Method for establishing fingerprint spectrum of lianqin zhenzhu dripping pill

Abstract

A method for establishing a fingerprint spectrum of a Lianqin Zhenzhu dripping pill, the method comprising determining active ingredients in a Lianqin Zhenzhu dripping pill by means of high-performance liquid chromatography, wherein the extraction solvent for the Lianqin Zhenzhu dripping pill is methanol-water, and the active ingredients include baicalin, geniposide, forsythiaside A and glycyrrhizic acid. By using the method for establishing a fingerprint spectrum of a Lianqin Zhenzhu dripping pill, the active ingredients in the preparation can be effectively extracted, and the main ingredients of the Lianqin Zhenzhu dripping pill can be effectively characterized. The method enables the rapid and accurate identification of the authenticity and uniformity of a finished preparation, and is of great significance for comprehensively controlling the quality of a product.

Description

A method for establishing a fingerprint spectrum of Lianqin Pearl Dropping Pills

[0001] This disclosure claims priority to a Chinese patent application filed with the Patent Office of China on January 29, 2024, entitled “A Method for Establishing a Fingerprint Spectrum of Lianqin Pearl Drops” and application number 202410121959.4. The entire contents of the above application are incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of traditional Chinese medicine fingerprints, and in particular to a method for establishing a fingerprint of Lianqin Zhenzhu Dropping Pills. Background Art

[0003] Lianqin Pearl Drops are a traditional Chinese medicine drop pill preparation made from 10 Chinese medicinal materials, including Forsythia suspensa, Scutellaria baicalensis, Gardenia jasminoides, and Licorice. They have the effects of clearing away heat and purging fire, detoxifying and relieving pain. They are mainly used to treat recurrent aphthous ulcers (mild aphthous ulcers and stomatitis aphthous ulcers) and heart and spleen heat accumulation syndrome. Lianqin Pearl Drops have a definite clinical efficacy, can promote the rapid healing of aphthous ulcers, significantly reduce the symptoms of spontaneous pain and irritation pain of ulcers, and reduce recurrence. Currently, Lianqin Pearl Drops have been included in the 2020 National Medical Insurance Catalogue as a traditional Chinese medicine oral medication. Its dosage form is the only traditional Chinese medicine drop pill dosage form in China.

[0004] Traditional Chinese medicine fingerprint technology is a comprehensive, quantifiable identification method primarily used to verify the authenticity and quality of traditional Chinese medicines and Chinese patent medicines, and to evaluate the uniformity and stability of the raw materials, semi-finished products, and finished products of traditional Chinese medicine preparations. Traditional Chinese medicines and their preparations are complex, multi-component systems, so their quality should be evaluated using appropriate detection methods that provide rich identification information. Establishing a traditional Chinese medicine fingerprint will comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicines and their preparations, thereby providing a holistic description and evaluation of drug quality.

[0005] Among them, high-performance liquid chromatography (HPLC) is one of the mainstream methods in traditional Chinese medicine fingerprint technology, with characteristics such as high separation efficiency, good reproducibility, high selectivity, high detection sensitivity, fast analysis speed, and strong analytical data reliability. The vast majority of traditional Chinese medicine ingredients can be analyzed and detected on HPLC, and a wealth of application experience has been accumulated. Therefore, HPLC has become the preferred method of traditional Chinese medicine fingerprint technology. Currently, there are no literature reports on methods for detecting the fingerprint of Lianqin Pearl Dropping Pills. Therefore, this field urgently needs to establish a method for the fingerprint of Lianqin Pearl Dropping Pills that is both easy to operate, stable and reproducible, and can comprehensively and accurately evaluate the quality and characteristics of the pill dosage form.

[0006] Summary of the Invention

[0007] Problems to be solved by the invention

[0008] In view of the above problems existing in the prior art, the purpose of the present invention is to provide a fingerprint spectrum of Lianqin Pearl Dropping Pills and a method for establishing the same, which can be used for the detection and quality control of the active ingredients of Lianqin Pearl Dropping Pills.

[0009] Solutions for solving problems

[0010] The present invention provides a method for establishing a fingerprint spectrum of Lianqin Pearl Dropping Pills, which comprises using high performance liquid chromatography to determine the effective ingredients in the Lianqin Pearl Dropping Pills; wherein, the extraction solvent of the Lianqin Pearl Dropping Pills is methanol-water, and the effective ingredients include baicalin, geniposide, forsythiaside A and glycyrrhizic acid.

