Construction of Qingbai Tongbi capsule fingerprint spectrum and application of Qingbai Tongbi capsule fingerprint spectrum in drug quality control

Abstract

The invention relates to construction of a Qingbai Tongbi capsule fingerprint spectrum and application of the Qingbai Tongbi capsule fingerprint spectrum in drug quality control. The HPLC fingerprint spectrum of the Qingbai Tongbi capsule has 24 characteristic fingerprint chromatographic peaks, wherein the peak 4 is catechin, the peak 10 is sinomenine, the peak 12 is gallic acid, the peak 13 is magnoflorine, the peak 14 is paeoniflorin, the peak 17 is liquiritin, the peak 18 is 1, 2, 3, 4, 6-O-pentagalloylglucose, and the peak 24 is ammonium glycyrrhizinate.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine analysis, and particularly relates to the construction of a fingerprint spectrum of Qingbai Tongbi Capsule and its application in drug quality control. Background Art

[0002] Qingbai Tongbi Capsules (QTC), a classic Chinese medicine formula meticulously developed by Shaanxi Provincial Hospital of Traditional Chinese Medicine (Shaanxi Medicine Registration Number: Z20130059), are specifically designed for the treatment of rheumatoid arthritis (Fengshi Bi). This unique treatment was meticulously developed by Professor Hu Xiaojuan, a renowned Shaanxi TCM practitioner, drawing on her extensive research into historical documents and a deep understanding of Fengshi Bi. She also drew on years of clinical experience and insights into the properties of classic formulas such as Guizhi Decoction and Shaoyao Gancao Decoction, incorporating advanced research findings from traditional Chinese medicine pharmacology. It significantly reduces symptoms of rheumatoid arthritis and other autoimmune joint diseases, including joint swelling, pain, cramps, and paralysis, thereby slowing disease progression. This formulation has been used in clinical practice for over 10 years and has demonstrated satisfactory results. As a compound preparation, the quality control of Qingbai Tongbi Capsules is currently primarily performed through two methods: thin-layer chromatography (TLC) to characterize Sinomenine and Paeonia lactiflora; and reverse-phase high-performance liquid chromatography (RP-HPLC) to precisely determine the sinomenine content. However, research on the overall properties of QTC remains limited, necessitating the establishment and improvement of relevant quality standards.

[0003] This study establishes a QTC HPLC fingerprint, combines it with chemical pattern recognition methods to initially screen for differential markers, and uses network pharmacology to identify potential targets and pathways, thereby predicting quality control indicators. The resulting indicators are closely integrated with clinical applications, providing a basis for holistic QTC quality control. Summary of the Invention

[0004] The present invention provides an HPLC fingerprint of Qingbai Tongbi Capsule (or a method for constructing it), which is characterized in that when the HPLC chromatographic conditions are as follows, the HPLC fingerprint of the Qingbai Tongbi Capsule is substantially the same as that of Figure 2 consistent;

[0005] HPLC chromatographic conditions are as follows:

[0006] The chromatographic column is Agilent 5TC-C 18Chromatographic column, specifications: 250 mm × 4.6 mm, 5 μm; mobile phase: acetonitrile as phase A, 0.1% phosphoric acid aqueous solution as phase B; gradient elution conditions: 0-5 min, 5%-8% A; 5-15 min, 8%-13% A; 15-25 min, 13%-15% A; 25-35 min, 15%-25% A; 35-45 min, 25%-35% A; 45-60 min, 35%-70% A; 60-70 min, 70%-95% A; volume flow rate 1.0 mL / min; column temperature 30°C; injection volume 10 μL; UV detection wavelength 210 nm.

[0007] Another embodiment of the present invention provides the HPLC fingerprint of the Qingbai Tongbi Capsule (or its construction method), characterized in that the HPLC fingerprint of the Qingbai Tongbi Capsule is substantially the same as that of the Figure 2 The results were consistent, with 24 characteristic fingerprint chromatographic peaks, of which peak 4 was catechin, peak 10 was sinomenine, peak 12 was gallic acid, peak 13 was magnolamine, peak 14 was paeoniflorin, peak 17 was liquiritin, peak 18 was 1,2,3,4,6-O-pentagalloylglucose, and peak 24 was ammonium glycyrrhizate.

