Quantitative detection method of active ingredients in Wen Tong Huoxue cream

Abstract

The application provides a quantitative detection method of active ingredients in Wending Huoxue cream, and belongs to the technical field of analysis and detection. The quantitative detection method of active ingredients in Wending Huoxue cream provided by the application screens the best mobile phase composition, mobile phase gradient elution program and the most sensitive mass spectrum analysis condition according to the polarity characteristics of main components in Wending Huoxue cream. The results of the embodiments of the application show that the established UPLC-MS method can separate multiple compounds with close polarity on a chromatographic column, so that the quantitative determination of nine chemical components with different types of structures in Wending Huoxue cream can be accurately performed, and the precision, repeatability and stability are good. The application has important significance for controlling the quality of Wending Huoxue cream and guaranteeing the clinical efficacy.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, specifically to a quantitative detection method for active ingredients in a warming and blood-activating cream. Background Technology

[0002] Wentong Huoxue Cream is an effective in-house preparation that has been used clinically for many years at the Affiliated Traditional Chinese Medicine Hospital of Xinjiang Medical University. It is composed of seven Chinese herbs, including Chuanxiong (Ligusticum striatum), Honghua (Carthamus tinctorius), and Baizhi (Angelica dahurica), and was modified based on the ancient formula Tongqiao Huoxue Decoction and clinical experience. It has the effects of warming the meridians and collaterals, promoting blood circulation and removing blood stasis, reducing swelling and relieving pain, and is mainly used to treat diabetic peripheral neuropathy. Modern pharmacological studies have confirmed that the key active ingredients in this preparation (such as coumarins in Chuanxiong, flavonoids in Honghua, and imperatorin, isoimperatorin, and ferulic acid in Baizhi) have core biological activities such as anti-inflammatory, analgesic, antioxidant, microcirculation improvement, and peripheral nerve repair. Previous studies have found that its local pharmacodynamic mechanism is related to regulating anti-inflammatory responses, reducing oxidative stress, and restoring redox balance. However, the chemical composition of traditional Chinese medicine compound preparations is complex, and there is currently a lack of systematic research on the pharmacodynamic material basis of Wentong Huoxue Cream; its material basis for treating diabetic peripheral neuropathy remains unclear. Summary of the Invention

[0003] The purpose of this invention is to provide a quantitative detection method for active ingredients in Wentong Huoxue Cream. This invention is based on UPLC-MS technology to simultaneously determine nine active ingredients in Wentong Huoxue Cream, and has high detection sensitivity, good selectivity and high separation efficiency, providing a scientific basis for subsequent quality control research.

[0004] To achieve the objectives of this invention, the following technical solutions are provided: A method for quantitative detection of active ingredients in a warming and blood-activating cream, comprising the following steps: The warming and blood-activating cream was dissolved in ethanol and extracted with ethanol to obtain the test solution; the active ingredients in the test solution include dihydrocapsaicin, imperatorin, isoimperatorin, hydrated oxyimperatorin, xanthotoxin, hydroxysafflower yellow A, chlorogenic acid, ferulic acid and ligustilide. The test solution was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry, and the quantitative determination results of the active ingredient were obtained based on the external standard method and mass spectrometry data. The conditions for the ultra-high performance liquid chromatography (UHPLC) include: a C18 silica gel reversed-phase column; mobile phase A is a 0.1% (v / v) formic acid aqueous solution, and mobile phase B is acetonitrile; the elution method is gradient elution; the gradient elution program is: 0–2 min, 90% mobile phase A; 2–7 min, 90%–10% mobile phase A; 7–8 min, 10% mobile phase A; 8–8.5 min, 10%–90% mobile phase A; 8.5–10 min, 90% mobile phase A. The mass spectrometry analysis was performed using multiple reaction monitoring (MRM) in both positive and negative ion modes. The conditions for the mass spectrometry analysis include: capillary voltage of 2-4 kV, cone voltage of 8-12 V, source offset voltage of 45-55 V, desolvation temperature of 480-520 °C, desolvation gas flow rate of 950-1050 L / Hr, cone gas flow rate of 140-160 L / Hr, and nebulizer gas pressure of 6-8 Bar; low mass end resolution 1 of 2.6-2.8, high mass end resolution 1 of 14-16, ion energy 1 of 0.3-0.5, low mass end resolution 2 of 2.7-3.0, high mass end resolution 2 of 14-16, ion energy 2 of 0.7-0.9, and collision gas flow rate of 0.08-0.1 mL / min.

