Method for measuring content of carpesium abrotanoides component

Abstract

The invention belongs to the field of quality analysis and control of traditional Chinese medicines, and discloses a method for determining the content of carpesium abrotanoides components, which comprises the following steps: by taking isochlorogenic acid A as an internal reference substance, calculating the content of chlorogenic acid, caffeic acid, galuteolin, 11 (13)-dehydroaxillary Ivalin, isochlorogenic acid C, 2, 3, 4, 5-tetramethyl-1, 3, 4, 5-tetramethyl-1, 3, 5-tetramethyl-1, 3, 5-tetramethyl-1, 3, 5-tetramethyl-1, 3, 5- According to the method, the content of 10 components of carabrone is rapidly and accurately detected according to the relative correction factors of 2, 3, 4, 5-tetracaffeoyl-D-glucaric acid, 2-deoxy-4-surface-gaillanin, carabrone and dellelactone, and the problem that the detection cost is high due to the fact that many reference substances are needed, are not easy to obtain and are high in price in carabrone content determination is solved.

Description

Technical Field

[0001] The present invention relates to the field of quality analysis and control of traditional Chinese medicines, and particularly to a method for determining the content of components in Carpesium abrotanoides L.. Background Art

[0002] Carpesium abrotanoides L. is a commonly used local ethnic medicine in Hunan Province. It is the dried whole herb of the Compositae plant Carpesium abrotanoides L., and has the effects of clearing heat and detoxifying, removing phlegm and stopping bleeding. Clinically, it is mainly used to treat toothache, oral erosion, herpes zoster, bacterial infection, skin pruritus, folliculitis, hepatitis, epidemic acute viral conjunctivitis and other diseases. Carpesium abrotanoides L. is included in the standards of various provinces, indicating the important role it plays in traditional medical practice. For example, the "Specifications for Processing of Traditional Chinese Medicine Decoction Pieces in Hunan Province" in 2021 edition, the "Specifications for Processing of Traditional Chinese Medicine Decoction Pieces in Jiangsu Province" in 2020 edition, the "Quality Standards for Chinese Medicinal Materials in Hubei Province" in 2018 edition, the "Specifications for Processing of Traditional Chinese Medicine in Zhejiang Province" in 2015 edition, the "Specifications for Processing of Traditional Chinese Medicine Decoction Pieces in Hebei Province" in 2003 edition, and the "Specifications for Processing of Traditional Chinese Medicine Decoction Pieces in Shanghai" in 2018 edition. However, it has not been included in the "Chinese Pharmacopoeia", and the determination method for its index components has not been established in the existing standards. The present invention can promote the improvement of the standards for Carpesium abrotanoides L., and ensure its curative effect in clinical practice or preparations, and can promote its application in clinical practice or preparations. This medicinal material is rich in components such as sesquiterpenes, sesquiterpene dimers, flavonoids and nitrogen-containing compounds. These components have been proven to have pharmacological activities such as anti-inflammatory, antibacterial, anti-parasitic, anti-tumor and antiviral. The present invention finds that Carpesium abrotanoides L., as a Compositae plant, also contains rich phenolic acid components. This type of component has functions such as anti-inflammatory, antioxidant, anti-tumor and bacteriostatic, and can be used as an important index to measure the quality of Carpesium abrotanoides L.. Summary of the Invention

[0003] According to the above problems, the technical problem to be solved by the present invention is to provide a method for determining the content of components in Carpesium abrotanoides L., which can accurately measure the content of multiple components in Carpesium abrotanoides L. at one time.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0005] A method for determining the content of components in Carpesium abrotanoides L. includes the following steps:

[0006] S1. Prepare the test solution of Carpesium abrotanoides L. and the mixed reference substance solution;

[0007] S2. Detect the fingerprint of Carpesium abrotanoides L. by ultra-high performance liquid chromatography. The ultra-high performance liquid chromatography includes mobile phase A, an aqueous phosphoric acid solution, and mobile phase B, an acetonitrile solution;

[0008] S3. Determination of relative correction factor: Precisely pipette the mixed reference solution, inject and determine it by the ultra-high performance liquid chromatography method, record the peak areas, use one of the components of Carpesium abrotanoides L. as the internal standard, and calculate the relative correction factors of other components to be determined of Carpesium abrotanoides L. relative to the internal standard: f i / s = f i / f s = (A i / W i ) / (A s / W s )

[0009] In the formula, f i / s is the relative correction factor, f i is the correction factor of the internal standard, f s is the correction factor of the component to be determined, A i is the peak area of the internal standard, W i is the amount of the internal standard, A s is the peak area of the component to be determined, W s is the amount of the component to be determined;

[0010] S4. Determination of relative retention time: Determine the relative retention time, that is, the ratio of the retention times of each component to be determined and the internal standard, to locate the chromatographic peaks;

[0011] S5. Content determination: Calculate the contents of the components to be determined respectively:

[0012] W x = f i / s × W i × A x / A i

[0013] In the formula, W x is the content of the component to be determined in the test solution, W i is the content of the internal standard, A x is the peak area of the component to be determined in the test solution, A i is the peak area of the internal standard.