[0008] Another embodiment of the present invention provides the application of the HPLC fingerprint of the Qingbai Tongbi Capsule (or its construction method) in the quality control and component analysis of the Qingbai Tongbi Capsule.

[0009] Another embodiment of the present invention provides an application of the HPLC fingerprint of the Qingbai Tongbi Capsule (or a method for constructing the same) in the quality control of the Qingbai Tongbi Capsule, characterized in that the application comprises the following steps:

[0010] (1) The contents of Qingbai Tongbi Capsules are prepared into a methanol solution of a certain concentration to obtain a test solution;

[0011] (2) Take the test solution obtained in step (1) and test it by HPLC to obtain an HPLC chart of the test sample, wherein the chromatographic conditions are as follows:

[0012] The chromatographic column is Agilent 5TC-C 18Chromatographic column, specifications: 250 mm × 4.6 mm, 5 μm; mobile phase: acetonitrile as phase A, 0.1% phosphoric acid aqueous solution as phase B; gradient elution conditions: 0-5 min, 5%-8% A; 5-15 min, 8%-13% A; 15-25 min, 13%-15% A; 25-35 min, 15%-25% A; 35-45 min, 25%-35% A; 45-60 min, 35%-70% A; 60-70 min, 70%-95% A; flow rate: 1.0 mL / min; column temperature: 30°C; injection volume: 10 μL; UV detection wavelengths: 210 nm and 260 nm.

[0013] (3) Compare the HPLC chromatogram of the test sample obtained in step (2) with the HPLC fingerprint of the Qingbai Tongbi Capsule described in the present invention. The Qingbai Tongbi Capsule with a similarity of more than 0.90 (preferably 0.93) is a qualified product.

[0014] The concentration of the test solution described in step (1) is preferably a 5-30 mg / mL methanol solution of the contents of Qingbai Tongbi Capsules, and more preferably 10-20 mg / mL; when preparing the test solution, it is preferred to first treat the contents of Qingbai Tongbi Capsules with an appropriate volume fraction of 60%-80% methanol solution, ultrasonically treat for 20-30 minutes, and then adjust the volume and filter to obtain the test solution.

[0015] Another embodiment of the present invention provides a method for analyzing the components of Qingbai Tongbi Capsules, characterized in that the method comprises the following steps:

[0016] (1) Preparation of reference solution:

[0017] Accurately weigh appropriate amounts of catechin, sinomenine, gallic acid, magnolamine, paeoniflorin, liquiritin, 1,2,3,4,6-O-pentagalloylglucose, and ammonium glycyrrhizate reference substances, dissolve them in 5% acetonitrile-95% 0.1% phosphoric acid aqueous solution, and prepare mixed reference substance solutions with mass concentrations of 0.5, 0.16, 0.07, 0.23, 0.53, 1, 0.18, and 0.125 mg / mL, respectively;

[0018] (2) Preparation of standard curves for each reference substance in step (1):

[0019] The mixed reference solution prepared in step (1) was diluted stepwise by 0, 2, 4, 8, 16, 32, and 64 times, and HPLC determination was performed according to the chromatographic conditions of the present invention. The peak area (Y) was linearly regressed against the reference concentration (X) to obtain a regression equation. The results were as follows: catechin Y = 125955.0X - 0.6727, r = 0.9999, linear range 0.78-50.00 μg / mL; sinomenine Y = 35705X - 1.4184, r = 0.9999, linear range 2.50-160.00 μg / mL; gallic acid Y = 82572X - 12.778, r = 0.9999, linear range 1.09-70.00 μg / mL; magnolia alkaloid Y = 44762X -35.045, r=0.9999, linear range: 3.59-230.00μg / mL; paeoniflorin Y=4311.2X-45.244, r=0.998, linear range: 8.28-530.00μg / mL; liquiritin Y=32812X+463.25, r=0.9999, linear range: 31.25-1000.00μg / mL; 1,2,3,4,6-O-pentagalloylglucose Y=27615X-29.289, r=0.9999, linear range: 2.81-180.00μg / mL; ammonium glycyrrhizate Y=6728.2X-18.574, r=0.998, linear range: 1.95-125.00μg / mL;

[0020] (3) Preparation of test solution: The contents of Qingbai Tongbi capsules were prepared into a methanol solution of a certain concentration to obtain the test solution;

