A method for constructing UPLC quantitative fingerprint of compound Lysimachia christinae granules

Abstract

This invention relates to a method for constructing a quantitative fingerprint spectrum of Compound Lysimachia christinae granules using UPLC, comprising the following steps: (1) preparation of a reference solution; (2) preparation of a test solution; (3) ultra-high performance liquid chromatography analysis; and (4) establishment of a quantitative fingerprint spectrum, by importing multiple batches of liquid chromatograms into a "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System," matching common peaks, generating a reference fingerprint spectrum, and combining this with the determination of the content of multiple indicator components to establish a quantitative fingerprint spectrum of Compound Lysimachia christinae granules. This invention can simultaneously achieve fingerprint spectrum detection and content determination of Compound Lysimachia christinae granules in a single analysis, enabling rapid, comprehensive, and accurate evaluation of the quality of Compound Lysimachia christinae granules, and providing technical support for the evaluation of the effectiveness, stability, and consistency of Compound Lysimachia christinae granules.

Description

Technical Field

[0001] This invention belongs to the field of quality testing technology for traditional Chinese medicine preparations, specifically relating to a method for constructing a UPLC quantitative fingerprint spectrum of compound Lysimachia christinae granules. Background Technology

[0002] Compound Lysimachia christinae granules are a compound traditional Chinese medicine preparation made from four main ingredients: Lysimachia christinae, Plantago asiatica, Pyrrosia lingua, and Zea mays. It is refined through processes such as decoction and concentration. It has the effects of clearing heat and dampness, promoting urination and expelling stones. Clinically, it is often used for urinary tract stones and urinary tract infections. Modern pharmacological studies have also shown that Compound Lysimachia christinae granules have diuretic, urinary tract stone formation inhibition, anti-inflammatory, and antioxidant effects, and can effectively inhibit crystalline kidney damage. Traditional Chinese medicine preparations are multi-component drugs that act on multiple targets in the body to exert therapeutic effects. Comprehensive quality control of traditional Chinese medicine preparations is more conducive to guiding the safety of clinical medication.

[0003] There are many studies on the pharmacological effects and clinical applications of Compound Lysimachia christinae Granules, but few studies on its quality control. The current Chinese Pharmacopoeia (2020 edition) only provides thin-layer qualitative identification of Lysimachia christinae and Pyrrosia lingua in Compound Lysimachia christinae Granules and sets content limits for mangiferin. There are no specific identification methods for Plantago asiatica and corn silk, and only the lower limit of the content of a single indicator component is specified. There is a lack of a comprehensive fingerprint spectral quality control method. Qiu Hongcong et al. established an HPLC fingerprint spectral of Compound Lysimachia christinae Granules, evaluated the similarity of 10 batches of Compound Lysimachia christinae Granules preparations, and assigned the characteristic peaks in the prescription to the medicinal flavors (Chinese Journal of Experimental Traditional Medical Formulae, 2016, 22(01):36-39). However, the article only identified mangiferin (the effective component in Pyrrosia lingua) and shampootaside (the effective component in Lysimachia christinae), without identifying other characteristic components in the prescription, without controlling the content of the components in the prescription, and the HPLC fingerprint spectral analysis time was too long. Ma Junhua et al. performed thin-layer chromatography qualitative identification of *Lysimachia christinae* and *Pyrrosia lingua* in three batches of compound *Lysimachia christinae* granules, and established a quality control method for compound *Lysimachia christinae* granules using mangiferin as a quantitative indicator (Modern Drugs and Clinical, 2014, 29(04):381-384). However, the article only studied two herbs in the prescription, and the number of batches studied was small. Quality research based solely on two herbs cannot comprehensively control the quality of the compound.

[0004] Existing research on the quality control of Compound Lysimachia christinae granules is limited, and the analysis time is too long, and the quality control methods are not comprehensive enough. Qualitative or quantitative control of only a few components is insufficient to comprehensively evaluate the quality characteristics of the preparation. This invention utilizes UPLC technology to establish a rapid and efficient analytical method for evaluating the quality of Compound Lysimachia christinae granules, providing a comprehensive quality assessment and offering guidance and reference for establishing more systematic and comprehensive quality control standards. Summary of the Invention

[0005] The purpose of this invention is to address the problems of limited research on the quality control of Compound Lysimachia christinae, excessively long analysis time, incomplete quality control methods, and difficulty in comprehensively evaluating the quality characteristics of Compound Lysimachia christinae granules by only qualitatively or quantitatively controlling a few components. This invention provides a method for constructing a UPLC quantitative fingerprint spectrum of Compound Lysimachia christinae granules.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for constructing a UPLC quantitative fingerprint of compound Lysimachia christinae granules, comprising the following steps:

[0008] a: Methods for constructing fingerprint maps

[0009] Step 1: Preparation of reference solutions: Take mangiferin, isovitexin, vesenacin-2, vesenacin-3, isomangiferin, isoharmonin, and shampooside as reference standards, and add 50-100% methanol (V / V) or 50-100% ethanol (V / V) to prepare reference solutions respectively.

