A method for determining the content of Tianmingjing components

Abstract

The application belongs to the field of traditional Chinese medicine quality analysis and control, and discloses a determination method of ingredients content of scabiosa comosa, which takes isochlorogenic acid A as an internal reference, calculates the relative correction factors of chlorogenic acid, caffeic acid, jinyingzhi, 11(13)-dehydroxy axillary ivy chrysanthemum, isochlorogenic acid C, 2,3,4,5-tetra caffeoyl-D-glucaric acid, 2-deoxy-4-tablet-Tianrenjuling, scabioside lactone ketone and terlene lactone, quickly and accurately detects the content of 10 kinds of ingredients of scabiosa comosa, and solves the problems of many required control products, difficulty in obtaining and high price in the determination of scabiosa comosa content, and high detection cost.

Description

Technical Field

[0001] This invention relates to the field of quality analysis and control of traditional Chinese medicine, specifically to a method for determining the content of Tianmingjing components. Background Technology

[0002] Carpesium abrotanoides L., a plant in the Asteraceae family, is a commonly used traditional Chinese medicine in Hunan Province. It is the dried whole herb and has the effects of clearing heat and detoxifying, resolving phlegm and stopping bleeding. Clinically, it is mainly used to treat toothache, oral erosion, herpes zoster, bacterial infections, pruritus, folliculitis, hepatitis, and epidemic acute viral conjunctivitis. The inclusion of Tianmingjing in the standards of various provinces demonstrates its important role in traditional medicine practice. These include standards such as the 2021 edition of the "Hunan Province Standard for Processing Traditional Chinese Medicine Pieces," the 2020 edition of the "Jiangsu Province Standard for Processing Traditional Chinese Medicine Pieces," the 2018 edition of the "Hubei Province Standard for Quality of Traditional Chinese Medicine Materials," the 2015 edition of the "Zhejiang Province Standard for Processing Traditional Chinese Medicine Pieces," the 2003 edition of the "Hebei Province Standard for Processing Traditional Chinese Medicine Pieces," and the 2018 edition of the "Shanghai Municipal Standard for Processing Traditional Chinese Medicine Pieces." However, it has not yet been included in the "Chinese Pharmacopoeia," and the standards already included have not yet established methods for determining its indicator components. This invention can promote the improvement of Tianmingjing standards and ensure its efficacy in clinical practice and pharmaceutical formulations. Therefore, this invention can promote the application of Tianmingjing in clinical practice and pharmaceutical formulations. This medicinal material is rich in sesquiterpenes, sesquiterpene dimers, flavonoids, and nitrogen-containing compounds. These components have been proven to have anti-inflammatory, antibacterial, antiparasitic, antitumor, and antiviral pharmacological activities. This invention also found that *Tianmingjing*, as a plant of the Asteraceae family, contains abundant phenolic acid components. These components have anti-inflammatory, antioxidant, antitumor, and antibacterial effects and can be used as an important indicator for measuring the quality of *Tianmingjing*. Summary of the Invention

[0003] Based on the problems raised above, the technical problem to be solved by the present invention is to provide a method for determining the content of multiple components in Tianmingjing, which can accurately measure the content of multiple components in Tianmingjing in one step.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] A method for determining the content of Tianmingjing components includes the following steps:

[0006] S1. Prepare the Tianmingjing test solution and the mixed reference solution;

[0007] S2. The fingerprint spectrum of Tianming was obtained by ultra-high performance liquid chromatography, wherein the ultra-high performance liquid chromatography method includes mobile phase A, phosphoric acid aqueous solution and mobile phase B, acetonitrile solution;

[0008] S3. Determination of Relative Correction Factor: Accurately pipette the mixed reference solution and inject it using the ultra-high performance liquid chromatography method. Record the peak area. Using one of the components of Tianmingjing as an internal standard, calculate the relative correction factor of the other analytes of Tianmingjing 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 f is the relative correction factor. i f is the internal standard correction factor. s A is the correction factor for the analyte. i W represents the peak area of ​​the internal standard. i For the internal standard quantity, A s W represents the peak area of ​​the component to be measured. s The amount of the component to be measured;

[0010] S4. Relative retention time determination: The relative retention time is the ratio of the retention time of each analyte to that of the internal standard, and the chromatographic peaks are located by measuring the relative retention time.

