Method for detecting chemical components in wintercreeper or compound wintercreeper mixture based on UPLC-QTRAP-MS / MS

Abstract

The invention belongs to the technical field of detection and analysis, and particularly relates to a method for detecting chemical components in euonymus fortunei or a compound euonymus fortunei mixture based on UPLC-QTRAP-MS / MS. The UPLC-QTRAP-MS / MS analysis method is adopted, the content of 15 components in the euonymus fortunei medicinal material or the content of 34 components in the compound euonymus fortunei mixture can be measured at the same time, and the method is applied to content measurement of multiple batches of compound euonymus fortunei mixtures. Through systematic methodology verification, the method has good precision, repeatability and accuracy, and can meet the requirements of multi-component quantitative analysis. The content determination result provides a powerful scientific basis for establishing a more scientific and reliable quality control method and comprehensively and systematically evaluating the clinical medication safety and rationality.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection and analysis, and particularly relates to a method for detecting chemical components in Eponymus fortunei or Eponymus fortunei compound mixture based on UPLC-QTRAP-MS / MS. Background Art

[0002] Fufangteng ( Euonymiherba ) is a plant of the Euonymus family, Euonymus spp. Euonymusfortunei (Turcz.) Hand.Mazz, Holly Euonymus Euonymus japonicus L. or sessile euonymus Euonymussubsessilis The dried aerial parts of Sprague. The Compendium of Materia Medica once described it as "mainly effective in treating all blood, qi, and cold conditions." Fufangteng (Ephedrine sprague) is a traditional ethnic medicine in Guangxi. It enters the liver, spleen, and kidney meridians and has the effects of tonifying qi and blood, relaxing tendons and activating collaterals, dispelling wind and dampness, and dispersing blood stasis and stopping bleeding. It is commonly used to treat qi and blood deficiency, lumbar muscle strain, rheumatic pain, fractures, traumatic bleeding, metrorrhagia, and uterine bleeding. Fufangteng Compound, with Fufangteng as the main ingredient and supplemented with red ginseng and astragalus, is widely used in clinical practice as a classic tonic formula. This preparation adheres to the principles of traditional Chinese medicine for 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 demonstrate definite efficacy in improving symptoms of both heart and spleen deficiency, regulating cardiovascular homeostasis, and correcting immune imbalances.

[0003] The 2021 edition of the "Guangxi Zhuang Autonomous Region Zhuang Medicine Quality Standard" only describes the biological identification method and thin-layer identification method for Fufang Teng, lacking a binary quality control specification based on "ingredient-content". In addition, the quality standard of the "Pharmacopoeia of the People's Republic of China" Part I (2020 edition) regarding compound Fufang Teng mixture only includes the thin-layer chromatography scanning method for the determination of astragaloside IV in Astragalus. Existing detection methods at home and abroad also mostly remain at the level of single-component qualitative and quantitative analysis, lacking a multi-index component simultaneous detection scheme based on chromatography-mass spectrometry technology. This situation restricts the in-depth development and rational clinical application of compound Fufang Teng mixture. Summary of the Invention

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

[0005] In order to achieve the above object, the present invention provides the following technical solutions: The present invention provides a method for detecting chemical components in Fufangteng or Fufangteng mixture based on UPLC-QTRAP-MS / MS, comprising the following steps: Extracting the Euonymus fortunei or the Euonymus fortunei compound mixture with methanol-water solution to obtain a test solution of the Euonymus fortunei or the Euonymus fortunei compound mixture; The test solution of Fufang Teng or Fufang Teng mixture is subjected to UPLC-QTRAP-MS / MS detection to obtain the peak area of ​​each test chemical component in Fufang Teng or Fufang Teng mixture; According to the peak area of ​​each chemical component to be tested in Fuyuan Teng or Fuyuan Teng compound mixture and the corresponding predetermined standard curve of the chemical component, the content of each chemical component to be tested in Fuyuan Teng or Fuyuan Teng compound mixture is obtained; the predetermined standard curve of each chemical component to be tested is the linear relationship between the concentration and peak area of ​​each chemical component to be tested.

[0006] During UPLC-QTRAP-MS / MS detection, the chromatographic separation conditions included: mobile phase A was formic acid aqueous solution; mobile phase B was acetonitrile; chromatographic column C 18 Column; the flow rate of mobile phase A and mobile phase B was 0.3 mL / min; the elution mode was gradient elution; The conditions for mass spectrometry detection include: ion source: electrospray ion source; detection mode: multiple reaction ion detection.

[0007] Preferably, the mass spectrometry detection conditions include: curtain gas: 45 psi; impingement gas: Low; ionization voltage: 5500 V; temperature: 450° C.; spray gas: 50 psi; and auxiliary heating gas: 20 psi.

