A method for constructing UPLC feature maps of heterogeneous particles and its application

Abstract

This invention discloses a method for constructing UPLC characteristic chromatograms of heterotrophic particles and its application, belonging to the field of pharmaceutical analysis technology. The construction method includes the following steps: precisely pipetting a mixed reference solution and a test solution, and determining them using ultra-high performance liquid chromatography (UHPLC) to establish UPLC characteristic chromatograms of heterotrophic particles; the UHPLC detection conditions include: mobile phases comprising mobile phase A and mobile phase B, with acetonitrile as mobile phase A and 0.08–0.15 wt% phosphoric acid aqueous solution as mobile phase B, performing gradient elution, and a detection wavelength of 199–210 nm. This invention establishes 19 characteristic peaks in heterotrophic particles, which can comprehensively and characteristically reflect the composition of heterotrophic particles, suitable for the quality control of heterotrophic particles. This invention can also detect the content of six components: isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rb2, with high detection efficiency, good repeatability, accuracy, precision, and stability.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a method for constructing UPLC characteristic maps of heterogeneous particles and its application. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Yigong granules are a traditional Chinese medicine compound granule composed of seven medicinal materials: ginseng, poria cocos, atractylodes macrocephala, tangerine peel, licorice, ginger, and jujube. It is mainly used for children with indigestion due to spleen deficiency and qi stagnation, and its efficacy is definite. However, its chemical composition is complex, and the existing quality standards are difficult to effectively control the intrinsic quality of the drug.

[0004] Patent CN 114216986B (authorization announcement date: April 7, 2023) proposes a method for constructing HPLC characteristic chromatograms of Yigongsan (a type of granulated powder), its HPLC standard fingerprint chromatogram, and its application. It synthesizes HPLC characteristic chromatograms of 15 batches of Yigongsan, generating an HPLC standard fingerprint chromatogram of Yigongsan at a wavelength of 203 nm, consisting of 8 characteristic peaks. However, this patent has relatively few characteristic peaks and a long detection time of 60 minutes, making it difficult to efficiently and comprehensively control the intrinsic quality of Yigongsan in the production of granulated powder. Summary of the Invention

[0005] In view of this, the present invention provides a method for constructing a UPLC feature map of heterogeneous particles and its application. The feature map established by the present invention is used for the quality detection of heterogeneous particles, which has good precision, stability and repeatability, and can comprehensively and effectively evaluate the quality of heterogeneous particles.

[0006] In a first aspect, the present invention provides a method for constructing a UPLC feature map of heterogeneous particles, comprising the following steps:

[0007] Accurately pipette the mixed reference solution and the test solution separately, and determine them by ultra-high performance liquid chromatography (UPLC) to establish a characteristic UPLC spectrum of heterogeneous particles.

[0008] The conditions for detection by ultra-high performance liquid chromatography include:

[0009] The mobile phase includes mobile phase A and mobile phase B. Acetonitrile is used as mobile phase A, and 0.08-0.15 wt% aqueous phosphoric acid solution is used as mobile phase B. Gradient elution is performed, and the detection wavelength is 199-210 nm.

[0010] The gradient elution procedure is as follows:

[0011] 0–8 min, mobile phase A: 19 wt%, mobile phase B: 81 wt%;

[0012] 8–16 min, mobile phase A: 19 wt% → 23 wt%, mobile phase B: 81 wt% → 77 wt%;

[0013] 16–34 min, mobile phase A: 23 wt% → 40 wt%, mobile phase B: 77 wt% → 60 wt%;

[0014] The specific method for preparing the mixed reference solution is as follows:

[0015] Accurately weigh the reference standards of isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rb2, and dissolve them in a solvent to prepare a mixed reference solution.

[0016] Preferably, the test solution is prepared by: grinding the heterogeneous particles into a fine powder, adding methanol, ultrasonicating, cooling, replenishing the weight loss with methanol, shaking well, and filtering with a filter membrane to obtain the test solution. Ultrasonic treatment helps to uniformly disperse the heterogeneous particles in methanol; however, the ultrasonic treatment process involves temperature increases, methanol volatilization, and increased concentration, leading to higher errors. Therefore, adding methanol after cooling reduces the impact of methanol volatilization and minimizes measurement errors.

[0017] Furthermore, the ultrasonic treatment time is 20-40 min; the preferred ratio of the mass (g) of the test sample raw material to the volume (mL) of the solvent is (0.5-1.5):(20-30).

[0018] Preferably, the chromatographic column is a Waters ACQUITY UPLC HSS T3 (100*2.1mm, 1.8μm), and the column temperature is 33-37℃.