[0005] Preferably, the warming and blood-activating cream comprises, by weight, 90-110 parts of Ligusticum chuanxiong, 90-110 parts of Cinnamomum cassia, 25-35 parts of Carthamus tinctorius, 100-110 parts of Angelica dahurica, 50-60 parts of Asarum heterotropoides, 20-30 parts of Pheretima aspergillum, and 50-60 parts of chili pepper.

[0006] Preferably, the volume content of the ethanol is 50-70%.

[0007] Preferably, the alcohol extraction is carried out under ultrasonic conditions; the ultrasonic power is 200~300W, the frequency is 30~50kHz, and the time is 30~60min.

[0008] Preferably, the external standard method includes the following steps: Dihydrocapsaicin, imperatorin, isoimperatorin, hydrated oxyimperatorin, xanthotoxin, hydroxysaffron yellow A, chlorogenic acid, ferulic acid and ligustilide were dissolved in ethanol to obtain a mixed reference solution. The mixed reference solution was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry. Linear regression was performed with the peak area of ​​each reference as the ordinate Y and the mass concentration as the abscissa X to obtain the regression equation for each reference.

[0009] Preferably, the conditions for ultra-high performance liquid chromatography also include: column temperature of 28℃~32℃; mobile phase flow rate of 0.25mL / min~0.35mL / min; injection volume of 1μL; and needle washing solution of MeOH and H2O, with a volume ratio of MeOH to H2O of 1:1.

[0010] Preferably, the linear ranges of the regression equations for dihydrocapsaicin, hydrated oxyimperatorin, imperatorin, isoimperatorin, xanthotoxin, hydroxysaffron yellow A, chlorogenic acid, ferulic acid, and ligustilide are 20.9–1045, 41.2–2060, 18.96–948, 37.84–1892, 1.12–56, 212.7–10635, 46.06–2303, 49.58–2479, and 493.08–24654 ng·mL, respectively. -1 .

[0011] Preferably, the positive / negative ion pairs of the dihydrocapsaicin, imperatorin, isoimperatorin, hydrated oxyimperatorin, xanthotoxin, hydroxysaffron yellow A, chlorogenic acid, ferulic acid, and ligustilide are 380.1 / 137.6, 271.3 / 203.2, 271.3 / 203.0, 97.1 / 202.2, 611.4 / 491.2, 353.2 / 191.2, 193.2 / 134.2, and 191.2 / 173.3, respectively.

[0012] This invention provides a quantitative detection method for active ingredients in a warming and blood-activating cream. Based on the polarity characteristics of the main components in the cream, this invention selects the optimal mobile phase composition, gradient elution program, and most sensitive mass spectrometry analysis conditions. Experimental results from the embodiments of this invention show that the established UPLC-MS method can effectively separate multiple compounds with similar polarities using the chromatographic column, thereby accurately determining nine chemical components of different structural types in the warming and blood-activating cream: dihydrocapsaicin, imperatorin, isoimperatorin, hydrated oxyimperatorin, xanthotoxin, hydroxysaffron yellow A, chlorogenic acid, ferulic acid, and ligustilide.

[0013] Methodological validation shows that the detection method provided by this invention is rapid, efficient, and highly sensitive, enabling simultaneous determination of the content of multiple components in Wentong Huoxue Cream. Its precision, repeatability, and stability are excellent. This method is of great significance for controlling the quality of Wentong Huoxue Cream and ensuring its clinical efficacy. Attached Figure Description

[0014] 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.

[0015] Figure 1 The extraction ion chromatogram (quantitative ion pair) of the mixed reference solution (A), test solution (B), and blank solvent (C) is shown. Figure 1From top to bottom, the compounds are ferulic acid, dihydrocapsaicin, ligustilide, xanthotoxin, chlorogenic acid, imperatorin, hydroxysaffron yellow A, hydrated oxyimperatorin, and isoimperatorin. Detailed Implementation