[0008] Preferably, the chromatographic column is a CORTECS UPLC C18 column; the column temperature is 35°C; and the injection volume is 2 μL.

[0009] Preferably, when detecting Fufangteng mixture, the gradient elution procedure is: 0-1 min: the volume percentage of the mobile phase B increased from 5% to 14%; 1-3 min: the volume percentage of the mobile phase B increased from 14% to 40%; 3-5 min: the volume percentage of the mobile phase B is 40%; 5-6 min, the volume percentage of the mobile phase B increased from 40% to 59%; In 6-8 minutes, the volume percentage of the mobile phase B increased from 59% to 95%.

[0010] Preferably, when detecting Euonymus fortunei, the gradient elution procedure is: 0-1 min: the volume percentage of the mobile phase B increased from 5% to 14%; 1-4 min: the volume percentage of the mobile phase B increased from 14% to 85%; After 4-5 minutes, the volume percentage of the mobile phase B increased from 85% to 95%.

[0011] Preferably, the chemical components to be tested in the compound Fufangteng mixture are galactol, pyrogallic acid, protocatechuic acid, p-hydroxybenzoic acid, gentisic acid, syringic acid, mogroflavin, camelliaside B, rutin, calycosin, kaempferol, kaempferol-3- O - At least two of the group consisting of rutinoside, syringaldehyde, ferulic acid, ginsenoside Re, ginsenoside Rg1, leucanthoside B, formononetin, salicylic acid, daidzein, quercetin, calycosin, ginsenoside Rb1, ginsenoside Rf, ginsenoside Rc, ginsenoside Ro, ginsenoside Rg2, ginsenoside Rh1, astragaloside IV, ginsenoside Rd, formononetin, ginsenoside Rg3, triptolide, and ginsenoside Rk1.

[0012] Preferably, the chemical components to be tested in the Euonymus fortunei are at least two of dactyl alcohol, rutin, camelliaside B, mogroflavin, syringaldehyde, triptolide, leucanthoside B, p-hydroxybenzoic acid, salicylic acid, gentisic acid, protocatechuic acid, ferulic acid, syringic acid, kaempferol and quercetin.

[0013] Preferably, when extracting Euonymus fortunei, the volume concentration of the methanol aqueous solution is 70%.

[0014] Preferably, when extracting the compound Fufangteng mixture, the volume concentration of the methanol-water solution is 50%.

[0015] Preferably, the volume concentration of formic acid in the formic acid aqueous solution is 0.1%.

[0016] The present invention uses a UPLC-QTRAP-MS / MS analysis method that can simultaneously determine the contents of 15 components in the medicinal material of Fufang Teng or the contents of 34 components in the compound Fufang Teng mixture, and can be applied to the content determination of multiple batches of the compound Fufang Teng mixture. Systematic methodological validation has shown that this method has good precision, repeatability, and accuracy, and can meet the requirements of multi-component quantitative analysis. The content determination results can provide a strong scientific basis for establishing a more scientific and reliable quality control method and comprehensively and systematically evaluating its clinical drug safety and rationality. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0018] Figure 1A is the MRM chromatogram of the mixed reference substance, and B is the MRM chromatogram of the test solution; 1. Dulcitol; 2. Pyrogallic acid; 3. Protocatechuic acid; 4. p-Hydroxybenzoic acid; 5. Gentisic acid; 6. Syringic acid; 7. Mogroflavin; 8. Camellia glycoside B; 9. Rutin; 10. Calycosin; 11. Kaempferol; 12. Kaempferol-3- O -rutinoside; 13. Syringaldehyde; 14. Ferulic acid; 15. Ginsenoside Re; 16. Ginsenoside Rg1; 17. Leucorhinol B; 18. Formononetin; 19. Salicylic acid; 20. Daidzein; 21. Quercetin; 22. Calycosin; 23. Ginsenoside Rb1; 24. Ginsenoside Rf; 25. Ginsenoside Rc; 26. Ginsenoside Ro; 27. Ginsenoside Rg2; 28. Ginsenoside Rh1; 29. ​​Astragaloside IV; 30. Ginsenoside Rd; 31. Formononetin; 32. Tripterygium wilfordii; 33. Ginsenoside Rg3; 34. Ginsenoside Rk1; Figure 2 A is the MRM chromatogram of the reference substance, and B is the MRM chromatogram of the Eucommia ulmoides sample, including 1. dulcimer; 2. protocatechuic acid; 3. p-hydroxybenzoic acid; 4. gentisic acid; 5. syringic acid; 6. mogroflavin; 7. camelliaside B; 8. rutin; 9. kaempferol; 10. syringaldehyde; 11. ferulic acid; 12. leucoside B; 13. salicylic acid; 14. quercetin; 15. triptolide. DETAILED DESCRIPTION