[0014] The multi-component determination method of this invention has good durability under different conditions and is feasible.

[0015] The method of this invention has good specificity, good linear relationship, good precision, good stability, good repeatability and good accuracy.

[0016] According to the embodiments of this invention, the invention can be further optimized. The following are the technical solutions formed after optimization:

[0017] In S3, using isochlorogenic acid A as the internal standard, calculate the relative correction factors of chlorogenic acid, caffeic acid, luteoloside, 11(13)-dehydroaxillarin, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-glucaric acid, 2-deoxy-4-epi-skyrin, carabrone, and telosmol.

[0018] In one preferred embodiment, in S3, the relative correction factors of chlorogenic acid, caffeic acid, luteoloside, 11(13)-dehydroaxillarin, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-glucaric acid, 2-deoxy-4-epi-skyrin, carabrone, and telosmol are 1.31 - 1.34, 0.11 - 0.81, 2.52 - 2.59, 3.54 - 3.71, 0.99 - 1.03, 0.95 - 1.01, 2.82 - 3.03, 1.91 - 2.05, and 3.00 - 3.21, respectively.

[0019] In one preferred embodiment, in S4, the relative retention times of chlorogenic acid, caffeic acid, luteoloside, 11(13)-dehydroaxillarin, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-glucaric acid, 2-deoxy-4-epi-skyrin, carabrone, and telosmol are 0.22 - 0.23, 0.29 - 0.31, 0.82 - 0.83, 0.94 - 0.98, 1.16 - 1.19, 1.70 - 1.73, 1.99 - 2.14, 2.07 - 2.19, and 2.20 - 2.37, respectively.

[0020] In one preferred embodiment, the detection conditions of the ultra-high performance liquid chromatography method include: the chromatographic column is an octadecylsilane-bonded silica gel chromatographic column, the mobile phase flow rate is 0.2 - 0.4 mL / min, the detection wavelength is 210 - 230 nm, the column temperature is 33 - 37 °C, and the injection volume is 0.5 - 1 μL.

[0021] The ultra-high performance liquid chromatography map of the carpesium abrotanoides sample obtained in the present invention is more complete and beautiful while meeting the requirements of content determination, with a stable baseline and symmetric peaks for each component.

[0022] In one preferred embodiment, the ultra-high performance liquid chromatography method uses gradient elution for elution; preferably, the gradient elution program includes:

[0023] From 0 to 2 min, the volume fraction of mobile phase B is 10%;

[0024] From 2 to 5 min, the volume fraction of mobile phase B increases from 10% to 14.5%;

[0025] From 5 to 15 min, the volume fraction of mobile phase B increases from 14.5% to 22%;

[0026] 15 - 17 min, the volume fraction of mobile phase B is increased from 22% to 26%;

[0027] 17 - 25 min, the volume fraction of mobile phase B is increased from 26% to 28%;

[0028] 25 - 30 min, the volume fraction of mobile phase B is increased from 28% to 37%.

[0029] In one preferred embodiment, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05 - 0.4%; preferably, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05 - 0.2%. Further preferably, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05 - 0.15%.

[0030] In one preferred embodiment, the preparation method of the Carpesium abrotanoides test sample solution comprises the following steps: extracting the components in Carpesium abrotanoides with an extractant, and the extractant comprises 50% - 80% methanol or 50% - 80% ethanol. Preferably, the extractant comprises 70% - 80% methanol or 70% - 80% ethanol.

[0031] In one preferred embodiment, the solid - liquid ratio of Carpesium abrotanoides to the extractant is 0.3 - 0.6 g: 15 - 25 mL; preferably, the solid - liquid ratio of Carpesium abrotanoides to the extractant is 0.45 - 0.55 g: 19.5 - 20.5 mL.

[0032] In one preferred embodiment, the extraction is carried out by ultrasonic extraction; the power of the ultrasonic extraction is 190 - 210 W, the frequency is 35 - 45 kHz, and the time is 50 - 70 min.

[0033] Preferably, in S1, the mixed reference substance solution comprises chlorogenic acid, caffeic acid, luteoloside, 11(13) - dehydroaxillarin, isochlorogenic acid A, isochlorogenic acid C, 2,3,4,5 - tetracaffeoyl - D - glucaric acid, 2 - deoxy - 4 - epi - tagitinin, carabrone, telekin; preferably, the mass concentration ratio of chlorogenic acid, caffeic acid, luteoloside, 11(13) - dehydroaxillarin, isochlorogenic acid A, isochlorogenic acid C, 2,3,4,5 - tetracaffeoyl - D - glucaric acid, 2 - deoxy - 4 - epi - tagitinin, carabrone, telekin is 150 - 160, 10 - 20, 140 - 150, 90 - 100, 440 - 450, 10 - 20, 70 - 80, 170 - 180, 60 - 70, 60 - 70 μg·mL -1 .

