HPLC (High Performance Liquid Chromatography) fingerprint spectrum detection method for

Abstract

The invention discloses an HPLC (High Performance Liquid Chromatography) fingerprint spectrum detection method of an acid-clearing tablet, and belongs to the technical field of medicine quality detection. The method comprises the following steps: S1, firstly taking acid-clearing tablet powder, adding a methanol solution as an extraction solvent, weighing, then carrying out ultrasonic treatment and cooling, weighing again, finally complementing the reduced weight with the methanol solution, uniformly shaking, filtering, and taking a subsequent filtrate as a test solution; s2, preparing a reference substance solution; and S3, sucking the test solution and the reference solution, carrying out high performance liquid chromatography determination, and establishing the HPLC fingerprint spectrum of the Xiaoshan tablet. According to the invention, the HPLC fingerprint spectrum of the Xianshu tablets is established, qualitative analysis of the Xianshu tablets is realized, a feasible method is provided for comprehensive quality evaluation of the Xianshu tablets, and a foundation is laid for basic research of subsequent pharmacodynamic substances of the Xianshu tablets; the method for comprehensively evaluating the quality of the acid-clearing tablets through the fingerprint spectrum is simple and convenient to operate, stable and reliable in result and high in repeatability, and can be used for comprehensively evaluating the overall quality of the acid-clearing tablets.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical quality detection, and particularly relates to a HPLC fingerprint detection method of Qing acid tablets. BACKGROUND

[0002] Qing acid tablets (approval number / letter number 1870 / DRNPRA / ISAF / 2024) are composed of peony leaf, smilax glabra, hovenia dulcis thunb, alisma orientale, plantago asiatica and chicory, and have the effects of reducing uric acid and treating idiopathic arthritis. Research reports that peony leaf contains gallic acid, methyl gallate, paeoniflorin, ellagic acid, 1,2,3,4,6-O-penta-galloyl glucose, mallotaside, apigenin-7-glucoside and the like, and has the effects of reducing uric acid, anti-inflammatory, treating gout and the like. The active components of smilax glabra mainly include flavonoids, phenylpropanoids and the like, and have the pharmacological effects of anti-inflammatory, reducing uric acid, immune regulation and the like. Hovenia dulcis thunb contains flavonoids, terpenes, phenylpropanoids and alkaloids and the like, and has the pharmacological activities of anti-oxidation, anti-hypoxia, anti-inflammatory, reducing uric acid and the like. Alisma orientale mainly contains terpenes, including triterpenes, sesquiterpenes, diterpenes and the like, and has the effects of diuresis, kidney protection, anti-inflammatory and the like. Chicory mainly contains flavonoids, polyphenols, sesquiterpene lactone and the like, and has the effects of significant anti-oxidation, anti-fatigue, anti-aging and the like.

[0003] As a quality control technology and method of a drug, the HPLC fingerprint can comprehensively reflect the overall quality of the drug. At present, there are few studies on the quality control of Qing acid tablets at home and abroad. As a drug with significant consumer response effect, it is urgent to provide a new method for establishing the HPLC fingerprint of Qing acid tablets, so as to effectively reflect the overall and complexity of the chemical components of Qing acid tablets, comprehensively evaluate and control the quality of the preparation, and then ensure the safety and effectiveness of clinical medication. SUMMARY

[0004] In order to solve the above technical problems, the purpose of the present application is to provide a HPLC fingerprint detection method of Qing acid tablets, so as to solve the problem that there is no effective method for quality detection and control of Qing acid tablets.

[0005] The technical scheme for solving the above technical problems of the present application is as follows: A HPLC fingerprint detection method of Qing acid tablets, comprising the following steps: S1: first take Qing acid tablet powder, add methanol solution as extraction solvent, weigh, then ultrasonic treatment and cool, weigh again, finally supplement the weight loss with methanol solution, shake well, filter, and take the filtrate as the test solution; S2: Gallic acid, p-hydroxybenzaldehyde, hydroxy paeonol, paeonolactoside, dihydromyricetin, paeonol, ferulic acid, plantainoside, tetragalloyl glucose, ellagic acid, jacobionoside, 1,2,3,4,6-O-penta-galloyl glucose and bigelovoside were used as reference substances, and methanol was added to prepare reference substance solutions; S3: The test sample solution obtained in S1 and the reference substance solution obtained in S2 were taken respectively for high performance liquid chromatography determination, and the HPLC fingerprint of Qing acid tablets was established.

[0006] Further, the volume fraction of the methanol solution in S1 is 60%-80%.

[0007] Further, the power of the ultrasonic treatment in S1 is 200-400 W, the frequency is 40-60 kHz, and the time is 20-40 min.