[0016] This invention provides a method for quantitative detection of active ingredients in a warming and blood-activating cream, comprising the following steps: The warming and blood-activating cream was dissolved in ethanol and extracted with ethanol to obtain the test solution; the active ingredients in the test solution include dihydrocapsaicin, imperatorin, isoimperatorin, hydrated oxyimperatorin, xanthotoxin, hydroxysafflower yellow A, chlorogenic acid, ferulic acid and ligustilide. The test solution was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry, and the quantitative determination results of the active ingredient were obtained based on the external standard method and mass spectrometry data. The conditions for the ultra-high performance liquid chromatography (UHPLC) include: a C18 silica gel reversed-phase column; mobile phase A is a 0.1% (v / v) formic acid aqueous solution, and mobile phase B is acetonitrile; the elution method is gradient elution; the gradient elution program is: 0–2 min, 90% mobile phase A; 2–7 min, 90%–10% mobile phase A; 7–8 min, 10% mobile phase A; 8–8.5 min, 10%–90% mobile phase A; 8.5–10 min, 90% mobile phase A. The mass spectrometry analysis was performed using multiple reaction monitoring (MRM) in both positive and negative ion modes. The conditions for the mass spectrometry analysis include: capillary voltage of 2-4 kV, cone voltage of 8-12 V, source offset voltage of 45-55 V, desolvation temperature of 480-520 °C, desolvation gas flow rate of 950-1050 L / Hr, cone gas flow rate of 140-160 L / Hr, and nebulizer gas pressure of 6-8 Bar; low mass end resolution 1 of 2.6-2.8, high mass end resolution 1 of 14-16, ion energy 1 of 0.3-0.5, low mass end resolution 2 of 2.7-3.0, high mass end resolution 2 of 14-16, ion energy 2 of 0.7-0.9, and collision gas flow rate of 0.08-0.1 mL / min.

[0017] Unless otherwise specified, all reagents used in this invention are commercially available products well known to those skilled in the art.

[0018] In this invention, the warming and blood-activating cream comprises, by weight, 90-110 parts of Ligusticum chuanxiong, 90-110 parts of Cinnamomum cassia, 25-35 parts of Carthamus tinctorius, 100-110 parts of Angelica dahurica, 50-60 parts of Asarum heterotropoides, 20-30 parts of Pheretima aspergillum, and 50-60 parts of Citrus aurantium, as specifically shown in Chinese Patent CN110812418A.

[0019] In this invention, the volume content of ethanol is 50-70%.

[0020] In this invention, the alcohol extraction is carried out under ultrasonic conditions; the power of the ultrasonic conditions is 200~300W, which can be 230 or 250W in specific embodiments, and the frequency is 30~50kHz, which can be 35 or 40kHz in specific embodiments.

[0021] In this embodiment of the invention, the preparation method of the test solution (sample solution) specifically includes: Weigh 20mg of Wentong Huoxue Cream, add an appropriate amount of 70% (v / v) ethanol, sonicate (power 250W, frequency 40kHz) for 30min, cool, and bring the volume to 10mL with 70% (v / v) ethanol. Shake well and sonicate at 12000rpm·min. -1 Centrifuge for 10 min, take the supernatant, dilute it 4 times with 70% (v / v) ethanol, vortex to mix, and obtain the test solution.

[0022] In this invention, the mass concentrations of capsaicin, imperatorin, isoimperatorin, hydrated oxyimperatorin, xanthotoxin, hydroxysaffron yellow A, chlorogenic acid, ferulic acid, and ligustilide in the mixed reference solution are 500-2000, 500-5000, 500-6000, 500-2500, 50-500, 5000-20000, 200-3000, 1000-4000, and 10000-40000 ng·mL, respectively. -1 In specific embodiments, the values ​​can be 1045, 948, 1892, 2060, 56, 10635, 2303, 249, and 24654 ng·mL. -1 .

[0023] In this invention, the ultra-high performance liquid chromatography (UHPLC) conditions also include: column temperature of 28℃~32℃; mobile phase flow rate of 0.25mL / min~0.35mL / min; injection volume of 1μL; and injection solution of MeOH and H2O with a volume ratio of 1:1. Specifically, the C18 silica gel reversed-phase column can be an HSS T3 column with dimensions of 100mm×2.1mm and 1.8µm.