[0034] The present invention quickly and accurately detects the contents of 10 components of Carpesium abrotanoides L., and solves the problem of high detection cost caused by the large number of reference substances required for the determination of the content of Carpesium abrotanoides L., which are difficult to obtain and expensive.

[0035] The beneficial effects of the present invention are as follows:

[0036] The present invention firstly creates an accurate and low-cost quality control and evaluation method for Carpesium abrotanoides L. By using the method of multi-component determination with a single reference substance, the contents of 10 components, namely chlorogenic acid, caffeic acid, luteoloside, 11(13)-dehydroaxillarin, isochlorogenic acid A, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-glucaric acid, 2-deoxy-4-epi-helianthenin, carabrone, and telekin, are simultaneously determined. That is, only one reference substance of isochlorogenic acid A is needed, and the contents of the other 9 components can be calculated through the relative correction factor.

[0037] The ultra-high performance liquid chromatography conditions established by the present invention have good specificity, and have good baseline separation for each component, ensuring the accuracy and reliability of the positioning of each chromatographic peak in the multi-component determination with a single reference substance.

[0038] The present invention fills the blank of the determination of physical indicators or active ingredient contents of Carpesium abrotanoides L. in the existing standards, and provides a scientific basis for solving the quality control problem of Carpesium abrotanoides L. Description of the Drawings

[0039] Figure 1 They are the chromatograms of blank (A), test sample (B), and mixed reference substance (C).

[0040] Figure 2 They are the high performance liquid chromatography diagrams of Carpesium abrotanoides L. in Comparative Example 1.

[0041] Figure 3 They are the ultra-high performance liquid chromatography diagrams of Carpesium abrotanoides L. in Comparative Example 2.

[0042] Figure 4 They are the ultra-high performance liquid chromatography diagrams of Carpesium abrotanoides L. in Comparative Example 3.

[0043] Figure 5 They are the ultra-high performance liquid chromatography diagrams of Carpesium abrotanoides L. with different extraction solvents in Comparative Example 4.

[0044] Figure 6 They are the ultra-high performance liquid chromatography diagrams of Carpesium abrotanoides L. with different wavelengths in Comparative Example 5. Detailed Embodiments

[0045] The present invention will be described in detail below in conjunction with specific embodiments.

[0046] The present invention uses the UPLC method with isochlorogenic acid A as the internal reference substance, and calculates the relative correction factors of chlorogenic acid, caffeic acid, luteoloside, 11(13)-dehydroaxillarin, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-glucaric acid, 2-deoxy-4-epi-skyrin, carabrone, and telesterone, including the following steps:

[0047] 1) Take the test solution and detect it by liquid chromatography;

[0048] The components include chlorogenic acid (CAS No.: 327-97-9), caffeic acid (CAS No.: 331-39-5), luteoloside (CAS No.: 5373-11-5), 11(13)-dehydroaxillarin (CAS No.: 87441-73-4), isochlorogenic acid A (CAS No.: 2450-53-5), isochlorogenic acid C (CAS No.: 57378-72-0), 2,3,4,5-tetracaffeoyl-D-glucaric acid (CAS No.: 1419478-52-6), 2-deoxy-4-epi-skyrin (CAS No.: 122872-03-1), carabrone (CAS No.: 1748-81-8), and telesterone (CAS No.: 6752-90-5);

[0049] 2) Using isochlorogenic acid A as the internal reference substance, by calculating the relative correction factors of chlorogenic acid, caffeic acid, luteoloside, 11(13)-dehydroaxillarin, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-glucaric acid, 2-deoxy-4-epi-skyrin, carabrone, and telesterone, the contents of 10 components in Carpesium abrotanoides L. were detected quickly and accurately;

[0050] 3) Relative correction factor: Precisely pipette the mixed reference solution, inject it for determination, record the peak area, and calculate the relative correction factor (fi / s)

[0051] Calculation formula: f i / s = f i / f s = (A i / W i ) / (A s / W s )

[0052] In the formula, W i is the amount of the internal standard substance, A i is the peak area of the internal standard substance, W s is the amount of the component to be measured, A s is the peak area of the component to be measured;

[0053] 4) Determination of relative retention time: The relative retention time, which is the ratio of the retention time of each component to be measured to that of isochlorogenic acid A, was used to locate the chromatographic peaks.