[0011] Preferably, the volume ratio of methanol to water is 10:90-90:10; more preferably, the volume ratio of methanol to water is 50:50-70:30; further preferably, the volume ratio of methanol to water is 55:45.

[0012] Preferably, the method comprises the following steps:

[0013] (1) preparing a test solution: extracting the Lianqin Pearl Dropping Pills with the extraction solvent, diluting with the extraction solvent and filtering to obtain the test solution;

[0014] (2) preparing a reference solution: using the extraction solvent to dissolve and dilute the reference substance containing the active ingredient, and filtering to obtain the reference solution;

[0015] (3) High performance liquid chromatography determination: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram;

[0016] Preferably, the extraction is ultrasonic extraction, and more preferably, the extraction time is 15-45 minutes.

[0017] Preferably, the chromatographic column of the high performance liquid chromatography method uses octadecylsilane bonded silica gel as a filler; more preferably, the chromatographic column is Phenomenex C18, 4.6 mm×250 mm, 5 μm.

[0018] Preferably, the mobile phase system of the HPLC method is 0.1% phosphoric acid solution-methanol; more preferably, the mobile phase A of the HPLC method is 0.1% phosphoric acid solution, and the mobile phase B is methanol; further preferably, the HPLC method includes gradient elution using the mobile phase; most preferably, the gradient elution is as follows:

[0019] Preferably, the flow rate of the mobile phase is 0.5-1.5 ml / min; more preferably, the flow rate of the mobile phase is 0.9-1.1 ml / min; further preferably, the flow rate of the mobile phase is 1 ml / min.

[0020] Preferably, the column temperature of the HPLC method is 25-45°C; more preferably, the column temperature of the HPLC method is 35°C.

[0021] Preferably, the detection wavelength of the high performance liquid chromatography is selected from one of 238 nm, 280 nm, and 335 nm; more preferably, the detection wavelength of the high performance liquid chromatography is 238 nm.

[0022] Preferably, the retention time of each chromatographic peak of the fingerprint is as follows:

[0023] Peak 1, average retention time RT is 2.799 min;

[0024] Peak 2, average retention time RT is 3.313 min;

[0025] Peak 3, average retention time RT is 6.252 min;

[0026] Peak 4, average retention time RT is 15.475 min;

[0027] Peak 5, average retention time RT is 22.752 min;

[0028] Peak 6, average retention time RT is 25.808 min;

[0029] Peak 7, average retention time RT is 26.374 min;

[0030] Peak 8, average retention time RT is 27.342 min;

[0031] Peak 9, average retention time RT is 32.166 min;

[0032] Peak 10, average retention time RT is 33.451 min;

[0033] Peak 11, average retention time RT is 37.805 min;

[0034] Peak 12, average retention time RT is 41.902 min;

[0035] Peak 13, average retention time RT is 42.329 min;

[0036] Peak 14, average retention time RT is 49.578 min;

[0037] Peak 15, average retention time RT is 54.907 min;

[0038] Peak 16, average retention time RT is 55.822min.

[0039] The present invention also provides an application of the method described in quality inspection of Lianqin Pearl Dropping Pills; preferably, the quality inspection includes identification of active ingredients.

[0040] Effects of the Invention

[0041] The technical solution of the present invention optimizes the extraction method of the active ingredients of Lianqin Pearl Dropping Pills, effectively extracting the active ingredients from the preparation. A method for establishing the fingerprint of Lianqin Pearl Dropping Pills using high-performance liquid chromatography is also established. This method can obtain a liquid fingerprint of Lianqin Pearl Dropping Pills, effectively characterizing the main components of Lianqin Pearl Dropping Pills. The fingerprint of the present invention can quickly and accurately identify the authenticity and uniformity of the finished preparation, which is of great significance for comprehensive control of product quality. It also has the advantages of simple operation, good reproducibility, high selectivity, high detection sensitivity, and high precision. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is a chromatogram obtained from the detection wavelength screening test.

[0043] Figure 2 is a chromatogram obtained from the mobile phase system screening test.

[0044] Figure 3 is a chromatogram obtained from the elution gradient screening experiment.

[0045] Figure 4 is a chromatogram obtained from the column screening test.

[0046] Figure 5 is a chromatogram obtained from the Lianqin Pearl Drops extraction solvent screening test.

