A method for constructing and evaluating the multi-component fingerprint of Jian'erle granules based on HPLC

Abstract

The application discloses a kind of based on HPLC Jian'erle granule multi-component fingerprint construction and active evaluation method, belong to the technical field of drug analysis.The application is based on ultra-high performance liquid chromatography technology, by optimizing drug extraction and chromatographic detection method, provide the fingerprint construction method of analysis Jian'erle granule drug quality;The method comprises the steps of ultrasonic dissolution Jian'erle granule, with 0.1% phosphoric acid and acetonitrile as mobile phase for liquid chromatography detection.According to the construction method provided by the application, a fingerprint consisting of 12 characteristic peaks is obtained, of which 10 characteristic peaks are identified to compounds.The results of the methodological experiment show that the construction method provided by the application has a similarity of >0.999 between 10 batches of Jian'erle granules, and has outstanding specificity, precision, repeatability and stability.The application provides a new strategy for efficient quality detection and quality control of Jian'erle granules, and has a broad application prospect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a method for constructing multi-component fingerprint profiles and evaluating the activity of Jianerle granules based on HPLC. Background Technology

[0002] Jianerle granules are a traditional Chinese medicine digestive aid. The prescription consists of six herbs: hawthorn, bamboo shoots, uncaria, white peony root, stevia, and chicken gizzard lining. It has the effects of strengthening the spleen and promoting digestion, clearing the heart and calming the mind. It is mainly used to treat anorexia and night crying caused by spleen deficiency and excessive heat in the heart and liver, with symptoms including poor appetite, indigestion, night terrors, and restless sleep. The current quality standard for Jianerle granules includes three thin-layer chromatography identification items: white peony root (white peony root reference material); stevia (stevioside); uncaria (uncaria reference material); and one content detection item (white peony root content calculated as paeoniflorin). However, the current quality standard does not fully reflect the overall quality of Jianerle granules.

[0003] According to the "Technical Guidelines for Research on Pharmaceutical Changes of Marketed Traditional Chinese Medicines", if drug standards cannot adequately reflect drug quality, or if the controllability of drug quality is low, and it is difficult to assess the impact of the change by conducting comparative studies of product quality before and after the change based solely on drug standards, quality and drug standard research should be carried out. Appropriate evaluation indicators and testing methods should be adopted according to product characteristics to conduct comparative quality studies, and the impact of the change on product quality should be objectively assessed based on the quality research results before and after the change.

[0004] Therefore, there is an urgent need to provide a stable analytical method for detecting the quality of Jianerle granules. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing and evaluating the multi-component fingerprint spectrum of Jianerle granules based on HPLC, so as to solve the problems existing in the prior art. The fingerprint spectrum construction method provided by this invention has a similarity of >0.999 among 10 batches of Jianerle granules, and has outstanding specificity, precision, repeatability and stability.

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

[0007] This invention provides a method for constructing a fingerprint spectrum of Jianerle particles, comprising the following steps:

[0008] Take the Jianerle granules, mix them with methanol solution, sonicate, cool, shake well, filter and collect the filtrate to obtain the test solution;

[0009] The fingerprint spectrum of the Jianerle granules was obtained by performing ultra-high performance liquid chromatography analysis on the test solution.

[0010] The packing material for the ultra-high performance liquid chromatography is octadecylsilane-bonded silica gel.

[0011] The mobile phase A of the ultra-high performance liquid chromatography is a 0.1% (v / v) phosphoric acid solution, and the mobile phase B is acetonitrile;

[0012] The mobile phase elution conditions for the ultra-high performance liquid chromatography are as follows:

[0013] 0-5 min, mobile phase A 95%, mobile phase B 5%;

[0014] 5-14 min, mobile phase A 95% → 88%, mobile phase B 5% → 12%;

[0015] 14-35 min, mobile phase A 88%→82%, mobile phase B 12%→18%;

[0016] 35-55 min, mobile phase A 82% → 30%, mobile phase B 18% → 70%;

[0017] 55-56 min, mobile phase A 30% → 95%, mobile phase B 70% → 5%;

[0018] 56-66 min, mobile phase A 95%, mobile phase B 5%.

[0019] Preferably, the volume fraction of the methanol solution is 10%; the mass-to-volume ratio of the Jianerle granules to the methanol solution is 1g:30mL.

[0020] Preferably, the ultrasonic treatment has a power of 1100W, a frequency of 40kHz, and a duration of 15min.

[0021] Preferably, the sample loading volume of the ultra-high performance liquid chromatography (UHPLC) is 1 μL.

[0022] Preferably, the flow rate of the ultra-high performance liquid chromatography is 0.3 mL / min.

[0023] Preferably, the column temperature of the ultra-high performance liquid chromatography is 30°C.

[0024] Preferably, the detection wavelength of the ultra-high performance liquid chromatography is 230 nm.

[0025] Preferably, the fingerprint spectrum of the Jianerle granules consists of 12 characteristic peaks; among which peaks 1, 5, 6, 7 and 12 are peaks specific to Paeonia lactiflora; peaks 2, 4, 8, 9, 10 and 11 are peaks specific to Stevia rebaudiana; and peak 3 is a common peak of Uncaria rhynchophylla and Stevia rebaudiana.