[0011] S5. Content determination: Calculate the content of the analyte separately.

[0012] W x =f i / s ×W i ×A x / A i

[0013] In the formula W x To determine the content of the analyte in the test solution, W i For the content of the internal standard, A x A represents the peak area of ​​the analyte in the test solution. i This represents the peak area of ​​the internal standard.

[0014] The one-test-multiple-evaluation method of the present invention has good durability under different conditions and is feasible.

[0015] The method of the present invention has good specificity, good linearity, good precision, good stability, good repeatability, and good accuracy.

[0016] According to embodiments of the present invention, the present invention can be further optimized, and the optimized technical solution is as follows:

[0017] In S3, using isochlorogenic acid A as an internal standard, the relative correction factors for chlorogenic acid, caffeic acid, luteolin, 11(13)-dehydroaxillary isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-gluconic acid, 2-deoxy-4-epio-terbinafine, terbinafine lactone, and terbinafine lactone were calculated.

[0018] In one preferred embodiment, the relative correction factors for chlorogenic acid, caffeic acid, luteolin, 11(13)-dehydroaxillary iwadin, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-gluconic acid, 2-deoxy-4-epiota-glucanol, terbinafine lactone, and telenolide in S3 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, the relative retention times of chlorogenic acid, caffeic acid, luteolin, 11(13)-dehydroaxillary ivanzidin, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-gluconic acid, 2-deoxy-4-epiota-glucan, terbinafine lactone, and telenolide in S4 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 include: an octadecylsilane-bonded silica column, a mobile phase flow rate of 0.2-0.4 mL / min, a detection wavelength of 210-230 nm, a column temperature of 33-37 °C, and an injection volume of 0.5-1 μL.

[0021] The ultra-high performance liquid chromatogram of the Tianmingjing sample obtained by this invention is more complete and aesthetically pleasing while meeting the requirements for content determination, with a stable baseline and symmetrical peaks for each component.

[0022] In one preferred embodiment, the ultra-high performance liquid chromatography employs gradient elution; preferably, the gradient elution procedure includes:

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

[0024] Over 2–5 minutes, the volume fraction of the mobile phase B increased from 10% to 14.5%.

[0025] Over 5–15 minutes, the volume fraction of the mobile phase B increased from 14.5% to 22%.

[0026] Within 15–17 minutes, the volume fraction of the mobile phase B increased from 22% to 26%.

[0027] Within 17–25 minutes, the volume fraction of the mobile phase B increased from 26% to 28%.

[0028] Over 25–30 minutes, the volume fraction of the mobile phase B 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%. More 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 Tianmingjing test solution includes the following steps: extracting the components in Tianmingjing using an extractant, wherein the extractant includes 50%-80% methanol or 50%-80% ethanol, preferably, the extractant includes 70%-80% methanol or 70%-80% ethanol.

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

[0032] In one preferred embodiment, the extraction is performed using ultrasound; the ultrasound extraction power is 190–210 W, the frequency is 35–45 kHz, and the time is 50–70 min.

[0033] Preferably, in S1, the mixed reference solution includes chlorogenic acid, caffeic acid, luteolin, 11(13)-dehydroaxillary iwazidin, isochlorogenic acid A, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-gluconic acid, 2-deoxy-4-epiota-glucan, gentamicin lactone, and telenolide; preferably, chlorogenic acid, caffeic acid, luteolin, and 11(13)-dehydroaxillary iwazidin are included. The mass concentration ratios of isochlorogenic acid A, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-gluconic acid, 2-deoxy-4-epiota-caryophyllene, gentamicin lactone, and telendrolactone are 150-160, 10-20, 140-150, 90-100, 440-450, 10-20, 70-80, 170-180, 60-70, and 60-70 μg·mL, respectively. -1 .