[0010] Step 2: Preparation of test solution: Take an appropriate amount of compound Lysimachia christinae granules sample, grind it finely, weigh an appropriate amount accurately, put it into a stoppered conical flask, add methanol or ethanol solvent with a volume concentration of 50% to 70%, the material-to-liquid ratio (the ratio of sample to methanol or ethanol solvent) is 1:1 to 1:50 g / mL (preferably 1:5 to 1:20 g / mL for 10g per bag (1), 1:30 to 1:50 g / mL for 3g per bag (2), more preferably 1:10 to 1:15 g / mL for 10g per bag (1), 1:40 to 1:45 g / mL for 3g per bag (2), stopper tightly, weigh, sonicate or reflux for 10 to 30 minutes, cool to room temperature, make up the lost weight with solvent, shake well, centrifuge, take the supernatant and filter to obtain the test solution of compound Lysimachia christinae granules;

[0011] Step 3: Ultra-high performance liquid chromatography (UPLC) analysis: Prepare three or more (preferably more than ten, more preferably more than twenty) different batches of Compound Lysimachia christinae granules test solutions according to the method in Step 2. Inject the reference solution and the test solution into the ultra-high performance liquid chromatograph separately and record the chromatograms separately.

[0012] Step 4: Establishment of fingerprint spectrum: Import the liquid chromatograms of at least 3 (preferably more than 10, more preferably more than 20) different batches of Compound Lysimachia christinae granules into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition", match common peaks to obtain reference fingerprint spectrum, identify common peaks and assign them to each herb, and establish the fingerprint spectrum of Compound Lysimachia christinae granules.

[0013] b: Construction method for content determination

[0014] Step 1: Preparation of reference solution: Weigh mangiferin and isomangiferin separately, and add 50-100% methanol (V / V) or 50-100% ethanol (V / V) to prepare a mixed reference solution of mangiferin and isomangiferin.

[0015] Step 2: Preparation of test solution: Take an appropriate amount of compound Lysimachia christinae granules sample, grind it finely, weigh an appropriate amount accurately, put it into a stoppered conical flask, add methanol or ethanol solvent with a volume concentration of 50% to 70%, the material-to-liquid ratio (the ratio of sample to methanol or ethanol solvent) is 1:1 to 1:50 g / mL (preferably 1:5 to 1:20 g / mL for 10g per bag (1), 1:30 to 1:50 g / mL for 3g per bag (2), more preferably 1:10 to 1:15 g / mL for 10g per bag (1), 1:40 to 1:45 g / mL for 3g per bag (2), stopper tightly, weigh, sonicate or reflux for 10 to 30 minutes, cool to room temperature, make up the lost weight with solvent, shake well, centrifuge, take the supernatant and filter to obtain the test solution of compound Lysimachia christinae granules;

[0016] Step 3: Ultra-high performance liquid chromatography (UPLC) analysis: Prepare three or more (preferably more than 10, more preferably more than 20) different batches of compound Lysimachia christinae granules test solutions according to the method in Step 2. Take the reference solution and the test solution respectively and inject them into the ultra-high performance liquid chromatograph to determine the corresponding peak areas of mangiferin and isomangiferin in the reference and the test solutions.

[0017] Step 4: Establishment of content determination method: Where A x Rf represents the peak area of ​​the analyte in the sample, Rf represents the correction factor, and m represents the sample weight. Calculate the content of mangiferin and isomangiferin in the compound Lysimachia christinae granules sample.

[0018] In a specific embodiment of the present invention, the concentrations of the reference solution in step 1 of step a are as follows: per 1 mL, mangiferin 80 μg, isovitexin 290 μg, veselin-3 180 μg, isomangiferin 60 μg, isoharonin 210 μg, shampodoside 290 μg, veselin-2 260 μg, and verbascoside 210 μg.

[0019] In a specific embodiment of the present invention, the ultra-high performance liquid chromatography (UHPLC) conditions described in a and b are as follows: the chromatographic column is a Waters Acquity UPLC BEH C18 (2.1×100mm, 1.7μm), the column temperature is 30-40℃, the flow rate is 0.3-0.5mL / min, the injection volume is 0.5-2μL, the detection wavelength is 350nm, acetonitrile is used as mobile phase A, and formic acid with a volume percentage concentration of 0.1-0.5% is used as mobile phase B for gradient elution. The gradient elution conditions are: 0-5min: 9% A; 5-10min: 9%-12% A; 10-18min: 12-20% A; 18-19min: 20-90% A; 19-23min: 90% A.