[0019] Preferably, the solvent for the mixed reference solution is methanol. Methanol has better solubility, which is beneficial for the dispersion of each compound, improves the detection efficiency of the detector, and helps to improve the stability of the detection.

[0020] Preferably, the flow rate of the mobile phase is 0.2–0.4 mL / min, and the injection volume is 2–4 μL.

[0021] Preferably, in the mixed reference solution, the concentrations of isoglycyrrhizin are 40–60 μg / mL, glycyrrhizin is 60–80 μg / mL, hesperidin is 40–60 μg / mL, ginsenoside Re is 220–240 μg / mL, ginsenoside Rb1 is 190–210 μg / mL, and ginsenoside Rb2 is 210–230 μg / mL.

[0022] Preferably, the process of establishing the characteristic spectrum is as follows: ultra-high performance liquid chromatography (UHPLC) is performed on different batches of heterogeneous particles, and the UHPLC chromatograms of different batches of heterogeneous particles are compared to obtain common peaks containing glycyrrhizin, isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, and ginsenoside Rb2 characteristic peaks. The characteristic spectrum of heterogeneous particles is constructed based on the common peaks.

[0023] Furthermore, the confirmation process for the characteristic peaks of glycyrrhizin, isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, and ginsenoside Rb2 is as follows: A series of reference solutions of different concentrations are prepared for each of the following: glycyrrhizin, isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, and ginsenoside Rb2. Ultra-high performance liquid chromatography (UHPLC) is used to detect the glycoparticles and the reference solutions, respectively. The corresponding characteristic peaks of the glycoparticles are determined based on the peak retention times of the reference solutions in UHPLC.

[0024] The characteristic spectrum obtained according to the above construction method of the present invention contains 19 characteristic peaks. Using glycyrrhizin (peak 8) as the reference peak S, the relative retention times of each characteristic peak and peak S are calculated. The relative retention times should be within ±10% of a specified value. The specified values ​​are: 0.245 (peak 1), 0.267 (peak 2), 0.384 (peak 3), 0.550 (peak 4), 0.623 (peak 5), 0.729 (peak 6), 0.898 (peak 7), 1.000 (peak S), 1.208 (peak 9), and 1.235 (peak 10). The peak values ​​were 1.309 (peak 11), 1.509 (peak 12), 1.563 (peak 13), 1.755 (peak 14), 1.773 (peak 15), 1.980 (peak 16), 2.016 (peak 17), 2.081 (peak 18), and 2.136 (peak 19). Among these, peak 2 is glycyrrhizin, peak 4 is hesperidin, peak 7 is isoglycyrrhizin, peak S is glycyrrhizin, peak 9 is ginsenoside Rg1, peak 10 is ginsenoside Re, peak 15 is ginsenoside Rf, peak 17 is ginsenoside Rb1, and peak 19 is ginsenoside Rb2.

[0025] Secondly, the present invention provides the application of the above-mentioned method for constructing UPLC feature maps of heterogeneous particles in the quality control of heterogeneous particles. The UPLC feature maps of heterogeneous particles are obtained according to the construction method. The similarity between the feature map of the sample to be tested and the UPLC feature map of the heterogeneous particles is evaluated. The sample to be tested with a relative retention time within ±10% of the specified value is a qualified product.

[0026] Thirdly, the present invention provides a method for detecting six chemical components in heterogeneous particles, comprising the following steps:

[0027] The test solution was analyzed by ultra-high performance liquid chromatography to obtain the content of six chemical components, which include isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Re, ginsenoside Rb1 and ginsenoside Rb2.

[0028] The conditions for detection by ultra-high performance liquid chromatography include:

[0029] The mobile phase includes mobile phase A and mobile phase B, with acetonitrile as mobile phase A and 0.08–0.15 wt% aqueous phosphoric acid solution as mobile phase B, for gradient elution;

[0030] The gradient elution procedure is as follows:

[0031] 0–8 min, mobile phase A: 19 wt%, mobile phase B: 81 wt%;

[0032] 8–16 min, mobile phase A: 19 wt% → 23 wt%, mobile phase B: 81 wt% → 77 wt%;

[0033] 16–34 min, mobile phase A: 23 wt% → 40 wt%, mobile phase B: 77 wt% → 60 wt%;

[0034] The ultra-high performance liquid chromatography method used a Waters ACQUITY UPLC HSS T3 column (100*2.1mm, 1.8μm), with a column temperature of 33–37℃; a mobile phase flow rate of 0.2–0.4 mL / min; and an injection volume of 2–4 μL.