[0034] This invention provides a rapid and accurate method for detecting the content of 10 components of Tianmingjing, solving the problems of high detection costs caused by the large number, difficulty in obtaining, and high price of reference standards required for the determination of Tianmingjing content.

[0035] The beneficial effects of this invention are:

[0036] This invention establishes for the first time an accurate and low-cost method for quality control and evaluation of Tianmingjing. It simultaneously determines the contents of 10 components, namely chlorogenic acid, caffeic acid, luteolin, 11(13)-dehydroaxillary isochlorogenic acid A, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-gluconic acid, 2-deoxy-4-epio-tianrenjulin, Tianmingjing lactone, and telerolactone, using a single-test-multiple-evaluation method. That is, only isochlorogenic acid A is needed as a reference standard, and the contents of the other 9 components can be calculated by relative correction factors.

[0037] The ultra-high performance liquid chromatography (UHPLC) conditions established in this invention have good specificity and good baseline separation for each component, ensuring accurate and reliable peak localization in multi-evaluation assays.

[0038] This invention fills the gap in the existing standards for the determination of the content of physical indicators or active ingredients in Tianmingjing, and provides a scientific basis for solving the quality control problem of Tianmingjing. Attached Figure Description

[0039] Figure 1 The graphs are for blank (A), test sample (B), and mixed control (C).

[0040] Figure 2 The image shows the high performance liquid chromatogram of Tianmingjing in Comparative Example 1.

[0041] Figure 3 The image shows the ultra-high performance liquid chromatogram of Tianmingjing in Comparative Example 2.

[0042] Figure 4 The image shows the ultra-high performance liquid chromatogram of Tianmingjing in Comparative Example 3.

[0043] Figure 5 The image shows the ultra-high performance liquid chromatograms of different extraction solvents for Tianmingjing in Comparative Example 4.

[0044] Figure 6 The image shows ultra-high performance liquid chromatograms of Tianmingjing at different wavelengths in Comparative Example 5. Detailed Implementation

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

[0046] This invention employs UPLC with isochlorogenic acid A as an internal reference, and calculates the relative correction factors for chlorogenic acid, caffeic acid, luteolin, 11(13)-dehydroaxillary isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-gluconic acid, 2-deoxy-4-epiota-glucan, gentamicin lactone, and telenolide. The process includes the following steps:

[0047] 1) Take the test solution and perform detection by liquid chromatography;

[0048] The ingredients include chlorogenic acid (CAS No.: 327-97-9), caffeic acid (CAS No.: 331-39-5), luteolin (CAS No.: 5373-11-5), 11(13)-dehydroaxillary ivyrin (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-gluconic acid (CAS No.: 1419478-52-6), 2-deoxy-4-epiota-galendrin (CAS No.: 122872-03-1), gentamicin lactone (CAS No.: 1748-81-8), and telene lactone (CAS No.: 6752-90-5);

[0049] 2) Using isochlorogenic acid A as an internal reference, the contents of 10 components of Tianmingjing were rapidly and accurately determined by calculating the relative correction factors of chlorogenic acid, caffeic acid, luteolin, 11(13)-dehydroaxillary iridoside, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-gluconic acid, 2-deoxy-4-epio-tianrenjulin, Tianmingjing lactone, and Tellerone.

[0050] 3) Relative correction factor: Accurately pipette the mixed reference solution, inject it for analysis, 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 For the internal standard quantity, A i W represents the peak area of ​​the internal standard. s For the amount of the component to be measured, A s The peak area of ​​the component to be measured;

[0053] 4) Relative retention time determination: The chromatographic peaks are located by using the relative retention time, which is the ratio of the retention time of each analyte to that of isochlorogenic acid A.