[0020] In a specific embodiment of the present invention, there are 12 common peaks in the fingerprint spectrum, of which peak 1 is mangiferin, peak 2 is isomangiferin, peak 3 is vezin-2, peak 4 is an isomer of vezin-3 and kaempferol-3-O-β-D-glucose-7-O-β-D-apigenin, peak 5 is isochorin, peak 6 is ochorin, peak 7 is shampodoside, peak 8 is an isomer of vezin-3, peak 9 is vezin-3, peak 10 is vitexin, peak 11 is verbascoside, and peak 12 is plantagoside.

[0021] Peaks 1 and 2 are exclusive components of *Gynostemma pentaphyllum*, peaks 3, 7, 8, and 9 are exclusive components of *Lysimachia christinae*, peak 10 is an exclusive component of corn silk, and peaks 11 and 12 are exclusive components of *Plantago asiatica*.

[0022] In a specific embodiment of the present invention, similarity matching is performed using 12 common peaks in step a, and the similarity between the chromatogram of the test sample and the reference chromatogram is calculated to be no less than 0.95.

[0023] In a specific embodiment of the present invention, the preparation method of the reference solution in step 1 of b is as follows: take an appropriate amount of mangiferin and isomangiferin, accurately weigh them, and add 50% to 70% methanol (V / V) to prepare a mixed reference solution containing 70 to 90 μg of mangiferin and 50 to 70 μg of isomangiferin per 1 mL.

[0024] In a specific embodiment of the present invention, each bag of the compound Lysimachia christinae granules described in b contains not less than 3.9 mg of mangiferin and not less than 3.8 mg of isomangiferin.

[0025] In a specific embodiment of the present invention, 12 common peaks were identified and assigned. Similarity matching was performed on the 12 common peaks to generate a reference spectrum of Compound Lysimachia christinae granules. Combined with the determination of the contents of two components, mangiferin and isomangiferin, a quantitative fingerprint spectrum of Compound Lysimachia christinae granules was established.

[0026] This invention enables simultaneous fingerprint spectral detection and content determination of Compound Lysimachia christinae granules in a single analysis, allowing for rapid, comprehensive, and accurate evaluation of the quality of Compound Lysimachia christinae granules and providing technical support for the evaluation of its effectiveness, stability, and consistency.

[0027] The present invention has the following advantages:

[0028] 1. The experimental operation of this invention is simple and the chromatographic conditions are easy to achieve; the detection time is short, reducing the amount of mobile phase used. Mobile phases often contain toxic solvents such as methanol or acetonitrile. Reducing the amount used greatly reduces environmental pollution, making it more environmentally friendly and lowering detection costs; it has high precision, and its stability and reproducibility are good; the detection wavelength used in this invention has more peaks, reflecting more complete information.

[0029] 2. For the first time, UPLC technology was used to conduct a quantitative fingerprint study on Compound Lysimachia christinae Granules, establishing a rapid and efficient quantitative fingerprinting method. Through a single analysis, the identification, fingerprint detection, and content determination of Compound Lysimachia christinae Granules can be achieved, enabling rapid, comprehensive, and accurate evaluation of the quality of Compound Lysimachia christinae Granules and providing technical support for the evaluation of the efficacy, stability, and consistency of Compound Lysimachia christinae Granules. Attached Figure Description

[0030] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0031] Figure 1 fingerprint overlay images of 22 batches of Compound Lysimachia christinae granules

[0032] Figure 2 To compare fingerprint spectra and common peaks

[0033] Figure 3 Mangiferin standard curve

[0034] Figure 4 Standard curve of isomangiol

[0035] Figure 5 This is a superimposed image of the chromatogram of the test sample and the fingerprint of the reference sample. Detailed Implementation

[0036] The following detailed description of the UPLC quantitative fingerprinting method for compound Lysimachia christinae granules of the present invention, with reference to specific embodiments, provides further details.