[0035] When the detection wavelength is 199–210 nm, the contents of ginsenoside Re, ginsenoside Rb1, and ginsenoside Rb2 are detected simultaneously; when the detection wavelength is 230–240 nm, the contents of isoglycyrrhizin, glycyrrhizin, and hesperidin are detected simultaneously.

[0036] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0037] (1) By selecting the mobile phase, the present invention enables better separation of more chemical components of heterogeneous particles and establishes 19 characteristic peaks in the UPLC spectrum of heterogeneous particles, which is more than the number of characteristic peaks in the prior art. It can more comprehensively and characteristically reflect the composition of heterogeneous particles, which is conducive to comprehensive quality control.

[0038] (2) The quality detection method provided by the present invention has good precision, stability and repeatability, while shortening the detection time of a single sample to within 34 minutes, and can efficiently and accurately control the quality of heterogeneous particles.

[0039] (3) This invention can not only establish characteristic spectra using nine components, namely glycyrrhizin, isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, and ginsenoside Rb2, as common peaks, but also simultaneously detect the content of three components, namely ginsenoside Re, ginsenoside Rb1, and ginsenoside Rb2. Furthermore, it can detect the content of isoglycyrrhizin, glycyrrhizin, and hesperidin under conditions of low interference. The detection efficiency is high, and the detection method has good repeatability, accuracy, precision, and stability, and exhibits good linearity within the detection range. Attached Figure Description

[0040] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0041] Figure 1 This is a comparison of ultra-high performance liquid chromatography (UHPLC) images of the test solution of the heterogeneous particles (bottom) and the mixed reference solution (top) of Example 1 of the present invention at a detection wavelength of 203 nm. In the figure, a, glycyrrhizin; b, hesperidin; c, isoglycyrrhizin; d, glycyrrhizin; e, ginsenoside Rg1; f, ginsenoside Re; g, ginsenoside Rf; h, ginsenoside Rb1; i, ginsenoside Rb2.

[0042] Figure 2 This is a comparison of ultra-high performance liquid chromatography (UHPLC) images of the test solution of the heterogeneous particles (bottom) and the mixed reference solution (top) of Example 1 of the present invention at a detection wavelength of 237 nm. In the figure, a is glycyrrhizin, b is hesperidin, c isoglycyrrhizin, and d is glycyrrhizin.

[0043] Figure 3 This is the standard UPLC characteristic spectrum of heterogeneous particles in Embodiment 1 of the present invention;

[0044] Figure 4 This is the ultra-high performance liquid chromatography (UHPLC) spectrum of Comparative Example 1 of this invention; in the figure, 1 represents glycyrrhizin, and 2 represents hesperidin.

[0045] Figure 5 This is the ultra-high performance liquid chromatography (UHPLC) spectrum of Comparative Example 2 of this invention; in the figure, 1 represents glycyrrhizin, and 2 represents hesperidin.

[0046] Figure 6This is the ultra-high performance liquid chromatography spectrum of Comparative Example 3 of this invention;

[0047] Figure 7 This is the ultra-high performance liquid chromatography (UHPLC) spectrum of Comparative Example 4 of this invention; in the spectrum, 1 represents glycyrrhizin, and 2 represents hesperidin.

[0048] Figure 8 This is the ultra-high performance liquid chromatography (UHPLC) spectrum of Comparative Example 5 of this invention; in the figure, 1 represents ginsenoside Rg1, and 2 represents ginsenoside Re. Detailed Implementation

[0049] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, 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 invention pertains.

[0050] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0051] Example 1

[0052] Instruments: Waters H-class ultra-high performance liquid chromatograph; 0.0001 g electronic balance, Mettler Toledo Instruments (Shanghai) Co., Ltd.; KQ-300DE CNC ultrasonic cleaner.

[0053] Test reagents: glycyrrhizin, isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, and ginsenoside Rb2. Reference standards were purchased from the China National Institutes for Food and Drug Control. Yigong granules were developed by Jinan Tonglu Pharmaceutical Co., Ltd., batch number 23040301. Methanol, acetonitrile, phosphoric acid, and water were all of chromatographic purity.

[0054] Chromatographic conditions: Waters ACQUITY UPLC HSS T3 (100*2.1mm, 1.8μm); mobile phase acetonitrile (A)-0.1% phosphoric acid (B), gradient elution sequence: 0–8 min, mobile phase A: 19 wt%, mobile phase B: 81 wt%; 8–16 min, mobile phase A: 19 wt% → 23 wt%, mobile phase B: 81 wt% → 77 wt%; 16–34 min, mobile phase A: 23 wt% → 40 wt%, mobile phase B: 77 wt% → 60 wt%; column temperature 35℃; injection volume 3 μL; flow rate 0.3 mL / min.