[0024] In this invention, the regression equation for dihydrocapsaicin is y1 = 100.56x1 + 6.4441. r 1 = 0.9998; the regression equation for imperatorin is y² = 90.474x² + 5.1164. r 2 = 0.9999; the regression equation for isoechopurin is y3 = 189.84x3 + 10.609. r3 = 0.9998; the regression equation for hydrated oxidized imperatorin is y4 = 184.92x4 + 24.976. r 4 = 0.9995; the regression equation for zanthoxylate is y5 = 5.5796x5 - 0.014. r 5 = 0.9999; the regression equation for hydroxysaffron yellow A is y6 = 1229.7x6 - 175.23. r y7 = 0.9991; the regression equation for chlorogenic acid is y7 = 256.6x7 - 21.363. r 7 = 0.9999; the regression equation for ferulic acid is y8 = 228.34x8 + 12.898. r y = 0.9996; the regression equation for ligustilide is y9 = 2222x9 + 238.6. r 9 = 0.9997.

[0025] In this invention, the mass spectrometry employs a Waters XEVO TQS tandem quadrupole mass spectrometer system.

[0026] In this invention, the positive / negative ion pairs of dihydrocapsaicin, imperatorin, isoimperatorin, hydrated oxyimperatorin, xanthotoxin, hydroxysaffron yellow A, chlorogenic acid, ferulic acid, and ligustilide are 380.1 / 137.6, 271.3 / 203.2, 271.3 / 203.0, 97.1 / 202.2, 611.4 / 491.2, 353.2 / 191.2, 193.2 / 134.2, and 191.2 / 173.3, respectively.

[0027] To further illustrate the present invention, the quantitative detection method for active ingredients in the warming and blood-activating cream provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0028] The sources and parameters of the reagents used in the following embodiments of the present invention are as follows: Dihydrocapsaicin (batch number LD30Z43) and xanthotoxin (batch number BD20056-1g) were purchased from Beijing Bailingwei Technology Co., Ltd.; imperatorin (batch number JB256320), isoimperatorin (content ≥98%, batch number JB256320) and hydroxysaffron yellow A (batch number JB258648) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; hydrated oxyimperatorin (batch number C17599512) and ferulic acid (batch number C17487265) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; chlorogenic acid (batch number KB363783) and ligustilide (batch number 24241028003) were purchased from Beijing Solarbio Technology Co., Ltd.

[0029] The Wentong Huoxue Cream was provided by the Affiliated Traditional Chinese Medicine Hospital of Xinjiang Medical University (the hospital preparation approval number is: New Drug Preparation No. Z20052604), and the batch numbers are 20240804, 20250701, 20250702, 20251022, and 20251023 respectively.

[0030] Ethanol (mass spectrometry grade, batch number LC50Z05) was purchased from Beijing Bailingwei Technology Co., Ltd., and acetonitrile (mass spectrometry grade, batch number F25P47201), methanol (mass spectrometry grade, batch number LF50Z32), and formic acid (mass spectrometry grade, batch number 226099) were purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0031] Example 1 Preparation of mixed reference solutions: Accurately weigh appropriate amounts of dihydrocapsaicin, imperatorin, isoimperatorin, hydrated oxyimperatorin, xanthotoxin, hydroxysaffron yellow A, chlorogenic acid, ferulic acid, and ligustilide reference standards. Dissolve and dilute to volume with 70% (v / v) ethanol to prepare reference stock solutions of specific concentrations. Accurately measure appropriate amounts of each reference stock solution and mix them to prepare mass concentrations of 1045, 948, 1892, 2060, 56, 10635, 2303, 249, and 24654 ng·mL. -1 The mixed reference stock solution was gradually diluted with 70% (v / v) ethanol to prepare a series of mixed reference solutions of different concentrations, and stored in a -20°C refrigerator for later use.

[0032] Preparation of the test solution: Accurately measure approximately 20 mg of each batch of Wentong Huoxue Cream, add an appropriate amount of 70% (v / v) ethanol, sonicate (power 250W, frequency 40kHz) for 30 min, allow to cool naturally, then dilute to 10 mL with 70% (v / v) ethanol, shake well, and sonicate at 12000 rpm·min. -1 Centrifuge for 10 min, take the supernatant, dilute it 4 times with 70% (v / v) ethanol, vortex to mix, and obtain the test solution.

[0033] Chromatographic conditions: Waters ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 µm), column temperature 30℃, flow rate 0.3 mL / min. -1 The injection volume was 1 μL; the mobile phase was 0.1% formic acid water (A)-acetonitrile (B), and the gradient elution program was: 0~2 min, 90% A; 2~7 min, 90%~10% A; 7~8 min, 10% A; 8~8.5 min, 10%~90% A; 8.5~10 min, 90% A.

[0034] The Waters XEVO TQS tandem quadrupole mass spectrometer system was used, and multiple reaction monitoring (MRM) was employed for monitoring in both positive and negative ion modes.