[0054] 5) Content determination: Take the test solution and the mixed reference solution, perform chromatographic analysis, and calculate the contents of orthocyanin, caffeic acid, luteolin, 11(13)-dehydroaxillary chlorophyll, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-gluconic acid, 2-deoxy-4-epio-terbinafine, terbinafine lactone, and terbinafine lactone according to the determined relative correction factor.

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

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

[0057] Example 1

[0058] 1. Sample Source

[0059] The specific information of the 17 batches of Tianmingjing medicinal materials is shown in Table 1. Among them, batches S4, 5, 9, 10, 11 and 12 are fresh medicinal materials. They were processed according to the 2021 edition of the "Hunan Province Standard for Processing Traditional Chinese Medicine Pieces", which involved removing impurities, washing, moistening, cutting and drying. They were identified by Associate Professor Wang Zhi of Hunan University of Traditional Chinese Medicine as the dried whole herb of Carpesium abrotanoides Linnaeus, a plant of the Asteraceae family.

[0060] Table 1 Information on Famous Medicinal Herbs

[0061]

[0062] 2. Chromatographic conditions

[0063] Waters ACQUITY BEH C 18The chromatographic column (2.1 × 100 mm, 1.7 μm) was used with a mobile phase of 0.1% phosphoric acid (A)-acetonitrile (B) and 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 35 °C and the flow rate was 0.3 mL / min. -1 The detection wavelength was 230 nm, and the injection volume was 0.5 μL.

[0064] 3. Solution preparation

[0065] 3.1 Preparation of reference solution

[0066] Accurately weigh appropriate amounts of chlorogenic acid, caffeic acid, luteolin, 11(13)-dehydro-adenosylpyridin, isochlorogenic acid A, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-gluconic acid, 2-deoxy-4-epiota-caryophyllene, terbinafine lactone, and telendrolactone reference standards, and dissolve them in 80% ethanol to prepare single reference standard stock solutions of a certain mass concentration. Take appropriate amounts of the above single reference standard stock solutions and place them in the same 10mL volumetric flask, add 80% ethanol to the mark, shake well, and obtain mass concentrations of 158.88, 19.86, 149.76, 95.70, 445.20, 19.96, 72.94, 173.50, 63.84, and 67.50 μg·mL, respectively. -1 A mixed reference standard stock solution.

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

[0068] 3.2 Preparation of the test solution

[0069] Accurately weigh 0.5g of Tianmingjing powder and place it in a stoppered conical flask. Accurately add 20mL of 80% ethanol, weigh it, and extract it by ultrasonication for 60min (power 200W, 40kHz). After cooling, weigh it again and make up the weight with 80% ethanol. Shake well and filter it through a 0.22μm organic microporous membrane to obtain the test solution.

[0070] 4. Methodological investigation of content determination

[0071] 4.1 Specificity Examination

[0072] Take blank solution, mixed reference solution, and test solution respectively, and inject them for analysis according to the above chromatographic conditions. The results are as follows: Figure 1 As shown in the figure. The results show that the retention times of the chromatographic peaks of the test solution are consistent with those of the mixed reference standard, and no chromatographic peaks appear at the corresponding positions in the blank control chromatogram, indicating that the method has good specificity.