[0055] Preparation of test solution:

[0056] Take the granules, grind them into a fine powder, weigh about 2g accurately, place them in a stoppered conical flask, add 50mL of methanol, sonicate for 30min, cool, replenish the lost weight with methanol, shake well, filter, and collect the filtrate to obtain the final product.

[0057] Preparation of mixed reference solution:

[0058] Accurately weigh the reference standards of glycyrrhizin, isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, and ginsenoside Rb2, and prepare a mixed reference solution with methanol.

[0059] Accurately pipette 3 μL each of the test sample and the mixed reference solution into the liquid chromatograph and determine the chromatogram; the chromatogram obtained at a detection wavelength of 203 nm is shown in the figure below. Figure 1 As shown, at a detection wavelength of 203 nm, the chemical composition information is abundant, and 19 common peaks can be obtained. Furthermore, the separation degree of ginsenoside Re, ginsenoside Rb1, and ginsenoside Rb2 is greater than 1.5, allowing for the simultaneous determination of the content of these three components.

[0060] The spectrum obtained at a detection wavelength of 237 nm is as follows Figure 2 As shown, isoglycyrrhizin, glycyrrhizin, and hesperidin have absorption peaks at a wavelength of 237 nm, and the separation is good with no interference from impurities. Therefore, 237 nm can be selected as the wavelength for determining the content of these components.

[0061] Establishment of the characteristic spectrum of heterogeneous particles:

[0062] Common peak identification: Ten batches of samples of Yigong Granules with batch numbers 23040301, 23040302, 23040401, 23040402, 23040601, 23040602, 23040701, 23040702, 23041001, and 23041002 were determined according to the chromatographic conditions specified in the technical scheme. The chromatograms at 203 nm were imported into the "Similarity Evaluation System for Chromatographic Characteristic Magnetism of Traditional Chinese Medicine" to generate common patterns of characteristic chromatograms, such as... Figure 3 As shown in Table 1, sample S1, which has more chemical composition information, was set as the reference spectrum. 19 common peaks were identified. Peak 8 was used as the reference peak S. The relative retention times of other common peaks and peak S were calculated.

[0063] Table 1 Relative Retention Time

[0064]

[0065]

[0066] Similarity evaluation: The similarity of the above 10 batches of Yigong granules was evaluated using the "Similarity Evaluation System for Chromatographic Characteristic Magnetism of Traditional Chinese Medicine". The similarity evaluations of S1-S10 are shown in Table 2. The similarity results show that the similarity between the 10 batches of Yigong granules and the control chromatogram is above 0.9 (see Table 2), indicating that the 10 batches of samples have good similarity.

[0067] Table 2 Similarity Evaluation of S1-S10

[0068] S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Comparison S1 1 S2 0.951 1 S3 0.922 0.943 1 S4 0.907 0.974 0.979 1 S5 0.933 0.955 0.983 0.998 1 S6 0.939 0.931 0.995 0.987 0.998 1 S7 0.923 0.982 0.993 0.988 0.992 0.996 1 S8 0.943 0.926 0.998 0.992 0.978 0.984 0.996 1 S9 0.910 0.962 0.960 0.998 0.989 0.989 0.992 0.993 1 S10 0.925 0.920 0.977 0.993 0.988 0.998 0.994 0.997 0.989 1 Comparison 0.924 0.955 0.989 0.995 0.993 0.997 0.993 0.992 0.991 0.998 1

[0069] Correlation between Yigong Granules and various medicinal materials:

[0070] Characteristic chromatogram analysis was performed on a negative sample of Yigong Granules (containing no flavor), and single-herb samples of ginseng, poria cocos, atractylodes macrocephala, tangerine peel, licorice, ginger, and jujube. Peaks 9, 10, 15, 17, 18, and 19 were found to originate from ginseng; peaks 3, 4, 5, 6, 11, 12, 13, and 14 from tangerine peel; and peaks 1, 2, 7, 8, 13, and 16 from licorice. Comparison with a mixed reference solution identified peak 2 as glycyrrhizin; peak 4 as hesperidin; peak 7 as isoglycyrrhizin; peak 8 as glycyrrhizin; peak 9 as ginsenoside Rg1; peak 10 as ginsenoside Re; peak 15 as ginsenoside Rf; peak 17 as ginsenoside Rb1; and peak 19 as ginsenoside Rb2.

