Quality control method for quantitative analysis of multiple components by single marker of honeysuckle

Abstract

The invention belongs to the technical field of medicinal material quality control, and discloses a honeysuckle flower QAMS (quantitative analysis of multi-components by single marker) quality control method, which comprises the following steps: preparing a single reference solution and a mixed reference solution of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C, and preparing a test solution; after high performance liquid chromatography detection, the relative correction factors of other components in the honeysuckle are calculated by taking chlorogenic acid as an internal reference substance through chromatographic peak positioning, and the contents of the components in the honeysuckle are obtained by utilizing the relative correction components, so that the synchronous determination of the multiple components in the honeysuckle is realized. The method has the advantages of cost saving, simplicity in operation, high detection accuracy and good stability, has great significance in quality control and clinical curative effect guarantee of the honeysuckle medicinal material, and provides a basis for establishing a quality evaluation standard of the honeysuckle medicinal material.

Description

Technical Field

[0001] This invention relates to the field of medicinal material quality control technology, specifically to a method for quality control of honeysuckle through multiple tests and evaluations. Background Technology

[0002] Honeysuckle is the dried flower bud or newly opened flower of *Lonicera japonica* Thunb., a plant in the Caprifoliaceae family. It is harvested in early summer before the flowers open and then dried. It is a typical representative of traditional Chinese medicine and a plant used for both food and medicine. Honeysuckle has antibacterial, antiviral, anti-inflammatory, immunomodulatory, hepatoprotective, and hemostatic effects, and is known as a Chinese medicine antibiotic.

[0003] Honeysuckle contains various pharmacologically active components, among which chlorogenic acids are the main active ingredients. Chlorogenic acids are phenolic acids formed by the esterification reaction of quinic acid and caffeic acid. They are phenylpropanoid compounds produced by plants during aerobic respiration. Depending on the esterification site, they can form various isomers, including neochlorogenic acid (5-caffeoylquinic acid), cryptochlorogenic acid (4-caffeoylquinic acid), isochlorogenic acid A (3,5-dicaffeoylquinic acid), isochlorogenic acid B (3,4-dicaffeoylquinic acid), and isochlorogenic acid C (4,5-dicaffeoylquinic acid). The content of active ingredients is an important indicator for the quality control of medicinal materials. The quality of the same variety of medicinal material varies due to differences in growth conditions, harvesting season, processing methods, and storage conditions, resulting in certain quality differences in finished traditional Chinese medicine preparations. Therefore, it is necessary to test and evaluate the quality of honeysuckle before it enters the production of traditional Chinese medicine preparations.

[0004] High-performance liquid chromatography (HPLC) is a classic method for determining the content of active ingredients. HPLC works by using a reference standard for the target ingredient, measuring its peak area at a series of concentrations, establishing a linear relationship between the peak area and the amount / concentration of the ingredient, and using this linear relationship to determine the peak area of ​​the target ingredient in the sample and calculate its content. The core of this technique is the use of a reference standard to establish a linear relationship between peak area and concentration / amount. This method is heavily reliant on reference standards, especially given the multi-component, multi-target nature of traditional Chinese medicine. When multiple indicators need to be determined, the availability and price of reference standards become significant constraints on the implementation of the research protocol. Chlorogenic acid, cryptochlorogenic acid, chlorogenic acid A, and other chlorogenic acid compounds are the main antiviral active ingredients in honeysuckle. Determining the content of these components is crucial for the quality control of honeysuckle. However, except for chlorogenic acid, the other reference standards are not only difficult to obtain but also expensive.

[0005] Therefore, the present invention provides a method for quality control of honeysuckle through multiple tests and evaluations. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention aims to establish a multi-evaluation quality control method for honeysuckle, which uses only chlorogenic acid as a reference standard to simultaneously determine six phenolic acid components in honeysuckle, including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C. This reduces dependence on reference standards, lowers detection costs, and simplifies experimental procedures.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] On the one hand, the present invention provides a quality control method for honeysuckle with one test and multiple evaluations, including the following steps:

[0009] S1. Preparation of reference solution

[0010] Accurately weigh chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C reference standards, dissolve them to obtain single reference standard solutions of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C, and mix the single reference standard solutions to obtain the reference standard mixed solution.

[0011] S2. Preparation of the test solution

[0012] Honeysuckle flowers are crushed, sieved, and honeysuckle powder is obtained. The honeysuckle powder is accurately weighed, added to solvent, ultrasonically extracted, filtered, and the test solution is obtained.

