Method for simultaneously and quantitatively detecting six components in radix impatiens and application thereof

Abstract

The present application belongs to the technical field of chemical component analysis and detection of traditional Chinese medicinal materials, and particularly relates to a method for simultaneously and quantitatively detecting six components in radix impatiens and application thereof. The method comprises the following steps: standard samples of six components, i.e. hydroxybenzoic acid, vanillic acid, syringic acid, 4-hydroxyacetophenone, 2,4-dihydroxyacetophenone and A-O-DCDT, are weighed respectively and prepared into a mixed control solution containing all the standard samples by using an organic solvent; an extracting agent is added to a test sample, and then the test sample is placed, ultrasonically extracted, cooled, fixed in volume, centrifuged, filtered, and dried to obtain a residue; the residue is redissolved and filtered to obtain a supernatant, which is the test sample solution; the mixed control solution and the test sample solution are detected by UPLC-DAD to obtain corresponding chromatograms; and the six components are qualitatively identified and quantitatively determined by comparing and analyzing the chromatograms of the mixed control solution and the test sample solution.

Description

Technical Field

[0001] This invention belongs to the field of chemical composition analysis and detection technology of traditional Chinese medicine, specifically involving a method for simultaneous quantitative detection of six components in Baiqian herb and its application. Background Technology

[0002] Baiqian is a commonly used herb in traditional Chinese medicine for relieving cough and asthma, with a long history of application. Its authentic origin is the willow-leaf Baiqian plant, belonging to the Asclepiadaceae family. Cynanchum Stauntonii (Decne.) Schltr. from Levi Or Daphne genkwa leaves Cynanchus glaucescens (Decne.) Hand.-Mazz. The dried rhizome and root of Cynanchum paniculatum. Traditional Chinese medicine believes it is slightly warm in nature, pungent and bitter in taste, and primarily enters the lung meridian. It excels at lowering qi, resolving phlegm, relieving cough and asthma, and is effective for symptoms such as lung qi stagnation, cough with excessive phlegm, and chest tightness. This efficacy was already evident in Zhang Zhongjing's *Synopsis of Prescriptions of the Golden Chamber* from the Han Dynasty, where his "Zeqi Decoction" combined Cynanchum paniculatum with other herbs was used to treat "coughing with shortness of breath and a deep pulse," demonstrating its long-standing clinical application in relieving cough and asthma.

[0003] With changes in society and the natural environment, the incidence of respiratory diseases such as chronic obstructive pulmonary disease and bronchitis is constantly rising, and the clinical demand for highly effective and safe antitussive, expectorant, and bronchodilator drugs is also increasing significantly. As a commonly used herb in traditional Chinese medicine for treating these conditions, the scientific and standardized quality control of Baiqian (Cynanchum paniculatum) is directly related to the stability of clinical efficacy and medication safety.

[0004] However, existing quality evaluation standards for Baiqian (Cynanchum paniculatum) are still incomplete. For example, the quality control items for Baiqian in the Chinese Pharmacopoeia (2025 edition) are mainly limited to appearance, processing, and routine inspections (such as moisture content), lacking quantitative detection standards for specific active ingredients, making it difficult to scientifically and comprehensively reflect the intrinsic quality of the medicinal material. Furthermore, current research focuses on the detection of a few components in Baiqian, such as fructose, glucose, carotene, and β-sitosterol, and these methods typically require different pretreatment steps and chromatographic conditions for separate determination. This method is complex, time-consuming, labor-intensive, and costly, making it difficult to achieve high-throughput, multi-index comprehensive quality evaluation. Studies have shown that various components in Baiqian, such as phenolic acids (e.g., p-hydroxybenzoic acid, vanillic acid, syringic acid), acetophenones (e.g., 4-hydroxyacetophenone, 2,4-dihydroxyacetophenone), and saponins, are closely related to its anti-inflammatory, antitussive, and antiasthmatic pharmacological activities. However, these components vary significantly in polarity, UV absorption characteristics, and chemical stability, making it difficult to achieve efficient separation and balanced detection of all target analytes in a single chromatographic system. Furthermore, the complex matrix background of traditional Chinese medicine severely interferes with the selectivity and accuracy of the analysis, particularly posing a challenge to the accurate quantification of trace components. This limits the possibility of conducting more in-depth quality research and effective control of Baiqian medicinal materials and their preparations. Summary of the Invention