[0019] The present invention provides a method for detecting chemical components in Fufangteng or Fufangteng mixture based on UPLC-QTRAP-MS / MS, comprising the following steps: Extracting the Euonymus fortunei or the Euonymus fortunei compound mixture with methanol-water solution to obtain a test solution of the Euonymus fortunei or the Euonymus fortunei compound mixture; The test solution of Fufang Teng or Fufang Teng mixture is subjected to UPLC-QTRAP-MS / MS detection to obtain the peak area of ​​each test chemical component in Fufang Teng or Fufang Teng mixture; According to the peak area of ​​each chemical component to be tested in Fuyuan Teng or Fuyuan Teng compound mixture and the corresponding predetermined standard curve of the chemical component, the content of each chemical component to be tested in Fuyuan Teng or Fuyuan Teng compound mixture is obtained; the predetermined standard curve of each chemical component to be tested is the linear relationship between the concentration and peak area of ​​each chemical component to be tested.

[0020] The present invention extracts the Euonymus fortunei or the Euonymus fortunei compound mixture through methanol aqueous solution to obtain a test solution of the Euonymus fortunei or the Euonymus fortunei compound mixture.

[0021] As an embodiment of the present invention, when extracting Euonymus fortunei, the volume concentration of the methanol aqueous solution can be 70%; when extracting the compound Euonymus fortunei mixture, the volume concentration of the methanol aqueous solution can be 50%.

[0022] The present invention performs UPLC-QTRAP-MS / MS detection on a test solution of Euonymus fortunei or a compound Euonymus fortunei mixture to obtain the peak area of ​​each test chemical component in the Euonymus fortunei or the compound Euonymus fortunei mixture.

[0023] As an embodiment of the present invention, the chemical components to be tested in the compound Fufangteng mixture can be dactyl alcohol, pyrogallic acid, protocatechuic acid, p-hydroxybenzoic acid, gentisic acid, syringic acid, mogroflavin, camelliaside B, rutin, calycosin isoflavone glycosides, kaempferol, kaempferol-3- O - At least two of the group consisting of rutinoside, syringaldehyde, ferulic acid, ginsenoside Re, ginsenoside Rg1, leucanthoside B, formononetin, salicylic acid, daidzein, quercetin, calycosin, ginsenoside Rb1, ginsenoside Rf, ginsenoside Rc, ginsenoside Ro, ginsenoside Rg2, ginsenoside Rh1, astragaloside IV, ginsenoside Rd, formononetin, ginsenoside Rg3, triptolide, and ginsenoside Rk1.

[0024] As an embodiment of the present invention, when UPLC-QTRAP-MS / MS is used to detect the test solution of Fufangteng mixture, the chromatographic separation conditions include: mobile phase A is a formic acid aqueous solution; the volume concentration of the formic acid aqueous solution can be 0.1%; mobile phase B is acetonitrile; the flow rate of mobile phase A and mobile phase B is 0.3 mL / min; the chromatographic column is C 18 The column can be a CORTECS UPLC C18 column, the column temperature can be 35°C, the injection volume can be 2 μL, and the elution method can be gradient elution; The procedure of the gradient elution can be: 0-1 min: the volume percentage of the mobile phase B increased from 5% to 14%; 1-3 min: the volume percentage of the mobile phase B increased from 14% to 40%; 3-5 min: the volume percentage of the mobile phase B is 40%; 5-6 min, the volume percentage of the mobile phase B increased from 40% to 59%; 6-8 min, the volume percentage of the mobile phase B increased from 59% to 95%; The mass spectrometry detection conditions include: ion source: electrospray ionization source; detection mode: multiple reaction monitoring (MRM); curtain gas (CUR): 45 psi; collision gas (CAD): Low; ionization voltage (IS): 5500 V; temperature (TEM): 450 °C; spray gas (GS1): 50 psi; auxiliary heating gas (GS2): 20 psi.

[0025] As an embodiment of the present invention, the chemical components to be tested in the Eucommia ulmoides can be at least two of dactyl alcohol, rutin, camelliaside B, mogroflavin, syringaldehyde, triptolide, leucoside B, p-hydroxybenzoic acid, salicylic acid, gentisic acid, protocatechuic acid, ferulic acid, syringic acid, kaempferol and quercetin.

[0026] As an embodiment of the present invention, when UPLC-QTRAP-MS / MS is used to detect the test solution of Fufang Teng, the gradient elution program can be: 0-1 min: the volume percentage of the mobile phase B increases from 5% to 14%; 1-4 min: the volume percentage of the mobile phase B increases from 14% to 85%; 4-5 min, the volume percentage of the mobile phase B increases from 85% to 95%. Other chromatographic separation conditions and mass spectrometry detection conditions are preferably the same as the conditions for detecting the test solution of Fufang Teng mixture, and will not be repeated.