[0071] Sample content determination:

[0072] Ten batches of ginsenoside Rb1, Rb2, and ginsenoside Rb2 were measured using the method described above at a detection wavelength of 203 nm. The contents of isoglycyrrhizin, glycyrrhizin, and hesperidin were also measured at a detection wavelength of 237 nm. The results are shown in Table 3.

[0073] Table 3. Results of sample content determination (unit: %)

[0074]

[0075] Methodological examination:

[0076] Feature map precision:

[0077] Weigh 2g of the Yigong granules and determine them according to the above method. Inject the sample six times consecutively and record the characteristic chromatograms. Using the retention time of glycyrrhizin (peak 8) as a reference, calculate the relative retention time of each common peak (see Table 4). The relative retention time of each common peak is less than 3%, indicating good precision.

[0078] Table 4 Relative Retention Time

[0079]

[0080] Feature map repeatability:

[0081] Weigh 2g of the Yigong granules and prepare 6 samples in parallel according to the above method. Record the characteristic spectra. Using the retention time of glycyrrhizin (peak 8) as a reference, calculate the relative retention time of each common peak (see Table 5 below). The relative retention time of each common peak is less than 3%, indicating good repeatability.

[0082] Table 5 Relative Retention Time

[0083]

[0084] Feature map stability:

[0085] Weigh 2g of the granules and prepare them according to the above method. Inject the sample at 0h, 2h, 4h, 8h, 12h, and 24h after preparation, and record the characteristic chromatograms. Using the retention time of glycyrrhizin (peak 8) as a reference, calculate the relative retention time of each common peak (see Table 6). The relative retention time of each common peak is less than 3%, indicating that the test solution has good stability within 24h.

[0086] Table 6 Relative Retention Time

[0087]

[0088] Linearity of content determination:

[0089] Take an appropriate amount of isoglycyrrhizin reference standard, dissolve it in methanol to prepare a reference standard solution of 60.32 μg / mL, and then dilute it with methanol to prepare reference standard dilutions with concentrations of 30.16, 15.08, 7.54 and 3.77 μg / mL, respectively.

[0090] Take an appropriate amount of glycyrrhizin reference standard, dissolve it in methanol to prepare a reference standard solution of 72.64 μg / mL, and then dilute it with methanol to prepare reference standard dilutions with concentrations of 36.32, 18.16, 9.08, and 4.54 μg / mL, respectively.

[0091] Take an appropriate amount of hesperidin reference standard, dissolve it in methanol to prepare a reference standard solution of 437.44 μg / mL, and then dilute it with methanol to prepare reference standard dilutions with concentrations of 218.72, 109.36, 54.68 and 27.34 μg / mL, respectively.

[0092] Take an appropriate amount of ginsenoside Re reference standard, dissolve it in methanol to prepare a reference standard solution of 429.76 μg / mL, and then dilute it with methanol to prepare reference standard dilutions with concentrations of 214.88, 107.44, 53.72 and 26.86 μg / mL, respectively.

[0093] Take an appropriate amount of ginsenoside Rb1 reference standard, dissolve it in methanol to prepare a reference standard solution of 403.04 mg / mL, and then dilute it with methanol to prepare reference standard dilutions with concentrations of 201.52, 100.76, 50.38, and 25.19 μg / mL, respectively.

[0094] Take an appropriate amount of ginsenoside Rb2 reference standard, dissolve it in methanol to prepare a reference standard solution of 457.44 mg / mL, and then dilute it with methanol to prepare reference standard dilutions with concentrations of 228.72, 114.36, 57.18, and 28.59 μg / mL, respectively.

[0095] Take 3 μL of each reference solution and determine them according to the above chromatographic method. Perform linear regression on the concentration and peak area of ​​each reference solution to obtain the regression equation, as shown in Table 7. The linear correlation coefficient r of the six components is greater than 0.999, indicating a good linear relationship within the determination range.

[0096] Table 7. Linear equations, correlation coefficients, and linear ranges for the six components.

[0097] name Linear equations r Linear range (μg) Isoliquiritin y = 23317x - 5381.3 1 0.011～0.181 glycyrrhizin y = 60798x - 92111 0.9999 0.014～0.218 hesperidin y = 12334x - 2015.8 1 0.082～1.312 Ginsenoside Re y = 2888.1x - 6063 1 0.081～1.289 <![CDATA[Ginsenoside Rb1]]> y = 2445.9x - 8434.9 1 0.076～1.209 <![CDATA[Ginsenoside Rb2]]> y = 2052x + 3995.1 0.9999 0.086～1.372

[0098] Precision of content determination

[0099] A mixed reference solution of six components—isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rb2—was prepared and analyzed using the method described above. Six consecutive injections were performed, and the peak areas of the six components were measured. An RSD of less than 3% for the peak areas of the six components indicates good precision.