[0021] (4) taking the test solution obtained in step (3), detecting it by HPLC, and calculating the contents of catechin, sinomenine, gallic acid, magnolia pine, paeoniflorin, liquiritin, 1,2,3,4,6-O-pentagalloyl glucose and ammonium glycyrrhizate in Qingbai Tongbi Capsule according to the concentrations of catechin, sinomenine, gallic acid, magnolia pine, paeoniflorin, liquiritin, 1,2,3,4,6-O-pentagalloyl glucose and ammonium glycyrrhizate reference substances and their retention times in the fingerprint and the linear equation in step (2);

[0022] The chromatographic conditions for HPLC detection in steps (2) and (4) are as follows:

[0023] The chromatographic column is Agilent 5TC-C 18Chromatographic column, specifications: 250 mm × 4.6 mm, 5 μm; mobile phase: acetonitrile as phase A, 0.1% phosphoric acid aqueous solution as phase B; gradient elution conditions: 0-5 min, 5%-8% A; 5-15 min, 8%-13% A; 15-25 min, 13%-15% A; 25-35 min, 15%-25% A; 35-45 min, 25%-35% A; 45-60 min, 35%-70% A; 60-70 min, 70%-95% A; volume flow rate 1.0 mL / min; column temperature 30°C; injection volume 10 μL; UV detection wavelength 210 nm.

[0024] The concentration of the test solution described in step (3) is preferably a 5-30 mg / mL methanol solution of the contents of Qingbai Tongbi Capsules, and more preferably 10-20 mg / mL; when preparing the test solution, it is preferred to first treat the contents of Qingbai Tongbi Capsules with an appropriate volume fraction of 60%-80% methanol solution, ultrasonically treat for 20-30 minutes, then adjust the volume and filter to obtain the test solution.

[0025] Unless otherwise specified, the ratio of the mobile phase in the mixed solution and chromatographic conditions in the present invention is by volume; 0.1% phosphoric acid aqueous solution refers to a phosphoric acid aqueous solution with a mass fraction of 0.1%.

[0026] Compared with the prior art, the advantages of the present invention are: (1) the present invention makes up for the deficiencies of the prior art, establishes a high performance liquid chromatography (HPLC) fingerprint of Qingbai Tongbi Capsule, and provides an effective method for the quality control of Qingbai Tongbi Capsule; (2) the present invention matches the chromatograms of 10 batches of Qingbai Tongbi Capsule samples to obtain a control spectrum. In the 10 batches of samples, a total of 24 chromatographic peaks are identified as common peaks in the fingerprint of Qingbai Tongbi Capsule, and the common pattern of the HPLC fingerprint of Qingbai Tongbi Capsule is determined; (3) the HPLC fingerprint method and content determination method of Qingbai Tongbi Capsule established by the present invention have good analytical and evaluation capabilities, and have the advantages of accuracy, simplicity, stability and reliability. Therefore, this method can be used as an effective evaluation method for the quality of Qingbai Tongbi Capsule. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 These are the HPLC fingerprints of 10 batches of Qingbai Tongbi Capsules samples (S1 to S10) and the reference fingerprint (R);

[0028] Figure 2 The reference fingerprints are generated by fitting the fingerprints of 10 batches of Qingbai Tongbi Capsules (S1 to S10) of the present invention;

[0029] Figure 3 It is the HPLC chart of the medicinal materials with common peaks;

[0030] Figure 4 This is the HPLC spectrum of the mixed reference solution, in which peak 4 is catechin, peak 10 is sinomenine, peak 12 is gallic acid, peak 13 is magnolamine, peak 14 is paeoniflorin, peak 17 is liquiritin, peak 18 is 1,2,3,4,6-O-pentagalloylglucose, and peak 24 is ammonium glycyrrhizate. DETAILED DESCRIPTION

[0031] Example 1

[0032] 1. Instruments and reagents

[0033] 1.1 Instruments

[0034] 1260 series high performance liquid chromatograph and VWD detector (Agilent, USA); KQ-100 ultrasonic cleaning machine (Kunshan Ultrasonic Instrument Co., Ltd.); BS210S 1 / 10,000 electronic analytical balance and BT25S 1 / 100,000 electronic analytical balance (Beijing Sartorius Scientific Instrument Co., Ltd.).

[0035] 1.1 Materials

[0036] 1.1.1 Drug testing

[0037] Chromatographic grade acetonitrile and methanol were purchased from Thermo Fisher Scientific (China) Co., Ltd.; chromatographic grade phosphoric acid was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; the rest were of analytical grade, and water was ultrapure water.