[0008] Further, the concentration of Qing acid tablets in the test sample solution in S1 is 3-6 mg / mL.

[0009] Further, the concentrations of the gallic acid, p-hydroxybenzaldehyde, hydroxy paeonol, paeonolactoside, dihydromyricetin, paeonol, ferulic acid, plantainoside, tetragalloyl glucose, ellagic acid, jacobionoside, 1,2,3,4,6-O-penta-galloyl glucose and bigelovoside reference substance solutions in S2 are 250-350 μg / mL, 5-20 μg / mL, 200-250 μg / mL, 230-260 μg / mL, 200-240 μg / mL, 200-230 μg / mL, 5-10 μg / mL, 50-90 μg / mL, 20-40 μg / mL, 15-20 μg / mL, 100-150 μg / mL, 100-200 μg / mL, 80-120 μg / mL, respectively.

[0010] Further, the concentrations of the gallic acid, p-hydroxybenzaldehyde, hydroxy paeonol, paeonolactoside, dihydromyricetin, paeonol, ferulic acid, plantainoside, tetragalloyl glucose, ellagic acid, jacobionoside, 1,2,3,4,6-O-penta-galloyl glucose and bigelovoside reference substance solutions in S2 are 293.9 μg / mL, 10 μg / mL, 234.33 μg / mL, 246.2 μg / mL, 220.2 μg / mL, 214.2 μg / mL, 8.2 μg / mL, 69.2 μg / mL, 30.2 μg / mL, 18.9 μg / mL, 124.98 μg / mL, 166.93 μg / mL, 97.40 μg / mL, respectively.

[0011] Further, the chromatographic column used in the high performance liquid chromatography in S3 is: a C18 chromatographic column with a specification of 250 mm*4.6 mm, 5 μm.

[0012] Further, the conditions of the high performance liquid chromatography in S3 are: the column temperature is 30 DEG C, the flow rate is 1.0 mL*min -1 , the detection wavelength is 270 nm, and the injection amount is 10 μL; The mobile phase A is acetonitrile, and the mobile phase B is a formic acid aqueous solution with a volume concentration of 0.1%; The elution conditions are: 0-10 min, the volume concentration of the mobile phase B is 93%→90%; 10-35 min, the volume concentration of the mobile phase B is 90%→83%; 35-80 min, the volume concentration of the mobile phase B is 83%→79%; and 80-90 min, the volume concentration of the mobile phase B is 79%→60%.

[0013] The present application has the following beneficial effects: (1) The present application establishes the HPLC fingerprint of Qing acid tablets by adopting the liquid chromatography technology, realizes the qualitative analysis of the common peak components of Qing acid tablets, provides a feasible method for the comprehensive quality evaluation of Qing acid tablets, and lays a foundation for the subsequent efficacy material basis research of the medicinal material.

[0014] (2) The present application establishes the HPLC fingerprint of Qing acid tablets in multiple batches, determines that the HPLC fingerprints of the multiple batches of Qing acid tablets all contain 13 common characteristic peaks from the reference substance by analysis and comparison, the 13 common characteristic peaks constitute the fingerprint characteristics of Qing acid tablets, and serve as the standard fingerprint of Qing acid tablets. The comprehensive quality evaluation method of Qing acid tablets by the fingerprint is simple in operation, stable and reliable in result, and strong in repeatability, and can be used for the comprehensive evaluation of the overall quality of Qing acid tablets. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is the HPLC control fingerprint of Qing acid tablets; Figure 2 is the HPLC fingerprint superimposition diagram of 10 batches of Qing acid tablets; Figure 3 is the HPLC chromatogram of the sample, the reference substance and each medicinal material. DETAILED DESCRIPTION

[0016] The principles and characteristics of the present application are described below in combination with the drawings, and the examples are only used for explaining the present application, and are not used for limiting the scope of the present application. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments not mentioned by the manufacturers are all conventional products that can be obtained by market purchase.

[0017] Example 1: A kind of HPLC fingerprint detection method of Qing acid tablet, comprising the following steps: S1: preparation of test solution First, take Qing acid tablet sample and crush, pass through No. 4 sieve;Then accurately weigh 0.45 g of Qing acid tablet powder, place in a 100 mL conical flask with a plug, accurately add 100 mL of 70% methanol, tightly plug, weigh;Then ultrasonic treat with 300 W power, 50 kHz frequency for 30 min, cool again and weigh;Finally, make up the weight loss with 70% methanol, shake, filter, take the filtrate as the test solution.