[0054] 5) Content determination: Absorb the test solution and the mixed reference solution, perform chromatographic analysis, and calculate the contents of chlorogenic acid, caffeic acid, luteoloside, 11(13)-dehydroaxillarin, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-glucaric acid, 2-deoxy-4-epi-skyrin, carabrone, and teloschistin according to the determined relative correction factors;

[0055] Calculation formula: W x =f i / s ×W i ×A x / A i

[0056] In the formula, A x and A i are the peak areas of the component to be measured and the internal standard substance in the test solution, respectively, W x and W i are the contents of the component to be measured and the internal standard substance in the test solution, respectively, and f i / s is the relative correction factor of the component to be measured.

[0057] Example 1

[0058] 1. Sample source

[0059] The specific information of 17 batches of Carpesium abrotanoides L. herbs is shown in Table 1. Among them, batches S4, 5, 9, 10, 11, and 12 are fresh herbs, which are obtained by removing impurities, washing, moistening thoroughly, cutting, and drying according to the processing method of "Hunan Provincial Traditional Chinese Medicine Decoction Pieces Processing Specification" (2021 edition), and are identified as the dried whole herb of the Compositae plant Carpesium abrotanoides Linnaeus by Associate Professor Wang Zhi of Hunan University of Chinese Medicine.

[0060] Table 1 Information table of Carpesium abrotanoides L. herbs

[0061]

[0062] 2. Chromatographic conditions

[0063] Waters ACQUITY BEH C 18Chromatographic column (2.1×100 mm, 1.7 μm), mobile phase: 0.1% phosphoric acid (A) - acetonitrile (B), gradient elution (0 - 2 min, 10% B; 2 - 5 min, 10% - 14.5% B; 5 - 15 min, 14.5% - 22% B; 15 - 17 min, 22% - 26% B; 17 - 25 min, 26% - 28% B; 25 - 30 min, 28% - 37% B), column temperature 35°C, flow rate 0.3 mL·min -1 , detection wavelength 230 nm, injection volume 0.5 μL.

[0064] 3. Preparation of solutions

[0065] 3.1 Preparation of reference substance solution

[0066] Weigh appropriate amounts of reference substances of chlorogenic acid, caffeic acid, luteoloside, 11(13)-dehydroaxillarin, isochlorogenic acid A, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-glucaric acid, 2-deoxy-4-epi-heliantheine, carabrone, and telesterone accurately, dissolve them in 80% ethanol to prepare single reference substance stock solutions with certain mass concentrations. Take appropriate amounts of the above single reference substance stock solutions and place them in the same 10 mL volumetric flask, dilute to the mark with 80% ethanol, shake well, to obtain a mixed reference substance stock solution with mass concentrations of 158.88, 19.86, 149.76, 95.70, 445.20, 19.96, 72.94, 173.50, 63.84, 67.50 μg·mL -1 respectively.

[0067] Precisely pipette 2 mL of the above mixed reference substance stock solution into a 10 mL volumetric flask, dilute to the mark with 80% ethanol, shake well, to obtain a mixed reference substance solution with mass concentrations of 31.78, 3.97, 29.95, 19.14, 89.04, 3.99, 14.59, 34.70, 12.77, 13.50 μg·mL -1 respectively.

[0068] 3.2 Preparation of test solution

[0069] Take 0.5 g of Carpesium abrotanoides L. powder, weigh accurately, place it in a stoppered conical flask, accurately add 20 mL of 80% ethanol, weigh, ultrasonically extract for 60 min (power 200 W, 40 kHz), cool, weigh again, make up the weight with 80% ethanol, shake well, filter through a 0.22 μm organic microporous membrane, to obtain the test solution.

[0070] 4. Methodology investigation for content determination

[0071] 4.1 Specificity investigation

[0072] Blank solution, mixed reference solution and test solution were respectively injected for analysis under the above chromatographic conditions, and the results were as Figure 1 shown. The results showed that the retention times of the chromatographic peaks in the test solution were consistent with those of the mixed reference, and no chromatographic peaks appeared at the corresponding positions in the blank control chromatogram, indicating that the method had good specificity.

[0073] 4.2 Investigation of linear relationship

[0074] Precisely pipette 0.25, 0.50, 1.00, 2.00, 5.00 mL of the mixed reference stock solution under item "3.1" respectively, and make up the volume to 5 mL with 80% ethanol to prepare 5 mixed reference solutions with different concentrations. Inject samples successively under the above chromatographic conditions, and record the peak areas of the chromatograms. With the peak areas of the corresponding components as the ordinate and the reference concentrations (μg·mL -1 ) as the abscissa, draw a standard curve to obtain the regression equations and linear ranges of 10 components including chlorogenic acid (peak 1), caffeic acid (peak 2), luteoloside (peak 7), 11(13)-dehydroaxillarin (peak 9), isochlorogenic acid A (peak 10), isochlorogenic acid C (peak 12), 2,3,4,5-tetracaffeoyl-D-glucaric acid (peak 13), 2-deoxy-4-epi-heliangolide (peak 15), carabrone (peak 16), and telekin (peak 17). Calculate the detection limits (LOD) and quantification limits (LOQ) of each component at signal-to-noise ratios (S / N) of 3 and 10 respectively, as shown in Table 2. The results showed that the correlation coefficients of each component were all above 0.9995, indicating good linear relationships.