[0038] 1.1.2 Reference substances

[0039] Reference substances 1,2,3,4,6-O-pentagalloylglucose (lot number PS011427), gallic acid (lot number PS012005), and catechin (lot number PS012222) were purchased from Chengdu Pusi Biotechnology Co., Ltd., with a mass fraction of ≥98%; paeoniflorin (lot number CHB230901), magnolamine (lot number CHB231226), ammonium glycyrrhizate (lot number CHB231005), sinomenine (lot number CHB201202), and liquiritin (lot number C HB231106) reference substances were purchased from Chengdu Kroma Biotechnology Co., Ltd., with a mass fraction of ≥98%; 10 batches of Qingbaitongbi capsules, batch numbers 20220701, 20220702, 20220703, 20230901, 20230902, 20230903, 20240401, 20240601, 20240602, 20240603, numbered S1 to S10, were obtained from Shaanxi Provincial Hospital of Traditional Chinese Medicine, 0.3 g × 45 capsules per bottle.

[0040] 2 Methods and Results

[0041] 2.1 Study on the HPLC fingerprint of Qingbai Tongbi Capsule

[0042] 2.1.1 Preparation of test solution

[0043] Accurately weigh the contents of Qingbai Tongbi Capsules (1.0 g), place it in a conical flask (50 mL), add 80% methanol solution by volume, plug it tightly, extract it by ultrasonic for 0.5 h, cool it, make it to volume again, shake it well, filter it, and take the filtrate to obtain the test solution.

[0044] 2.1.2 Preparation of mixed reference solution

[0045] Accurately weigh appropriate amounts of catechin, sinomenine, gallic acid, magnolamine, paeoniflorin, liquiritin, 1,2,3,4,6-O-pentagalloylglucose, and ammonium glycyrrhizate reference substances, dissolve them in 5% acetonitrile-95% 0.1% phosphoric acid aqueous solution, and prepare mixed reference substance solutions with mass concentrations of 0.5, 0.16, 0.07, 0.23, 0.53, 1, 0.18, and 0.125 mg / mL, respectively.

[0046] 2.1.3 Preparation of single herbal medicine solutions

[0047] Weigh Sinomene Sinensis, White Peony Root, and Radix Glycyrrhizae Radix according to the QTC (capsule) prescription ratio, and prepare single-ingredient solutions according to the method under "2.1.1".

[0048] 2.1.4 Chromatographic conditions

[0049] The chromatographic column is Agilent 5TC-C 18 Chromatographic column (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile (A)-0.1 wt% phosphoric acid aqueous solution (B); gradient elution conditions: 0-5 min, 5%-8% A; 5-15 min, 8%-13% A; 15-25 min, 13%-15% A; 25-35 min, 15%-25% A; 35-45 min, 25%-35% A; 45-60 min, 35%-70% A; 60-70 min, 70%-95% A; volume flow rate: 1.0 mL / min; column temperature: 30°C; injection volume: 10 μL; UV detection wavelength: 210 nm.

[0050] 2.1.5 Precision test, stability test, and repeatability test

[0051] The same batch of QTC (capsule) samples (S4) were taken and injected continuously for 6 times. The RSDs of the relative retention times and relative peak areas of the 24 common peaks were less than 0.67% and 2.95%, respectively, indicating that the instrument and experimental method had good precision; the same batch of QTC (capsule) samples (S4) were taken and placed at room temperature for 2, 4, 8, 12, 16, and 24 hours. The RSDs of the relative retention times and relative peak areas of the 24 common peaks were less than 1.99% and 2.89%, respectively, indicating that the test solution had good stability within 24 hours at room temperature; the same batch of QTC (capsule) samples (S4) were taken and 6 test solutions were prepared in parallel. The RSDs of the relative retention times and relative peak areas of the 24 common peaks were less than 2.05% and 2.94%, respectively, indicating that the extraction method had good repeatability.