[0013] S3. Set chromatographic conditions

[0014] C18 column; column temperature: 35~38 ℃; flow rate: 0.8~1.0 ml·min -1 UV wavelength: 350 nm; Mobile phase: Gradient elution was performed using acetonitrile as mobile phase A and 0.3% formic acid water as mobile phase B. The gradient elution program is as follows:

[0015]

[0016] S4. Content determination

[0017] Using chlorogenic acid as an internal reference, the relative correction factor coefficients of chlorogenic acid with neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C were determined. The chlorogenic acid reference solution and the test solution were accurately pipetted into the high-performance liquid chromatograph, and the chromatographic peak areas were recorded under the chromatographic conditions of step S3. The contents of neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C were calculated using the established relative correction factors.

[0018] Preferably, the preparation of the reference solution specifically includes the following steps:

[0019] S11. Accurately weigh chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C reference standards, and place them in volumetric flasks respectively.

[0020] S12. Dissolve in an appropriate amount of methanol solution, and make up to volume to obtain single reference solutions; the concentrations of single reference solutions of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C are 0.035~1.75 mg / ml, 0.002~0.11 mg / ml, 0.002~0.081 mg / ml, 0.002~0.11 mg / ml, 0.006~0.29 mg / ml and 0.002~0.09 mg / ml, respectively.

[0021] S13. Accurately pipette each single reference solution, mix and shake well, dilute with methanol, and prepare mixed reference solutions of different concentrations.

[0022] Preferably, the concentrations of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C in the reference standard mixed solution are 8.59~291.6 μg / ml, 0.47~18.33 μg / ml, 0.36~15.00 μg / ml, 0.47~18.33 μg / ml, 1.41~48.33 μg / ml, and 0.36~15.00 μg / ml, respectively.

[0023] Preferably, the preparation of the test solution specifically includes the following steps:

[0024] S21. Crush the honeysuckle flowers, sieve them, and obtain honeysuckle powder.

[0025] S22. Accurately weigh the honeysuckle powder and place it in an Erlenmeyer flask. Add solvent and weigh it.

[0026] S23. Ultrasonic extraction for 30-40 minutes, followed by cooling and reweighting.

[0027] S24. Filter through a 0.45 μm microporous membrane and collect the filtrate for later use.

[0028] Preferably, the honeysuckle has a mass of 30-60 mg, and the mass-to-volume ratio of the honeysuckle to the solvent is 6 mg: 5-7 ml.

[0029] Preferably, the solvent is a methanol solution with a volume fraction of 70% to 80%.

[0030] Preferably, the sieve mesh size is 60-80 mesh.

[0031] Preferably, the method for determining the relative correction factor coefficients of neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C is as follows: accurately pipette the reference solution from step S1 and inject it into a high-performance liquid chromatograph. Under the chromatographic conditions of step S3, record the chromatographic peak area. Using chlorogenic acid as an internal reference, calculate the relative correction factors of neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C based on the chromatographic peak areas of neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C presented in the characteristic chromatogram of the test solution. Formula (1) is as follows:

[0032]

[0033] Where Ci is the mass concentration of other components, Ai is the peak area of ​​other components; Cs is the mass concentration of internal standard chlorogenic acid, and As is the peak area of ​​chlorogenic acid.

[0034] Preferably, the C18 column is a Diamonsil C18 (2) column: 4.6 mm × 250 mm, 5 μm or a Kromasil C18 column: 4.6 mm × 250 mm, 5 μm or an Elite Hypersil C18 column: 4.6 mm × 250 mm, 5 μm.

[0035] Preferably, the column temperature is 38 °C; the flow rate is 0.8 ml·min. -1 .

[0036] Preferably, the injection volume is 10-20 μL.

[0037] Preferably, the calculation formula (2) for the content of neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C is as follows:

[0038]

[0039] Wherein, RCF is the relative correction coefficient between each component and chlorogenic acid; A sample is the peak area of ​​the component to be tested in the sample; C green: the concentration of chlorogenic acid reference standard; A green: the peak area of ​​chlorogenic acid reference standard; weighing: the sample weight.

[0040] On the other hand, the present invention also provides an application of a multi-evaluation quality control method for honeysuckle in detecting the content of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C in honeysuckle.