[0027] Table 1 Mass spectrometry parameters of chemical components in Fufangteng mixture or Fufangteng

[0028] In order to further illustrate the present invention, the scheme of the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be understood as limiting the scope of protection of the present invention.

[0029] Instruments and reagents involved in the examples ① Instruments A60 / 220.R2 1 / 100,000 balance (Radwag); 5424R high-speed centrifuge (Eppendorf); SCIEX QTRAP6500+ liquid chromatography-mass spectrometry (SCIEX); Milli-Q7005 ultrapure water preparation instrument (Millipore); Analyst analysis software (SCIEX).

[0030] ②Drug testing Reference substances: rutin (lot number: DST210520-017), ginsenoside Rg3 (lot number: DST191107-011(S)), formononetin (lot number: MUST-17041101), camelliaside B (lot number: DSTDS008001), mogroflavin (lot number: DSTDL003001), ginsenoside Rf (lot number: MUST-13101010), syringaldehyde (lot number: DSTDD030901), triptolide (lot number: DST220812-078 ), Ginsenoside Rg1 (Batch No.: DST190828-009), Ginsenoside B (Batch No.: DST240424-078), Ginsenoside Re (Batch No.: MUST-13041201), p-Hydroxybenzoic Acid (Batch No.: DSTDD011401), Ginsenoside Rk1 (Batch No.: DST200409-034), Salicylic Acid (Batch No.: DSTDS012401), Gentisic Acid (Batch No.: DSTDL001301), Protocatechuic Acid (Batch No.: DSTDY008101), Ginseng Saponin Rg2 (Batch No.: DST190517-010), Dulcitol (Batch No.: DSTDW002701), Ferulic Acid (Batch No.: DST191112-001), Syringic Acid (Batch No.: DST200927-057), Kaempferol (Batch No.: DST230914-003), Ginsenoside Rd (Batch No.: DSTDRO01502), Quercetin (Batch No.: DSTDH002802), Ginsenoside Rh1 (Batch No.: DST170405-035(S)), Daidzein ( Batch number: DST180128-020), ginsenoside Ro (Batch number: DST221230-031), formononetin (Batch number: DSTDM001102), ginsenoside Rb1 (Batch number: DST191212-006), calycosin (Batch number: DSTDM001301), astragaloside IV (Batch number: DSTDH001503), pyrogallol (Batch number: DJ0172-0020), calycosin (Batch number: DST220226-012), kaempferol-3- O -Rutinoside (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 chromatographically pure and purchased from Fisher; ultrapure water was prepared using an ultrapure water instrument.

[0031] The 10 batches of compound Fufangteng mixture in the example were provided by Guangxi Traditional Chinese Medicine Bai Nian Le Pharmaceutical Co., Ltd., which were numbered S1-S10, and the batch numbers of S1-S10 were 20230905, 20230301, 20230302, 20230303, 20230304, 20230504, 20230505, 20231011, 20231001, and 20240304, respectively.

[0032] The origin of the medicinal material Eucommia ulmoides is Yulin City, Guangxi Zhuang Autonomous Region, and the batch number is 20230204.

[0033] Example 1 1. Solution Preparation 1.1. Preparation of reference solution Accurately weigh the reference substances rutin, ginsenoside Rg3, formononetin, camelliaside B, mogroflavin, ginsenoside Rf, syringaldehyde, triptolide, ginsenoside Rg1, leucoside B, ginsenoside Re, p-hydroxybenzoic acid, ginsenoside Rk1, salicylic acid, gentisic acid, protocatechuic acid, ginsenoside Rg2, ferulic acid, syringic acid, kaempferol, ginsenoside Rd, quercetin, ginsenoside Rh1, daidzein, ginsenoside Ro, formononetin, ginsenoside Rb1, calycosin, astragaloside IV, pyrogallol, calycosin, kaempferol-3- O -An appropriate amount of rutinoside and ginsenoside Rc was dissolved in methanol to prepare a 1 mg / mL reference solution; accurately weighed the reference substance dulcitol, dissolved in ultrapure water to prepare a 1 mg / mL reference solution.

[0034] 1.2 Preparation of test solution Pipette 1 mL of Fufangteng mixture and place it in a 50 mL volumetric flask. Add 50% methanol to the scale line and ultrasonicate for 30 min (power 300 W, frequency 40 kHz). Cool and fill the liquid level to the scale line. Pass the solution through a 0.22 μm microporous filter 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 scale line to obtain the diluted test solution.