[0005] The purpose of this invention is to provide a method for the simultaneous quantitative detection of six components in Baiqian herb and its application, thereby overcoming the shortcomings of the prior art. It is essential to establish a method for the simultaneous detection of phenolic acids, acetophenones, and saponins in Baiqian herb, so as to achieve the requirements of method stability, simple operation, and high accuracy.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for simultaneously and quantitatively detecting six components in Baiqian medicinal material, comprising the following steps: (1) Weigh out the standards of six components, namely hydroxybenzoic acid, vanillic acid, syringic acid, 4-hydroxyacetophenone, 2,4-dihydroxyacetophenone and AO-DCDT, and prepare a mixed reference solution containing each standard in an organic solvent. (2) Add the extractant to the sample, let stand, extract by ultrasound, cool, make up to volume, centrifuge and filter to obtain the filtrate; evaporate the filtrate to dryness to obtain the residue, redissolve the residue and filter, and the supernatant obtained is the sample solution. (3) The mixed reference solution and the test solution were detected by UPLC-DAD, and the corresponding chromatograms were obtained respectively; The UPLC detection conditions were as follows: Infinity LabPoroshell 120 AQ-C18 column; mobile phase A was phosphoric acid aqueous solution; mobile phase B was acetonitrile; and the gradient elution program was as follows: 0–5 min, 10% B; 5–12 min, 10%–12% B; 12–16 min, 12% B; 16–35 min, 12%–30% B; 35–49 min, 30%–53% B; 49–65 min, 53%–82% B; 65–69 min, 82%–10% B; 69–70 min, 10% B; and the flow rate was 0.4–0.6 mL / min. -1 The column temperature was 30-35℃, the injection volume was 6-8μL, and the detection wavelength was 210-274nm. (4) Qualitative identification and quantitative determination of the six components were performed by comparing the spectra of the mixed reference solution and the test solution.

[0008] In some other embodiments, in step (3), the UPLC detection conditions are: mobile phase A is a 0.1% aqueous phosphoric acid solution, mobile phase B is acetonitrile, and the flow rate is 0.5 mL·min. -1 The column temperature was 30 °C and the injection volume was 7 μL.

[0009] In some other embodiments, in step (3), the detection wavelength is 260 nm for 0-14 min, 274 nm for 14-52 min, and 210 nm for 52-70 min.

[0010] In some other embodiments, the organic solvent in step (1) includes methanol and ethanol.

[0011] In some other embodiments, in step (2), the extractant includes water-saturated ethyl acetate, and 15-25 mL of extractant is added per gram of sample; the standing time is 10-15 h, the ultrasonic extraction time is 30-60 min, the solvent used for redissolution includes methanol and ethanol, and the filter membrane used for filtration has a pore size of 0.2-0.25 μm.

[0012] In some other embodiments, in step (2), 20 mL of extractant is added per gram of sample; the standing time is 12 h, the ultrasonic extraction time is 40 min, the solvent used for reconstitution is methanol, and the filter membrane used for filtration has a pore size of 0.22 μm.

[0013] In some other embodiments, in step (2), the test sample is dried cyperus chinensis powder, which is the rhizome of cyperus chinensis with a particle size of 50-60 mesh, and the drying temperature is constant at 50-60℃.

[0014] In some other embodiments, in step (4), qualitative identification is performed based on a comparison of the chromatograms of the mixed reference solution and the test solution; The content of the six compounds in the test solution was calculated based on the ratio of the peak areas of the chromatograms of the mixed reference solution and the test solution.

[0015] In some other embodiments, in step (4), during quantitative determination, the concentration at S / N=3 is used as the detection limit, and the concentration at S / N=10 is used as the quantification limit.