[0073] 4.2 Examination of Linear Relationships

[0074] Accurately transfer 0.25, 0.50, 1.00, 2.00, and 5.00 mL of the mixed reference standard stock solution from section "3.1", and dilute to 5 mL with 80% ethanol to prepare five aliquots of mixed reference standard solutions of different concentrations. Inject the solutions sequentially under the chromatographic conditions described above, and record the peak areas of the chromatograms. Plot the peak area of ​​the corresponding component on the ordinate, and the reference standard concentration (μg·mL⁻¹) on the ordinate. -1 Using α as the x-axis, a standard curve was plotted to obtain the regression equations and linear ranges for 10 components: chlorogenic acid (peak 1), caffeic acid (peak 2), luteolin (peak 7), 11(13)-dehydro-axillary ivanzidin (peak 9), isochlorogenic acid A (peak 10), isochlorogenic acid C (peak 12), 2,3,4,5-tetracaffeoyl-D-gluconic acid (peak 13), 2-deoxy-4-epio-terbinafine (peak 15), terbinafine lactone (peak 16), and terbinafine lactone (peak 17). The limits of detection (LOD) and quantitation (LOQ) for each component were calculated at concentrations with 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 a good linear relationship.

[0075] Table 2. Linear Relationship of 10 Components in Tianming Essence

[0076]

[0077]

[0078] 4.3 Precision Test

[0079] Take Tianmingjing powder and prepare the test solution according to the method in section "3.2". Inject the sample 6 times continuously under the above chromatographic conditions. The retention time RSD of each index component is calculated to be <2.43% and the peak area RSD is <0.21%, indicating that the instrument has good precision.

[0080] Table 3. Precision test results

[0081]

[0082]

[0083] 4.4 Stability Test

[0084] Take Tianmingjing powder and prepare the test solution according to the method in section "3.2". Inject and analyze under the above chromatographic conditions. Inject and detect at 0, 2, 4, 8, 12 and 24 h respectively. The RSD of the retention time of each index component is all <0.39% and the RSD of the peak area is all <1.45%, indicating that the method has good stability.

[0085] Table 4. Stability test results

[0086]

[0087]

[0088] 4.5 Repeatability Test

[0089] Six batches of Tianmingjing powder were taken, and test solutions were prepared according to the method in section "3.2". The samples were injected and detected at 0, 2, 4, 8, 12 and 24 h under the above chromatographic conditions. The retention time RSD of each index component was calculated to be <0.25% and the content RSD was <2.75%, indicating that the method has good repeatability.

[0090] Table 5 Results of Repeatability Testing

[0091]

[0092]

[0093] 4.6 Spiking Recovery Test

[0094] Take 6 portions of Tianmingjing medicinal powder (S17) with known component content, about 0.5 g each, accurately weigh them, place them in conical flasks, add each reference solution at a component content ratio of about 1:1, prepare the test solution according to the method under "3.2", inject and analyze under the chromatographic conditions under "2.", and calculate the average recovery rate of each component. The results showed that the average recoveries of chlorogenic acid, caffeic acid, luteolin, 11(13)-dehydroaxillary isochlorogenic acid A, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-gluconic acid, 2-deoxy-4-epiota-glucan, terbinafine lactone, and terbinafine lactone were 98.71%, 102.26%, 101.47%, 98.12%, 99.43%, 94.65%, 95.48%, 103.06%, 97.57%, and 98.84%, respectively, with RSDs all < 2.79%, indicating that the method has good accuracy.

[0095] Table 6. Results of recovery after spiking

[0096]

[0097]

[0098]

[0099] 5. Research on One Test with Multiple Assessments

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

[0101] Five mixed reference solutions of different concentrations were precisely pipetted and injected under the above chromatographic conditions. The peak areas were recorded. Using isochlorogenic acid A as an internal standard, the relative correction factors (f) of chlorogenic acid, caffeic acid, luteolin, 11(13)-dehydroadenosylpyridin, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-gluconic acid, 2-deoxy-4-epiota-caryophyllene, terbinafine lactone, and telenolide were calculated according to formula (1). 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 For the internal standard quantity, A i W represents the peak area of ​​the internal standard. s Let A be the amount of the substance to be measured. s The peak area is the sample to be measured. 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] Accurately pipette the mixed reference solution and, under the chromatographic conditions described above, test two Waters H-Class ultra-high performance liquid chromatographs of different batches, four columns (specific models and batch numbers are shown in Table 8), and flow rates (0.2, 0.3, 0.4 mL·min). -1 The effects of column temperature (33, 35, 37℃) on the relative correction factor are shown in Tables 8-9. The results show that the relative correction factor RSD for each index component under different conditions is <3.34%, indicating that the established one-measure-multiple-evaluation method has good robustness under different conditions.