[0075] Table 2 Investigation of linear relationships of 10 components in Carpesium abrotanoides L.

[0076]

[0077]

[0078] 4.3 Precision test

[0079] Take the powder of Carpesium abrotanoides L., prepare the test solution according to the method under item "3.2", inject samples continuously 6 times under the above chromatographic conditions, and calculate that the RSDs of the retention times of each target component are all < 2.43%, and the RSDs of the peak areas are all < 0.21%, indicating good precision of the instrument.

[0080] Table 3 Results of precision investigation

[0081]

[0082]

[0083] 4.4 Stability test

[0084] Take the Carpesium abrotanoides L. powder, prepare the test solution according to the method under item "3.2", inject for analysis under the above chromatographic conditions, inject and detect at 0, 2, 4, 8, 12, and 24 h respectively. Calculate that the RSDs of the retention times of each index component are all < 0.39%, and the RSDs of the peak areas are all < 1.45%, indicating that this method has good stability.

[0085] Table 4 Results of stability investigation

[0086]

[0087]

[0088] 4.5 Repeatability test

[0089] Take 6 batches of Carpesium abrotanoides L. powder, prepare the test solution according to the method under item "3.2", inject and detect at 0, 2, 4, 8, 12, and 24 h respectively under the above chromatographic conditions. Calculate that the RSDs of the retention times of each index component are all < 0.25%, and the RSDs of the contents are all < 2.75%, indicating that this method has good repeatability.

[0090] Table 5 Results of repeatability investigation

[0091]

[0092]

[0093] 4.6 Spike recovery test

[0094] Take 6 portions of Carpesium abrotanoides L. medicinal material powder (S17) with known component contents, about 0.5 g, weigh accurately, place in a conical flask, add each reference substance solution according to the component content ratio of about 1:1, prepare the test solution according to the method under item "3.2", inject for analysis under the chromatographic conditions of item "2.", and calculate the average spike recoveries of each component. The results show that the average spike recoveries of chlorogenic acid, caffeic acid, luteoloside, 11(13)-dehydroabrotanin, isochlorogenic acid A, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-glucaric acid, 2-deoxy-4-epi-helianthenin, carabrone, and telesterone are 98.71%, 102.26%, 101.47%, 98.12%, 99.43%, 94.65%, 95.48%, 103.06%, 97.57%, and 98.84% respectively, and the RSDs are all < 2.79%, indicating that this method has good accuracy.

[0095] Table 6 Spike recovery results

[0096]

[0097]

[0098]

[0099] 5. One - measure - multiple - evaluation study

[0100] 5.1 Calculation of relative correction factor (f)

[0101] Precisely pipette the mixed reference substance solutions with 5 different concentrations and inject them for determination under the above chromatographic conditions. Record the peak areas. Using isochlorogenic acid A as the internal standard substance, calculate the relative correction factors (f) of chlorogenic acid, caffeic acid, luteoloside, 11(13) - dehydroaxillarin, isochlorogenic acid C, 2,3,4,5 - tetracaffeoyl - D - glucaric acid, 2 - deoxy - 4 - epi - tagitinin, carabrone, and telekin i / s )

[0102] f i / s = f i / f s =(A i / W i ) / (A s / W s ) (1)

[0103] In the formula, W i is the amount of the internal standard substance, A i is the peak area of the internal standard substance, W s is the amount of the analyte, and A s is the peak area of the analyte. The results are shown in Table 7

[0104] Table 7 Relative correction factors of isochlorogenic acid A for each component

[0105]

[0106] 5.2 System suitability test

[0107] Precisely pipette the mixed reference substance solution and, under the above chromatographic conditions, investigate the effects of two different batch numbers of Waters H - Class ultra - high - performance liquid chromatographs, 4 chromatographic columns (specific models and batch numbers are shown in Table 8), flow rates (0.2, 0.3, 0.4 mL·min -1 )), and column temperatures (33, 35, 37 °C) on the relative correction factor. See Tables 8 - 9. The results show that the RSDs of the relative correction factors of each target component under different conditions are all < 3.34%, indicating that the established one - measure - multiple - evaluation method has good durability under different conditions

[0108] Table 8 Effects of different chromatographs and chromatographic columns on the relative correction factor

[0109]

[0110]