[0047] Figure 6 is a chromatogram obtained from the Lianqin Pearl Dropping Pills extraction time screening test.

[0048] Figure 7 is a chromatogram obtained from the Lianqin Pearl Dropping Pills filter membrane screening test.

[0049] FIG8 is a chromatogram obtained from the concentration screening test of Lianqin Pearl Dropping Pills samples.

[0050] Figure 9 is a chromatogram obtained from the column temperature and flow rate method durability test.

[0051] Figure 10 is a chromatogram obtained from the chromatographic peak identification experiment.

[0052] Figure 11 is a chromatogram obtained from the experiment of obtaining the fingerprint of Lianqin Pearl Drops. DETAILED DESCRIPTION

[0053] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following detailed description is given by way of specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly illustrate the details of the local features. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0054] As used herein, the term "fingerprint" refers to a characteristic pattern or image of a component population obtained by spectral or chromatographic determination.

[0055] As used herein, the term "mobile phase system" refers to the substance that carries the components to be analyzed forward during the chromatography process.

[0056] The present invention provides a method for establishing a fingerprint spectrum of Lianqin Pearl Dropping Pills, which comprises using high performance liquid chromatography to determine the effective ingredients in the Lianqin Pearl Dropping Pills; wherein, the extraction solvent of the Lianqin Pearl Dropping Pills is methanol-water, and the effective ingredients include baicalin, geniposide, forsythiaside A and glycyrrhizic acid.

[0057] In certain embodiments, the volume ratio of methanol to water is 10:90-90:10.

[0058] In certain embodiments, the volume ratio of methanol to water is 10:90, or 15:85, or 20:80, or 25:75, or 30:70, or 35:65, or 40:60, or 45:55, or 50:50, or 55:45, or 60:40, or 65:35, or 70:30, or 75:25, or 80:20, or 85:15, or 90:10.

[0059] In certain embodiments, the volume ratio of methanol to water is 50:50-70:30.

[0060] In certain embodiments, the volume ratio of methanol to water is 55:45.

[0061] In certain embodiments, the method comprises the steps of:

[0062] (1) preparing a test solution: extracting the Lianqin Pearl Dropping Pills with the extraction solvent, diluting with the extraction solvent and filtering to obtain the test solution;

[0063] (2) preparing a reference solution: using the extraction solvent to dissolve and dilute the reference substance containing the active ingredient, and filtering to obtain the reference solution;

[0064] (3) High performance liquid chromatography determination: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0065] In certain embodiments, the extraction is ultrasonic extraction.

[0066] In certain embodiments, the extraction takes 15-45 minutes.

[0067] In some embodiments, the extraction time is 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, or 20 minutes, or 21 minutes, or 22 minutes, or 23 minutes, or 24 minutes, or 25 minutes, or 26 minutes, or 27 minutes, or 28 minutes, or 29 minutes, or 30 minutes, or 31 minutes, or 32 minutes, or 33 minutes, or 34 minutes, or 35 minutes, or 36 minutes, or 37 minutes, or 38 minutes, or 39 minutes, or 40 minutes, or 41 minutes, or 42 minutes, or 43 minutes, or 44 minutes, or 45 minutes.

[0068] In certain embodiments, the concentration of the test solution in step (1) is 0.2 to 0.4 pellets / ml.

[0069] In certain embodiments, the concentration of the test solution in step (1) is 0.2 pill / ml, 0.21 pill / ml, 0.22 pill / ml, 0.23 pill / ml, 0.24 pill / ml, 0.25 pill / ml, 0.26 pill / ml, 0.27 pill / ml, 0.28 pill / ml, 0.29 pill / ml, or 0.3 pill / ml, or 0.31 pill / ml, or 0.32 pill / ml, or 0.33 pill / ml, or 0.34 pill / ml, or 0.35 pill / ml, or 0.36 pill / ml, or 0.37 pill / ml, or 0.38 pill / ml, or 0.39 pill / ml, or 0.4 pill / ml.

[0070] In certain embodiments, the filter membrane used for the filtration is selected from one or more of a PTFE membrane, a PVDF membrane, and a NY membrane.

[0071] In certain embodiments, the filter membrane used in the filtration is a PTFE membrane.

[0072] In certain embodiments, the filter membrane used in the filtration is a PVDF membrane.

[0073] In certain embodiments, the filter membrane used in the filtration is a NY membrane.