[0026] Preferably, the identification result of the characteristic peak is:

[0027] Peak 1 is gallic acid; Peak 2 is neochlorogenic acid; Peak 3 is chlorogenic acid; Peak 4 is cryptochlorogenic acid; Peak 5 is paeoniflorin; Peak 6 is paeoniflorin; Peak 8 is isochlorogenic acid B; Peak 10 is 3,5-O-dicaffeoylquinic acid; Peak 11 is 4,5-O-dicaffeoylquinic acid; Peak 12 is benzoylpaeoniflorin.

[0028] The present invention also provides an application of the fingerprint spectrum obtained according to the above construction method in identifying the quality of Jianerle granules.

[0029] The present invention discloses the following technical effects:

[0030] This invention, based on ultra-high performance liquid chromatography (UHPLC), provides a fingerprint chromatogram construction method for analyzing the quality of Jianerle granules by optimizing drug extraction and chromatographic detection methods. The method includes ultrasonic dissolution of Jianerle granules and liquid chromatographic detection using 0.1% phosphoric acid and acetonitrile as the mobile phase. According to the construction method provided by this invention, a fingerprint chromatogram consisting of 12 characteristic peaks was obtained, of which 10 characteristic peaks clearly identified the compound. Methodological experimental results show that the construction method provided by this invention has a similarity of >0.999 among 10 batches of Jianerle granules, and exhibits outstanding specificity, precision, repeatability, and stability. This invention provides a new strategy for efficient quality detection and quality control of Jianerle granules, and has broad application prospects. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 The chromatogram shows the elution conditions; where each curve, from the x-axis to the positive direction of the y-axis, represents elution condition 1, elution condition 2, elution condition 3, elution condition 4, and elution condition 5, respectively.

[0033] Figure 2 Chromatograms were selected for the mobile phase components; where each curve, from the x-axis to the positive y-axis, represents 0.1% phosphoric acid-methanol and 0.1% phosphoric acid-acetonitrile, respectively.

[0034] Figure 3 Chromatograms were selected for the acid types; the curves, from the x-axis to the positive y-axis, represented 0.1% formic acid-acetonitrile, 0.1% glacial acetic acid-acetonitrile, and 0.1% phosphoric acid-acetonitrile, respectively.

[0035] Figure 4Chromatograms were selected based on acid concentration; the curves, from the x-axis to the positive y-axis, represented 0.1% phosphate-acetonitrile, 0.15% phosphate-acetonitrile, and 0.05% phosphate-acetonitrile, respectively.

[0036] Figure 5 Chromatograms were selected based on flow rates; the flow rates of each curve from the x-axis to the positive y-axis were 0.2 mL / min, 0.3 mL / min, 0.4 mL / min, and 0.5 mL / min, respectively.

[0037] Figure 6 Select the chromatogram for column temperature; where the curves are 25℃, 30℃, 35℃ and 40℃ respectively from the x-axis to the positive y-axis.

[0038] Figure 7 These are chromatograms at different wavelengths; where each curve, from the x-axis to the positive y-axis, represents 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 330nm, 350nm, 370nm, and 390nm respectively.

[0039] Figure 8 To maximize the information, examine the chromatograms; where, from the x-axis to the positive direction of the y-axis, each curve represents, in order, the test sample-extended time, the test sample-normal time, and the blank-extended time.

[0040] Figure 9 The graph shows a comparison of different concentrations of methanol extraction solvents; the curves, from the x-axis to the positive direction of the y-axis, represent 10% methanol, 50% methanol, 70% methanol, and anhydrous methanol, respectively.

[0041] Figure 10 The graph shows a comparison of different concentrations of ethanol extraction solvents; the curves, from the x-axis to the positive direction of the y-axis, represent water, 10% ethanol, 50% ethanol, 70% ethanol, and anhydrous ethanol, respectively.

[0042] Figure 11 The graph shows the comparison of solvent usage for extraction; the curves are 20 mL, 25 mL, 30 mL, and 50 mL respectively, with the x-axis pointing towards the positive direction of the y-axis.

[0043] Figure 12 The comparison chart shows the extraction methods; where the curves, from the horizontal axis to the positive direction of the vertical axis, represent ultrasonic and reflux, respectively.

[0044] Figure 13 The extraction time is used to examine and compare the graphs; where the curves are 15 min, 30 min, and 45 min respectively from the x-axis to the positive y-axis.

[0045] Figure 14 This is a comparison chart of injection volume; where the curves represent 1 μL, 2 μL, and 3 μL respectively from the x-axis to the positive y-axis.

[0046] Figure 15 Comparison chart of medicinal flavor peak attribution - white peony root;

[0047] Figure 16 Comparison chart of medicinal flavor peak attribution and hawthorn;

[0048] Figure 17 Comparison of medicinal flavor peak attribution and bamboo leaf core;

[0049] Figure 18 Comparison diagram of the attribution of medicinal flavor peaks and Uncaria rhynchophylla;

[0050] Figure 19 Comparison chart of stevia and its medicinal flavor peak classification;

[0051] Figure 20 Comparison chart of medicinal flavor peak attribution and chicken gizzard lining;

[0052] Figure 21 A comparison chart of chromatographic peak identification;

[0053] Figure 22 This is a stacked fingerprint image of the marketed Jianerle granules; where R is the control fingerprint image; S2 is 231101; S3 is 231102; S4 is 231103; S5 is 231104; S6 is 231201; S7 is 231202; S8 ​​is 231203; S9 ​​is 231204; S10 is 231205; S11 is 231206.