[0018] S2: preparation of control solution Respectively weigh gallic acid, p-hydroxybenzaldehyde, hydroxy peony glycoside, peony lactone glycoside, dihydro myricetin, paeoniflorin, ferulic acid, plantain glycoside, tetragalloyl glucose, ellagic acid, luteoloside, 1,2,3,4,6-O-penta-galloyl glucose, big Persian glycoside as control, add methanol to prepare control solution containing 293.90, 10.00, 234.33, 246.20, 220.20, 214.20, 8.20, 69.20, 30.20, 18.90, 124.98, 166.93, 97.40 μg per 1 mL respectively.

[0019] S3: HPLC detection The test solution obtained in S1 and the control solution obtained in S2 are passed into a high performance liquid chromatograph for detection, and an HPLC fingerprint is established.

[0020] The detection conditions are: the chromatographic column is Kromasil C 18 (250 mm x 4.6 mm, 5 μm), the mobile phase A is acetonitrile, and the mobile phase B is 0.1% formic acid aqueous solution;The column temperature is 30℃, the flow rate is 1.0 mL·min -1 , the detection wavelength is 270 nm, and the injection amount is 10 μL; The gradient elution conditions are: 0-10 min, the volume concentration of mobile phase B is 93%→90%;10-35 min, the volume concentration of mobile phase B is 90%→83%;35-80 min, the volume concentration of mobile phase B is 83%→79%;80-90 min, the volume concentration of mobile phase B is 79%→60%.

[0021] (1) precision experiment The test sample solution was prepared according to the S1 method, and 6 times of continuous injection was carried out according to the S3 chromatographic condition, with 1, 2, 3, 4, 6-O-penta-galloyl glucose as a reference peak, the relative standard deviation (RSD) of the relative retention time of the common peak was less than 0.5%, and the RSD of the relative peak area was less than 5.0%, indicating that the precision was good.

[0022] (2) Reproducibility test The test sample solution was prepared according to the S1 method, and 6 times of continuous injection was carried out according to the S3 chromatographic condition, with 1, 2, 3, 4, 6-O-penta-galloyl glucose as a reference peak, the relative standard deviation (RSD) of the relative retention time of the common peak was less than 0.5%, and the RSD of the relative peak area was less than 5.0%, indicating that the precision was good.

[0023] (3) Establishment of control fingerprint The test sample solution of Qing acid tablets of 10 batches (S1-S10) was prepared according to the S1 method, and then injection analysis was carried out according to the HPLC detection condition of S3. The data was analyzed by using the important chromatographic fingerprint similarity evaluation system (2012 version), with S1 as the reference spectrum, the time window width range was 1 min, and the control fingerprint was generated by using the average method, multi-point correction and full spectrum peak matching, as shown in Figure 1 and Figure 2 .

[0024] Figure 1 The control fingerprint of Qing acid tablets in

[0025] (2) Difference analysis of relative retention time and relative peak area of different batches of preparations The relative retention time and relative peak area of the results of 10 batches of preparations were determined, and the experimental results are shown in Tables 1 and 2.

[0026] Table 1 Determination results of relative retention time of 10 batches of preparations

[0027] Table 2 Determination results of relative peak area of 10 batches of preparations

[0028] The results show that the chemical components of the 10 batches of preparations are relatively uniform, the relative standard deviation RSD of the retention time is less than 0.5%, and the relative standard deviation RSD of the relative peak area is less than 5.0%.

[0029] (4) Similarity analysis The similarity of the chromatograms of the 10 batches of samples was determined by taking the control fingerprint in Table 2 as a reference, and the experimental results are shown in Table 3. Figure 1

[0030] Table 3 Similarity of chromatograms of 10 batches of preparations

[0031] The results show that the similarity of the chromatograms of the 10 batches of preparations is 0.994, 0.997, 0.998, 0.997, 0.998, 0.996, 0.995, 0.994, 0.995 and 0.997, respectively, all greater than 0.90, indicating that the quality of the ten batches of Qing acid tablets is stable, and the HPLC fingerprint method established by the present application is suitable for the quality evaluation and control of Qing acid tablets.