[0074] In certain embodiments, the HPLC column uses octadecylsilane bonded silica gel as a filler.

[0075] In certain embodiments, the chromatography column is Phenomenex C18, 4.6 mm×250 mm, 5 μm.

[0076] In certain embodiments, the mobile phase system of the high performance liquid chromatography is 0.1% phosphoric acid solution-methanol.

[0077] In certain embodiments, the mobile phase A of the HPLC method is 0.1% phosphoric acid solution, and the mobile phase B is methanol.

[0078] In certain embodiments, the high performance liquid chromatography method comprises gradient elution using the mobile phase.

[0079] In certain embodiments, the gradient elution is as follows:

[0080] In certain embodiments, the flow rate of the mobile phase is 0.5-1.5 ml / min.

[0081] In certain embodiments, the flow rate of the mobile phase is 0.5 ml / min, or 0.6 ml / min, or 0.7 ml / min, or 0.8 ml / min, or 0.9 ml / min, or 1.0 ml / min, or 1.1 ml / min, or 1.2 ml / min, or 1.3 ml / min, or 1.4 ml / min, or 1.5 ml / min.

[0082] In certain embodiments, the flow rate of the mobile phase is 0.9-1.1 ml / min.

[0083] In certain embodiments, the flow rate of the mobile phase is 1 ml / min.

[0084] In certain embodiments, the column temperature of the HPLC method is 25-45°C.

[0085] In certain embodiments, the column temperature of the HPLC method is 25°C, or 26°C, or 27°C, or 28°C, or 29°C, or 30°C, or 31°C, or 32°C, or 33°C, or 34°C, or 35°C, or 36°C, or 37°C, or 38°C, or 39°C, or 40°C, or 41°C, or 42°C, or 43°C, or 44°C, or 45°C.

[0086] In certain embodiments, the column temperature of the HPLC method is 35°C.

[0087] In certain embodiments, the detection wavelength of the high performance liquid chromatography is selected from one of 238 nm, 280 nm, and 335 nm.

[0088] In certain embodiments, the detection wavelength of the high performance liquid chromatography is 238 nm.

[0089] In certain embodiments, the detection wavelength of the high performance liquid chromatography is 280 nm.

[0090] In certain embodiments, the detection wavelength of the high performance liquid chromatography is 335 nm.

[0091] In certain embodiments, the retention time of each chromatographic peak of the fingerprint is as follows:

[0092] Peak 1, average retention time RT is 2.799 min;

[0093] Peak 2, average retention time RT is 3.313 min;

[0094] Peak 3, average retention time RT is 6.252 min;

[0095] Peak 4, average retention time RT is 15.475 min;

[0096] Peak 5, average retention time RT is 22.752 min;

[0097] Peak 6, average retention time RT is 25.808 min;

[0098] Peak 7, average retention time RT is 26.374 min;

[0099] Peak 8, average retention time RT is 27.342 min;

[0100] Peak 9, average retention time RT is 32.166 min;

[0101] Peak 10, average retention time RT is 33.451 min;

[0102] Peak 11, average retention time RT is 37.805 min;

[0103] Peak 12, average retention time RT is 41.902 min;

[0104] Peak 13, average retention time RT is 42.329 min;

[0105] Peak 14, average retention time RT is 49.578 min;

[0106] Peak 15, average retention time RT is 54.907 min;

[0107] Peak 16, average retention time RT is 55.822min.

[0108] The present invention also provides an application of the method in quality detection of Lianqin Pearl Dropping Pills.

[0109] In certain embodiments, the quality testing includes identification of the active ingredient.

[0110] Unless otherwise stated, the experimental methods in the following examples without specific conditions are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight. Unless otherwise stated, the room temperature described in the present invention is 20°C to 30°C. Unless otherwise stated, the various materials and reagents used in the present invention can be obtained using conventional methods in the art or through commercial channels, including but not limited to the following instruments and reagents:

[0111] Vanquish Core high-performance liquid chromatograph (Thermo Fisher Scientific, USA);

[0112] DAD UV detector (Thermo Fisher Scientific, USA);

[0113] Mettler XPR2 / A one-millionth electronic balance (Mettler-Toledo International Ltd., Switzerland);

[0114] XJ-700KT ultrasonic cleaning machine (Shanghai Fangxu Technology Co., Ltd.);

[0115] Mettler S220 multi-parameter tester (Mettler-Toledo International GmbH, Switzerland);