[0041] Compared with the prior art, the beneficial effects of the present invention are:

[0042] 1. This invention is based on high performance liquid chromatography (HPLC) technology and combines a single-test-multiple-evaluation method for the determination of the content of multiple components in honeysuckle medicinal materials. This is beneficial for the comprehensive quality control of honeysuckle medicinal materials and related preparations. Moreover, this method has high detection sensitivity, good stability, low cost, simple operation, easy mastery, and is easy to promote further.

[0043] 2. This invention uses chlorogenic acid as an internal reference. The relative correction factor coefficients of neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C are calculated, and their contents are then calculated using these coefficients. Since chlorogenic acid is readily available and inexpensive, this invention significantly reduces testing costs and time. Furthermore, the method is rapid and accurate, allowing for more comprehensive quality control of honeysuckle. This is of great significance for the quality control of honeysuckle medicinal materials and ensuring its clinical efficacy, providing a basis for establishing quality evaluation standards for honeysuckle medicinal materials. Attached Figure Description

[0044] Figure 1 This is a high-performance liquid chromatogram for the determination of multiple indicators of honeysuckle (A is the chromatogram of the reference standard; B is the chromatogram of the honeysuckle sample; 1-neochlorogenic acid, 2-chlorogenic acid, 3-cryptochlorogenic acid, 4-isochlorogenic acid B, 5-isochlorogenic acid A, 6-isochlorogenic acid C). Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0046] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0047] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods. Unless otherwise specified, the materials and reagents used in the embodiments of this invention are commercially available. Experimental methods without detailed specifications are performed according to conventional experimental methods or the operating instructions recommended by the supplier. Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0048] This invention provides a method for quality control of honeysuckle using a single-test, multi-evaluation approach. This method determines the content of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C in honeysuckle using this established method, which can provide a basis for establishing quality evaluation standards for honeysuckle medicinal materials. The method includes the following steps:

[0049] S1. Preparation of the reference standard mixed solution

[0050] S11. Accurately weigh chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C reference standards, place them in volumetric flasks, dissolve them in methanol, and dilute to volume to obtain single reference standard solutions with concentrations of chlorogenic acid 1.70±0.05 mg / ml, neochlorogenic acid 0.10±0.01 mg / ml, cryptochlorogenic acid 0.08±0.01 mg / ml, isochlorogenic acid B 0.10±0.01 mg / ml, isochlorogenic acid A 0.28±0.01 mg / ml, and isochlorogenic acid C 0.08±0.01 mg / ml.

[0051] S12. Accurately pipette 200 μL of each single reference solution, mix and shake well to obtain a mixed reference solution. In the mixed reference solution, the concentrations of chlorogenic acid are 8.59~291.6 μg / ml, neochlorogenic acid is 0.47~18.33 μg / ml, cryptochlorogenic acid is 0.36~15.00 μg / ml, isochlorogenic acid B is 0.47~18.33 μg / ml, isochlorogenic acid A is 1.41~48.33 μg / ml, and isochlorogenic acid C is 0.36~15.00 μg / ml.

[0052] S2. Preparation of the test solution

[0053] Crush honeysuckle into powder, pass through a 60-80 mesh sieve, and accurately weigh 30-60 mg into a 50 mL Erlenmeyer flask. Use 70%-80% methanol as the extraction solvent, adding it at a material-to-liquid ratio of 6:5-7 (w / v, mg / mL). Extract using ultrasound for 30 min. Remove the flask, cool to room temperature, and re-weigh. Pipette an appropriate amount and filter through a 0.45 μm microporous membrane, reserving the filtrate for later use.

[0054] S3. Setting chromatographic conditions

[0055] The C18 column (4.6 mm × 250 mm, 5 μm) was selected from one of the following: Diamonsil C18 (2) column, Kromasil C18 column, and Elite Hypersil C18 column; column temperature: 35~38 ℃; flow rate: 0.8~1.0 ml·min -1 Ultraviolet light: 200~400 nm, preferably 350 nm; Mobile phase: Gradient elution was performed using acetonitrile as mobile phase A and 0.3% formic acid water as mobile phase B. The gradient elution program is shown in Table 1.