[0108] Table 8. Effects of different chromatograms and columns on relative correction factors.

[0109]

[0110]

[0111] Note: Column 1: Waters ACQUITY BEH C18

[0112] (2.1×100mm, 1.7μm, S / N: 03493930915175), Column 2 (Waters ACQUITY) BEH C18 (2.1×100mm, 1.7μm, S / N: 02473428215748), Column 3 (Waters ACQUITY) BEH C18 (2.1×100mm, 1.7μm, S / N: 03493930915184), 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 the relative correction factor.

[0114]

[0115] 5.3 Peak localization of the analyte

[0116] The chromatographic peaks were located using the relative retention time, which is the ratio of the retention time of each analyte to that of isochlorogenic acid A. The results are shown in Table 10. The RSD of the relative retention time of each analyte was <3.05%, indicating that the relative retention time of each analyte fluctuated little and had no significant difference.

[0117] Table 10 Relative retention times of each component

[0118]

[0119] 5.4 Determination of sample content

[0120] The contents of the above 10 components in 17 batches of Tianmingjing samples were determined by the Quality Assay Multiple Evaluation (QAMS) method 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] In the formula, Xi represents the result determined by the QAMS method; This represents the average value of the results obtained using the two methods.

[0123] The calculation results are shown in Table 11. The results indicate that the one-measurement-multiple-evaluation method is feasible for evaluating the content of multiple index components in Tianmingjing.

[0124] Table 11 Comparison of content using the single-test-multiple-evaluation method and the external standard method (mg·g) -1 (n=3)

[0125]

[0126] Note: If the N / A ratio is below the limit of quantitation, quantitative analysis cannot be performed.

[0127] The method for Tianmingjing established in this invention differs from related technologies in that it uses different chromatographic instruments, columns, mobile phases, gradient elution programs, wavelength detection programs, flow rates, column temperatures, and injection volumes. The types of Tianmingjing components established using these chromatographic conditions also differ from related technologies. The identified components include five phenolic acids (chlorogenic acid, caffeic acid, isochlorogenic acid A, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-gluconic acid), four sesquiterpenes (11(13)-dehydro-adenosylpyridin, 2-deoxy-4-epio-tianrenjulin, Tianmingjing lactone, and telerone), and one flavonoid (luteolin). The resulting ultra-high performance liquid chromatogram of the Tianmingjing sample is more complete and aesthetically pleasing while meeting the requirements for content determination, with a stable baseline and symmetrical peaks for each component.

[0128] Comparative Example 1

[0129] This comparative example demonstrates an HPLC separation method for Tianmingjing, with the specific HPLC conditions as follows:

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

[0131] The high-performance liquid chromatography (HPLC) chromatogram of the Tianmingjing test solution in this comparative example is shown below. Figure 2 As shown in the figure, HPLC is time-consuming and requires a large amount of mobile phase and sample. Therefore, UPLC was subsequently used for separation.

[0132] Comparative Example 2

[0133] This comparative example demonstrates a Tianmingjing UPLC separation method, with the specific liquid phase conditions as follows:

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

[0135] The UPLC chromatogram of the Tianmingjing test solution in this comparative example is shown below. Figure 3 ,Depend on Figure 3 It was learned that this method has a shorter separation time and faster speed than Example 2, with peaks basically completely eluted within 20 minutes and a large number of peaks. However, the use of formic acid water in this method causes uneven baseline, and the peak separation effect is not ideal. The liquid phase conditions need to be optimized.