[0116] Milli-Q ultrapure water instrument (Millipore, USA);

[0117] Methanol (chromatographic grade, Merck, Germany);

[0118] Acetonitrile (chromatographic grade, Wokai);

[0119] Orthophosphoric acid (chromatographically pure, Tedia, USA);

[0120] Ultrapure water (chromatographic grade, prepared immediately before use using Milli-Q EQ7000);

[0121] 0.22 μm PTFE membrane (Shanghai Anpu Laboratory Technology Co., Ltd.);

[0122] 0.22 μm PVDF membrane (Shanghai Anpu Laboratory Technology Co., Ltd.);

[0123] 0.22 μm Nylon membrane (Nap Analytical Technology Co., Ltd.);

[0124] Glycyrrhizic acid (Adamas, batch number P1850507, purity ≥95%);

[0125] Baicalin (Adamas, batch number P1452747, purity ≥90%);

[0126] Forsythiaside A (BiDe Pharmaceutical, batch number DTX078, purity ≥98%);

[0127] Geniposide (Adamas, batch number P2307377, purity ≥98%);

[0128] Lianqin Pearl Dropping Pills (Batch No.: 221010, 221024, 221201, Suzhou Pharmaceutical Factory, Jiangsu Wuzhong Pharmaceutical Group Co., Ltd.).

[0129] Example 1: Screening of detection wavelength

[0130] Referring to the detection methods for various Chinese herbal ingredients in the pharmacopoeia and combining the results of a full-wavelength UV scan from 190 nm to 750 nm, the responses of the chromatographic peaks at 238 nm, 280 nm, and 335 nm were analyzed. As shown in Figure 1, the chromatographic baselines at all selected wavelengths were stable, but geniposide only responded at 238 nm. This wavelength also exhibited the highest number of chromatographic peaks and good peak shape, so the detection wavelength of 238 nm was selected.

[0131] Example 2: Screening of chromatographic columns

[0132] The same sample solution was injected and analyzed using two C18 columns, the Phenomenex Gemini C18 (4.6mm×250mm, 5μm) and the Dikma Plasitic ODS (4.6mm×250mm, 5μm), under otherwise identical testing conditions. The fingerprints of Lianqin Pearl Dropping Pills were compared. As shown in Figure 4, the retention of the major components by the two C18 columns was essentially identical. However, the baseline was more stable using the Phenomenex Gemini C18 (4.6mm×250mm, 5μm) column, so this column was selected for subsequent analysis.

[0133] Example 3: Screening of mobile phase system

[0134] Fingerprint analysis was performed using four different mobile phase systems: 0.1% phosphoric acid solution-methanol, 0.1% phosphoric acid solution-acetonitrile, 0.01 mol / L potassium dihydrogen phosphate solution-methanol, and 0.01 mol / L sodium dihydrogen phosphate solution-methanol. The same sample solution was used, and all other detection conditions were consistent. As shown in Figure 2, 0.1% phosphoric acid solution-methanol was superior to the other mobile phase systems in terms of the number of chromatographic peaks, resolution, and peak shape. Therefore, 0.1% phosphoric acid solution-methanol was selected as the mobile phase system.

[0135] Example 4: Screening of elution gradients

[0136] In order to achieve better separation between spectral peaks, seven elution gradients were screened, and the specific gradients are shown in Table 1.

[0137] Table 1: Elution gradient screening

[0138] The results are shown in Figure 3. Gradient 7 is superior to the other six gradients in terms of the number of chromatographic peaks, separation, and peak shape. Therefore, gradient 7 was selected as the elution gradient.

[0139] According to the screening results of Examples 1-4, the chromatographic conditions for the optimal fingerprint of Lianqin Pearl Dropping Pills are as follows: using octadecylsilane bonded silica gel as the filler (Phenomenex C18, 4.6 mm × 250 mm, 5 μm or a chromatographic column with equivalent performance); using 0.1% phosphoric acid solution as mobile phase A and methanol as mobile phase B; performing gradient elution according to Table 2; the flow rate is 1.0 ml per minute; the column temperature is 35°C; the injector temperature is 6°C; the detection wavelength is 238 nm; and the injection volume is 10 μl.