[0032] (5) Identification and identification of characteristic fingerprint peaks According to the 13 characteristic peaks identified in step (1) and the comparison with the control, they include: peak 2 is gallic acid, peak 3 is p-hydroxybenzaldehyde, peak 6 is hydroxy peony glycoside, peak 10 is peony lactone glycoside, peak 11 is dihydro myricetin, peak 14 is peony glycoside, peak 16 is ferulic acid, peak 17 is da chejian glycoside, peak 18 is 1,2,3,6-O-tetragalloyl glucose, peak 20 is ellagic acid, peak 22 is osmanthus glycoside, peak 23 is 1,2,3,4,6-O-penta galloyl glucose, and peak 26 is da pueraria glycoside. The sources of each characteristic peak were identified by HPLC chromatogram determination of the test sample, the mixed control and six kinds of medicinal materials of peony leaf, plantain, smilax glabra, jujube fruit, chicory and alisma orientale, and the experimental results are shown in Table 4. Figure 3

[0033] Table 4 Sources of each characteristic peak

[0034] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.​​

Claims

1. A method for detecting the fingerprint of Qingsuan tablets using HPLC, characterized in that, Includes the following steps: S1: First, take the powder of Qing Suan Pian, add methanol solution as the extraction solvent, weigh it, then sonicate and cool it, weigh it again, finally make up the weight loss with methanol solution, shake well, filter, and take the filtrate as the test solution. S2: Using gallic acid, p-hydroxybenzaldehyde, hydroxypaeoniflorin, paeoniflorin lactone, dihydromyricetin, paeoniflorin, ferulic acid, psyllium glycoside, tetragalloyl glucose, ellagic acid, luteolin, 1,2,3,4,6-O-pentagalloyl glucose and cosmososide as reference standards, reference solutions were prepared by adding methanol to each. S3: Take the test solution obtained in S1 and the reference solution obtained in S2 respectively and perform high performance liquid chromatography to determine the HPLC fingerprint of Qingsuan tablets.

2. The HPLC fingerprint detection method for Qing Suan tablets according to claim 1, characterized in that, The volume fraction of methanol in S1 is 60%-80%.

3. The HPLC fingerprint detection method for Qing Suan tablets according to claim 1, characterized in that, The ultrasonic treatment in S1 has a power of 200-400 W, a frequency of 40-60 kHz, and a duration of 20-40 min.

4. The HPLC fingerprint detection method for Qing Suan tablets according to claim 1, characterized in that, The concentration of Qing Suan tablets in the test solution in S1 is 3-6 mg / mL.

5. The HPLC fingerprint detection method for Qing Suan tablets according to claim 1, characterized in that, The concentrations of gallic acid, p-hydroxybenzaldehyde, hydroxypaeoniflorin, paeoniflorin lactone, dihydromyricetin, paeoniflorin, ferulic acid, psyllium glycoside, tetragalloyl glucose, ellagic acid, luteolin, 1,2,3,4,6-O-pentagalloyl glucose, and cosmososide reference solutions in S2 are 250-350 μg / mL, 5-20 μg / mL, 200-250 μg / mL, 230-260 μg / mL, 200-240 μg / mL, 200-230 μg / mL, 5-10 μg / mL, 50-90 μg / mL, 20-40 μg / mL, 15-20 μg / mL, 100-150 μg / mL, 100-200 μg / mL, and 80-120 μg / mL, respectively.

6. The HPLC fingerprint detection method for Qing Suan tablets according to claim 5, characterized in that, The concentrations of gallic acid, p-hydroxybenzaldehyde, hydroxypaeoniflorin, paeoniflorin lactone, dihydromyricetin, paeoniflorin, ferulic acid, psyllium glycoside, tetragalloyl glucose, ellagic acid, luteolin, 1,2,3,4,6-O-pentagalloyl glucose, and cosmososide reference solutions in S2 were 293.9 μg / mL, 10 μg / mL, 234.33 μg / mL, 246.2 μg / mL, 220.2 μg / mL, 214.2 μg / mL, 8.2 μg / mL, 69.2 μg / mL, 30.2 μg / mL, 18.9 μg / mL, 124.98 μg / mL, 166.93 μg / mL, and 97.40 μg / mL, respectively.

7. The HPLC fingerprint detection method for Qingsuan tablets according to claim 1, characterized in that, The high-performance liquid chromatography column used in S3 is a C18 column with dimensions of 250 mm × 4.6 mm and 5 μm.

8. The HPLC fingerprint detection method for Qing Suan tablets according to claim 1, characterized in that, The high-performance liquid chromatography (HPLC) conditions in S3 are as follows: column temperature 30℃, flow rate 1.0 mL / min. -1 The detection wavelength was 270 nm, and the injection volume was 10 μL. Mobile phase A is acetonitrile, and mobile phase B is a 0.1% (v / v) aqueous solution of formic acid; The elution conditions were as follows: 0-10 min, mobile phase B volume concentration 93%→90%; 10-35 min, mobile phase B volume concentration 90%→83%; 35-80 min, mobile phase B volume concentration 83%→79%; 80-90 min, mobile phase B volume concentration 79%→60%.

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