[0054] Figure 23 For reference fingerprint spectrum; Peak 2: neochlorogenic acid, Peak 3: chlorogenic acid, Peak 6: paeoniflorin, Peak 8: isochlorogenic acid B, Peak 11: 4,5-O-dicaffeoylquinic acid, Peak 12: benzoylpaeoniflorin;

[0055] Figure 24 To specifically investigate the HPLC comparison chromatograms;

[0056] Figure 25 The chromatograms are for precision testing; where R is the reference fingerprint chromatogram; S2-S7 are the results of the first precision chromatogram (precision 1, 2, 3, 4, 5, 6); S8-S13 are the results of the second precision chromatogram (precision 1, 2, 3, 4, 5, 6).

[0057] Figure 26The HPLC comparison chromatograms are used for repeatability testing; where R is the reference fingerprint chromatogram; S2-S7 are the results of the first repeatability chromatogram (repeat 1, 2, 3, 4, 5, 6); S8-S13 are the results of the second repeatability chromatogram (repeat 1, 2, 3, 4, 5, 6).

[0058] Figure 27 Chromatograms for stability testing; where R is the reference fingerprint chromatogram; S2 indicates stability at 0 h; S3 indicates stability at 4.4 h; S4 indicates stability at 8.8 h; S5 indicates stability at 12 h; S6 indicates stability at 20 h; S7 indicates stability at 24 h.

[0059] Figure 28 Chromatograms were examined for reagents from different manufacturers; R represents the reference fingerprint chromatogram; S2 represents phosphoric acid (Fuyu) and acetonitrile (CINC); S3 represents phosphoric acid (Daomao) and acetonitrile (Fulton).

[0060] Figure 29 Chromatograms were examined for different columns; where R is the reference fingerprint chromatogram; S2 is the ZORBAX EclipseXDB-C... 18 S3 is Hypersil GOLD. Detailed Implementation

[0061] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0062] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0063] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0064] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0065] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0066] This invention collected 10 batches of commercially available Jianerle granules and established a UPLC fingerprint analysis method to more comprehensively evaluate product quality. This provides an assessment basis for the consistency of product quality before and after process and site changes, and assesses the degree of impact of process and site changes on product quality.

[0067] Example 1

[0068] 1. Experimental Materials

[0069] 1.1 Instruments and Reagents

[0070] Liquid Chromatography System: ACQUITY UPLC (Waters);

[0071] Column: ZORBAX Eclipse XDB-C 18 (2.1×100mm, 1.8μm);

[0072] Hypersil GOLD C 18 (2.1×100mm, 1.9μm);

[0073] Shim-pack Scepter HD-C 18 -80 (2.1×100mm, 1.9μm);

[0074] Electronic analytical balance: XPR36DR / A (parts per million) Mettler;

[0075] ATX224 (1 / 10,000) from Shimadzu, Japan;

[0076] BCE423-1CCN (1 / 1000) Sartorius;

[0077] CNC ultrasonic cleaner: SN-QX-300D Sunne;

[0078] Ultrapure water purifier: Exceed-Ad-40 ECO Laboratory Ultrapure Water purifier;

[0079] Acetonitrile, chromatographic grade, CINC Shanghai Xingkeji and DWSCI Fulton;

[0080] Methanol, analytical grade, Tianjin Fuyu Reagent Co., Ltd.

[0081] Methanol, chromatographic grade, CINC Shanghai Xingke;

[0082] Phosphoric acid, analytical grade, Tianjin Damao Chemical Reagent Co., Ltd. and Fuyu Reagent;

[0083] Formic acid, analytical grade, Tianjin Damao Chemical Reagent Co., Ltd.;

[0084] Glacial acetic acid, analytical grade, Tianjin Damao Chemical Reagent Co., Ltd.;

[0085] The water is ultrapure water, which was made in-house using a laboratory ultrapure water system.

[0086] 1.2 Standard Reference Materials

[0087] 3,5-O-dicaffeoylquinic acid, batch number: 111782-202309, specification: 20mg, purity: 95.0%;

[0088] Isoquercetin, batch number: 111809-202205, specification: 20mg, purity: 96.3%;

[0089] 4,5-O-dicaffeoylquinic acid, batch number: 111894-202205, specification: 20mg, purity: 94.9%;

[0090] Stevioside, batch number: 111515-202104, specification: 20mg, purity: for identification purposes;

[0091] Quercetin, batch number: 111538-202308, specification: 20mg, purity: 95.3%;

[0092] Gallic acid, batch number: 110831-202407, specification: 30mg, purity: 90.8%;

[0093] New chlorogenic acid, batch number: 112110-202401, specification: 20mg, purity: 99.2%;

[0094] Paeoniflorin, batch number: 110736-202447, specification: 20mg, purity: 98.1%;

[0095] Hyperoside, batch number: 111521-202310, specification: 20mg, purity: 94.7%;

[0096] Dehydrouncisine, batch number: 112088-202101, specification: 10mg, purity: for identification purposes;

[0097] Chlorogenic acid, batch number: 110753-202119, specification: 20mg, purity: 96.3%;

[0098] Catechins, batch number: 110877-202306, specification: 20mg, purity: 96.6%;

[0099] Cryptochlorogenic acid, batch number: DSTDY003503, specification: 20mg, purity: 98.29%;

[0100] Paeoniflorin, batch number: DST220412-071, specification: 20mg, purity: 98.18%;

[0101] Benzoyl paeoniflorin, batch number: DST240524-053, specification: 20mg, purity: 98.35%;

[0102] Uncariae, batch number: 112028-201601, specification: 10mg, purity: for identification purposes;

[0103] Isorhododendronine, batch number: 111927-201804, specification: 20mg, purity: for identification purposes;

[0104] Isochlorogenic acid B, batch number: DST240925-037, specification: 20mg, purity: 98.08%;

[0105] 1,2,3,4,6-Pentagalloglucoside, Batch No.: DSTDW000103, Specification: 20mg, Purity: 98.29%;

[0106] Xiguogan, batch number: DSDDX010801, specification: 20mg, purity: 99.72%.