[0035] 1.3 Test conditions The chromatographic separation conditions included: mobile phase A was formic acid aqueous solution; mobile phase B was acetonitrile; the chromatographic column was a CORTECS UPLC C18 column (2.1×100 mm, 1.6 μm); the flow rate of mobile phase A and mobile phase B was 0.3 mL / min; the column temperature was 35°C; the injection volume was 2 μL; the volume concentration of formic acid in the formic acid aqueous solution was 0.1%; the elution method was gradient elution; and the gradient elution program was as follows: 0-1 min: the volume percentage of the mobile phase B increased from 5% to 14%; 1-3 min: the volume percentage of the mobile phase B increased from 14% to 40%; 3-5 min: the volume percentage of the mobile phase B is 40%; 5-6 min, the volume percentage of the mobile phase B increased from 40% to 59%; In 6-8 minutes, the volume percentage of the mobile phase B increased from 59% to 95%.

[0036] The mass spectrometry detection conditions include: ion source: electrospray ionization source; detection mode: multiple reaction ion detection; curtain gas (CUR): 45 psi; collision gas (CAD): Low; ionization voltage (IS): 5500 V; temperature (TEM): 450 °C; spray gas (GS1): 50 psi; auxiliary heating gas (GS2): 20 psi.

[0037] 2. Methodological Investigation 2.1. Standard curve, detection line, and limit of quantification Accurately measure an appropriate amount of the reference substance stock solution and prepare it with methanol to contain 2000 ng / mL of dulcitol, ginsenoside Rb1, 1600 ng / mL of kaempferol-3- O -A mixed reference solution with a concentration of 1500 ng / mL for rutinoside, 1000 ng / mL for ginsenoside Rg1, ginsenoside Rc, ginsenoside Ro, and ginsenoside Rg3, 800 ng / mL for formononetin, ferulic acid, protocatechuic acid, gentisic acid, pyrogallic acid, mogroflavin, camelliaside B, ginsenoside Rf, ginsenoside Rh1, ginsenoside Rk1, calycosin, ginsenoside Rg2, ginsenoside Re, syringic acid, p-hydroxybenzoic acid, and salicylic acid, 600 ng / mL for calycosin and astragaloside IV, 400 ng / mL for syringaldehyde and leucin B, and 200 ng / mL formononetin, daidzein, rutin, quercetin, triptolide, and ginsenoside Rd. The mixed reference solution was diluted with methanol in sequence at 2, 2, 2, 2.5, 2, 2.5, 2, and 2 times to obtain nine concentrations of mixed reference solution. Samples were injected and analyzed. The regression equation and correlation coefficient were calculated using the analyte peak area (Y) as the ordinate and the analyte concentration (X) as the abscissa. The mixed reference solution was diluted in a stepwise manner, and the detection and quantification limits were determined using a signal-to-noise ratio (S / N) ≥ 3 and the reference concentration corresponding to the lowest point in the linear range, respectively. The results are shown in Table 2. Figure 1 A is the MRM chromatogram of the mixed reference substance, and B is the MRM chromatogram of the test solution. Figure 1It can be seen that the target compounds do not interfere with each other and have good peak shapes.

[0038] Table 2 Standard curve regression equations, detection limits, and quantification limits of 34 chemical components in Fufang Fufangteng mixture

[0039] 2.2. Precision experiment Intra-day precision: 1 mL of Fufangteng mixture sample (S1) was prepared using the method described in "1.2 Preparation of Test Solution." Six consecutive injections were made according to the chromatographic conditions, and the compound peak areas were recorded. The RSD values ​​for the peak areas of the 34 compounds were calculated. The results are shown in Table 3. All RSD values ​​were less than 9.94%, indicating good intra-day precision of the instrument.

[0040] Table 3 Intra-day precision of the 34 components of Fufangteng mixture (n=6)

[0041] 2.3. Inter-day precision A 1 mL sample of Fufangteng mixture (S1) was prepared using the method described in "1.2 Preparation of Test Solution." Samples were injected continuously for three days according to the chromatographic conditions, with two injections per day. The peak areas of the compounds were recorded. The RSDs of the peak areas for the 34 compounds were calculated, and the results are shown in Table 4. All RSDs were less than 9.21%, indicating good inter-day precision of the instrument.

[0042] Table 4 Inter-day precision of the 34 components of Fufangteng mixture (n=6)

[0043] 2.4. Repeatability Experiment A 1 mL sample of Fufangteng mixture (S1) was prepared in parallel using the method described in "1.2 Preparation of Test Solution." Six sample solutions were injected separately and the compound content was calculated. The RSD values ​​for the contents of the 34 compounds were calculated, and the results are shown in Table 5. Table 5 shows that the RSD values ​​were all less than 8.99%, indicating good reproducibility of the method.