[0016] Secondly, the present invention provides the application of the method for simultaneous quantitative detection of six components in Baiqian medicinal material as described in the first aspect in the quality evaluation of Baiqian medicinal material.

[0017] The beneficial effects of this invention are: (1) The detection method of the present invention successfully overcomes the technical difficulty of large differences in the physicochemical properties of different categories of active ingredients in Baiqian. It can simultaneously separate and determine the content of six key active ingredients, including phenolic acids (p-hydroxybenzoic acid, vanillic acid, syringic acid), acetophenones (4-hydroxyacetophenone, 2,4-dihydroxyacetophenone), and saponins (AO-DCDT), in a single run. The method has high sensitivity, low detection limit, high accuracy, good reproducibility and stability, and provides an efficient and reliable quantitative analysis tool for comprehensively and scientifically evaluating the intrinsic quality of Baiqian medicinal materials.

[0018] (2) By systematically optimizing the ultra-high performance liquid chromatography (UPLC) conditions, especially by utilizing a phosphoric acid-water-acetonitrile gradient elution system combined with variable wavelength detection, efficient baseline separation of six components under the same chromatographic conditions was achieved. This method significantly shortens the single analysis time, reduces solvent consumption, and effectively avoids matrix interference by utilizing the full-wavelength scanning function of a diode array detector (DAD), greatly improving analytical efficiency and method specificity. It is suitable for rapid qualitative and quantitative analysis and quality control of Baiqian medicinal materials and their preparations. Attached Figure Description

[0019] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0020] Figure 1 The above are UPLC chromatograms of six phenolic acids, acetophenones, and saponins in Example 1 of this invention. In A, the test solution is a test solution and B is a mixed reference solution. In A and B, 1 is p-hydroxybenzoic acid, 2 is vanillic acid, 3 is syringic acid, 4 is 4-hydroxyacetophenone, 5 is 2,4-dihydroxyacetophenone, and 6 is AO-DCDT. Detailed Implementation

[0021] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Where specific conditions are not specified for the instruments and equipment used, they shall be performed under conventional conditions or conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0022] As mentioned earlier, the various components of *Cynanchum paniculatum*, including phenolic acids, acetophenones, and saponins, exhibit significant differences in polarity, UV absorption characteristics, and chemical stability, making it difficult to achieve efficient separation and balanced detection of all target analytes in a single chromatographic system. Furthermore, the complex matrix background of traditional Chinese medicine severely interferes with the selectivity and accuracy of the analysis, particularly posing a challenge to the accurate quantification of trace components.

[0023] This invention utilizes optimized UPLC technology with variable wavelengths to detect six active ingredients under gradient elution conditions in the same phosphoric acid-water-acetonitrile system. The method is highly efficient and can simultaneously separate and quantitatively detect six components, including p-hydroxybenzoic acid, vanillic acid, syringic acid, 4-hydroxyacetophenone, 2,4-dihydroxyacetophenone, and AO-DCDT. It has advantages such as high sensitivity, low detection limit, high accuracy, and good stability, enabling efficient qualitative and quantitative analysis of Baiqian medicinal materials.

[0024] Example 1 This embodiment provides the plotting of standard curves for six components, specifically including the following steps: Preparation of mixed reference solutions: Accurately weigh appropriate amounts of six reference standards: p-hydroxybenzoic acid, vanillic acid, syringic acid, 4-hydroxyacetophenone, 2,4-dihydroxyacetophenone, and AO-DCDT. Dissolve them in methanol to prepare solutions with concentrations of 20.6, 101.6, 153.1, 20.8, 71.2, and 116.8 μg·mL, respectively. -1 Prepare the mixed reference solution stock solution by shaking well before use.

[0025] The stock solution of the mixed reference solution was accurately pipetted and diluted using the fractional dilution method to obtain a series of reference solutions with seven different mass concentrations. The peak areas of the six components at different concentrations were determined using ultra-high performance liquid chromatography (UHPLC).