[0111] Note: Chromatographic column 1: Waters ACQUITY BEH C18

[0112] (2.1×100mm, 1.7μm, S / N: 03493930915175), Chromatographic column 2 (Waters ACQUITY BEH C18 (2.1×100mm, 1.7μm, S / N: 02473428215748), Chromatographic column 3 (Waters ACQUITY BEH C18 (2.1×100mm, 1.7μm, S / N: 03493930915184), Chromatographic column 4 (Waters CORTECS UPLC T3, 1.6μm, 2.1mm×100mm, S / N: 01153932315931)

[0113] Table 9 Effects of different flow rates and column temperatures on relative correction factors

[0114]

[0115] 5.3 Chromatographic peak localization of the components to be measured

[0116] The relative retention time, i.e., the ratio of the retention time of each component to be measured to that of isochlorogenic acid A, was used to localize the chromatographic peaks. The results are shown in the table. The RSD of the relative retention time of each component to be measured was < 3.05%, indicating that the relative retention time of each component to be measured had little fluctuation and no significant difference. The results are shown in Table 10

[0117] Table 10 Relative retention times of each component

[0118]

[0119] 5.4 Determination of the content of samples

[0120] The contents of the above 10 components in 17 batches of Carpesium abrotanoides samples were determined by the method of quantitative analysis of multi-components by single marker (QAMS) and the external standard method (ESM) respectively. The accuracy of the results of the two methods was evaluated by the relative average deviation

[0121]

[0122] Wherein, Xi is the result determined by the QAMS method is the average value of the results determined by the two methods

[0123] The calculation results are shown in Table 11, indicating that the application of the QAMS method in the evaluation of the contents of multiple index components in Carpesium abrotanoides is feasible

[0124] Table 11 Comparison of the contents determined by multiple components in one assay and external standard method (mg·g -1 , n = 3)

[0125]

[0126] Note: N / A is below the quantitation limit and quantitative analysis cannot be performed.

[0127] The method for Carpesium abrotanoides established in the present invention is different from the related technologies, including the chromatographic instrument, chromatographic column, mobile phase, gradient elution program, variable wavelength detection program, flow rate, column temperature, and injection volume used. The types of components of Carpesium abrotanoides established under these chromatographic conditions are also different from those of the related technologies. The identified components include 5 phenolic acids (chlorogenic acid, caffeic acid, isochlorogenic acid A, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-glucaric acid), 4 sesquiterpenoids (11(13)-dehydroaxillarin, 2-deoxy-4-epi-heliangolide, carabrone, telesterone), and 1 flavonoid (luteoloside). The ultra-high performance liquid chromatography (UPLC) chromatogram of the Carpesium abrotanoides sample obtained is more complete and beautiful while meeting the requirements of content determination, with a stable baseline and symmetric peak shapes of each component.

[0128] Comparative Example 1

[0129] This comparative example is a HPLC separation method for Carpesium abrotanoides. The specific liquid phase conditions are as follows:

[0130] The instrument is an Agilent high performance liquid chromatograph and a DAD detector; the chromatographic column is SunFire TM C 18 (250 mm × 4.6 mm, 5 μm); the mobile phase is water (A)-acetonitrile (B), with gradient elution (0 - 30 min, 5% - 95% B); the column temperature is 35°C; the wavelength is 230 nm; the injection volume is 10 μL; the flow rate is 1 mL / min.

[0131] The HPLC detection chromatogram of the test solution of Carpesium abrotanoides in this comparative example is shown in Figure 2 . It can be seen from the figure that HPLC takes a long time and requires a large amount of mobile phase and sample. Subsequently, it was changed to UPLC for separation.

[0132] Comparative Example 2

[0133] This comparative example is a UPLC separation method for Carpesium abrotanoides. The specific liquid phase conditions are as follows:

[0134] The instrument is a Waters H-Class ultra-high performance liquid chromatograph and a PDA detector; the chromatographic column is Waters ACQUITY BEH C 18(2.1×100 mm, 1.7 μm); The mobile phase was 0.1% formic acid in water (A) - acetonitrile (B), with gradient elution (0 - 2 min, 5% - 22% B; 2 - 25 min, 22% - 65% B; 25 - 30 min, 65% - 5% B); The column temperature was 30 °C; The wavelength was 230 nm; The injection volume was 1 μL; The flow rate was 0.3 mL / min.

[0135] The UPLC chromatogram of the Carpesium abrotanoides L. test solution in this comparative example is shown in Figure 3 , from Figure 3 it can be seen that compared with Example 2, this method has a shorter separation time and faster speed, and basically all the peaks elute completely within 20 minutes, with a large number of peaks. However, this method uses formic acid in water, resulting in an uneven baseline and less than ideal peak separation effect. The liquid phase conditions still need to be optimized.