[0037] Example 1: Establishment of UPLC fingerprinting method for compound Lysimachia christinae granules

[0038] 1.1 Instruments, reagents, and sources

[0039] Instruments: Waters Acquity Class I ultra-high performance liquid chromatograph; Agilent Infinity II 1290-6545Q-TOF LC-MS quadrupole time-of-flight mass spectrometer; ML204T / 02 analytical balance; XSE105 analytical balance; KQ-500DV ultrasonic instrument; Waters Acquity UPLC BEH C18 column (2.1×100mm, 1.7μm) and pre-column; SOrvall ST8 high-speed centrifuge;

[0040] Reagents: Acetonitrile (Thermo) and formic acid (Thermo) are mass spectrometry grade; formic acid (Macklin) and acetonitrile (Fisher) are chromatographic grade; methanol (Energy Chemical) is analytical grade; ultrapure water;

[0041] Test drugs: mangiferin and isovitexin were purchased from Chengdu Purifa Technology Development Co., Ltd.; vezin-2, vezin-3, isomangiferin, isovitexin, and shambutol were purchased from Chengdu Pusi Biotechnology Co., Ltd.

[0042] The samples were from Guangxi Wantong Pharmaceutical Co., Ltd., 10g [specification (1)] and 3g [specification (2)]. For details, please refer to Table 1.

[0043] Table 1

[0044]

[0045] 1.2 Solution Preparation

[0046] 1.2.1 Preparation of reference solution

[0047] Preparation of mixed standard solutions of compound Lysimachia christinae granules: Take appropriate amounts of mangiferin, vitexin, vesenacin-3, isomangiferin, isochorin, shampodoside, vesenacin-2, and verbascoside reference standards, mix them with 50% methanol (V / V), and prepare reference solutions with concentrations of 80 μg mangiferin, 290 μg isomangiferin, 180 μg vesenacin-3, 60 μg isomangiferin, 210 μg isochorin, 290 μg shampodoside, 260 μg vesenacin-2, and 210 μg verbascoside per 1 mL.

[0048] 1.2.2 Preparation of test solution

[0049] Take 10 bags of the compound Lysimachia christinae granules from the "Variation of Content" section, mix them well, take an appropriate amount, grind them finely, take 2g [specification (1)] or 0.6g [specification (2)], accurately weigh them, put them into a stoppered conical flask, add 25mL of 50% methanol (V / V), seal tightly, weigh, sonicate for 10 minutes, cool to room temperature, make up the lost weight with 50% methanol (V / V), shake well, 10000r·min -1 Centrifuge for 10 minutes, then filter the supernatant to obtain the final product.

[0050] 1.3 Chromatographic conditions

[0051] Chromatographic column: Waters Acquity UPLC BEH C18 (2.1×100mm, 1.7μm)

[0052] Mobile phase: A: Acetonitrile; B: 0.1% formic acid / water (v / v)

[0053] Flow rate: 0.4 mL / min

[0054] Column temperature: 35℃

[0055] Injection volume: 1 μL

[0056] Detection wavelength: 350nm

[0057] Gradient elution conditions: 0-5 min: 9% A; 5-10 min: 9%-12% A; 10-18 min: 12-20% A; 18-19 min: 20-90% A; 19-23 min: 90% A

[0058] 1.4 Investigation of fingerprint mapping methodology

[0059] 1.4.1 Precision Examination

[0060] For the compound Lysimachia christinae granules with batch number 201026 / 2 (due to variations in fill volume), prepare the test solution according to the procedure in 1.2.2 above, inject the sample six times consecutively, and record the fingerprint spectrum. Using peak 2 (isomangiferin) as the reference peak (S), calculate the relative retention time RSD (relative standard deviation) values ​​of the 12 common peaks (the 12 common peaks identified in 1.5, the same below). All values ​​are less than 1%, and the relative peak area RSD values ​​are less than 5%, indicating good instrument precision.

[0061] 1.4.2 Repeatability Test

[0062] Six parallel test solutions of Compound Lysimachia christinae granules (batch number 201026 / 2, with varying fill weight) were prepared according to procedure 1.2.2 above. These solutions were injected, and fingerprint chromatograms were recorded. Using peak 2 (isomangiferin) as the reference peak (S), the relative retention time (RSD) values ​​of the 12 common peaks were all less than 1%, and the relative peak area (RSD) values ​​were all less than 5%, indicating good method repeatability.

[0063] 1.4.3 Stability Assessment

[0064] Take the compound Lysimachia christinae granules (batch number 201026 / 2) with varying fill weights, prepare the test solution according to the procedure in 1.2.2 above, and inject it into the liquid chromatograph after standing at room temperature for 0h, 2h, 4h, 8h, 12h, and 24h, respectively, and record the fingerprint chromatogram. Using peak 2 (isomangiferin) as the reference peak (S), the relative retention time RSD values ​​of the 12 common peaks were all less than 1%, and the relative peak area RSD values ​​were all less than 5%, indicating that the test solution had good stability within 24h.