[0136] Comparative Example 3

[0137] This comparative example demonstrates a Tianming-based UPLC separation method, with the following specific gradient:

[0138] The instrument used was a Waters H-Class ultra-high performance liquid chromatograph with a PDA detector; the chromatographic column was a Waters ACQUITY. BEH C 18 (2.1×100mm, 1.7μm); mobile phase: 0.1% phosphoric acid (A)-acetonitrile (B), 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); column temperature: 30℃; wavelength: 230nm; injection volume: 0.5μL; flow rate: 0.3mL / min.

[0139] The UPLC chromatogram of the Tianmingjing test solution in this comparative example is shown below. Figure 4 ,Depend on Figure 4 It was learned that when the method was changed to phosphoric acid water, the baseline was more stable than that of formic acid water, and most components had been separated, but some components still needed to be separated further.

[0140] Comparative Example 4

[0141] This comparative example, which investigates the extraction solvent of the Tianmingjing test sample, consists of the following steps:

[0142] 1) Preparation of the test solution

[0143] Accurately weigh 0.5 g of Tianmingjing powder (passed through a 60-mesh sieve) and 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 the solution, and extract with ultrasound for 60 min. After cooling, weigh the solution again and make up the weight with the corresponding extraction solvent. Shake well and filter through a 0.22 μm microporous membrane to obtain the test solution.

[0144] 2) Liquid phase conditions

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

[0146] The ultra-high performance liquid chromatography (UHPLC) chromatogram of the Tianmingjing test solution in this comparative example is shown below. Figure 5 ,Depend on Figure 5 It was found that when the extraction solvent was water, 30% methanol / ethanol, or methanol / ethanol, the number of components was small or the peak area was small. When the extraction solvent was 50-80% methanol / ethanol, there was no significant difference in the number of components and the peak area.

[0147] Comparative Example 5

[0148] This comparative example, which investigates the wavelength of the UPLC chromatographic method of Tianming Precision, consists of the following steps:

[0149] 1) Preparation of the test solution

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

[0151] 2) Liquid phase conditions

[0152] The chromatographic column was Waters ACQUITY. BEH C 18(2.1×100mm, 1.7μm); 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: 30℃; wavelength: 230nm; injection volume: 0.5μL; flow rate: 0.3mL / min.

[0153] The ultra-high performance liquid chromatography (UHPLC) chromatograms of the Tianmingjing test solution at different wavelengths in this comparative example are shown below. Figure 6 ,Depend on Figure 6 It was found that among the 19 characteristic peaks, peaks 9, 15, 16, and 17 ellipse at 210-230 nm, while the remaining components ellipse at 201-380 nm.

[0154] In summary, this invention uses isochlorogenic acid A, which is relatively inexpensive and readily available, as an internal standard. Based on the established UPLC conditions, a method for simultaneously determining the content of 10 components in gentian violet extract was developed. This method effectively solves the problems of the large number of reference standards required for the determination of gentian violet extract content, which are difficult to obtain and expensive. It provides a reference for the quantitative evaluation of gentian violet extract components, saves detection costs and time, and demonstrates good linearity, high precision, good repeatability, and good stability.