[0107] 2. Establishment of analytical methods

[0108] 2.1 Fingerprint pattern establishment

[0109] Preparation of the test solution: Take about 1g of the powder of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of 70% methanol, weigh it, sonicate it (power 1100W, frequency 40kHz) for 15min, take it out, cool it, make up the weight, shake it well, filter it, and take the filtrate to obtain the test solution.

[0110] Assay: Accurately pipette 1 μL of the test solution and inject it into the liquid chromatograph, then collect the chromatogram.

[0111] 2.1.1 Screening based on mobile phase ratio

[0112] Using octadecylsilane-bonded silica gel as a filler (ZORBAX Eclipse XDB-C)18 The column was 2.1 × 100 mm (1.8 μm); using 0.1% phosphoric acid solution as mobile phase A and acetonitrile as mobile phase B, gradient elution was investigated according to the mobile phase elution conditions shown in Tables 1-5; the flow rate was 0.3 mL / min; the column temperature was 30 ℃; and the detection wavelength was 230 nm. Suitable mobile phase elution conditions were screened.

[0113] Table 1 Mobile phase elution conditions 1

[0114]

[0115] Table 2 Mobile phase elution conditions 2

[0116]

[0117] Table 3 Mobile phase elution conditions 3

[0118]

[0119] Table 4 Mobile phase elution conditions

[0120]

[0121] Table 5 Mobile phase elution conditions

[0122]

[0123] like Figure 1 As shown, the experimental results indicate that the separation effect of each component in the chromatogram corresponding to mobile phase elution condition 5 is better than that of other elution conditions, and it can fully reflect its main components. Therefore, this is used as the gradient elution chromatographic condition.

[0124] 2.1.2 Investigation of mobile phase composition

[0125] Using octadecylsilane-bonded silica gel as a filler (ZORBAX Eclipse XDB-C) 18 The mobile phase consisted of a 2.1 × 100 mm, 1.8 μm column; 0.1% phosphoric acid solution was used as mobile phase A, and acetonitrile and methanol were used as mobile phase B. Gradient elution was performed according to the mobile phase elution conditions in Table 5; the flow rate was 0.3 mL / min; the column temperature was 30 ℃; and the detection wavelength was 230 nm. Suitable organic phase components in the mobile phase were screened by comparing the peaks in the chromatogram.

[0126] like Figure 2 As shown, the experimental results indicate that, compared to the 0.1% phosphoric acid-acetonitrile system, the chromatogram baseline of the 0.1% phosphoric acid-methanol system is unstable, with fewer peaks and poorer peak shapes. Therefore, the mobile phase composition is tentatively set as the 0.1% phosphoric acid-acetonitrile system.

[0127] 2.1.3 Selection of Acid Types

[0128] Using octadecylsilane-bonded silica gel as a filler (ZORBAX Eclipse XDB-C) 18 The column was 2.1 × 100 mm (1.8 μm); acetonitrile was used as mobile phase B, and 0.1% phosphoric acid solution, 0.1% formic acid solution, and 0.1% glacial acetic acid solution were used as mobile phase A, respectively, according to the mobile phase elution conditions in Table 5; the flow rate was 0.30 mL / min; the column temperature was 30℃; and the detection wavelength was 230 nm. Suitable acid types in the mobile phase were screened by comparing the peaks in the chromatograms.

[0129] like Figure 3 As shown, the experimental results indicate that the baseline of 0.1% phosphoric acid solution-acetonitrile is relatively stable, and 0.1% phosphoric acid solution is tentatively selected as the mobile phase.

[0130] 2.1.4 Selection of Acid Concentration

[0131] Using octadecylsilane-bonded silica gel as a filler (ZORBAX Eclipse XDB-C) 18 The column was 2.1 × 100 mm (1.8 μm). Acetonitrile was used as mobile phase B, and 0.1% phosphoric acid solution, 0.05% phosphoric acid solution, and 0.15% phosphoric acid solution were used as mobile phase A, respectively. Gradient elution was performed according to the mobile phase elution conditions in Table 5. The flow rate was 0.30 mL / min, the column temperature was 30 ℃, and the detection wavelength was 230 nm. The concentration of acid in the mobile phase was investigated by comparing the peaks in the chromatogram.

[0132] like Figure 4 As shown, the experimental results indicate that the baseline of 0.1% phosphoric acid solution-acetonitrile is relatively stable, the chromatographic peaks have good shapes, and the overall distribution is uniform. Therefore, the acid concentration is tentatively set at 0.1% phosphoric acid solution.

[0133] 2.1.5 Selection of Flow Rate

[0134] Using octadecylsilane-bonded silica gel as a filler (ZORBAX Eclipse XDB-C) 18 The column was 2.1 × 100 mm (1.8 μm); gradient elution was performed using 0.1% phosphoric acid solution as mobile phase A and acetonitrile as mobile phase B, according to the mobile phase elution conditions in Table 5; flow rates of 0.2 mL / min, 0.3 mL / min, 0.4 mL / min, and 0.5 mL / min were investigated; the column temperature was 30 ℃; and the detection wavelength was 230 nm. A suitable flow rate was selected by comparing the peaks in the chromatogram.