[0065] 1.4.4 Intermediate Precision Examination

[0066] For the compound Lysimachia christinae granules with batch number 201026 / 2, which had different filling volumes, two personnel prepared test solutions according to the procedure in 1.2.2 above. Fingerprint spectra were obtained on two Waters Acquity I Class detectors (both diode array detectors, 190–400 nm). Figure I-1 (4-15). The results showed that there were no significant differences in the chromatograms obtained by instruments from the same manufacturer. Using peak 2 (isomangiferin) as the reference peak (S), the relative retention time RSD values ​​of the 12 common peaks were all less than 5%, and the relative peak area RSD values ​​were all less than 10%. Therefore, this method is applicable to ultra-high performance liquid chromatographs from the same manufacturer, and the intermediate precision is good.

[0067] 1.5 Establishment of fingerprint spectrum and identification of common peaks

[0068] Following the established chromatographic analysis method in 1.3, precisely pipette 1 μL of each of the 22 batches of Compound Lysimachia christinae granules test solution (prepared according to the procedure in 1.2.2 above) and inject them into the ultra-high performance liquid chromatograph, recording the chromatographic peak information. Generate an overlay image in the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" (…). Figure 1 ), compared with fingerprint spectrum R ( Figure 2), of which there are 12 peaks. Liquid chromatography-mass spectrometry (LC-MS) was used, combined with literature ([1] Zhang Xueqin et al. Research progress on chemical components and pharmacological effects of Plantago asiatica [J]. Strait Pharmaceutical Journal, 2013, 25(11): 1-8. [2] Zhong Ming et al. Research progress on chemical components and pharmacological effects of Lysimachia christinae [J]. Guangxi Medical Journal, 2018, 40(01): 80-82+91. etc.), and database (SciFinder). n The samples were compared with reference standards (such as ChemSpider, etc.) and identified 12 peaks, which were then assigned to various medicinal ingredients. The results are shown in Table 2. Using the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, similarity matching was performed using the 12 common peaks. The similarity between the fingerprints of 22 batches of samples and the reference fingerprints was calculated, and all values ​​were greater than 0.95. The results are shown in Table 3. Using peak 2 as the reference peak, the relative retention time of the reference fingerprint was calculated. The results are shown in Table 4.

[0069] Table 2. Results of common peak identification and herbal ingredient attribution in the fingerprint spectrum of Compound Lysimachia christinae granules.

[0070]

[0071] * Indicates comparison with a control.

[0072] Table 3. Similarity between 22 batches of samples and the control chromatograms

[0073]

[0074] Table 4. Relative retention times of the fingerprint profiles.

[0075]

[0076] Example 2: Establishment of UPLC method for content determination of compound Lysimachia christinae granules

[0077] 1.1 Instruments, reagents, and sources

[0078] Instruments: Waters Acquity Class I ultra-high performance liquid chromatograph; ML204T / 02 analytical balance (0.0001 g); XSE105 analytical balance (0.0001 g); KQ-500DV ultrasonic instrument; Waters Acquity UPLC BEH C18 column (2.1×100 mm, 1.7 μm) and pre-column; SOrvall ST8 high-speed centrifuge;

[0079] Reagents: Formic acid (Macklin) and acetonitrile (Fisher) are chromatographic grade; methanol (Energy Chemical) is analytical grade; ultrapure water;

[0080] Test reagents: Mangiferin (batch number 111607-201704, specification 20mg, purity 98.1%) was purchased from the National Institutes for Food and Drug Control; Isomangiferin (batch number PS010037, specification 25mg, purity 98.0%) was purchased from Chengdu Pusi Biotechnology Co., Ltd.

[0081] The sample source is the same as in Example 1.

[0082] 1.2 Solution Preparation

[0083] 1.2.1 Preparation of reference solution

[0084] Preparation of mixed standard solution of compound Lysimachia christinae granules: Accurately weigh mangiferin and isomangiferin reference standards, mix with 50% methanol (V / V) to prepare solutions with concentrations of 80 μg mangiferin and 60 μg isomangiferin per 1 mL, respectively, as mixed standard solutions. The concentrations of the standard solutions are shown in Table 5.

[0085] Table 5 Concentration of Reference Solution

[0086]

[0087] 1.2.2 Preparation of test solution

[0088] Same as in Example 1 for the preparation of the test solution.

[0089] 1.3 Chromatographic conditions

[0090] Same chromatographic conditions as in Example 1

[0091] 1.4 Methodological Investigation of Content Determination

[0092] 1.4.1 Precision Examination

[0093] One batch of Compound Lysimachia christinae Granules (batch number 201026 / 2) was injected six times consecutively. The RSD values ​​of mangiferin and isomangiferin were both less than 1%, indicating that the instrument precision was good. The results are shown in Table 6.