[0056] Table 1 Elution gradients in liquid chromatography

[0057]

[0058] S4. Content determination

[0059] S41. Determination of the Relative Correction Factor (RCF)

[0060] Accurately pipette the mixed solution of reference standards from step S12 to prepare a series of reference standard solutions with different concentrations, as shown in Table 2. Accurately pipette 10-20 μL of each solution and inject it into the high-performance liquid chromatograph (HPLC-DAD). Under the chromatographic conditions of step S3, record the peak areas. Using chlorogenic acid as an internal reference, calculate the relative correction factors of chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C based on the peak areas of neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C in the characteristic chromatogram of the test solution. Formula (1) is as follows:

[0061]

[0062] Where Ci is the mass concentration of other components, Ai is the peak area of ​​other components; Cs is the mass concentration of internal standard chlorogenic acid, and As is the peak area of ​​chlorogenic acid.

[0063] S42. Determination of honeysuckle content in samples

[0064] Accurately pipette the chlorogenic acid reference solution and the test solution into the high-performance liquid chromatograph. Under the chromatographic conditions of step S3, record the peak areas. Calculate the contents of neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C using relative correction factors. The calculation formula (2) is as follows:

[0065]

[0066] Wherein, RCF is the relative correction coefficient between each component and chlorogenic acid; A sample is the peak area of ​​the component to be tested in the sample; C green: the concentration of chlorogenic acid reference standard; A green: the peak area of ​​chlorogenic acid reference standard; weighing: the sample weight.

[0067] The equipment and reagents used in the following embodiments are as follows:

[0068] Instruments and equipment: Sartorius SQP electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd., Beijing), Shimadzu LC-20A high-performance liquid chromatograph [equipped with SPD-M20A diode array detector] (Shimadzu Corporation, Japan)

[0069] Materials and Reagents: Honeysuckle (origin: Pingyi County and Jining County, Shandong Province; commercially available in Gansu, Henan, and Hebei provinces), and chlorogenic acid reference standard (batch number: RFS-L00701908029) were purchased from Chengdu Ruifensidedan Biotechnology Co., Ltd. (Chengdu, Sichuan). Neochlorogenic acid reference standard (batch number: BYC-000316-202008), cryptochlorogenic acid (batch number: BCY-000317-202012), isochlorogenic acid B (batch number: BCY-000319-202008), isochlorogenic acid A (batch number: BCY-000318-202007), and isochlorogenic acid C (batch number: BCY-000320-202007) were all purchased from Jiangxi Baicaoyuan Biotechnology Co., Ltd. (Nanchang, Jiangxi). All reference standards were tested by high-performance liquid chromatography (HPLC) and the purity was greater than 98%. Methanol and acetonitrile were both chromatographic grade and purchased from Thermo Fisher Scientific (China) Co., Ltd. (Shanghai). Formic acid (mass spectrometry grade) was purchased from Thermo Fisher Scientific Co., Ltd., and Wahaha purified water was purchased from Hangzhou Wahaha Group Co., Ltd. (Hangzhou).

[0070] Example 1: Method Validation of External Standard Method for Content Determination

[0071] The method for determining the content of various components in honeysuckle using a single measurement and multiple evaluation method established in this invention was validated methodologically as follows:

[0072] 1. Establishing linear equations

[0073] 1.1 Preparation of the reference standard mixed solution

[0074] Accurately weigh 8.40 mg of chlorogenic acid reference standard, dissolve it in methanol and dilute to volume in a 5 mL volumetric flask; accurately weigh 2.43 mg, 1.84 mg, 2.70 mg, 6.91 mg, and 2.18 mg of neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C reference standards respectively, place them in 25 mL volumetric flasks, dissolve them in methanol and dilute to volume to obtain stock solutions of each reference standard;

[0075] Accurately pipette 200 μL of each stock solution, mix and shake well to obtain a mixed reference solution; use this solution as the starting concentration, dilute by equal volume, and repeat the dilution 5 times to obtain a series of reference solutions. The concentrations of the reference solutions are shown in Table 2.

[0076] Table 2. Series of concentrations of standard solution mixtures used for standard curve studies and RCF calculations. (Unit: μg / mL)

[0077]

[0078] 1.2 Liquid Chromatography Conditions

[0079] The following liquid chromatography conditions were used to determine the concentrations of a series of reference solutions. A Shimadzu LC-20A high-performance liquid chromatograph was used, employing a Diamonsil C18 (2) column (4.6 mm × 250 mm, 5 μm), a column temperature of 38 ℃, and a flow rate of 0.8 ml·min. -1 A gradient elution system consisting of acetonitrile (A) and 0.3% formic acid solution (B) was used, with gradient conditions shown in Table 1. The injection volume was 20 μL. Chromatographic data were recorded using UV full-wavelength monitoring mode (wavelength range 200–400 nm), and a detection wavelength of 350 nm was ultimately selected for subsequent experiments.

