount of mixed reference solution. Six test solutions were prepared in parallel using the method described in section "3.2 Preparation of Test Solutions". Each solution was injected for analysis, and the recovery rate was calculated. The results are shown in Table 15. The average recovery rates of each component ranged from 93.11% to 119.81%.

[0153] Table 15 Recovery rates of 15 components from Euonymus fortunei (n=6)

[0154]

[0155] 4.7. Dilution Effect Experiment

[0156] Accurately measure the stock solution of the reference standard and prepare a mixed reference solution containing 6000 ng / mL of euonymus alcohol and 1000 ng / mL of mogroflavin. Dilute the above solution 10, 20, and 40 times, repeating this dilution six times. Inject the solutions for analysis, record the concentrations of each compound, and compare the results with the prepared concentrations to determine the accuracy. The results are shown in Table 16; the accuracy of each component ranged from 0.33% to 9.33%.

[0157] Table 16. Accuracy of dilution effect of two components in Euonymus fortunei (n=6)

[0158]

[0159] 4.5. Content determination

[0160] Weigh 500.0 mg of Euonymus fortunei powder and prepare the test solution according to the method in "3.2 Preparation of test solution". Determine the concentration of 15 compounds using "3.3 Detection conditions", record the concentration of each compound, and calculate the content of each compound according to the standard curve. The results are shown in Table 17.

[0161] Table 17 Content of 15 chemical components in Euonymus fortunei (μg / g, n=3)

[0162]

[0163] 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. Other embodiments can be obtained 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 chemical components in Euonymus fortunei or compound Euonymus fortunei mixture based on UPLC-QTRAP-MS / MS, characterized in that, Includes the following steps: Extracting Euonymus fortunei or compound Euonymus fortunei mixture with methanol-water solution to obtain the test solution of Euonymus fortunei or compound Euonymus fortunei mixture; The test solution of Euonymus fortunei or compound Euonymus fortunei mixture was analyzed by UPLC-QTRAP-MS / MS to obtain the peak area of ​​each chemical component to be tested in Euonymus fortunei or compound Euonymus fortunei mixture. Based on the peak area of ​​each chemical component to be tested in Euonymus fortunei or Compound Euonymus fortunei Mixture and the corresponding predetermined standard curve of the chemical component, the content of each chemical component to be tested in Euonymus fortunei or Compound Euonymus fortunei Mixture is obtained; the predetermined standard curve of each chemical component to be tested is a linear relationship between the concentration and peak area of ​​each chemical component to be tested. For UPLC-QTRAP-MS / MS detection, the chromatographic separation conditions included: mobile phase A was formic acid aqueous solution; mobile phase B was acetonitrile; the chromatographic column was a C18 column; the flow rates of mobile phase A and mobile phase B were 0.3 mL / min; and the elution method was gradient elution. The conditions for mass spectrometry detection include: ion source: electrospray ionization source; detection mode: multiple reaction ion detection; The chemical components to be tested in the compound Fufangteng mixture are: eurygol, pyrogallol, protocatechuic acid, p-hydroxybenzoic acid, gentian acid, syringic acid, mogroflavonoids, kaempferol B, rutin, verrucoside, kaempferol-3-O-rutinoside, eugenol, ferulic acid, ginsenoside Re, ginsenoside Rg1, cyprodinone B, argentin, salicylic acid, daidzein, quercetin, verrucoside, ginsenoside Rb1, ginsenoside Rf, ginsenoside Rc, ginsenoside Ro, ginsenoside Rg2, ginsenoside Rh1, astragaloside A, ginsenoside Rd, argentin, ginsenoside Rg3, tripterygine, and ginsenoside Rk1. The chemical components to be tested in Euonymus fortunei are euonymol, rutin, kaempferol B, mogroflavin, syringaldehyde, tripterygine, cyprotinin B, p-hydroxybenzoic acid, salicylic acid, gentian acid, protocatechuic acid, ferulic acid, syringic acid, kaempferol and quercetin. When testing Compound Euonymus fortunei Mixture, the gradient elution procedure is as follows: 0-1 min: The volume percentage of mobile phase B increases from 5% to 14%; 1-3 min: The volume percentage of mobile phase B increases from 14% to 40%; 3-5 min: The volume percentage of mobile phase B is 40%; In 5-6 minutes, the volume percentage of the dynamic phase B increased from 40% to 59%. Over 6-8 minutes, the volume percentage of the dynamic phase B increased from 59% to 95%. When detecting Euonymus fortunei, the gradient elution procedure is as follows: 0-1 min: The volume percentage of mobile phase B increases from 5% to 14%; 1-4 min: The volume percentage of mobile phase B increases from 14% to 85%; In 4-5 minutes, the volume percentage of the dynamic phase B increased from 85% to 95%.

2. The method as described in claim 1, characterized in that, The conditions for the mass spectrometry detection include: curtain gas: 45 psi; jet gas: Low; ionization voltage: 5500 V; temperature: 450 ℃; spray gas: 50 psi; auxiliary heating gas: 20 psi.

3. The method as described in claim 1, characterized in that, The chromatographic column was a CORTECS UPLC C18 column; the column temperature was 35 ℃; and the injection volume was 2 μL.

4. The method as described in claim 1, characterized in that, When extracting Euonymus fortunei, the volume concentration of the methanol-water solution is 70%.

5. The method as described in claim 1, characterized in that, When extracting compound Euonymus fortunei mixture, the volume concentration of methanol aqueous solution is 50%.

6. The method as described in claim 1, characterized in that, The volume concentration of formic acid in the formic acid aqueous solution is 0.1%.

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