[0094] 1.4.2 Repeatability Test

[0095] Six test solutions were prepared in parallel from batch number 201026 / 2 of Compound Lysimachia christinae granules and analyzed separately. The RSD of the contents of the two index components were both less than 1%, indicating that the method has good repeatability. The results are shown in Table 6.

[0096] 1.4.3 Stability Assessment

[0097] One sample solution with batch number 201026 / 2 was taken and analyzed after being placed at room temperature for 0h, 2h, 4h, 8h, 12h, and 24h, respectively, to examine the room temperature stability of the sample solution. The results showed that the RSD of the peak areas of both indicators was less than 1%, indicating that the sample solution had good stability within 24h. The results are shown in Table 6.

[0098] Table 6. Results of the content determination and precision stability test.

[0099]

[0100] 1.4.4 Examination of Linear Relationships

[0101] Precisely pipette mixed standard solutions containing mangiferin and isomangiferin at concentrations of 133.2276, 104.6170 μg / mL; 106.5821, 83.6936 μg / mL; 79.9366, 62.7702 μg / mL; 53.2911, 41.8468 μg / mL; 26.6455, 20.9234 μg / mL; 13.3228, 10.4617 μg / mL; and 5.3291, 4.1847 μg / mL, respectively, and inject them for analysis. Linear regression was performed on peak area Y against solution concentration X to generate standard curves for the two quantitative indicators. The results are shown in Table 7. Figures 3-4 .

[0102] Table 7 Standard curves for mangiferin and isomangiferin

[0103]

[0104] 1.4.5 Recovery Experiment

[0105] Accurately weigh approximately 1g of Compound Lysimachia christinae Granules (batch number 201026 / 2) with known content. Add known amounts of a mixed solution of two reference standards at concentration levels of 50%, 100%, and 150% for mangiferin (866.24 μg per gram of sample) and isomangiferin (691.92 μg per gram of sample). Prepare nine test solutions (three portions each of low, medium, and high concentrations) according to the test solution preparation method in section 1.2.2. Inject each solution into the solution for analysis. The recoveries of the two quantitative indicators (recovery rate % = (measured amount - content in sample) / amount of added reference standard * 100%) were all within 90–108%, with RSD less than 5%. The results are shown in Table 8.

[0106] Table 8. Recovery rates of mangiferin and isomangiferin

[0107]

[0108] 1.4.6 Intermediate Precision Examination

[0109] Two separate individuals prepared six test solutions from the same batch of Compound Lysimachia christinae granules, for a total of 12 test solutions. These solutions were then injected and analyzed. The RSD of the content of the two indicator components was less than 5%, indicating that this method is applicable to ultra-high performance liquid chromatographs from the same manufacturer and has good intermediate precision. The results are shown in Table 9.

[0110] Table 9. Results of intermediate precision experiments for mangiferin and isomangiferin

[0111]

[0112] 1.5 Calculation of correction factors for mangiferin and isomangiferin

[0113] Following the analytical method established in section 1.3, inject the mixed reference solution of batch JQCHB-210817-1 six times consecutively, and the mixed reference solution of batch JQCHB-210817-2 three times consecutively. Calculate the correction factor. The mean and RSD values ​​are shown in Table 10. The results show that the correction factor RSD values ​​for mangiferin and isomangiferin are both less than 2%, which meets the requirements.

[0114] Table 10 Calculation of correction factors for mangiferin and isomangiferin

[0115]

[0116] 1.6 Content determination results of each batch of samples

[0117] Twenty-two batches of Compound Lysimachia christinae granules were taken and analyzed using the established chromatographic method, employing the external standard method. Where A x Rf represents the peak area of ​​the analyte in the sample, m represents the sample weight, and the dilution factor is 25. The contents of mangiferin and isomangiferin were calculated, and the contents of the two components are shown in Table 11. In each bag of sample, the content of mangiferin (Cm) is calculated as follows: [Table data omitted for brevity]. 19 H 18 O 11 The dosage shall not be less than 3.9 mg, calculated as isomangoside (C). 19 H 18 O 11 The dosage shall not be less than 3.8 mg.