[0155] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for determining the content of a lobelia ingredient, characterized by, The method comprises the following steps: S1, preparing a test sample solution of swertiae and a mixed control sample solution; S2, detecting a fingerprint of swertiae by using an ultra-high performance liquid chromatography, wherein the ultra-high performance liquid chromatography comprises a mobile phase A of phosphoric acid water solution and a mobile phase B of acetonitrile solution; S3, relative correction factor determination: precisely taking the mixed control sample solution, determining by using the ultra-high performance liquid chromatography, recording peak area, taking one component of swertiae as an internal standard, and calculating relative correction factors of other components of swertiae relative to the internal standard; wherein f i / s is a relative correction factor, f i is an internal standard correction factor, f s is a test component correction factor, A i is an internal standard peak area, W i is an internal standard amount, A s is a test component peak area, W s is a test component amount; S4, relative retention time determination: determining relative retention time, i.e. the ratio of the retention time of each component to be detected to that of the internal standard, and positioning the chromatographic peak; S5, content determination: calculating the content of each component to be detected; wherein 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; In S3, taking isochlorogenic acid A as the internal standard, and calculating the relative correction factors of chlorogenic acid, caffeic acid, luteolin-7-O-β-D-glucuronide, 11(13)-dehydroxy-vasikirarin, isochlorogenic acid C, 2,3,4,5-tetra-O-caffeoyl-D-glucaric acid, 2-deoxy-4-epi-tanacetone, swertipicrin, and terulactone; The detection conditions of the ultra-high performance liquid chromatography include: the chromatographic column is Waters ACQUITY UPLC ® BEH C 18 , the specification of the chromatographic column is 2.1 x 100 mm, 1.7 μm, and the detection wavelength is 210-230 nm; The ultra-high performance liquid chromatography is eluted by using a gradient elution mode; the gradient elution mode comprises the following procedures: 0-2 min, the volume fraction of the mobile phase B is 10%; 2-5 min, the volume fraction of the mobile phase B is increased from 10% to 14.5%; 5-15 min, the volume fraction of the mobile phase B is increased from 14.5% to 22%; 15-17 min, the volume fraction of the mobile phase B is increased from 22% to 26%; 17-25 min, the volume fraction of the mobile phase B is increased from 26% to 28%; 25-30 min, the volume fraction of the mobile phase B is increased from 28% to 37%; The volume fraction of phosphoric acid in the phosphoric acid water solution is 0.05-0.4%; The preparation method of the test sample solution of swertiae comprises the following steps: extracting components in swertiae by using an extracting agent, wherein the extracting agent comprises 50%-80% methanol or 50%-80% ethanol.

2. The method of determining asafetida content according to claim 1, wherein, In S3, the relative correction factors of chlorogenic acid, caffeic acid, luteolin-7-O-β-D-glucuronide, 11(13)-dehydroxy-vasikirarin, isochlorogenic acid C, 2,3,4,5-tetra-O-caffeoyl-D-glucaric acid, 2-deoxy-4-epi-tanacetone, swertipicrin, and terulactone 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.

3. The method for determining the content of Tianmingjing components according to claim 1, characterized in that, In S4, the relative retention times of chlorogenic acid, caffeic acid, luteolin, 11(13) dehydroxyaxillaryvera, isochlorogenic acid C, 2,3,4,5-tetra caffeoyl-D-glucaric acid, 2-deoxy-4-epi-tanacetol, tanacetol, and terenol 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.

4. The method for determining the content of asperosaponins according to any one of claims 1 to 3, characterized in that, The detection conditions of the ultra-high performance liquid chromatography include: a flow rate of the mobile phase is 0.2-0.4 mL / min, a column temperature is 33-37 ℃, and a sample injection amount is 0.5-1 μL.

5. The method for determining the content of asperosaponins according to any one of claims 1 to 3, characterized in that, The volume fraction of phosphoric acid in the aqueous phosphoric acid solution is 0.05-0.2%.

6. The method of determining the content of asperosaponins according to any one of claims 1 to 3, characterized in that, The extraction agent includes 70%-80% methanol or 70%-80% ethanol.

7. The method of determining the content of asperosaponins according to claim 6, characterized in that, The solid-liquid ratio of the tanacetum to the extraction agent is 0.3-0.6 g: 15-25 mL.

8. The method for determining the content of Tianmingjing components according to claim 7, characterized in that, The solid-liquid ratio of the tanacetum to the extraction agent is 0.45-0.55 g: 19.5-20.5 mL.

9. The method for determining the content of Tianmingjing components according to claim 6, characterized in that, The extraction is performed by ultrasonic extraction, and the ultrasonic extraction is performed at a power of 190-210 W, a frequency of 35-45 kHz, and for a time of 50-70 min.

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