[0140] Table 2: Optimal elution gradient

[0141] Example 5: Screening of extraction solvents

[0142] Combined with the extraction process and mobile phase system of the preparation, different proportions of methanol-water and ethanol-water were screened for solvents. The specific method is as follows:

[0143] Take 5 pills of Lianqin Pearl Dropping Pills (batch number 221201), place them in a 25 ml volumetric flask, add about 20 ml each of the solvents methanol-water (50:50), methanol-water (70:30), methanol-water (90:10), ethanol-water (50:50), ethanol-water (70:30) and ethanol-water (90:10), perform ultrasonic extraction for 30 minutes, let cool, dilute to the scale with the corresponding solvent, shake well, filter, and take the filtrate for sampling and analysis.

[0144] The results showed that methanol-water (50:50) and methanol-water (70:30) were superior to the other solvent groups in terms of the number of chromatographic peaks, resolution, and peak shape. Further refinement of the extraction solvents was conducted, with methanol-water (55:45), methanol-water (60:40), and methanol-water (65:35) being considered. Sample preparation was performed using the same procedures as described above. As shown in Figure 5, extraction effects were excellent with methanol-water (50:50) to methanol-water (70:30), with the number of chromatographic peaks, resolution, and peak shape being excellent. Therefore, methanol-water (50:50) to methanol-water (70:30) were selected as the extraction solvents.

[0145] Example 6: Screening of extraction time

[0146] During the sample extraction process, ultrasonic treatment was performed using an ultrasonic cleaning machine (power 700W, frequency 40kHz). The effects of ultrasonication times of 15, 30, and 45 minutes on sample extraction were investigated. Five Lianqin Pearl Dropping Pills (batch number 221201) were placed in a 25ml volumetric flask. Approximately 20ml of methanol-water (55:45) was added and ultrasonicated for different times. The mixture was cooled and diluted to the mark with methanol-water (55:45). The mixture was shaken and filtered. The filtrate was sampled and analyzed using the same chromatographic conditions as described in Example 5.

[0147] The results are shown in FIG6 . When the ultrasonic time is 15 to 45 minutes, there is no significant effect on the separation and peak shape of the sample chromatographic peaks, so the ultrasonic time is selected to be 15 to 45 minutes.

[0148] Example 7: Screening of filter membranes

[0149] In order to prevent insoluble particles from entering the chromatographic system and clogging the chromatographic column and liquid phase system, the test solution needs to be filtered through a microporous filter membrane before sampling and analysis. The effects of three different microporous filter membranes (PTFE membrane, PVDF membrane and NY membrane) on the results were investigated. Take 5 Lianqin Pearl Dropping Pills (batch number 221201), place them in a 25ml volumetric flask, add about 20ml of methanol-water (55:45), ultrasonicate for about 30 minutes, cool, dilute to the scale with methanol-water (55:45), shake well, filter using three filter membranes respectively (discard 2ml of each filter), take the filtrate for sampling and analysis, and the chromatographic conditions are as shown in Example 5.

[0150] The results are shown in Figure 7. The three filter membrane materials (PTFE membrane, PVDF membrane and NY membrane) have no significant effect on the number of detected active ingredients, separation and peak shape of the sample. Therefore, PTFE membrane, PVDF membrane and NY membrane were selected as the filter membrane materials.

[0151] Example 8: Screening of sample concentration

[0152] The fingerprint detection of samples at concentrations of 0.1 pill / ml, 0.2 pill / ml, and 0.4 pill / ml were investigated respectively, and the chromatographic conditions were as shown in Example 5.

[0153] The results are shown in Figure 8. When the sample concentration is 0.2 pill / ml and 0.4 pill / ml, the fingerprint of Lianqin Pearl Dropping Pills can be clearly detected, and the separation between chromatographic peaks is good, so the sample concentration is selected to be 0.2-0.4 pill / ml.

[0154] According to the screening results of Examples 5-8, the optimal method for preparing the test solution was determined as follows: take 5 to 10 Lianqin Pearl Dropping Pills, place them in a 25 ml volumetric flask, add about 20 ml of methanol-water (50:50) to methanol-water (70:30), ultrasonicate (power 700 W, frequency 40 kHz) for 15 to 45 minutes, cool, dilute to the scale with methanol-water (50:50) to methanol-water (70:30), shake well, filter, and take the filtrate.