[0100] Table 8. Peak areas and RSDs of the six components

[0101]

[0102]

[0103] Content determination repeatability

[0104] Weigh 2g of Yigong granules and prepare 6 samples in parallel according to the above method. Measure the peak area of ​​the 6 components and calculate their content (%). The RSD of the contents of the 6 components is less than 3%, indicating good repeatability.

[0105] Table 9. Content and RSD of the 6 components

[0106]

[0107] Content determination stability

[0108] Weigh 2g of Yigong granules and prepare them according to the above method. Inject the sample at 0h, 2h, 4h, 8h, 12h and 24h after sample preparation and measure the peak area of ​​the six components. The RSD of the peak area of ​​the six components at different measurement times is less than 3%, indicating that the test solution has good stability within 24h.

[0109] Table 10 Peak areas of the six components at different determination times

[0110]

[0111] Content determination accuracy

[0112] Six 1g portions of the isoglycyrrhizin granules were weighed, and reference standards with approximately the same content as those in the sample (isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rb2) were added to each portion. The chromatographic peak areas of the six compounds (isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rb2) were recorded according to the above method. The contents of the six compounds were calculated, and the recoveries and RSDs of each compound were also calculated. The results are shown in Tables 11-16. The average recoveries and RSDs of the six components met the requirements, indicating that the method is accurate.

[0113] Table 11 Results of isoliquiritigenin Recovery

[0114]

[0115] Table 12 Results of Glycyrrhizin Recovery Rate

[0116]

[0117]

[0118] Table 13 Results of Hesperidin Recovery Rate

[0119]

[0120] Table 14 Results of Ginsenoside Re Recovery Rate

[0121]

[0122] Table 15 Results of Ginsenoside Rb1 Recovery Rate

[0123]

[0124] Table 16 Results of Ginsenoside Rb2 Recovery Rate

[0125]

[0126] Comparative Example 1

[0127] Instruments: Waters H-class ultra-high performance liquid chromatograph; 0.0001 g electronic balance, Mettler Toledo Instruments (Shanghai) Co., Ltd.; KQ-300DE CNC ultrasonic cleaner.

[0128] Reagents: glycyrrhizin, isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rb2. Reference standards were purchased from the China National Institutes for Food and Drug Control. Yigong granules were developed by Jinan Tonglu Pharmaceutical, batch number 23040301. Methanol was chromatographically pure; acetonitrile was chromatographically pure; potassium dihydrogen phosphate was analytically pure; phosphoric acid was chromatographically pure; water was ultrapure water.

[0129] Chromatographic conditions: Horizon C18 column (100*2.1mm, 1.6μm); mobile phase: acetonitrile (A) - 0.03mol / L potassium dihydrogen phosphate (pH adjusted to 3 with phosphoric acid) (B); gradient elution sequence: 0–15 min, 17% A; 15–20 min, 17%–22% A; 20–40 min, 22%–40% A; column temperature: 35℃; flow rate: 0.3 mL / min; detection wavelength: 203 nm. Glycyrrhizin (peak 1) and hesperidin (peak 2) showed broadened peak shapes, were not separated, and had excessively long detection times, failing to achieve the goal of rapid detection of characteristic chromatograms. Detection results are as follows... Figure 4 As shown.

[0130] Comparative Example 2

[0131] Instruments: Waters H-class ultra-high performance liquid chromatograph; 0.0001 g electronic balance, Mettler Toledo Instruments (Shanghai) Co., Ltd.; KQ-300DE CNC ultrasonic cleaner.

[0132] Reagents: glycyrrhizin, isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rb2. Reference standards were purchased from the China National Institutes for Food and Drug Control. Yigong granules were developed by Jinan Tonglu Pharmaceutical, batch number 23040301. Methanol was chromatographically pure; acetonitrile was chromatographically pure; potassium dihydrogen phosphate was analytically pure; phosphoric acid was chromatographically pure; water was ultrapure water.

[0133] Chromatographic conditions: Waters ACQUITY UPLC HSS T3 column (100*2.1mm, 1.8μm); mobile phase: acetonitrile (A) - 0.03mol / L potassium dihydrogen phosphate (adjusted to pH 3 with phosphoric acid) (B); gradient elution sequence: 0–15 min, 17% A; 15–20 min, 17%–22% A; 20–40 min, 22%–40% A; column temperature: 35℃; flow rate: 0.3 mL / min; detection wavelength: 203 nm. Detection results are as follows: Figure 5 As shown, this indicates that glycyrrhizin (peak 1) and hesperidin (peak 2) are significantly delayed and not completely separated.