[0135] like Figure 5As shown in the chromatogram of the test sample, different flow rates have a significant impact on the chromatographic peaks, especially the peaks within the red box. The separation effect is best at 0.3 mL / min, while at other flow rates, there are either peak inclusions or no separation. Considering the overall separation effect and balance, a flow rate of 0.3 mL / min is tentatively set.

[0136] 2.1.6 Selection of Column Temperature

[0137] Using octadecylsilane-bonded silica gel as a filler (ZORBAX Eclipse XDB-C) 18 The column was 2.1 × 100 mm (1.8 μm); 0.1% phosphoric acid solution was used as mobile phase A, and acetonitrile was used as mobile phase B. Gradient elution was performed according to the mobile phase elution conditions in Table 5; the flow rate was 0.3 mL; the column temperatures of 25℃, 30℃, 35℃, and 40℃ were investigated; the detection wavelength was 230 nm. The appropriate column temperature was screened by comparing the peaks in the chromatogram.

[0138] like Figure 6 As shown in the chromatogram, when the column temperature is 30℃, the chromatographic peaks in the sample have good shapes and are uniformly distributed. The column temperature is tentatively set at 30℃.

[0139] 2.1.7 Selection of detection wavelength

[0140] Accurately pipette the test solution and inject it into the liquid chromatograph. Use a diode array detector to perform a full wavelength scan in the range of 190-390 nm. Compare the chromatograms at different detection wavelengths.

[0141] like Figure 7 As shown in the figure, the experimental results indicate that there is a relatively large amount of chromatographic peak information when the detection wavelength is 200-300 nm. There are fewer chromatographic peaks above 300 nm. Considering factors such as the cutoff absorption wavelength of acetonitrile, and taking into account peak shape, number of peaks, and separation, a detection wavelength of 230 nm is tentatively proposed.

[0142] 2.1.8 Information Maximization Consideration

[0143] To determine whether the main components of the test sample were reflected in the chromatogram and whether the principle of maximizing effective information was met, 0.1% phosphoric acid solution was used as mobile phase A, acetonitrile as mobile phase B, and the flow rate was 0.3 mL / min; the column temperature was 30℃; the detection wavelength was 230 nm; gradient elution was performed according to the mobile phase elution conditions shown in Table 6, and the acquisition time was increased to 121 min. The test sample solution was injected, and the chromatogram was recorded.

[0144] Table 6 Mobile phase elution conditions

[0145]

[0146] like Figure 8 As shown, the experimental results indicate that extending the elution time of a high proportion of acetonitrile does not result in any other chromatographic peaks in the test solution, and the information collected by this method satisfies the principle of maximizing information.

[0147] 2.2 Investigation of the preparation method of the test sample

[0148] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (ZORBAX Eclipse XDB-C). 18 2.1×100mm,

[0149] 1.8 μm); using 0.1% phosphoric acid solution as mobile phase A and acetonitrile as mobile phase B, gradient elution was performed according to the mobile phase elution conditions in Table 5; the flow rate was 0.3 mL / min; the column temperature was 30 ℃; and the detection wavelength was 230 nm.

[0150] 2.2.1 Investigation of extraction solvent

[0151] Grind the granules of this product into a fine powder, divide into 9 portions, each approximately 1g, and accurately weigh them. Place each portion in a stoppered conical flask, and accurately add 25mL each of methanol, 70% methanol, 50% methanol, 10% methanol, anhydrous ethanol, 70% ethanol, 50% ethanol, 10% ethanol, and water. Weigh the flasks, sonicate (1100W, 40kHz) for 15 minutes, remove, cool, and weigh again. Make up the weight loss with the appropriate solvent, shake well, and filter to obtain the final product. Accurately inject each of the above test solutions into a liquid chromatograph and collect the chromatograms.

[0152] like Figure 10 As shown in the chromatogram, when water, anhydrous ethanol, 70% ethanol, 50% ethanol, and 10% ethanol are used as solvents, there are cases of chromatographic peak loss and poor peak shape. Therefore, they are not included in the peak area comparison. There is no significant difference in the chromatographic peaks in the chromatograms of other solvents.

[0153] Table 7 Comparison of peak areas in fingerprint spectra for different solvents

[0154]

[0155] like Figure 9 As shown in Table 7, the experimental results show that the relative standard deviation (RSD) of the total peak area / sample weight is 6.1% when methanol is used as the extraction solvent at different concentrations. This indicates that there are significant differences among different concentrations of methanol as the extraction solvent. When the methanol concentration is 10%, the total peak area / sample weight is the largest, indicating that 10% methanol is better for extracting the sample. Therefore, 10% methanol is tentatively set as the solvent for the extraction solution.

[0156] 2.2.2 Investigation on the amount of extraction solvent used

[0157] Grind the granules of this product into a fine powder, divide into four portions, each approximately 1 g, and accurately weigh them. Place each portion in a stoppered conical flask, and accurately add 20 mL, 25 mL, 30 mL, and 50 mL of 10% methanol respectively. Weigh the flasks, sonicate (1100 W, 40 kHz) for 15 min, remove, cool, and weigh again. Make up the weight loss with the appropriate solvent, shake well, and filter to obtain the final product. Accurately inject each of the above test solutions into a liquid chromatograph and collect the chromatogram.

[0158] Table 8 Comparison of peak areas in fingerprint spectral data based on extraction solvent dosage.