[0035] Mass spectrometry conditions: capillary voltage 3 kV, cone voltage 10 V, source offset voltage 50 V, desolvation temperature 500 °C, desolvation gas flow rate 1000 L / Hr, cone gas flow rate 150 L / Hr, nebulizer gas pressure 7 Bar, low mass end resolution 1 2.74, high mass end resolution 1 15.53, ion energy 1 0.4, low mass end resolution 2 2.83, high mass end resolution 2 15.15, ion energy 2 0.8, collision gas flow rate 0.08 mL / min.

[0036] The parameters of the nine analytes are shown in Table 1; the chromatograms of the extracted ion currents (quantitative ion pairs) of the mixed reference standard, test solution, and blank solution are shown in Table 1. Figure 1 As shown.

[0037] Table 1. Mass spectrometry parameters of nine components in Wentong Huoxue Cream

[0038] 1) Methodological validation Establishment of standard curve The above-mentioned mixed reference solutions of different concentrations were sampled and measured. Linear regression was performed with the peak area of ​​each reference as the ordinate (Y) and the mass concentration of each reference as the abscissa (X). The results are shown in Table 2.

[0039] Table 2. Regression equations, correlation coefficients, and linear ranges for the nine compounds.

[0040] 2) Precision verification Accurately pipette a mixed reference solution of medium mass concentration and inject it six times consecutively under the chromatographic conditions described above. Record the peak area and calculate the RSD of the ratio of the peak area of ​​each component to the peak area of ​​the corresponding internal standard. The results are shown in Table 3. The RSD of the peak areas obtained for all nine components is less than 10%, indicating good instrument precision.

[0041] 3) Repeatability verification Six samples from the same batch were taken, and test solutions were prepared according to the above method. The samples were then injected and analyzed under the above chromatographic conditions. Six parallel determinations were performed, and the peak areas were recorded. The RSD of each component was calculated. The results are shown in Table 3. The RSDs of all nine components were below 10%, indicating good repeatability of the method.

[0042] 4) Stability verification The same batch of test solutions were injected and measured at 0, 2, 4, 8, 12, and 24 hours under the chromatographic conditions described above. The peak areas were recorded, and the RSD of the ratio of the peak area of ​​each component to the peak area of ​​the corresponding internal standard was calculated. The results are shown in Table 3. The RSD of the peak areas of all nine components was less than 6%, indicating that the test solutions were stable within 24 hours.

[0043] Table 3. Precision, repeatability, and stability test results of the nine components.

[0044] 5) Recovery experiment Take 0.5 mL of the known content of Wentong Huoxue Cream sample (S5), and accurately add an appropriate amount of mixed reference solution (70% (v / v) ethanol as solvent). Prepare three parallel test solutions for low, medium, and high levels, respectively. Then, inject and determine the samples under the above chromatographic conditions, record the peak area, and calculate the recovery rate. The results are shown in Table 4. The average recovery rates of the nine components are all between 88.85% and 102.48%, and the RSD is between 0.25% and 3.36%, indicating that the method has good accuracy.

[0045] Table 4. Results of the recovery tests for the nine components.

[0046] Content determination of the test sample: Take 3 batches of Wentong Huoxue Cream, prepare 3 test sample solutions for each batch in parallel, and then inject and determine the content of each component according to the above chromatographic conditions. The content determination results of each component are calculated according to the standard curve equation and are shown in Table 5.

[0047] Table 5. Results of determination of the content of nine components in each batch of Wentong Huoxue Cream (ng·mL) -1 (n=6)

[0048] This invention quantitatively determined nine components in Wentong Huoxue Cream using UPLC-MS / MS. The content of chemical components in Wentong Huoxue Cream varied considerably. Furthermore, the content of each component differed significantly among six batches of Wentong Huoxue Cream samples. The results showed that Wentong Huoxue Cream contained high levels of ligustilide, imperatorin, and isoimperatorin. Each milligram of Wentong Huoxue Cream contained 4930.8 ng of ligustilide, 2127 ng of hydroxysaffron yellow A, and 412 ng of hydrated oxyimperatorin. This method can comprehensively and objectively detect the nine main components in Wentong Huoxue Cream, providing a scientific basis for ensuring its clinical efficacy.