[0118] Table 11. Determination of the content of Compound Lysimachia christinae Granules in different batches (SD: standard deviation of the sample)

[0119]

[0120] Example 3: Application of UPLC quantitative fingerprinting of Compound Lysimachia christinae granules

[0121] 1.1 Instruments, reagents, and sources

[0122] Instruments: Waters Acquity Class I ultra-high performance liquid chromatograph; ML204T / 02 analytical balance (0.0001 g); XSE105 analytical balance (0.0001 g); KO-500DV ultrasonic instrument; Waters Acquity UPLC BEH C18 column (2.1×100 mm, 1.7 μm) and pre-column; SOrvall ST8 high-speed centrifuge;

[0123] Reagents: Formic acid (Macklin) and acetonitrile (Fisher) are chromatographic grade; methanol (Energy Chemical) is analytical grade; ultrapure water;

[0124] Test reagents: Mangiferin (batch number 111607-201704, specification 20mg, purity 98.1%) was purchased from the National Institutes for Food and Drug Control; Isomangiferin (batch number PS010037, specification 25mg, purity 98.0%) was purchased from Chengdu Pusi Biotechnology Co., Ltd.

[0125] The sample source is the same as in Example 1.

[0126] 1.2 Solution Preparation

[0127] 1.2.1 Preparation of reference solution

[0128] Preparation of the reference solution as in Example 2

[0129] 1.2.2 Preparation of test solution

[0130] Same as in Example 1 for the preparation of the test solution.

[0131] 1.3 Chromatographic conditions

[0132] Same chromatographic conditions as in Example 1

[0133] 1.4 Application of quantitative fingerprinting of compound Lysimachia christinae granules

[0134] 1.4.1 Application of the fingerprinting method for Compound Lysimachia christinae granules

[0135] According to the established chromatographic analysis method, accurately pipette 1 μL of the test solution of Compound Lysimachia christinae granules (batch number 201026 / 2, specification 10*36 bags / box) (prepared according to the above procedure 1.2.2), inject it into the ultra-high performance liquid chromatograph, record the chromatographic peak information, and generate an overlay image with the control fingerprint spectrum generated in Example 1. Figure 5The fingerprint chromatogram of the test sample solution showed 12 characteristic peaks, and the retention times of these peaks corresponded to those of two chromatographic peaks in the control fingerprint chromatogram. Using the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, the similarity between the test sample and the control fingerprint chromatogram was matched based on these 12 common peaks. The results are shown in Table 12. The similarity between the test sample fingerprint chromatogram and the control fingerprint chromatogram was 0.999, which is greater than 0.95, and the results meet the requirements.

[0136] Table 12 Similarity results between the chromatograms of the test sample and the reference fingerprint chromatograms

[0137]

[0138] 1.4.2 Application of the method for determining the content of compound Lysimachia christinae granules

[0139] Following the established chromatographic analysis method, precisely pipette the reference solution and test solution separately for liquid chromatography analysis, determine the corresponding peak areas, and use the external standard method. Where A x The peak area of ​​the analyte in the sample is represented by Rf, the mean correction factor of the analyte is represented by m, and the dilution factor is 25 (the mean correction factor for mangiferin is 3146.24, and the mean correction factor for isomangiferin is 2801.68). The contents of mangiferin and isomangiferin in the test sample were calculated, and the contents of the two components were shown in Table 13. Each bag of the test sample contained 8.7 mg of mangiferin and 6.9 mg of isomangiferin, both of which meet the requirements.

[0140] Table 13 Results of content determination of test samples

[0141]

[0142] The technical solution provided by this invention is compared with the 2020 edition of the Chinese Pharmacopoeia as follows:

[0143]

[0144] As can be seen from the table, the method of the present invention has strong specificity, increased number of test indicators, more peak identifications, and complete information reflected by the fingerprint spectrum. The use of liquid chromatography-mass spectrometry (LC-MS) technology to perform qualitative analysis on common chromatographic peaks clarifies the material basis of compound Lysimachia christinae granules.

[0145] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes or modifications made in accordance with the scope of the present invention patent shall fall within the scope of the claims of the present invention patent.