[0134] Comparative Example 3

[0135] Instruments: Waters H-class ultra-high performance liquid chromatograph; 0.0001 g electronic balance, Mettler Toledo Instruments (Shanghai) Co., Ltd.; KQ-300DE CNC ultrasonic cleaner.

[0136] Reagents: glycyrrhizin, isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rb2. Reference standards were purchased from the China National Institutes for Food and Drug Control. Yigong granules were developed by Jinan Tonglu Pharmaceutical, batch number 23040301. Methanol was chromatographically pure; acetonitrile was chromatographically pure; potassium dihydrogen phosphate was analytically pure; phosphoric acid was chromatographically pure; water was ultrapure water.

[0137] Chromatographic conditions: Waters ACQUITY UPLC HSS T3 (100*2.1mm, 1.8μm); mobile phase: acetonitrile (A) - 0.03mol / L potassium dihydrogen phosphate (adjusted to pH 2 with phosphoric acid) (B); gradient elution sequence: 0–15 min, 10% A; 15–20 min, 10%–22% A; 20–40 min, 22%–40% A; column temperature: 35℃; injection volume: 5μl; flow rate: 0.5mL / min; detection wavelength: 203nm. Detection results are as follows: Figure 6 As shown, the peak elution time is later, and all components are clustered together and cannot be separated.

[0138] Comparative Example 4

[0139] Instruments: Waters H-class ultra-high performance liquid chromatograph; 0.0001 g electronic balance, Mettler Toledo Instruments (Shanghai) Co., Ltd.; KQ-300DE CNC ultrasonic cleaner.

[0140] Test reagents: glycyrrhizin, isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, and ginsenoside Rb2. Reference standards were purchased from the China National Institutes for Food and Drug Control. Yigong granules were developed by Jinan Tonglu Pharmaceutical Co., Ltd., batch number 23040301. Methanol, acetonitrile, phosphoric acid, and water were all of chromatographic purity.

[0141] Chromatographic conditions: Waters ACQUITY UPLC HSS T3 (100*2.1mm, 1.8μm); mobile phase acetonitrile (A)-0.1% phosphoric acid (B), gradient elution sequence: 0–6 min, 18% A; 6–10 min, 18%–23% A; 10–20 min, 23%–40% A; column temperature 35℃; injection volume 5 μl; flow rate 0.6 mL / min; detection wavelength 203 nm. Detection results are as follows: Figure 7 As shown, this indicates that glycyrrhizin (peak 1) and hesperidin (peak 2) were interfered with by impurities and were not separated.

[0142] Comparative Example 5

[0143] Instruments: Waters H-class ultra-high performance liquid chromatograph; 0.0001 g electronic balance, Mettler Toledo Instruments (Shanghai) Co., Ltd.; KQ-300DE CNC ultrasonic cleaner.

[0144] Test reagents: glycyrrhizin, isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, and ginsenoside Rb2. Reference standards were purchased from the China National Institutes for Food and Drug Control. Yigong granules were developed by Jinan Tonglu Pharmaceutical Co., Ltd., batch number 23040301. Methanol, acetonitrile, phosphoric acid, and water were all of chromatographic purity.

[0145] Chromatographic conditions: Waters ACQUITY UPLC HSS T3 (100*2.1mm, 1.8μm); mobile phase acetonitrile (A)-0.1% phosphoric acid (B), gradient elution sequence: 0–10 min, 16% A; 10–18 min, 16%–23% A; 18–36 min, 23%–40% A; column temperature 35℃; injection volume 3μl; flow rate 0.3mL / min; detection wavelength 203nm. Detection results are as follows: Figure 8 As shown, this indicates that ginsenoside Rg1 (peak 1) and ginsenoside Re (peak 2) are not completely separated.