[0080] 1.3 Establishment of the linear equation for the reference standard

[0081] A series of reference standard solutions with varying concentrations were determined. A detection wavelength of 350 nm was selected, and linear regression was performed with the reference standard concentration as the abscissa and the peak area as the ordinate to obtain the linear equation for each reference standard. The results are shown in Table 3.

[0082] Table 3 Linear equations for each reference standard

[0083]

[0084] 2. Precision and stability studies

[0085] 2.1 Precision test: Accurately pipette the mixed standard solution numbered "3" under the preparation of the reference standard mixed solution in "1.1", and perform 6 consecutive determinations using the chromatographic conditions in "1.2". Calculate the RSD value of the peak area of ​​each compound in the 6 determinations to evaluate the precision of the liquid chromatography determination. The results are shown in Table 4.

[0086] 2.2 Stability Study: Accurately pipette the mixed standard solution (number "3") under the preparation section of "1.1" for the reference standard mixed solution, and measure the solution at 0, 2, 24, 26, 48, and 56 h using the chromatographic conditions described in "1.2". Calculate the RSD of the peak area to examine the stability of the assay method within 56 h. The RSD value of the peak area is used as the evaluation index. The results are shown in Table 4.

[0087] Table 4. RSD values ​​for precision and stability studies of liquid chromatography methods

[0088]

[0089] The precision and stability data in Table 4 show that the RSD values ​​of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C are all below 2%, indicating that the instrument precision is good and the mixed solution of reference standards is stable within 56 h.

[0090] 3. Repeatability and recovery experiments

[0091] 3.1 Preparation of the test solution: Accurately weigh 30 mg of honeysuckle powder (passed through a 65-mesh sieve) and place it in a 50 mL Erlenmeyer flask. Accurately add 25 mL of 75% methanol and weigh. Extract by sonication for 30 min. Remove, cool to room temperature, and weigh again. Pipette an appropriate amount and filter through a 0.45 μm microporous membrane, reserving the filtrate for later use.

[0092] 3.2 Repeatability Experiment: Following the preparation method of the test solution in "3.1", six parallel test solutions were prepared from honeysuckle samples of the same batch number. The peak areas of each target component in the six test solutions were determined using the chromatographic conditions in "1.2", and the content and RSD values ​​of the content were calculated. The experimental results are shown in Table 5. The liquid chromatograms of the reference standard and the sample are shown in Table 5. Figure 1 .

[0093] 3.3 Recovery Experiment: Accurately weigh 15 mg of honeysuckle powder and transfer it to a 50 mL stoppered conical flask. Then accurately add 25 mL of a mixed reference solution dissolved in 75% methanol (containing 0.8 μg / mL). -1 Neochlorogenic acid, 19.6 μg·mL -1 Chlorogenic acid, 0.252 μg·mL -1 Cryptochlorogenic acid, 0.157 μg·mL -1 Isochlorogenic acid B, 9.8 μg·mL -1 Isochlorogenic acid A, 0.93 μg·mL -1 Isochlorogenic acid (C). Ultrasonic extraction was performed for 30 min, then removed, cooled to room temperature, and the weight was added. Six parallel treatments were performed. An appropriate amount of the extract was filtered through a 0.45 μm microporous membrane, and the filtrate was collected for analysis. The experimental results are shown in Table 5.

[0094] Table 5. Experimental results on repeatability and recovery rate

[0095]

[0096] The repeatability and recovery data in Table 5 show that the RSD values ​​of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C are all below 4%, indicating that the method of the present invention has good repeatability and accuracy.

[0097] Example 2: Generation of Relative Correction Factor (RCF)

[0098] Accurately pipette the mixed standard solutions numbered "1 to 6" under the preparation of the reference mixed solution in "1.1" of Example 1, inject them into the high-performance liquid chromatograph, and determine them using the chromatographic conditions in "1.2" of Example 1, recording the chromatographic peak areas. Using chlorogenic acid as an internal reference, calculate the correction factor for each component to chlorogenic acid according to formula (1) based on the corresponding chromatographic peak areas of chlorogenic acid at each concentration at a detection wavelength of 350 nm. The correction factors at each concentration are shown in Table 6.