[0052] 2.1.6 Fingerprint establishment and similarity analysis

[0053] Take 10 batches of QTC (capsules) samples (S1-S10), prepare the test solution according to the method under "2.1.1", analyze the test solution according to the chromatographic conditions under "2.1.4", and record the generated HPLC graph. The above spectra were processed and analyzed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)". Using chromatogram S1 as the reference spectrum, the median method was used with a time window of 0.1 min to fit the fingerprints of 10 batches of QTC (capsules) (S1-S10) to generate the reference fingerprint spectrum ( Figure 1 Middle R, Figure 2 ), a total of 24 common peaks were calibrated based on the stability and resolution of the peak area, with magnolamine as the reference peak. Figure 1 The fingerprints of 10 batches of QTC (capsules) samples (S1 to S10) were automatically matched with the reference fingerprints, and the similarities were 0.934 to 1.000. The results are shown in Table 1. This shows that the quality of these 10 batches of capsules is relatively stable and is consistent with the reference fingerprints ( Figure 2 ) is a qualified product if the similarity is above 0.90 (preferably 0.93).

[0054] Table 1 Similarity calculation results of fingerprints of 10 batches of QTC (capsules)

[0055]

[0056]

[0057] 2.1.7 Identification of shared peaks and drug attribution

[0058] Take the test sample solution, mixed reference solution and single herbal medicine solution under "2.1.1", "2.1.2" and "2.1.3" and analyze them according to the chromatographic conditions under "2.1.4". By comparison, the ownership of the 24 common peaks (1 to 24) in QTC (capsule) in the three herbs was determined. The results are shown in Figure 3 、 4 and Table 2.

[0059] Table 2 Common peak identification of QTC

[0060]

[0061] Example 2 Determination of the contents of 8 ingredients in QTC (capsules)

[0062] The preparation of the test solution and the mixed reference solution and the chromatographic conditions were the same as in Example 1.

[0063] The test solution and the mixed reference solution were taken and sampled and measured according to the liquid chromatography conditions in Example 1. The results showed that the separation between the chromatographic peaks of the eight index components and their adjacent chromatographic peaks was greater than 1.5, and the chromatograms of the negative sample solutions had no interference at the peak position of the component to be tested, indicating that the method had good specificity for the eight components.

[0064] 1. Linear relationship investigation

[0065] The mixed reference solution in Example 1 was diluted stepwise by 0, 2, 4, 8, 16, 32, and 64 times, and the assay was performed according to the chromatographic conditions in Example 1. The peak area (Y) was used to linearly regress the reference substance concentration (X), and the regression equations were obtained. The results were as follows: catechin Y = 125955.0X-0.6727, r = 0.9999, linear range 0.78-50.00 μg / mL; sinomenine Y = 35705X-1.4184, r = 0.9999, linear range 2.50-160.00 μg / mL; gallic acid Y = 82572X-12.778, r = 0.9999, linear range 1.09-70.00 μg / mL; magnolamine Y = 44762X-35.045, r = 0.9999, linear range 3.59-230.00 μg / mL; paeoniflorin Y = 35705X-1.4184, r = 0.9999, linear range 2.50-160.00 μg / mL; =4311.2X-45.244, r=0.998, linear range 8.28~530.00μg / mL; glycyrrhizin Y=32812X+463.25, r=0.9999, linear range 31.25~1000.00μg / mL; 1,2,3,4,6-O-pentagalloylglucose Y=27615X-29.289, r=0.9999, linear range 2.81~180.00μg / mL; ammonium glycyrrhizate Y=6728.2X-18.574, r=0.998, linear range 1.95~125.00μg / mL; the results showed that the eight components had good linear relationships within their respective mass concentration ranges.

[0066] 2. Precision test

[0067] A mixed reference solution was prepared according to the method in Example 1, 10 μL of the same mixed reference solution was accurately pipetted, and the sample was continuously injected 6 times according to the chromatographic conditions in Example 1, with 10 μL injected each time. The peak area RSDs of catechin, sinomenine, gallic acid, magnolialine, paeoniflorin, liquiritin, 1,2,3,4,6-O-pentagalloylglucose, and ammonium glycyrrhizate were 0.27%, 0.49%, 0.45%, 0.16%, 0.37%, 0.73%, 0.23%, and 0.94%, respectively, and the RSDs were all ≤3.0%, indicating that the instrument had good precision.