[0026] Chromatographic conditions: Infinity LabPoroshell 120 AQ-C18 column (4.6 mm × 100 mm, 2.7 μm); mobile phase: acetonitrile (B) - 0.1% phosphoric acid aqueous solution (A), gradient elution (0–5 min, 10% B; 5–12 min, 10%–12% B; 12–16 min, 12% B; 16–35 min, 12%–30% B; 35–49 min, 30%–53% B; 49–65 min, 53%–82% B; 65–69 min, 82%–10% B; 69–70 min, 10% B); detection wavelength: 260 nm (0–14 min), 274 nm (14–52 min), 210 nm (52–70 min); flow rate: 0.5 mL·min -1Column temperature: 30 ℃; injection volume: 7 μL.

[0027] A standard curve was plotted with the mass concentration of the reference standard as the abscissa (X) and the peak area as the ordinate (Y). Linear regression was then performed to obtain the standard curve equation. The standard curve equation and its corresponding linear range are shown in Table 1.

[0028] Table 1. Linear regression equations, correlation coefficients, and linear ranges of the six components of Baiqian.

[0029] Example 2 This embodiment provides the preparation of a pre-white sample solution, specifically including the following steps: Take about 1 g of Baiqian powder, accurately weigh it, place it in a 50 mL stoppered conical flask, accurately add 20.0 mL of water-saturated ethyl acetate, seal tightly, weigh it, let it stand for 12 h, then sonicate it for 40 min, cool it, weigh it again, replenish the weight lost during sonication with water-saturated ethyl acetate, shake well, filter it, evaporate the filtrate to dryness, dissolve it in 2 mL of methanol, and filter it through a 0.22 μm microporous membrane to obtain the final product.

[0030] Example 3 This embodiment provides a methodological review of the first six quantitative detection methods for components, as detailed below: (1) Limit of detection and limit of quantitation: The mixed reference solution provided in Example 1 was used as the sample, and it was diluted stepwise. The method provided in Example 1 was used for detection. The concentration of the target compound when S / N=3 was taken as its limit of detection, and the concentration of the target compound when S / N=10 was taken as its limit of quantitation. The limit of detection and limit of quantitation of each component are shown in Table 2.

[0031] (2) Precision: Take the mixed reference solution and inject it continuously for 6 times. Detect the content according to the chromatographic conditions in Example 1. The relative standard deviation of the determination results is shown in Table 2. As can be seen from the table, the relative standard deviation of the precision of each component is less than 1.12% (n=6), indicating that the instrument has good precision.

[0032] (3) Stability: Take the sample solution of Baiqian and detect the content at 0h, 2h, 4h, 8h, 12h and 24h respectively according to the chromatographic conditions in Example 1. The relative standard deviation of the determination results is shown in Table 2. As can be seen from the table, the relative standard deviation of the stability of each component within 24h is less than 1.18% (n=6), indicating that the method has good stability within 24h.

[0033] (4) Repeatability: Take the same batch of Baiqian powder and prepare 6 samples in parallel according to the Baiqian sample solution preparation method. Detect the content according to the chromatographic conditions in Example 1. The relative standard deviation of the determination results is shown in Table 2. As can be seen from the table, the relative standard deviation of the repeatability of each component is less than 1.58% (n=6), indicating that the method has good repeatability.

[0034] (5) Spiking recovery filtration: Take six parallel portions of Baiqian powder with known contents of each component, add equal amounts of reference standard to each, and detect the contents according to the preparation of Baiqian sample solution and the chromatographic conditions in Example 1. The average recovery rate is shown in Table 2. As can be seen from the table, the average recovery rate of each component is between 97.74% and 102.81%, indicating that the recovery rate of this method is good.

[0035] Table 2. Detection limits, quantitation limits, repeatability, precision, stability, and recovery rates of the six components of Baiqian.