[0099] Table 6. RCF of each reference standard at different concentrations

[0100]

[0101] The results showed that, within the linear range, the relative standard deviation (RSD) of the RCF values ​​of the reference standards for each compound at different concentrations was less than 2.58%. This indicates that, under the defined chromatographic conditions, within the linear range, the RCF values ​​of each compound fluctuated minimally with changes in concentration, meeting the technical requirements for quantitative calculations.

[0102] In summary, the method for simultaneous determination of six components in honeysuckle established in this invention has, through linear range testing in this embodiment, determined the linear range of each component; through precision testing, confirmed the precision of the method; through stability testing, determined the accurate time range for determination of each component; and through repeatability and recovery testing, proved the accuracy of the determination method. The research content of this embodiment confirms that the HPLC method for simultaneous determination of six components in honeysuckle established in this invention is accurate and reliable. Furthermore, based on the peak area of ​​each reference standard at different concentrations, the relative correction coefficients (RCF) of five representative chlorogenic acid components in honeysuckle with chlorogenic acid as an internal reference can be obtained according to formula (1). In addition, within the linear range determined in this embodiment, the RSD value of the RCF calculated from the reference standards at different concentrations is less than 4.53%, indicating that the change in the concentration of the reference standards has little effect on the RCF value.

[0103] Example 3: Robustness of Relative Correction Factor

[0104] Based on Example 2, this example investigated the effects of different chromatographic columns, flow rates, and column temperatures on the correction factor.

[0105] 1.1 The effect of different chromatographic columns on the correction factor

[0106] In this embodiment, the mixed standard solution numbered "3" under the preparation of the reference standard mixed solution in "1.1" of Example 1 was precisely pipetted and measured using Diamonsil C18 (2), Kromasil C18, and Elite Hypersil C18 columns, respectively, according to the chromatographic conditions in "1.2" of Example 1. The correction factor for each reference standard was calculated according to the correction factor calculation formula in Example 3. The effect of different chromatographic columns on the correction factor was investigated. The results are shown in Table 7.

[0107] Table 7. Effect of different chromatographic columns on correction factors

[0108]

[0109] The results showed that the RCF values ​​remained stable using different chromatographic columns; the RSD values ​​of the RCF were all less than 3.15%, indicating that different chromatographic columns had no significant effect on the RCF values.

[0110] 1.2 Effects of different flow rates and column temperatures on the correction factor

[0111] In this embodiment, the mixed standard solution numbered "3" under the preparation of the reference standard mixed solution in "1.1" of Example 1 was accurately pipetted and determined using the chromatographic conditions in "1.2" of Example 1. The results were compared when the mobile phase flow rate was 0.8 ml·min. -1 1.0 ml·min -1 The changes in RCF values ​​under the specified conditions were investigated, and the changes in RCF values ​​at column temperatures of 35 ℃ and 38 ℃ were compared. The results are shown in Table 8.

[0112] Table 8. RCF values ​​obtained under different flow rates and column temperatures.

[0113]

[0114] The results showed no significant difference in RCF values ​​when using different flow rates and column temperatures; at 1.0 and 0.8 ml·min -1 The RCF deviations of several chemical components obtained at different flow rates were less than 2.2%, indicating that the optimal flow rate is within the range of 0.8–1.0 ml·min. -1 Within the flow rate range, the RCF value has little effect; within the column temperature range of 35~38 ℃, the RCF value deviation of each compound is less than 4.5%, and the column temperature has little effect on the RCF.

[0115] 1.3 Determination of Relative Retention Time

[0116] Chromatographic peak localization is performed using relative retention time. The formula (3) for calculating relative retention time is as follows:

[0117]

[0118] Among them, t s t represents the retention time of chlorogenic acid. i It is the retention time of other chemical components, t i / s These are relative retention times. The relative retention times of each component with chlorogenic acid were investigated using different chromatographic columns. The results are shown in Table 9.

[0119] Table 9. Calculation results of relative retention values ​​of each component using different chromatographic columns.

[0120]

[0121] In summary, using chromatographic columns from three different manufacturers, the effects of different columns on RCF values ​​were compared. It was found that, based on the established liquid chromatography method, the RSD value of RCF values ​​obtained under different column conditions was less than 3.15%, indicating that different columns have no significant effect on RCF values. Furthermore, fluctuations in column temperature and flow rate also had no significant impact on RCF values.

[0122] Example 4: Determination of Sample Content

[0123] 1. Sample collection

[0124] (1) Six batches of honeysuckle medicinal materials were collected from Pingyi County and Jining County, Shandong Province.