[0068] 3. Stability test

[0069] About 1 g of sample (S4) was taken, and a test solution was prepared according to the method in Example 1. After standing at room temperature for 0, 2, 4, 8, 12, and 24 h, the sample was injected and analyzed according to the chromatographic conditions under "2.4.3". The peak area RSDs of catechin, sinomenine, gallic acid, magnolia pine, paeoniflorin, liquiritin, 1,2,3,4,6-O-pentagalloylglucose, and ammonium glycyrrhizate were 0.68%, 1.43%, 2.35%, 0.52%, 2.23%, 0.71%, 1.31%, and 0.48%, respectively. The RSDs were all ≤3.0%, indicating that the test solution had good stability within 24 h.

[0070] 4. Repeatability test

[0071] Take samples from the same batch (S4), each about 1 g, and prepare 6 test solutions in parallel according to the method under "2.4.2". The samples were injected and analyzed according to the chromatographic conditions in Example 1. The RSDs of the contents of catechin, sinomenine, gallic acid, magnolamine, paeoniflorin, liquiritin, 1,2,3,4,6-O-pentagalloylglucose, and ammonium glycyrrhizate were 1.17%, 1.48%, 1.88%, 0.70%, 1.09%, 2.90%, 2.03%, and 0.46%, respectively. The RSDs were all ≤3.0%, indicating that the extraction method had good repeatability.

[0072] 5. Sample recovery test

[0073] Six portions of QTC capsules with known contents (approximately 0.5 g each) were accurately weighed and appropriate amounts of reference solution of known concentration were added to each portion. Test solutions were prepared according to the method in Example 1, and the recoveries were calculated according to the chromatographic conditions in Example 1. The results showed that the average recoveries of catechin, sinomenine, gallic acid, magnolialine, paeoniflorin, liquiritin, 1,2,3,4,6-O-pentagalloylglucose, and ammonium glycyrrhizate were 103.44%, 101.41%, 100.63%, 99.66%, 96.81%, 102.90%, 97.11%, and 98.80%, respectively, with RSDs of 1.79%, 2.33%, 2.25%, 0.6%, 2.67%, 2.08%, 1.74%, and 3.70%, respectively, indicating good accuracy of the extraction method.

[0074] 6. Determination of test sample content

[0075] 1 g of each of 10 batches of QTC (capsule) samples (S1 to S10) were taken, and test solutions were prepared according to the method in Example 1. The samples were injected and measured according to the chromatographic conditions in Example 1, the peak areas were recorded, and the content was calculated using the external standard method. The results are shown in Table 3. The results showed that the contents of catechin in 10 batches of QTC (capsule) samples (S1~S10) were 0.9071~1.1893 mg / g, sinomenine was 2.1833~3.1186 mg / g, gallic acid was 0.3970~1.4276 mg / g, magnoliavine was 3.5079~5.4466 mg / g, paeoniflorin was 14.2077~19.5701 mg / g, liquiritin was 1.4128~3.3610 mg / g, 1,2,3,4,6-O-pentagalloylglucose was 0.4420~1.6977 mg / g, and ammonium glycyrrhizate was 2.7388~4.7612 mg / g. The average contents of the eight components in the 10 batches of samples fluctuated within a small range, indicating that the preparation process was stable.

[0076] Table 3 Content determination results of 8 ingredients in 10 batches of QTC (capsules) (n=3)

[0077]

[0078] Example 3 Optimization of chromatographic conditions

[0079] The present invention investigated different mobile phases, including methanol-water, acetonitrile-water, and acetonitrile-0.1wt% phosphoric acid aqueous solution. The results showed that the acetonitrile-0.1wt% phosphoric acid aqueous solution system had the best peak separation effect, so the acetonitrile-0.1% phosphoric acid aqueous solution was selected as the mobile phase. Simultaneously, an investigation of the detection wavelength revealed that at 210nm, the number of chromatographic peaks was relatively large, and the absorption of each peak was relatively uniform, which can reflect the integrity and fuzziness of the fingerprint spectrum.

[0080] Secondly, when optimizing the preparation method of the test solution, the present invention investigated the effects of extraction solvents (ethanol, methanol, 40% methanol, 60% methanol, 80% methanol), extraction methods (ultrasonic extraction, heating reflux extraction), extraction time (15, 30, 45 min), and material-liquid ratio (1:50, 1:100, 1:200) on the extraction effect, and determined the optimal extraction conditions: the solvent corresponding to 1 g of capsule content is 50 mL of 80% methanol solution by volume, and ultrasonic extraction is performed for 30 min.