[0159]

[0160] like Figure 11 As shown in Table 8, the experimental results indicate that when the solvent volume of the test sample reaches 30 mL, the overall response value of the chromatographic peak is good; and the concentration ratio is already saturated. Therefore, 30 mL is tentatively set as the extraction solvent volume of the test sample.

[0161] 2.2.3 Examination of Extraction Methods

[0162] Grind the granules of this product into a fine powder, divide into two portions, each approximately 1 g, and accurately weigh them. Place each portion in a stoppered conical flask, and accurately add 30 mL of 10% methanol to each flask. Weigh the flasks and test the extraction methods: ultrasonic treatment (1100 W, 40 kHz) and reflux extraction, respectively, for 15 min. Remove the flasks, cool them, and weigh them again. Make up the weight loss with the appropriate solvent, shake well, and filter to obtain the final product. Accurately inject each of the above test solutions into the liquid chromatograph and collect the chromatograms.

[0163] Table 9 Comparison of fingerprint peak areas based on extraction methods

[0164]

[0165] like Figure 12 As shown in Table 9, the experimental results indicate that the total peak area / sample weight ratio of ultrasonic extraction is higher than that of reflux extraction, suggesting that ultrasonic extraction is superior. Therefore, ultrasonic extraction is tentatively proposed as the extraction method for the test sample solution.

[0166] 2.2.4 Examination of extraction time

[0167] Grind the granules of this product into a fine powder, divide into three portions, each approximately 1 g, and accurately weigh them. Place each portion in a stoppered conical flask, and accurately add 30 mL of 10% methanol to each flask. Weigh the flasks, and sonicate them (1100 W, 40 kHz) for 15 min, 30 min, and 45 min respectively. Remove the flasks, cool them, and weigh them again. Make up the weight loss with the appropriate solvent, shake well, and filter to obtain the final product. Accurately inject each of the above test solutions into a liquid chromatograph and collect the chromatograms.

[0168] Table 10 Comparison of fingerprint peak areas based on extraction time.

[0169]

[0170] Note: The "total peak area / sample volume" data at 45 min of ultrasound differs significantly from that at other ultrasound times, therefore it is not included in the RSD value calculation.

[0171] like Figure 13 As shown in Table 10, the experimental results show that the RAD values ​​for 15 min and 30 min of ultrasound are 1.0%, which is lower than 2.0%, indicating that there is no significant difference between 15 min and 30 min of ultrasound. Therefore, 15 min is tentatively set as the extraction time for the test solution.

[0172] 2.2.5 Injection Volume Examination

[0173] Accurately inject 1 μL, 2 μL, and 3 μL of the above test solution into the liquid chromatograph and collect chromatograms.

[0174] Table 11 Comparison of peak areas in fingerprint chromatograms based on injection volume.

[0175]

[0176] Note: Peak loss may occur when the injection volume is 3 μL, therefore it is not included in the RAD value calculation.

[0177] like Figure 14 As shown in Table 11, the experimental results show that the RAD values ​​for injection volumes of 1 μL and 2 μL are 0.2%, which is less than 2%, indicating that there is no significant difference between injection volumes of 1 μL and 2 μL, and the chromatographic peak information and response values ​​are basically the same. Considering all aspects, 1 μL is tentatively set as the injection volume of the test solution.

[0178] 2.3 Peak Attribution and Reference Selection

[0179] 2.3.1 Attribution of Medicinal Flavor Peaks

[0180] According to the established method, a single-herb decoction solution was prepared, and the determination was carried out under provisional chromatographic conditions to identify the chromatographic peaks in the sample fingerprint spectrum.

[0181] Table 12 Results of chromatographic peak retention times in fingerprint chromatograms of various medicinal herbs.

[0182]

[0183] like Figures 15-20 As shown in Table 12, the results indicate that, based on the comparison between the determination results of each single herb slice and the test sample, the conclusion is that peaks 1, 5, 6, and 7 are from white peony root, peaks 2, 4, 8, 9, 10, 11, and 12 are from stevia, and peak 3 is from both Uncaria rhynchophylla and stevia, which are common peaks.

[0184] 2.3.2 Chromatographic Peak Identification

[0185] like Figure 21 As shown in Table 13, peak 8 was found to have an undesirable peak shape during peak assignment and was therefore excluded from subsequent analysis. Further comparison with the reference solutions revealed that the peak at 41.814 min was benzoylpaeoniflorin, which had a better peak shape and higher response value; therefore, this peak was added as peak 12. Additionally, during comparison, gallic acid (peak 1) was found at 1.451 min, neochlorogenic acid (peak 2) at 4.490 min, and chlorogenic acid (peak 3) at 9.369 min. The chromatographic peak at 10.808 min is cryptochlorogenic acid (peak 4), the chromatographic peak at 14.063 min is paeoniflorin (peak 5), the chromatographic peak at 15.501 min is paeoniflorin (peak 6), the chromatographic peak at 26.342 min is isochlorogenic acid B (peak 8), the chromatographic peak at 27.354 min is 3,5-O-dicaffeoylquinic acid (peak 10), the chromatographic peak at 31.751 min is 4,5-O-dicaffeoylquinic acid (peak 11), and peaks 7 and 9 were not identified.

[0186] Table 13. Attribution of medicinal flavors to chromatographic peaks in fingerprint chromatograms.

[0187]

[0188] 2.4 Preliminary determination of methods

[0189] Determined by high performance liquid chromatography (General Chapter 0512, Part IV, Chinese Pharmacopoeia 2020 Edition).