[0155] Example 9: Preparation of reference solution

[0156] Based on the optimal solvent used to prepare the test solution, and in combination with the responses of geniposide, forsythiaside A, baicalin, and glycyrrhizic acid at a wavelength of 238 nm, a reference solution was prepared. Appropriate amounts of geniposide, forsythiaside A, baicalin, and glycyrrhizic acid reference substances were dissolved and diluted in methanol-water (50:50) to methanol-water (70:30) to produce a mixed solution containing approximately 50 μg / ml each of geniposide and forsythiaside A, 30 μg / ml of baicalin, and 85 μg / ml of glycyrrhizic acid per 1 ml.

[0157] The reference solution and the test solution were taken separately to compare the retention times of the chromatographic peaks. The results are shown in Figure 10. The test solution contained chromatographic peaks with the same retention times as those of geniposide, forsythiaside A, baicalin, and glycyrrhizic acid.

[0158] Example 10: Precision Test

[0159] Six parallel aliquots of Lianqin Pearl Dropping Pills (Batch No. 221201) were prepared according to the optimal method described above. Six precise injections of the sample solution were performed, and the relative standard deviations of the peak areas of the main chromatographic peaks (peaks 2, 3, 4, 5, 7, 8, 11, 13, and 16) were calculated. The results are shown in Table 3.

[0160] Table 3: Summary of peak area results of precision tests

[0161] The test solution was injected six times continuously, and the relative standard deviations of the peak areas of the main chromatographic peaks (peaks 2, 3, 4, 5, 7, 8, 11, 13 and 16) were all less than 10%, indicating that the injection precision of this method was good.

[0162] Example 11: Repeatability test

[0163] Lianqin Pearl Dropping Pills (Batch No. 221201) were used to prepare 6 test sample solutions in parallel according to the above optimal method to investigate the repeatability of the analytical method. The results are shown in Table 4.

[0164] Table 4: Summary of peak area results of repeatability test

[0165] The relative standard deviations of the peak areas of the main chromatographic peaks (peaks 2, 3, 4, 5, 7, 8, 11, 13 and 16) in the six test solutions were all less than 5%, indicating that the method had good repeatability.

[0166] Example 12: Durability Test

[0167] Lianqin Pearl Dropping Pills (Batch No. 221201) were used to prepare the test solution according to the above-mentioned optimal method. The effects of different column temperatures (25°C, 35°C, and 45°C) and flow rates (0.9ml / min, 1.0ml / min, and 1.1ml / min) on fingerprint detection were investigated. As shown in Figure 9, the peak shape and separation effect of the fingerprint obtained were good when the column temperature was 35±10°C and the flow rate was 1.0±0.1ml / min. In other words, the method has good durability when the column temperature is 35±10°C and the flow rate is 1.0±0.1ml / min.

[0168] Example 13: Solution stability test

[0169] Lianqin Pearl Dropping Pills (Batch No. 221201) were used to prepare the test solution according to the above optimal method. The fingerprints of the solution at 0, 10, 14 and 28 hours after being placed at 6°C were examined. The results are shown in Table 5.

[0170] Table 5: Summary of peak area results of solution stability test

[0171] When the test solution was placed at 6°C for 28 hours, the ratios of the peak areas of the main chromatographic peaks (peaks 2, 3, 4, 5, 7, 8, 11, 13 and 16) to the peak area at 0 time were all within the range of 0.9 to 1.1, and other chromatographic peaks not counted were clearly identifiable, indicating that the test solution had good stability when placed at 6°C for 28 hours.

[0172] Example 14: Generation of fingerprints for Lianqin Pearl Dropping Pills

[0173] The homogeneity of different batches of samples was investigated using the established analytical method. Three batches of Lianqin Pearl Dropping Pills (batch numbers: 221010, 221024, and 221201) were prepared using the optimal method described above. The optimized chromatographic conditions were used for analysis and detection, resulting in their fingerprint spectra. The results are shown in Figure 11. There are 16 common characteristic peaks on the spectrum. Chromatographic peaks 4, 8, 11, and 16 correspond to geniposide, forsythiaside A, baicalin, and glycyrrhizic acid, respectively. Peak 8 forsythiaside A has the strongest response. The retention times of the peaks in the fingerprint spectra are as follows:

[0174] Peak 1, average retention time RT is 2.799 min;

[0175] Peak 2, average retention time RT is 3.313 min;

[0176] Peak 3, average retention time RT is 6.252 min;

[0177] Peak 4, average retention time RT is 15.475 min;

[0178] Peak 5, average retention time RT is 22.752 min;