Claims

1. An HPLC fingerprint of Qingbai Tongbi Capsule, characterized in that When the HPLC chromatographic conditions are as follows, the HPLC fingerprint of the Qingbai Tongbi Capsule is substantially consistent with that in FIG2 ; HPLC chromatographic conditions are as follows: chromatographic column is Agilent 5TC-C 18 Chromatographic column, specifications: 250mm×4.6mm, 5μm; mobile phase: acetonitrile as phase A, 0.1% phosphoric acid aqueous solution as phase B; gradient elution conditions: 0-5min, 5%-8%A; 5-15min, 8%-13%A; 15-25min, 13%-15%A; 25-35min, 15%-25%A; 35-45min, 25%-35%A; 45-60min, 35%-70%A; 60-70min, 70%-95%A; volume flow rate 1.0mL / min; column temperature 30℃; injection volume 10μL; UV detection wavelength is 210nm.

2. The HPLC fingerprint of the Qingbai Tongbi Capsule according to claim 1 is characterized in that The HPLC fingerprint of the Qingbai Tongbi capsule has 24 characteristic fingerprint chromatographic peaks, wherein peak 4 is catechin, peak 10 is sinomenine, peak 12 is gallic acid, peak 13 is magnolia pine, peak 14 is paeoniflorin, peak 17 is liquiritin, peak 18 is 1,2,3,4,6-O-pentagalloylglucose, and peak 24 is ammonium glycyrrhizate.

3. The method for constructing the HPLC fingerprint of Qingbai Tongbi Capsule according to any one of claims 1 to 2, characterized in that When the HPLC chromatographic conditions are as follows, the HPLC fingerprint of the Qingbai Tongbi Capsule has 24 characteristic fingerprint chromatographic peaks, wherein peak 4 is catechin, peak 10 is sinomenine, peak 12 is gallic acid, peak 13 is magnolia pine, peak 14 is paeoniflorin, peak 17 is liquiritin, peak 18 is 1,2,3,4,6-O-pentagalloylglucose, and peak 24 is ammonium glycyrrhizate; HPLC chromatographic conditions are as follows: chromatographic column is Agilent 5TC-C 18 Chromatographic column, specifications: 250 mm × 4.6 mm, 5 μm; mobile phase: acetonitrile as phase A, 0.1% phosphoric acid aqueous solution as phase B; gradient elution conditions: 0-5 min, 5%-8% A; 5-15 min, 8%-13% A; 15-25 min, 13%-15% A; 25-35 min, 15%-25% A; 35-45 min, 25%-35% A; 45-60 min, 35%-70% A; 60-70 min, 70%-95% A; volume flow rate 1.0 mL / min; column temperature 30°C; injection volume 10 μL; UV detection wavelength 210 nm.

4. Application of the HPLC fingerprint of Qingbai Tongbi Capsules according to any one of claims 1 to 3 or the method for constructing the same in quality control and component analysis of Qingbai Tongbi Capsules.

5. Application of the HPLC fingerprint of Qingbai Tongbi Capsules or the method for constructing the same according to any one of claims 1 to 3 in the quality control of Qingbai Tongbi Capsules, characterized in that The application comprises the following steps: (1) The contents of Qingbai Tongbi Capsules are prepared into a methanol solution of a certain concentration to obtain a test solution; (2) Take the test solution obtained in step (1) and test it by HPLC to obtain the HPLC chart of the test product. The chromatographic conditions are as follows: the chromatographic column is Agilent 5TC-C 18 Chromatographic column, specifications: 250 mm × 4.6 mm, 5 μm; mobile phase: acetonitrile as phase A, 0.1% phosphoric acid aqueous solution as phase B; gradient elution conditions: 0-5 min, 5%-8% A; 5-15 min, 8%-13% A; 15-25 min, 13%-15% A; 25-35 min, 15%-25% A; 35-45 min, 25%-35% A; 45-60 min, 35%-70% A; 60-70 min, 70%-95% A; flow rate 1.0 mL / min; column temperature 30°C; injection volume 10 μL; UV detection wavelength 210 nm; (3) Compare the HPLC chromatogram of the test sample obtained in step (2) with the HPLC fingerprint of the Qingbai Tongbi Capsule described in the present invention. The Qingbai Tongbi Capsule with a similarity of more than 0.90 is a qualified product.