[0136] Comparative Example 3

[0137] This comparative example is a Carpesium abrotanoides L. UPLC separation method, with the specific gradient as follows:

[0138] The instrument was a Waters H-Class ultra-high performance liquid chromatograph and a PDA detector; The chromatographic column was a Waters ACQUITY BEH C 18 (2.1×100 mm, 1.7 μm); The mobile phase was 0.1% phosphoric acid in water (A) - acetonitrile (B), with gradient elution (0 - 2 min, 10% - 10% B; 2 - 5 min, 10% - 14.5% B; 5 - 10 min, 14.5% - 18% B; 10 - 15 min, 18% - 22% B; 15 - 30 min, 22% - 50% B); The column temperature was 30 °C; The wavelength was 230 nm; The injection volume was 0.5 μL; The flow rate was 0.3 mL / min.

[0139] The UPLC chromatogram of the Carpesium abrotanoides L. test solution in this comparative example is shown in Figure 4 , from Figure 4 it can be seen that when this method was changed to phosphoric acid in water, the baseline was more stable than that of formic acid in water, and most of the components had been separated, but some components still needed further exploration for separation.

[0140] Comparative Example 4

[0141] This comparative example was an investigation of the extraction solvent for the Carpesium abrotanoides L. test solution, consisting of the following steps:

[0142] 1) Preparation of the test solution

[0143] Take 0.5 g of Carpesium abrotanoides powder (passed through a 60-mesh sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 20 mL of the corresponding extraction solvent (water, 30% methanol / ethanol, 50% methanol / ethanol, 70% methanol / ethanol, 80% methanol / ethanol, methanol / ethanol), weigh it, ultrasonically extract for 60 min, let it cool and then weigh it again, make up the weight with the corresponding extraction solvent, shake well, filter through a 0.22-μm microporous membrane to obtain the test solution.

[0144] 2) Liquid phase conditions

[0145] The chromatographic column is Waters ACQUITY BEH C 18 (2.1×100 mm, 1.7 μm); the mobile phase is 0.1% phosphoric acid water (A) - acetonitrile (B), gradient elution (0 - 30 min, 10% - 65% B); the column temperature is 30 °C; the wavelength is 230 nm; the injection volume is 1 μL; the flow rate is 0.3 mL / min.

[0146] The ultra-high performance liquid chromatography detection chart of the Carpesium abrotanoides test solution in this comparative example is shown in Figure 5 , and it can be known from Figure 5 that when the extraction solvent is water, 30% methanol / ethanol, and methanol / ethanol, the components are few or the peak areas are small, and there is no significant difference in the number and peak areas of the components of 50 - 80% methanol / ethanol.

[0147] Comparative Example 5

[0148] This comparative example is for the investigation of the wavelength of the Carpesium abrotanoides UPLC chromatographic method, and it consists of the following steps:

[0149] 1) Preparation of the test solution

[0150] Take 0.5 g of Carpesium abrotanoides powder (passed through a 60-mesh sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 20 mL of 80% ethanol, weigh it, ultrasonically extract for 60 min, let it cool and then weigh it again, make up the weight with 80% ethanol, shake well, filter through a 0.22-μm microporous membrane to obtain the test solution.

[0151] 2) Liquid phase conditions

[0152] The chromatographic column is Waters ACQUITY BEH C 18(2.1×100 mm, 1.7 μm); The mobile phase was 0.1% phosphoric acid (A) - acetonitrile (B), with gradient elution (0 - 2 min, 10% B; 2 - 5 min, 10% - 14.5% B; 5 - 15 min, 14.5% - 22% B; 15 - 17 min, 22% - 26% B; 17 - 25 min, 26% - 28% B; 25 - 30 min, 28% - 37% B); The column temperature was 30 °C; The wavelength was 230 nm; The injection volume was 0.5 μL; The flow rate was 0.3 mL / min.

[0153] The ultra - performance liquid chromatography detection graphs of the Carpesium abrotanoides L. test solution at different wavelengths in this comparative example are shown in Figure 6 , and it can be seen from Figure 6 that among the 19 characteristic peaks, peaks 9, 15, 16, and 17 eluted at 210 - 230 nm, and the remaining components eluted at 201 - 380 nm.

[0154] In summary, in the present invention, isochlorogenic acid A, which is relatively moderate in price and easily available, was used as the internal standard substance. Based on the established UPLC conditions, a method for simultaneous determination of the contents of 10 components in Carpesium abrotanoides L. was established by multi - component quantification with a single internal standard. This method well solves the problems of requiring a large number of reference substances, being difficult to obtain, and being expensive in the content determination of Carpesium abrotanoides L., provides a reference for the quantitative evaluation research of Carpesium abrotanoides L. components, saves detection costs and time, and has good linear relationships, high precision, good repeatability, and good stability.