[0036] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for simultaneous quantitative detection of six components in Baiqian medicinal material, characterized in that, Includes the following steps: (1) Weigh out the standards of six components, namely hydroxybenzoic acid, vanillic acid, syringic acid, 4-hydroxyacetophenone, 2,4-dihydroxyacetophenone and AO-DCDT, and prepare a mixed reference solution containing each standard in an organic solvent. (2) Add the extractant to the sample, let stand, extract by ultrasound, cool, make up to volume, centrifuge and filter to obtain the filtrate; evaporate the filtrate to dryness to obtain the residue, redissolve the residue and filter, and the supernatant obtained is the sample solution. (3) The mixed reference solution and the test solution were detected by UPLC-DAD, and the corresponding chromatograms were obtained respectively; The UPLC detection conditions were as follows: Infinity LabPoroshell 120 AQ-C18 column; mobile phase A was phosphoric acid aqueous solution; mobile phase B was acetonitrile; and the gradient elution program was as follows: 0–5 min, 10% B; 5–12 min, 10%–12% B; 12–16 min, 12% B; 16–35 min, 12%–30% B; 35–49 min, 30%–53% B; 49–65 min, 53%–82% B; 65–69 min, 82%–10% B; 69–70 min, 10% B; and the flow rate was 0.4–0.6 mL / min. -1 The column temperature was 30-35 ℃, the injection volume was 6-8 μL, and the detection wavelength was 210-274 nm. (4) Qualitative identification and quantitative determination of the six components were performed by comparing the spectra of the mixed reference solution and the test solution.

2. The method for simultaneous quantitative detection of six components in Baiqian medicinal material according to claim 1, characterized in that, In step (3), the UPLC detection conditions are as follows: mobile phase A is 0.1% phosphoric acid aqueous solution, mobile phase B is acetonitrile, column temperature is 30 ℃, and injection volume is 7 μL.

3. The method for simultaneous quantitative detection of six components in Baiqian medicinal material according to claim 1, characterized in that, In step (3), the detection wavelengths are: 260 nm for 0~14 min, 274 nm for 14~52 min, and 210 nm for 52~70 min.

4. The method for simultaneous quantitative detection of six components in Baiqian medicinal material according to claim 1, characterized in that, In step (1), the organic solvent includes methanol and ethanol.

5. The method for simultaneous quantitative detection of six components in Baiqian medicinal material according to claim 1, characterized in that, In step (2), the extractant includes water-saturated ethyl acetate, and 15-25 mL of extractant is added per gram of test sample; the standing time is 10-15 h, the ultrasonic extraction time is 30-60 min, the solvent used for redissolution includes methanol and ethanol, and the filter membrane used for filtration has a pore size of 0.2-0.25 μm.

6. The method for simultaneous quantitative detection of six components in Baiqian medicinal material according to claim 5, characterized in that, In step (2), 20 mL of extractant is added per gram of sample; the standing time is 12 h, the ultrasonic extraction time is 40 min, the solvent used for redissolution is methanol, and the filter membrane used for filtration has a pore size of 0.22 μm.

7. The method for simultaneous quantitative detection of six components in Baiqian medicinal material according to claim 1, characterized in that, In step (2), the test sample is dried Baiqian powder, which is the rhizome of Baiqian with a particle size of 50-60 mesh and is dried at a constant temperature of 50-60℃.

8. The method for simultaneous quantitative detection of six components in Baiqian medicinal material according to claim 1, characterized in that, In step (4), qualitative identification is performed by comparing the chromatograms of the mixed reference solution and the test solution; The content of the six compounds in the test solution was calculated based on the ratio of the peak areas of the chromatograms of the mixed reference solution and the test solution.

9. The method for simultaneous quantitative detection of six components in Baiqian medicinal material according to claim 8, characterized in that, In step (4), during quantitative determination, the concentration at S / N=3 is taken as the detection limit, and the concentration at S / N=10 is taken as the quantitation limit.

10. The application of the method for simultaneous quantitative detection of six components in Baiqian medicinal material according to any one of claims 1-9 in the quality evaluation of Baiqian medicinal material.

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