[0146] The above description is merely a preferred embodiment of the present invention and is not intended to limit the 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 constructing a UPLC feature map of heterogeneous particles, characterized in that, Includes the following steps: Accurately pipette the mixed reference solution and the test solution separately, and determine them by ultra-high performance liquid chromatography (UPLC) to establish a characteristic UPLC spectrum of heterogeneous particles. The conditions for detection by ultra-high performance liquid chromatography include: The mobile phase consists of mobile phase A and mobile phase B. Acetonitrile is used as mobile phase A, and 0.08-0.15 wt% aqueous phosphoric acid solution is used as mobile phase B. Gradient elution is performed, and the detection wavelength is 199-210 nm. The chromatographic column is a Waters ACQUITY UPLC HSS T3, 100×2.1 mm, 1.8 μm. The gradient elution procedure is as follows: 0~8min, mobile phase A: 19wt%, mobile phase B: 81wt%; 8~16 min, mobile phase A: 19wt%→23wt%, mobile phase B: 81wt%→77wt%; 16~34 min, mobile phase A: 23wt%→40wt%, mobile phase B: 77wt%→60wt%; The specific method for preparing the mixed reference solution is as follows: Accurately weigh the reference standards of glycyrrhizin, isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, and ginsenoside Rb2, and dissolve them in a solvent to prepare a mixed reference solution. The test solution is prepared by grinding the heterogeneous particles into a fine powder, adding methanol, ultrasonic treatment, cooling, replenishing the weight loss with methanol, shaking well, and filtering with a filter membrane to obtain the test solution.

2. The construction method as described in claim 1, characterized in that, The ultrasonic treatment time is 20-40 min; the ratio of the mass of the test sample raw material (g) to the volume of the solvent (mL) is (0.5-1.5):(20-30).

3. The construction method as described in claim 1, characterized in that, The column temperature of the chromatographic column is 33~37℃.

4. The construction method as described in claim 1, characterized in that, The solvent for the mixed reference solution is methanol; the flow rate of the mobile phase is 0.2~0.4 mL / min, and the injection volume is 2~4 μL.

5. The construction method as described in claim 1, characterized in that, The process of establishing the characteristic spectrum is as follows: ultra-high performance liquid chromatography (UHPLC) is used to detect different batches of heterogeneous particles. The UHPLC chromatograms of different batches of heterogeneous particles are compared to obtain common peaks containing glycyrrhizin, isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, and ginsenoside Rb2 characteristic peaks. The characteristic spectrum of heterogeneous particles is constructed based on the common peaks.

6. The construction method as described in claim 5, characterized in that, The confirmation process for the characteristic peaks of glycyrrhizin, isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, and ginsenoside Rb2 is as follows: A series of reference solutions of different concentrations are prepared using glycyrrhizin, isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, and ginsenoside Rb2. Ultra-high performance liquid chromatography (UHPLC) is used to detect the test solution and the reference solution, respectively. The corresponding characteristic peaks of the heterogeneous particles are determined based on the peak retention times of the reference HPLC peaks.

7. The application of the method for constructing UPLC characteristic maps of heterogeneous particles as described in any one of claims 1 to 6 in the quality detection of heterogeneous particles, characterized in that, The UPLC feature map of heterogeneous particles was obtained according to the construction method described above, and the similarity between the feature map of the sample to be tested and the UPLC feature map of heterogeneous particles was evaluated.

8. A method for detecting six chemical components in heterogeneous particles, characterized in that, Includes the following steps: The test solution was analyzed by ultra-high performance liquid chromatography to obtain the content of six chemical components, which include isoglycyrrhizin, glycyrrhizin, hesperidin, ginsenoside Re, ginsenoside Rb1 and ginsenoside Rb2. The preparation method of the test solution is as follows: grind the heterogeneous particles into fine powder, add methanol, sonicate, cool, replenish the weight loss with methanol, shake well, and filter with a filter membrane to obtain the test solution. The conditions for detection by ultra-high performance liquid chromatography include: The mobile phase includes mobile phase A and mobile phase B, with acetonitrile as mobile phase A and 0.08~0.15wt% aqueous phosphoric acid solution as mobile phase B, for gradient elution; The gradient elution procedure is as follows: 0~8min, mobile phase A: 19wt%, mobile phase B: 81wt%; 8~16 min, mobile phase A: 19wt%→23wt%, mobile phase B: 81wt%→77wt%; 16~34 min, mobile phase A: 23wt%→40wt%, mobile phase B: 77wt%→60wt%; The ultra-high performance liquid chromatography method used a Waters ACQUITY UPLC HSS T3 column, 100×2.1mm, 1.8μm, with a column temperature of 33~37℃; the mobile phase flow rate was 0.2~0.4 mL / min, and the injection volume was 2~4μL. When the detection wavelength is 199~210nm, the contents of ginsenoside Re, ginsenoside Rb1 and ginsenoside Rb2 are detected simultaneously; when the detection wavelength is 230~240nm, the contents of isoglycyrrhizin, glycyrrhizin and hesperidin are detected simultaneously.

Images