[0155] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for determining the content of Tianmingjing ingredients, characterized in that: The following steps are involved: S1. Prepare the Tianmingjing test solution and mixed reference solution; S2. Using ultra-high performance liquid chromatography to obtain the fingerprint of Tianmingjing, wherein the ultra-high performance liquid chromatography includes mobile phase A, phosphoric acid aqueous solution and mobile phase B, acetonitrile solution; S3. Determination of relative correction factor: accurately aspirate the mixed reference solution, adopt the ultra-high performance liquid chromatography method to measure, record the peak area, take one of the components of Tianmingjing as the internal standard, and calculate the relative correction factor of other components of Tianmingjing to be measured relative to the internal standard: f i / s =f i / f s =(A i / W i ) / (A s / W s ) Where f i / s is the relative correction factor, f i is the internal standard correction factor, f s is the correction factor of the component to be measured, A i is the peak area of ​​the internal standard, W i is the amount of internal standard, A s is the peak area of ​​the component to be measured, W s is the amount of the component to be measured; S4. Relative retention time determination: Determine the relative retention time, i.e., the ratio of the retention time of each component to be measured to the retention time of the internal standard, and locate the chromatographic peak; S5. Content determination: Calculate the content of the components to be tested respectively: W x =f i / s ×W i ×A x / A i Where W x is the content of the component to be tested in the test solution, W i is the content of internal standard, A x A is the peak area of ​​the component to be tested in the test solution. i is the peak area of ​​the internal standard.

2. The method for determining the content of Tianmingjing ingredients according to claim 1, characterized in that: In S3, isochlorogenic acid A was used as the internal standard to calculate the relative correction factors of chlorogenic acid, caffeic acid, olivine, 11(13)-dehydro-axillin, isochlorogenic acid C, 2,3,4,5-tetra-caffeoyl-D-glucaric acid, 2-deoxy-4-epi-gavariolin, gavarinolide, and telolactone.

3. The method for determining the content of Tianmingjing ingredients according to claim 2, characterized in that: In S3, the relative correction factors of chlorogenic acid, caffeic acid, galanthus glycoside, 11(13)dehydroaxylvacetin, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-glucaric acid, 2-deoxy-4-epi-galanthus, galanthus lactone, and telolactone were 1.31-1.34, 0.11-0.81, 2.52-2.59, 3.54-3.71, 0.99-1.03, 0.95-1.01, 2.82-3.03, 1.91-2.05, and 3.00-3.21, respectively.

4. The method for determining the content of Tianmingjing ingredients according to claim 2, characterized in that: In S4, the relative retention times of chlorogenic acid, caffeic acid, galanthus glycoside, 11(13)dehydroaxylvacetin, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-glucaric acid, 2-deoxy-4-epi-galanthus, galanthus lactone, and telolactone were 0.22-0.23, 0.29-0.31, 0.82-0.83, 0.94-0.98, 1.16-1.19, 1.70-1.73, 1.99-2.14, 2.07-2.19, and 2.20-2.37, respectively.

5. The method for determining the content of Tianmingjing components according to any one of claims 1 to 4, characterized in that: The detection conditions of the ultra-high performance liquid chromatography method include: the chromatographic column is an octadecylsilane bonded silica gel chromatographic column, the mobile phase flow rate is 0.2-0.4mL / min, the detection wavelength is 210-230nm, the column temperature is 33-37°C, and the injection volume is 0.5-1μL.

6. The method for determining the content of Tianmingjing components according to any one of claims 1 to 4, characterized in that: The ultra-high performance liquid chromatography method adopts gradient elution for elution; preferably, the procedure of the gradient elution includes: 0-2min, the volume fraction of the mobile phase B is 10%; 2-5 min, the volume fraction of the mobile phase B increases from 10% to 14.5%; 5-15 min, the volume fraction of the mobile phase B increases from 14.5% to 22%; 15-17 min, the volume fraction of the mobile phase B increased from 22% to 26%; 17-25 min, the volume fraction of the mobile phase B increased from 26% to 28%; From 25 to 30 minutes, the volume fraction of the mobile phase B increases from 28% to 37%.

7. The method for determining the content of Tianmingjing components according to any one of claims 1 to 4, characterized in that: The volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05-0.4%; preferably, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05-0.2%.

8. The method for determining the content of Tianmingjing components according to any one of claims 1 to 4, characterized in that: The method for preparing the Tianmingjing test solution comprises the following steps: extracting the components in Tianmingjing with an extractant, wherein the extractant comprises 50%-80% methanol or 50%-80% ethanol, and preferably, the extractant comprises 70%-80% methanol or 70%-80% ethanol.

9. The method for determining the content of Tianmingjing ingredients according to claim 8, characterized in that: The solid-liquid ratio of the Tianmingjing to the extractant is 0.3-0.6 g:15-25 mL; preferably, the solid-liquid ratio of the Tianmingjing to the extractant is 0.45-0.55 g:19.5-20.5 mL.

10. The method for determining the content of Tianmingjing ingredients according to claim 8, characterized in that: The extraction adopts ultrasonic extraction; the power of the ultrasonic extraction is 190-210W, the frequency is 35-45kHz, and the time is 50-70min.

Images