[0049] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for quantitative detection of active ingredients in a warming and blood-activating cream, characterized in that, Includes the following steps: The warming and blood-activating cream was dissolved in ethanol and extracted with ethanol to obtain the test solution; the active ingredients in the test solution include dihydrocapsaicin, imperatorin, isoimperatorin, hydrated oxyimperatorin, xanthotoxin, hydroxysafflower yellow A, chlorogenic acid, ferulic acid and ligustilide. The test solution was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry, and the quantitative determination results of the active ingredient were obtained based on the external standard method and mass spectrometry data. The conditions for the ultra-high performance liquid chromatography (UHPLC) include: a C18 silica gel reversed-phase column; mobile phase A is a 0.1% (v / v) formic acid aqueous solution, and mobile phase B is acetonitrile; the elution method is gradient elution; the gradient elution program is: 0–2 min, 90% mobile phase A; 2–7 min, 90%–10% mobile phase A; 7–8 min, 10% mobile phase A; 8–8.5 min, 10%–90% mobile phase A; 8.5–10 min, 90% mobile phase A. The mass spectrometry analysis was performed using multiple reaction monitoring in both positive and negative ion modes. The conditions for the mass spectrometry analysis include: capillary voltage of 2-4 kV, cone voltage of 8-12 V, source offset voltage of 45-55 V, desolvation temperature of 480-520 °C, desolvation gas flow rate of 950-1050 L / Hr, cone gas flow rate of 140-160 L / Hr, and nebulizer gas pressure of 6-8 Bar; low mass end resolution 1 of 2.6-2.8, high mass end resolution 1 of 14-16, ion energy 1 of 0.3-0.5, low mass end resolution 2 of 2.7-3.0, high mass end resolution 2 of 14-16, ion energy 2 of 0.7-0.9, and collision gas flow rate of 0.08-0.1 mL / min.

2. The quantitative detection method according to claim 1, characterized in that, The warming and blood-activating cream, by weight, includes 90-110 parts of Ligusticum chuanxiong, 90-110 parts of Cinnamomum cassia, 25-35 parts of Carthamus tinctorius, 100-110 parts of Angelica dahurica, 50-60 parts of Asarum heterotropoides, 20-30 parts of Pheretima aspergillum, and 50-60 parts of Citrus aurantium.

3. The quantitative detection method according to claim 1, characterized in that, The volume content of the ethanol is 50-70%.

4. The quantitative detection method according to claim 1, characterized in that, The alcohol extraction is carried out under ultrasonic conditions; the ultrasonic power is 200~300W, the frequency is 30~50kHz, and the time is 30~60min.

5. The quantitative detection method according to claim 1, characterized in that, The external standard method includes the following steps: Dihydrocapsaicin, imperatorin, isoimperatorin, hydrated oxyimperatorin, xanthotoxin, hydroxysaffron yellow A, chlorogenic acid, ferulic acid and ligustilide were dissolved in ethanol to obtain a mixed reference solution. The mixed reference solution was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry. Linear regression was performed with the peak area of ​​each reference as the ordinate Y and the mass concentration as the abscissa X to obtain the regression equation for each reference.

6. The quantitative detection method according to claim 1, characterized in that, The conditions for ultra-high performance liquid chromatography also include: column temperature of 28℃~32℃; mobile phase flow rate of 0.25mL / min~0.35mL / min; injection volume of 1μL; and needle washing solution of MeOH and H2O with a volume ratio of 1:

1.

7. The quantitative detection method according to claim 1, characterized in that, The linear ranges of the regression equations for dihydrocapsaicin, hydrated oxyimperatorin, imperatorin, isoimperatorin, xanthotoxin, hydroxysaffron yellow A, chlorogenic acid, ferulic acid, and ligustilide were 20.9–1045, 41.2–2060, 18.96–948, 37.84–1892, 1.12–56, 212.7–10635, 46.06–2303, 49.58–2479, and 493.08–24654 ng·mL, respectively. -1 .

8. The quantitative detection method according to claim 1, characterized in that, The positive / negative ion pairs of the dihydrocapsaicin, imperatorin, isoimperatorin, hydrated oxyimperatorin, xanthotoxin, hydroxysafflower yellow A, chlorogenic acid, ferulic acid, and ligustilide are 380.1 / 137.6, 271.3 / 203.2, 271.3 / 203.0, 97.1 / 202.2, 611.4 / 491.2, 353.2 / 191.2, 193.2 / 134.2, and 191.2 / 173.3, respectively.

Images