6. The use according to claim 5, characterized in that The concentration of the test solution described in step (1) is preferably a 5-30 mg / mL methanol solution of the contents of Qingbai Tongbi Capsules, and more preferably 10-20 mg / mL; when preparing the test solution, it is preferred to first treat the contents of Qingbai Tongbi Capsules with an appropriate volume fraction of 60%-80% methanol solution, ultrasonically treat for 20-30 minutes, and then adjust the volume and filter to obtain the test solution.

7. A method for analyzing the ingredients of Qingbai Tongbi Capsules, characterized in that The method comprises the following steps: (1) Preparation of mixed reference solution: Accurately weigh appropriate amounts of catechin, sinomenine, gallic acid, magnolamine, paeoniflorin, liquiritin, 1,2,3,4,6-O-pentagalloylglucose, and ammonium glycyrrhizate reference substances, dissolve them in 5% acetonitrile-95% 0.1% phosphoric acid aqueous solution, and prepare mixed reference substance solutions with mass concentrations of 0.5, 0.16, 0.07, 0.23, 0.53, 1, 0.18, and 0.125 mg / mL, respectively; (2) Preparation of standard curves for each reference substance in step (1): The mixed reference solution prepared in step (1) was diluted stepwise by 0, 2, 4, 8, 16, 32, and 64 times, and HPLC determination was performed according to the chromatographic conditions. The peak area (Y) was linearly regressed against the reference concentration (X) to obtain the regression equation. The results were as follows: catechin Y = 125955.0X - 0.6727, r = 0.9999, linear range 0.78-50.00 μg / mL; sinomenine Y = 35705X - 1.4184, r = 0.9999, linear range 2.50-160.00 μg / mL; gallic acid Y = 82572X - 12.778, r = 0.9999, linear range 1.09-70.00 μg / mL; magnolia alkaloid Y = 44762X - 1.4184, r = 0.9999, linear range 2.50-160.00 μg / mL; 35.045, r = 0.9999, linear range 3.59 ~ 230.00 μg / mL; paeoniflorin Y = 4311.2X - 45.244, r = 0.998, linear range 8.28 ~ 530.00 μg / mL; liquiritin Y = 32812X + 463.25, r = 0.9999, linear range 31.25 ~ 1000.00 μg / mL; 1,2,3,4,6-O-pentagalloylglucose Y = 27615X - 29.289, r = 0.9999, linear range 2.81 ~ 180.00 μg / mL; ammonium glycyrrhizate Y = 6728.2X - 18.574, r = 0.998, linear range 1.95 ~ 125.00 μg / mL; (3) Preparation of test solution: The contents of Qingbai Tongbi capsules were prepared into a methanol solution of a certain concentration to obtain the test solution; (4) The test solution obtained in step (3) was taken and tested by HPLC. The contents of catechin, sinomenine, gallic acid, magnolia pine, paeoniflorin, liquiritin, 1,2,3,4,6-O-pentagalloyl glucose and ammonium glycyrrhizate in Qingbai Tongbi Capsules were calculated based on the concentrations of catechin, sinomenine, gallic acid, magnolia pine, paeoniflorin, liquiritin, 1,2,3,4,6-O-pentagalloyl glucose and ammonium glycyrrhizate reference substances and their retention times in the fingerprint and the linear equation in step (2).

8. The method according to claim 7, characterized in that The chromatographic conditions of the HPLC detection in steps (2) and (4) are as follows: the chromatographic column is Agilent 5TC-C 18 Chromatographic column, specifications: 250mm×4.6mm, 5μm; mobile phase: acetonitrile as phase A, 0.1% phosphoric acid aqueous solution as phase B; gradient elution conditions: 0-5min, 5%-8%A; 5-15min, 8%-13%A; 15-25min, 13%-15%A; 25-35min, 15%-25%A; 35-45min, 25%-35%A; 45-60min, 35%-70%A; 60-70min, 70%-95%A; volume flow rate 1.0mL / min; column temperature 30℃; injection volume 10μL; UV detection wavelength is 210nm.

9. The method according to any one of claims 7 to 8, characterized in that The concentration of the test solution described in step (3) is preferably a 5-30 mg / mL methanol solution of the contents of Qingbai Tongbi Capsules, and more preferably 10-20 mg / mL; when preparing the test solution, it is preferred to first treat the contents of Qingbai Tongbi Capsules with an appropriate volume fraction of 60%-80% methanol solution, ultrasonically treat for 20-30 minutes, and then adjust the volume and filter to obtain the test solution.

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