[0044] Table 5 Repeatability of the 34 components of Fufangteng mixture (μg / mL, n=6)

[0045] Stability test A 1 mL sample of Fufangteng mixture (S1) was prepared using the method described in "1.2 Preparation of Test Solution." Samples were injected at 0, 2, 4, 8, 12, and 24 hours, and the compound peak areas were recorded. The RSDs of the peak areas for the 34 compounds were calculated. The results are shown in Table 6. As shown in Table 6, all RSDs were less than 9.13%, indicating good stability of the test solution over a 24-hour period.

[0046] Table 6 Stability of 34 components of Fufangteng mixture (n=6)

[0047] 2.6. Sample recovery experiment Six 0.5 mL portions of Fufangteng mixture (S1) were added to a certain amount of the mixed reference solution. Six parallel aliquots of the test solution were prepared using the method described in "1.2 Preparation of Test Solution." Each aliquot was injected and analyzed, and the recovery was calculated. The results are shown in Table 7. The average recovery of each component ranged from 81.71% to 119.68%.

[0048] Table 7 Recovery rates of 34 components of Fufangteng mixture (n=6)

[0049] 2.7. Dilution effect experiment Precisely measure the reference stock solution to prepare a mixed reference solution containing 2000 ng / mL of dulcitol and ginsenoside Rb1, 1600 ng / mL of kaempferol, 800 ng / mL of ginsenoside Rk1, and 600 ng / mL of calycosin and astragaloside IV. This solution was diluted 5-, 10-, and 20-fold, and diluted six times using the same method. Samples were injected and analyzed, and the concentrations of each compound were recorded and compared with the prepared concentrations to determine accuracy. The results are shown in Table 8. The accuracy of each component ranged from -3.44% to 6.36%.

[0050] Table 8 Accuracy of the dilution effect of the six components of Fufangteng mixture (n=6)

[0051] 2.8. Content determination 1 mL of Fufangteng mixture from 10 batches was taken respectively, and the test solution was prepared according to the method in "1.2 Preparation of test solution". The test was performed using "1.3 Detection Conditions". The concentrations of 34 compounds were recorded, and their contents were calculated according to the standard curve. The results are shown in Table 9.

[0052] Table 9 Contents of 34 chemical components in Fufangteng mixture (batches S1-S10) (μg / mL, n=3)

[0053] Note: C1. Dulcitol; C2. Pyrogallic acid; C3. Protocatechuic acid; C4. p-Hydroxybenzoic acid; C5. Gentisic acid; C6. Syringic acid; C7. Mogroflavin; C8. Camelliaside B; C9. Rutin; C10. Calycosides; C11. Kaempferol; C12. Kaempferol-3- O -rutinoside; C13. Syringaldehyde; C14. Ferulic acid; C15. Ginsenoside Re; C16. Ginsenoside Rg1; C17. Leucanthoside B; C18. Formononetin; C19. Salicylic acid; C20. Daidzein; C21. Quercetin; C22. Calycosin; C23. Ginsenoside Rb1; C24. Ginsenoside Rf; C25. Ginsenoside Rc; C26. Ginsenoside Ro; C27. Ginsenoside Rg2; C28. Ginsenoside Rh1; C29. Astragaloside IV; C30. Ginsenoside Rd; C31. Formononetin; C32. Tripterygium wilfordii; C33. Ginsenoside Rg3; C34. Ginsenoside Rk1.

[0054] Example 2 3. Solution Preparation 3.1. Preparation of reference solution Accurately weigh appropriate amounts of the reference substances rutin, camelliaside B, mogroflavin, syringaldehyde, triptolide, leucoside B, p-hydroxybenzoic acid, salicylic acid, gentisic acid, protocatechuic acid, ferulic acid, syringic acid, kaempferol, and quercetin, dissolve them in methanol to prepare a 1 mg / mL reference solution; accurately weigh the reference substance dulcitol, dissolve it in ultrapure water to prepare a 1 mg / mL reference solution.

[0055] 3.2. Preparation of test solution Weigh 500.0 mg of Eucommia ulmoides powder and place it in a 50 mL volumetric flask. Add 70% methanol to the scale line and ultrasonicate for 40 min (power 300 W, frequency 40 kHz). Cool and fill the liquid level to the scale line. 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 scale line to obtain the diluted test solution.

[0056] 3.3. Test conditions The chromatographic separation conditions included: mobile phase A was 0.1% formic acid in water; mobile phase B was acetonitrile; the chromatographic column was a CORTECS UPLC C18 column; the flow rate of mobile phase A and mobile phase B was 0.3 mL / min; the column temperature was 35°C; the injection volume was 2 μL; and the elution method was gradient elution. The gradient elution program is as follows: 0-1 min: the volume percentage of the mobile phase B increased from 5% to 14%; 1-4 min: the volume percentage of the mobile phase B increased from 14% to 85%; 4-5 min, the volume percentage of the mobile phase B increased from 85% to 95%.