[0125] (2) Six batches of samples of golden chrysanthemum and honeysuckle sold in Gansu, Henan and Hebei provinces were collected.

[0126] 2. Sample solution preparation method: Accurately weigh 30 mg of honeysuckle powder (passed through a 65-mesh sieve) and place it in a 50 mL Erlenmeyer flask. Accurately add 25 mL of 75% methanol and weigh. Extract by sonication for 30 min. Remove, cool to room temperature, and weigh again. Take an appropriate amount and filter through a 0.45 μm microporous membrane, reserving the filtrate for later use.

[0127] 3. Liquid chromatography conditions: A Shimadzu LC-20A high-performance liquid chromatograph (Shimadzu Corporation, Japan) was used with a Diamonsil C18 (2) column (4.6 mm × 250 mm, 5 μm). Gradient elution was performed using an elution system consisting of acetonitrile (A) and 0.3% formic acid water (B). The gradient conditions are shown in Table 1. The column temperature was 38 ℃, and the flow rate was 0.8 ml·min. -1 Injection volume: 20 μL. Chromatographic data were recorded using UV full-wavelength (wavelength range 200 ~ 400 nm) monitoring mode, and the peak area of ​​each compound was extracted at 350 nm.

[0128] 4. Content determination results

[0129] (1) Calculation of content using the external standard method: The content of each component in the sample was calculated using the concentration of the reference standard in Table 10 and the peak area of ​​the chromatographic peak according to the following formula:

[0130]

[0131] Wherein, A sample: the chromatographic peak area of ​​the sample; C standard: the concentration of the reference standard of the analyte; A standard: the peak area of ​​the reference standard of the analyte; weighing: the sample weight.

[0132] (2) RCF method to calculate content: In the absence of reference standards for other components besides chlorogenic acid, the content of five compounds, including neochlorogenic acid and cryptochlorogenic acid, is calculated by RCF method using formula (2).

[0133] The contents of each component in the sample calculated using the external standard method and the RCF method are shown in Table 11-12.

[0134] Table 10. Concentration, peak area, and RCF of reference standards used in sample content determination.

[0135]

[0136] Table 11. Determination of the content of various components in honeysuckle medicinal materials from Shandong Province (Unit: mg / g)

[0137]

[0138] Table 12. Determination of the content of various components in honeysuckle medicinal materials from Gansu, Henan and Hebei provinces (Unit: mg / g)

[0139]

[0140] This embodiment measures the content of each component in eight batches of honeysuckle medicinal materials from Shandong's authentic producing areas, as well as commercially available honeysuckle slices from Shandong, Henan, and Hebei provinces, comparing the results of the classic external standard method and the RCF method (Tables 11 and 12). The results show that the contents of the five components in the honeysuckle medicinal materials and slices determined by the two methods are similar, or even the same, demonstrating the feasibility of the RCF method.

[0141] In summary, this invention solves the bottleneck problem of quantifying these components in honeysuckle when reference standards for other components are lacking by establishing a relative correction factor between the commonly used reference standard chlorogenic acid and five other chlorogenic acid-like components, while also reducing the cost of purchasing reference standards.

[0142] The specific embodiments described in this application are merely illustrative of the invention and are not intended to limit it. Those skilled in the art can make modifications to these embodiments without contributing any inventive step after reading this specification, but such modifications are protected by patent law as long as they fall within the scope of the claims of this invention.

Claims

1. A method for quality control of honeysuckle through multiple evaluations and one test, characterized in that, Includes the following steps: S1. Preparation of reference solution Accurately weigh chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C reference standards, dissolve them to obtain single reference standard solutions of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C, and mix the single reference standard solutions to obtain the reference standard mixed solution. S2. Preparation of the test solution The honeysuckle was crushed, sieved, and honeysuckle powder was obtained. The honeysuckle powder was accurately weighed, added to a solvent, and extracted by ultrasonication. The mixture was then filtered to obtain the test solution. S3. Set chromatographic conditions C18 column; column temperature: 35~38 ℃; flow rate: 0.8~1.0 ml·min -1 UV wavelength: 200~400 nm; Mobile phase: Gradient elution was performed using acetonitrile as mobile phase A and 0.3% formic acid water as mobile phase B. The gradient elution program is as follows: S4. Content determination Using chlorogenic acid as an internal reference, the relative correction factor coefficients of chlorogenic acid with neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C were determined. The chlorogenic acid reference solution and the test solution were accurately pipetted into the high-performance liquid chromatograph, and the chromatographic peak areas were recorded under the chromatographic conditions of step S3. The contents of neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C were calculated using the established relative correction factors.