Claims

1. A method for UPLC quantitative analysis and / or fingerprinting construction of compound Lysimachia christinae granules, characterized in that, Includes the following steps: a: Methods for constructing fingerprint maps Step 1: Preparation of reference solutions: Take mangiferin, isovitexin, vezin-2, vezin-3, isomangiferin, isoharonin, and shampooside as reference standards, and add 50-100% V / V methanol or 50-100% V / V ethanol to prepare reference solutions respectively. Step 2: Preparation of the test solution: Take the compound Lysimachia christinae granules sample, add methanol solvent with a volume concentration of 50%~70% or ethanol solvent with a volume concentration of 50%~70%, the material-to-liquid ratio is 1:1~1:50 g / mL, sonicate or reflux for 10~30 min, make up the weight loss with solvent, shake well, centrifuge, take the supernatant and filter to obtain the compound Lysimachia christinae granules test solution; Step 3: Ultra-high performance liquid chromatography (UPLC) analysis: Prepare three or more different batches of compound Lysimachia christinae granules test solutions according to the method in Step 2. Inject the reference solution and test solution into the ultra-high performance liquid chromatograph separately and record the chromatograms. Step 4: Establishment of fingerprint spectrum: Import the liquid chromatograms of at least 3 different batches of Compound Lysimachia christinae granules into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition", match common peaks to obtain reference fingerprint spectrum, identify common peaks and assign them to each herb, and establish the fingerprint spectrum of Compound Lysimachia christinae granules. b: Construction method for content determination Step 1: Preparation of reference solution: Weigh mangiferin and isomangiferin separately, and add 50-100% V / V methanol or 50-100% V / V ethanol to prepare a mixed reference solution of mangiferin and isomangiferin. Step 2: Preparation of test solution: Take the compound Lysimachia christinae granules sample, add methanol solvent with a volume concentration of 50%~70% or ethanol solvent with a volume concentration of 50%~70%, the material-to-liquid ratio is 1:1~1:50 g / mL, sonicate or reflux for 10~30 min, make up the weight loss with solvent, shake well, centrifuge, take the supernatant and filter to obtain the compound Lysimachia christinae granules test solution; Step 3: Ultra-high performance liquid chromatography (UPLC) analysis: Prepare three or more different batches of compound Lysimachia christinae granules test solutions according to the method in Step 2. Take the reference solution and test solution respectively and inject them into the ultra-high performance liquid chromatograph to determine the corresponding peak areas of mangiferin and isomangiferin in the reference solution and test solution. Step 4: Establishment of content determination method: using external standard method, A x Rf represents the peak area of ​​the analyte in the sample, Rf represents the correction factor, and m represents the sample weight. Calculate the contents of mangiferin and isomangiferin in the compound Lysimachia christinae granules sample; The ultra-high performance liquid chromatography (UHPLC) conditions described in a and b are as follows: a C18 2.1×100 mm, 1.7 µm WatersAcquity UPLC BEH column; a column temperature of 30–40 °C; a flow rate of 0.3–0.5 mL / min; an injection volume of 0.5–2 μL; a detection wavelength of 350 nm; and gradient elution using acetonitrile as mobile phase A and formic acid with a volume percentage concentration of 0.1–0.5% as mobile phase B. The gradient elution is as follows: 0-5 min: 9% A; 5-10 min: 9%-12% A; 10-18 min: 12-20% A.

2. The construction method according to claim 1, characterized in that: In step 1 described in section a, the concentrations of the prepared reference solutions are as follows: per 1 mL, mangiferin 70-90 μg, isovitexin 280-300 μg, veselin-3 170-200 μg, isomangiferin 50-70 μg, isoharonin 200-220 μg, shampodoside 270-300 μg, veselin-2 250-280 μg, and verbascoside 200-240 μg.

3. The construction method according to claim 1, characterized in that, The material-to-liquid ratio in both a and b, i.e., the ratio of the sample to methanol or ethanol solvent, is 1:5~1:20 g / mL for 10 g per bag of specification 1 and 1:30~1:50 g / mL for 3 g per bag of specification 2.

4. The construction method according to claim 1, characterized in that: The reference fingerprint spectrum contains 12 common peaks, of which peak 1 is mangiferin, peak 2 is isomangiferin, peak 3 is vezin-2, peak 4 is an isomer of vezin-3 and kaempferol-3-O-β-D-glucose-7-O-β-D-apigenin, peak 5 is isochorin, peak 6 isochorin, peak 7 is shampodoside, peak 8 is an isomer of vezin-3, peak 9 is vezin-3, peak 10 is vitexin, peak 11 is verbascoside, and peak 12 is plantagoside. Peaks 1 and 2 are exclusive components of *Gynostemma pentaphyllum*, peaks 3, 7, 8, and 9 are exclusive components of *Lysimachia christinae*, peak 10 is an exclusive component of corn silk, and peaks 11 and 12 are exclusive components of *Plantago asiatica*.

5. The construction method according to any one of claims 1-4, characterized in that, In step a, the similarity was calculated using 12 common peaks, and the similarity between the chromatogram of the test sample and the reference chromatogram was not less than 0.

95.

6. The construction method according to claim 1, characterized in that, In step 1 of section b, mangiferin and isomangiferin are weighed and mixed with 50%~70% V / V methanol or 50%~70% V / V ethanol to prepare a mixed reference solution containing 70~90 μg of mangiferin and 50~70 μg of isomangiferin per 1 mL.

7. The construction method according to claim 1, characterized in that, The compound Lysimachia christinae granules sample described in b contains 3.9~20.0 mg of mangiferin and 3.8~19.0 mg of isomangiferin per bag.

Images