[0057] The mass spectrometry detection conditions include: ion source: electrospray ionization source; detection mode: multiple reaction ion detection; curtain gas (CUR): 45 psi; collision gas (CAD): Low; ionization voltage (IS): 5500 V; temperature (TEM): 450 °C; spray gas (GS1): 50 psi; auxiliary heating gas (GS2): 20 psi.

[0058] 4. Methodological Investigation 4.1. Standard curve, detection line, and limit of quantification Accurately measure an appropriate amount of reference solution and prepare a mixed reference solution with methanol containing 6000 ng / mL of dulcitol, 1600 ng / mL of kaempferol, 1000 ng / mL of mogroflavin, 800 ng / mL of protocatechuic acid, gentisic acid, camelliaside B, rutin, ferulic acid, syringic acid, p-hydroxybenzoic acid, and salicylic acid, 400 ng / mL of syringaldehyde and leucine B, and 200 ng / mL of quercetin and triptolide. This solution was diluted with methanol in the following order: 2-fold, 2-fold, 2-fold, 2.5-fold, 2-fold, 2.5-fold, 2-fold, and 2-fold to obtain nine mixed reference solutions. Samples were injected and analyzed. Regression equations and correlation coefficients were calculated using the analyte peak area (Y) as the ordinate and the analyte concentration (X) as the abscissa. The mixed reference solution was gradually diluted, and the reference concentration corresponding to the signal-to-noise ratio S / N ≥ 3 and the lowest point in the linear range was used as the detection line and quantification limit, respectively. The results are shown in Table 10. The compounds do not interfere with each other, and the MRM diagram is shown in Figure 2 , Figure 2 A is the MRM chromatogram of the reference substance, 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.

[0059] Table 10 Standard curve regression equations, detection limits, and quantification limits of 15 chemical components of Euonymus fortunei

[0060] 4.2. Precision experiment Intra-day precision: Weigh 500.0 mg of Eucommia foenum-fang tsingtao powder and prepare the test solution using the method described in "3.2 Preparation of Test Solution." Six consecutive injections were made according to the chromatographic conditions, and the compound peak areas were recorded. The RSD values ​​of the peak areas for each 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.

[0061] Table 11 Intra-day precision of 15 components of Euonymus fortunei (n=6)

[0062] 4.3. Inter-day precision Weigh 500.0 mg of Eucommia foenum-fang tsingtao powder and prepare the test solution using the method described in "3.2 Test Solution Preparation." Samples were injected continuously for three days, repeating twice daily according to the chromatographic conditions. The peak areas of the compounds were recorded. The RSD values ​​of the peak areas for the 15 compounds were calculated, and the results are shown in Table 12. All RSD values ​​were less than 5.86%, indicating good inter-day precision of the instrument.

[0063] Table 12 Inter-day precision of 15 components of Euonymus fortunei (n=6)

[0064] 4.4. Repeatability Experiment Weigh 500.0 mg of Eucommia foenum-shaped herb powder and prepare six test solutions in parallel using the method described in "3.2 Preparation of Test Solutions." Each solution was injected and the compound content was calculated. The RSD values ​​for the contents of the 15 compounds were calculated and are shown in Table 13. All RSD values ​​were less than 8.05%, indicating good reproducibility of the method.

[0065] Table 13 Repeatability of 15 components of Euonymus fortunei (μg / g, n=6)

[0066] 4.5. Stability test Weigh 500.0 mg of Eucommia ulmoides powder and prepare test solutions using the method described in "3.2 Test Solution Preparation." Samples were injected at 0, 2, 4, 8, 12, and 24 hours, and the compound peak areas were recorded. The RSDs of the peak areas for the 15 compounds were calculated, and the results are shown in Table 14. All RSDs were less than 5.05%, indicating good stability of the test solutions over a 24-hour period.

[0067] Table 14 Stability of 15 components of Euonymus fortunei (n=6)

[0068] 4.6. Sample recovery experiment Six 250 mg portions of Euphorbia fangiana powder were weighed and added to a predetermined amount of a mixed reference solution. Six parallel test solutions were prepared using the method described in "3.2 Test Solution Preparation." Samples were injected and analyzed separately, and the recovery rates were calculated. The results are shown in Table 15. The average recovery rates for each component ranged from 93.11% to 119.81%.

[0069] Table 15 Recovery rates of 15 components of Euonymus fortunei (n=6)

[0070] 4.7. Dilution effect experiment Precisely measure the reference stock solution to prepare a mixed reference solution containing 6000 ng / mL of dulcitol and 1000 ng / mL of mogroflavin. Dilute this solution 10-, 20-, and 40-fold, and repeat the same dilution method six times. Samples were injected and analyzed, and the concentrations of each compound were recorded and compared with the prepared concentrations to determine accuracy. The results are shown in Table 16. The accuracy of each component ranged from 0.33% to 9.33%.