[0190] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase (100 mm in length, 2.1 mm in inner diameter, and 1.8 μm in particle size). 0.1% phosphoric acid solution was used as mobile phase A, and acetonitrile was used as mobile phase B. Gradient elution was performed according to the mobile phase elution conditions specified in Table 5; the flow rate was 0.3 mL / min; the column temperature was 30 °C; and the detection wavelength was 230 nm. The theoretical plate number, calculated based on the paeoniflorin peak, should be no less than 3000.

[0191] Preparation of reference solution: Take appropriate amounts of neochlorogenic acid, chlorogenic acid, paeoniflorin, isochlorogenic acid B, 4,5-O-dicaffeoylquinic acid, and benzoylpaeoniflorin, accurately weigh them, and add 10% methanol to prepare a mixed solution containing 20 μg of each in 1 mL, which is used as the reference solution.

[0192] Preparation of the test solution: Take an appropriate amount of the product granules, grind them into a fine powder, weigh about 1g accurately, place them in a stoppered conical flask, accurately add 30mL of 10% methanol, stopper tightly, weigh, sonicate (power 1100W, frequency 40kHz) for 15min, remove, cool, make up the weight, shake well, filter, and take the filtrate to obtain the test solution.

[0193] Determination method: Accurately pipette 1 μL of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0194] 2.5 Establishment of Comparison Fingerprints

[0195] Ten batches of marketed Jianerle granules were collected, and test solutions were prepared according to the method described in section 2.4, "Preliminary Determination of Methods." The chromatograms were analyzed and recorded sequentially, as shown in the figure below. A common pattern was obtained using fingerprint chromatogram similarity evaluation software as a control fingerprint chromatogram, as shown below. Figure 22 As shown, a total of 12 peaks were detected in the fingerprint spectra of 10 batches of samples.

[0196] 2.6 Similarity Evaluation

[0197] The similarity of fingerprint chromatograms of 10 batches of commercially available Jianerle granules was evaluated using the Pharmacopoeia Commission's "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)". Mark peak matching was performed using common peaks to generate a common pattern diagram, and a control chromatogram was established for similarity calculation.

[0198] As shown in Table 14, the similarity of 10 batches of commercially available Jianerle granules was all between 0.999 and 1.000.

[0199] Table 14. Similarity results of Jianerle granules before market launch.

[0200]

[0201] Example 2 Methodological Validation

[0202] 1. Specificity test

[0203] To investigate whether blank solvents and excipients interfere with the fingerprint chromatogram of Jianerle granules, under the proposed chromatographic conditions, 10% methanol solution, silicon dioxide (SiO2), sweet orange flavoring, sucrose, reference solution, and test solution were precisely injected into the liquid chromatograph, and the chromatograms were recorded.

[0204] like Figure 24 As shown in the figure, the experimental results indicate that the blank solvent and each excipient have no interfering peaks at the chromatographic peak positions, indicating good specificity.

[0205] 2. Precision test

[0206] Take the test solution and inject it 6 times consecutively, following the determination method under "2.4 Preliminary determination of method" in Example 1.

[0207] Table 15 Similarity Results of Precision Examination

[0208]

[0209] like Figure 25 As shown in Table 15, the experimental results indicate that, using Mark peak matching and the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, the similarity between the fingerprint chromatogram of the test sample and the control fingerprint chromatogram is above 0.999. The National Drug Standards Working Manual (4th Edition) requires that the precision similarity of fingerprint chromatograms should not be lower than 0.95, indicating that the instrument precision is good.

[0210] 3. Repeatability test

[0211] Take about 1.0g of this product and prepare 6 parallel portions. Accurately weigh them and operate according to the preparation method and determination method of the test solution under "2.5 Preliminary determination of method" in Example 1.

[0212] Table 16. Similarity Results of Repeatability Tests

[0213]

[0214] like Figure 26 As shown in Table 16, the experimental results indicate that, using Mark peak matching and the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, the similarity between the fingerprint chromatogram of the test sample and the control fingerprint chromatogram is above 0.999. The National Drug Standards Working Manual (4th Edition) requires that the repeatability similarity of fingerprint chromatograms should not be lower than 0.95, indicating good repeatability.

[0215] 4. Stability

[0216] Take the repeatability sample 1 and place it for 0, 4.4, 8.8, 12, 20 and 24 hours after preparation, respectively, and perform the determination method under "2.5 Preliminary determination of method" in Example 1.

[0217] Table 17 Similarity Results of Stability Test

[0218]

[0219] like Figure 27As shown in Table 17, the experimental results indicate that, using Mark peak matching and the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, the similarity between the fingerprint chromatogram of the test sample and the control fingerprint chromatogram is above 0.999. The National Drug Standards Working Manual (4th Edition) requires that the stability similarity of fingerprint chromatograms not be lower than 0.95, and the results show that the test sample solution exhibits good stability within 24 hours.

[0220] 5. Durability test

[0221] 5.1 Reagents from different manufacturers

[0222] Take about 1.0 g of this product, accurately weigh it, and operate according to the preparation method and determination method of the test solution under "2.4 Preliminary determination of method" in Example 1.

[0223] Table 18. Similarity results of reagents from different manufacturers.

[0224]

[0225] like Figure 28 As shown in Table 18, the experimental results indicate that, using Mark peak matching and the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, the similarity between the fingerprints of the test samples and the control fingerprints determined by reagents from different manufacturers was 1.000. The "National Drug Standards Working Manual" (4th edition) requires that the similarity of fingerprint chromatograms obtained by reagents from different manufacturers should not be lower than 0.90, indicating that the reagents from different manufacturers have good durability.