[0179] Peak 6, average retention time RT is 25.808 min;

[0180] Peak 7, average retention time RT is 26.374 min;

[0181] Peak 8, average retention time RT is 27.342 min;

[0182] Peak 9, average retention time RT is 32.166 min;

[0183] Peak 10, average retention time RT is 33.451 min;

[0184] Peak 11, average retention time RT is 37.805 min;

[0185] Peak 12, average retention time RT is 41.902 min;

[0186] Peak 13, average retention time RT is 42.329 min;

[0187] Peak 14, average retention time RT is 49.578 min;

[0188] Peak 15, average retention time RT is 54.907 min;

[0189] Peak 16, average retention time RT is 55.822min.

[0190] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations of the claims. Various modifications and variations may be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments merely illustrate several implementations of the present invention and do not limit the scope of protection of the patent of this invention.

Claims

1. A method for establishing a fingerprint spectrum of Lianqin Pearl Dropping Pills, characterized in that: The method comprises using high performance liquid chromatography to determine the effective ingredients in the Lianqin Pearl Dropping Pills; wherein, the extraction solvent of the Lianqin Pearl Dropping Pills is methanol-water, and the effective ingredients comprise baicalin, gardenia jasminoides, forsythiaside A and glycyrrhizic acid.

2. The method according to claim 1, characterized in that The volume ratio of methanol to water is 10:90-90:10; preferably, the volume ratio of methanol to water is 50:50-70:30; more preferably, the volume ratio of methanol to water is 55:

45.

3. The method according to claim 1, characterized in that The method comprises the following steps: (1) preparing a test solution: extracting the Lianqin Pearl Dropping Pills with the extraction solvent, diluting with the extraction solvent and filtering to obtain the test solution; (2) preparing a reference solution: using the extraction solvent to dissolve and dilute the reference substance containing the active ingredient, and filtering to obtain the reference solution; (3) High performance liquid chromatography determination: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram; Preferably, the extraction is ultrasonic extraction, and more preferably, the extraction time is 15-45 minutes.

4. The method according to claim 3, characterized in that The chromatographic column of the high performance liquid chromatography method uses octadecylsilane bonded silica gel as a filler; preferably, the chromatographic column is Phenomenex C18, 4.6 mm×250 mm, 5 μm.

5. The method according to claim 3, characterized in that The mobile phase system of the high performance liquid chromatography method is 0.1% phosphoric acid solution-methanol; preferably, the mobile phase A of the high performance liquid chromatography method is 0.1% phosphoric acid solution, and the mobile phase B is methanol; more preferably, the high performance liquid chromatography method includes gradient elution using the mobile phase; further preferably, the gradient elution is as follows:

6. The method according to claim 5, characterized in that The flow rate of the mobile phase is 0.5-1.5 ml / min; preferably, the flow rate of the mobile phase is 0.9-1.1 ml / min; more preferably, the flow rate of the mobile phase is 1 ml / min.

7. The method according to claim 3, characterized in that The column temperature of the HPLC method is 25-45°C; preferably, the column temperature of the HPLC method is 35°C.

8. The method according to claim 3, characterized in that The detection wavelength of the high performance liquid chromatography is selected from one of 238 nm, 280 nm, and 335 nm; preferably, the detection wavelength of the high performance liquid chromatography is 238 nm.

9. The method according to any one of claims 1 to 8, characterized in that The retention time of each chromatographic peak of the fingerprint is as follows: Peak 1, average retention time RT is 2.799 min; Peak 2, average retention time RT is 3.313 min; Peak 3, average retention time RT is 6.252 min; Peak 4, average retention time RT is 15.475 min; Peak 5, average retention time RT is 22.752 min; Peak 6, average retention time RT is 25.808 min; Peak 7, average retention time RT is 26.374 min; Peak 8, average retention time RT is 27.342 min; Peak 9, average retention time RT is 32.166 min; Peak 10, average retention time RT is 33.451 min; Peak 11, average retention time RT is 37.805 min; Peak 12, average retention time RT is 41.902 min; Peak 13, average retention time RT is 42.329 min; Peak 14, average retention time RT is 49.578 min; Peak 15, average retention time RT is 54.907 min; Peak 16, average retention time RT is 55.822min.

10. Use of the method according to any one of claims 1 to 9 in quality testing of Lianqin Pearl Dropping Pills; preferably, the quality testing includes identification of active ingredients.

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