[0071] Table 16 Accuracy of dilution effect of two components of Euonymus fortunei (n=6)

[0072] 4.5. Content determination Weigh 500.0 mg of Eucommia ulmoides powder and prepare the test solution according to the method in “3.2 Preparation of Test Solution”. Perform the assay using “3.3 Assay Conditions”. Record the concentrations of the 15 compounds and calculate their contents based on the standard curve. The results are shown in Table 17.

[0073] Table 17 Contents of 15 chemical components in Euonymus fortunei (μg / g, n=3)

[0074] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for detecting chemical components in Fufangteng Teng or Fufangteng Teng mixture based on UPLC-QTRAP-MS / MS, characterized in that: The following steps are involved: Extracting the Euonymus fortunei or the Euonymus fortunei compound mixture with methanol-water solution to obtain a test solution of the Euonymus fortunei or the Euonymus fortunei compound mixture; The test solution of Fufang Teng or Fufang Teng mixture is subjected to UPLC-QTRAP-MS / MS detection to obtain the peak area of ​​each test chemical component in Fufang Teng or Fufang Teng mixture; Obtaining the content of each chemical component to be tested in the Fufang Teng or Fufang Teng mixture according to the peak area of ​​each chemical component to be tested in the Fufang Teng or Fufang Teng mixture and a predetermined standard curve of the corresponding chemical component; 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; During UPLC-QTRAP-MS / MS detection, the chromatographic separation conditions included: mobile phase A was formic acid aqueous solution; mobile phase B was acetonitrile; chromatographic column C 18 Column; the flow rate of mobile phase A and mobile phase B was 0.3 mL / min; the elution mode was gradient elution; The conditions for mass spectrometry detection include: ion source: electrospray ion source; detection mode: multiple reaction ion detection.

2. The method according to claim 1, wherein The mass spectrometry detection conditions include: curtain gas: 45 psi; impingement gas: Low; ionization voltage: 5500 V; temperature: 450° C.; spray gas: 50 psi; and auxiliary heating gas: 20 psi.

3. The method according to claim 1, wherein The chromatographic column was a CORTECS UPLC C18 column; the column temperature was 35°C; and the injection volume was 2 μL.

4. The method according to claim 1, wherein When detecting Fufangteng mixture, the gradient elution procedure is as follows: 0-1 min: the volume percentage of the mobile phase B increased from 5% to 14%; 1-3 min: the volume percentage of the mobile phase B increased from 14% to 40%; 3-5 min: the volume percentage of the mobile phase B is 40%; 5-6 min, the volume percentage of the mobile phase B increased from 40% to 59%; In 6-8 minutes, the volume percentage of the mobile phase B increased from 59% to 95%.

5. The method according to claim 1, wherein When detecting Euonymus fortunei, the gradient elution procedure is as follows: 0-1 min: the volume percentage of the mobile phase B increased from 5% to 14%; 1-4 min: the volume percentage of the mobile phase B increased from 14% to 85%; After 4-5 minutes, the volume percentage of the mobile phase B increased from 85% to 95%.

6. The method according to claim 1, wherein The chemical components to be tested in the compound Fufangteng mixture are dactyl alcohol, pyrogallic acid, protocatechuic acid, p-hydroxybenzoic acid, gentisic acid, syringic acid, mogroflavin, camelliaside B, rutin, calycosin, kaempferol, kaempferol-3- O - At least two of the group consisting of rutinoside, syringaldehyde, ferulic acid, ginsenoside Re, ginsenoside Rg1, leucanthoside B, formononetin, salicylic acid, daidzein, quercetin, calycosin, ginsenoside Rb1, ginsenoside Rf, ginsenoside Rc, ginsenoside Ro, ginsenoside Rg2, ginsenoside Rh1, astragaloside IV, ginsenoside Rd, formononetin, ginsenoside Rg3, triptolide, and ginsenoside Rk1.

7. The method according to claim 1, wherein The chemical components to be tested in the Euonymus fortunei are at least two of dactyl alcohol, rutin, camelliaside B, mogroflavin, syringaldehyde, triptolide, leucanthoside B, p-hydroxybenzoic acid, salicylic acid, gentisic acid, protocatechuic acid, ferulic acid, syringic acid, kaempferol and quercetin.

8. The method according to claim 1, wherein When extracting Euonymus fortunei, the volume concentration of methanol aqueous solution is 70%.

9. The method according to claim 1, wherein When extracting the compound Fufangteng mixture, the volume concentration of the methanol aqueous solution is 50%.

10. The method according to claim 1, wherein The volume concentration of formic acid in the formic acid aqueous solution is 0.1%.

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