[0226] 5.2 Different chromatographic columns

[0227] Take about 1.0 g of this product, accurately weigh it, and operate according to the preparation method and determination method of the test solution under "2.4 Preliminary determination of method" in Example 1 to investigate different brands of chromatographic columns.

[0228] Table 19. Similarity results of different chromatographic columns

[0229]

[0230] like Figure 29 As shown in Table 19, the experimental results indicate that, using Mak peak matching and the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, the fingerprint chromatograms of the test samples, according to the ZORBAX Eclipse XDB-C system, are suitable for fingerprint analysis. 18 The similarity between the fingerprint chromatograms of the Hypersil GOLD column and the control fingerprint chromatograms was 1.000 and 0.992, respectively. The National Drug Standards Working Manual (4th Edition) requires that the similarity of fingerprint chromatograms between different chromatographic columns should not be lower than 0.90, and the results indicate that the columns of different brands have good durability.

[0231] Example 3: Determination of fingerprint mapping method

[0232] The determination was performed according to high performance liquid chromatography (General Chapter 0512, Part IV, Chinese Pharmacopoeia 2020 Edition).

[0233] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material (100 mm in length, 2.1 mm in inner diameter, and 1.8 μm in particle size); 0.1% phosphoric acid solution was used as mobile phase A, and acetonitrile was used as mobile phase B, with gradient elution performed according to the mobile phase elution conditions in Table 5; the flow rate was 0.3 mL / min; the column temperature was 30℃; and the detection wavelength was 230 nm. The theoretical plate number, calculated based on the paeoniflorin peak, should be no less than 3000.

[0234] Preparation of reference solution: Take appropriate amounts of neochlorogenic acid, chlorogenic acid, paeoniflorin, isochlorogenic acid B, 4,5-O-dicaffeoylquinic acid, and benzoylpaeoniflorin, accurately weigh them, and add 10% methanol to prepare a mixed solution containing 20 μg of each in 1 mL, which is used as the reference solution.

[0235] Preparation of the test solution: Take an appropriate amount of the product granules, grind them into a fine powder, weigh about 1g accurately, place them in a stoppered conical flask, accurately add 30mL of 10% methanol, stopper tightly, weigh, sonicate (power 1100W, frequency 40kHz) for 15min, remove, cool, make up the weight, shake well, filter, and take the filtrate to obtain the test solution.

[0236] Determination method: Accurately pipette 1 μL of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0237] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of Jianerle particles, characterized in that, Includes the following steps: Take the Jianerle granules, mix them with methanol solution, sonicate, cool, shake well, filter and collect the filtrate to obtain the test solution; The fingerprint spectrum of the Jianerle granules was obtained by performing ultra-high performance liquid chromatography analysis on the test solution. The packing material for the ultra-high performance liquid chromatography is octadecylsilane-bonded silica gel. The mobile phase A of the ultra-high performance liquid chromatography is a 0.1% (v / v) phosphoric acid solution, and the mobile phase B is acetonitrile; The mobile phase elution conditions for the ultra-high performance liquid chromatography are as follows: 0-5 min, mobile phase A 95%, mobile phase B 5%; 5-14 min, mobile phase A 95%→88%, mobile phase B 5%→12%; 14-35 min, mobile phase A 88%→82%, mobile phase B 12%→18%; 35-55 min, mobile phase A 82%→30%, mobile phase B 18%→70%; 55-56 min, mobile phase A 30%→95%, mobile phase B 70%→5%; 56-66 min, mobile phase A 95%, mobile phase B 5%; The detection wavelength of the ultra-high performance liquid chromatography is 230 nm.

2. The construction method as described in claim 1, characterized in that, The volume fraction of the methanol solution is 10%; the mass-to-volume ratio of the Jianerle granules to the methanol solution is 1g:30mL.

3. The construction method as described in claim 1, characterized in that, The ultrasonic treatment had a power of 1100W, a frequency of 40kHz, and a duration of 15min.

4. The construction method as described in claim 1, characterized in that, The sample loading volume for the ultra-high performance liquid chromatography is 1 μL.

5. The construction method as described in claim 1, characterized in that, The flow rate of the ultra-high performance liquid chromatography was 0.3 mL / min.

6. The construction method as described in claim 1, characterized in that, The column temperature of the ultra-high performance liquid chromatography is 30℃.

7. The construction method as described in claim 1, characterized in that, The fingerprint spectrum of Jianerle granules consists of 12 characteristic peaks; among them, peaks 1, 5, 6, 7 and 12 are peaks unique to Paeonia lactiflora; peaks 2, 4, 8, 9, 10 and 11 are peaks unique to Stevia rebaudiana; and peak 3 is a common peak of Uncaria rhynchophylla and Stevia rebaudiana.

8. The construction method as described in claim 7, characterized in that, The identification result of the characteristic peak is as follows: Peak 1 is gallic acid; Peak 2 is neochlorogenic acid; Peak 3 is chlorogenic acid; Peak 4 is cryptochlorogenic acid; Peak 5 is paeoniflorin; Peak 6 is paeoniflorin; Peak 8 is isochlorogenic acid B; Peak 10 is 3,5-O-dicaffeoylquinic acid; Peak 11 is 4,5-O-dicaffeoylquinic acid; Peak 12 is benzoylpaeoniflorin.

9. The application of a fingerprint spectrum obtained by the construction method according to any one of claims 1-8 in the quality detection and / or quality control of Jianerle granules.

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