2. The honeysuckle quality control method with one test and multiple evaluations according to claim 1, characterized in that, The preparation of the reference solution specifically includes the following steps: S11. Accurately weigh chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C reference standards, and place them in volumetric flasks respectively. S12. Dissolve in an appropriate amount of methanol solution, and make up to volume to obtain single reference solutions; the concentrations of single reference solutions of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C are 0.035~1.75 mg / ml, 0.002~0.11 mg / ml, 0.002~0.081 mg / ml, 0.002~0.11 mg / ml, 0.006~0.29 mg / ml and 0.002~0.09 mg / ml, respectively. S13. Accurately pipette each single reference solution, mix and shake well, dilute with methanol, and prepare mixed reference solutions of different concentrations.

3. The honeysuckle quality control method with one test and multiple evaluations according to claim 2, characterized in that, The concentrations of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C in the reference standard mixed solution were 8.59~291.6 μg / ml, 0.47~18.33 μg / ml, 0.36~15.00 μg / ml, 0.47~18.33 μg / ml, 1.41~48.33 μg / ml, and 0.36~15.00 μg / ml, respectively.

4. The honeysuckle quality control method with one test and multiple evaluations according to claim 1, characterized in that, The preparation of the test solution specifically includes the following steps: S21. Crush the honeysuckle flowers, sieve them, and obtain honeysuckle powder. S22. Accurately weigh the honeysuckle powder and place it in an Erlenmeyer flask. Add solvent and weigh it. S23. Ultrasonic extraction for 30-40 minutes, followed by cooling and reweighting. S24. Filter through a 0.45 μm microporous membrane and collect the filtrate for later use.

5. The honeysuckle quality control method with one test and multiple evaluations according to claim 1, characterized in that, The honeysuckle has a mass of 30-60 mg, and the mass-to-volume ratio of the honeysuckle to the solvent is 6 mg: 5-7 ml.

6. The honeysuckle quality control method with one test and multiple evaluations according to claim 5, characterized in that, The solvent is a methanol solution with a volume fraction of 70% to 80%.

7. The honeysuckle quality control method with one test and multiple evaluations according to claim 1, characterized in that, The sieve mesh size is 60-80 mesh.

8. The honeysuckle quality control method with one test and multiple evaluations according to claim 1, characterized in that, The method for determining the relative correction factor coefficients of neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C is as follows: accurately pipette the reference solution from step S1 and inject it into the high-performance liquid chromatograph. Under the chromatographic conditions of step S3, record the chromatographic peak area. Using chlorogenic acid as an internal reference, calculate the relative correction factors of neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C based on the chromatographic peak areas of neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C presented in the characteristic chromatogram of the test solution. Formula (1) is as follows: Where Ci is the mass concentration of other components, Ai is the peak area of ​​other components; Cs is the mass concentration of internal standard chlorogenic acid, and As is the peak area of ​​chlorogenic acid.

9. The honeysuckle quality control method with one test and multiple evaluations according to claim 8, characterized in that, The C18 column is a Diamonsil C18 (2) column: 4.6 mm × 250 mm, 5 μm or a Kromasil C18 column: 4.6 mm × 250 mm, 5 μm or an Elite Hypersil C18 column: 4.6 mm × 250 mm, 5 μm.

10. The honeysuckle quality control method with one test and multiple evaluations according to claim 1, characterized in that, The column temperature was 38 °C; the flow rate was 0.8 ml·min. -1 The detection wavelength is 350nm.

11. A method for quality control of honeysuckle with one test and multiple evaluations according to claim 1 or 8, characterized in that, The injection volume is 10-20 μL.

12. The honeysuckle quality control method with one test and multiple evaluations according to claim 1, characterized in that, The calculation formula (2) for the contents of neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C is as follows: Wherein, RCF is the relative correction coefficient between each component and chlorogenic acid; A sample is the peak area of ​​the component to be tested in the sample; C green: the concentration of chlorogenic acid reference standard; A green: the peak area of ​​chlorogenic acid reference standard; weighing: the sample weight.

13. The application of the honeysuckle one-test-multiple-evaluation quality control method as described in any one of claims 1-12 in the detection of the content of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C in honeysuckle.

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