Quality detection method of Zhongtongshu preparation

Abstract

This invention relates to the field of drug detection technology, specifically to a quality detection method for a swelling and pain relief preparation. The quality detection method includes the determination of lupinone and curcumin content, and specifically includes the following steps: preparation of the test solution, preparation of the reference solution, and HPLC determination. This invention establishes a quality detection method for a swelling and pain relief preparation, and methodological experiments have confirmed that this method has high accuracy, good stability, and simple operation, further improving the quality standard of the swelling and pain relief preparation and enabling better control of drug quality.

Description

Technical Field

[0001] This invention relates to the field of drug testing technology, specifically to a quality testing method for a swelling and pain relief preparation. Background Technology

[0002] Zhongtongshu preparation is a traditional Chinese medicine preparation made from banana root, wood sorrel and turmeric as the main raw materials. It has the effects of promoting blood circulation, removing blood stasis, reducing swelling and relieving pain. Clinically, it is often used to treat traumatic injuries, swelling and pain due to blood stasis, and soft tissue contusions, and has good therapeutic effects.

[0003] The current quality standard for the swelling and pain relief preparation (standard number WS-10540(ZD-0540)-2002-2012Z) includes thin-layer chromatography identification of banana root, thin-layer chromatography identification of turmeric, and determination of curcumin content. Specifically, the preparation method for the curcumin content determination test solution is as follows: accurately measure 5 mL of the sample, evaporate to near dryness, mix with a small amount of macroporous resin D101 and apply to a macroporous adsorption resin column D101 (inner diameter 1.5 cm, column height 15 cm), elute with 80 mL of water, discard the aqueous solution, continue eluting with 100 mL of ethanol, collect the eluent, evaporate to dryness, dissolve in acetonitrile and transfer to a 5 mL volumetric flask, add acetonitrile to the mark, shake well, filter, and collect the filtrate. The preparation method of the above test solution is relatively complex, has high experimental costs, requires a high level of technical skill from the experimenter, and is prone to significant human error. When the curcumin content was determined according to the method in the standard, the chromatographic peak of curcumin in the obtained HPLC chromatogram was asymmetrical and showed tailing phenomenon. Moreover, the content determination method is only for turmeric in the preparation and cannot achieve a more comprehensive control over the quality of the drug.

[0004] Based on the above problems, this invention establishes a quality detection method for swelling and pain relief preparations using lupinone and curcumin content as indicators and high performance liquid chromatography. The aim is to further improve the quality standards of swelling and pain relief preparations, increase the detection efficiency of laboratory personnel, reduce detection costs, and achieve better control over drug quality. Summary of the Invention

[0005] The purpose of this invention is to provide a quality testing method for a swelling and pain relief preparation.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The swelling and pain relief preparation of the present invention, based on the preparation of 1000mL of medicinal liquid, has the following effective components: 63g of banana root, 17g of wood sorrel, and 20g of turmeric. The preparation process of the swelling and pain relief preparation is as follows: the above three ingredients are pulverized into coarse powder, and according to the percolation method under the section on fluid extracts and extracts in the Chinese Pharmacopoeia, 45% ethanol is used as solvent. After soaking for 48 hours, the percolation is slowly carried out, and the percolate is collected at a rate of 1-2mL per minute to 1000mL. After standing, it is filtered to obtain the product.

[0008] The quality testing method for the swelling and pain relief preparation of the present invention includes the determination of the contents of lupinone and curcumin, as follows:

[0009] 1) Preparation of test solution: Accurately weigh 25-35g of drug solution, evaporate the solvent by rotary evaporation at 50℃, dissolve the residue in an appropriate amount of ethanol and transfer it to a 10mL volumetric flask, make up to volume, shake well, filter, and the test solution is obtained.

[0010] 2) Preparation of reference solution: Accurately weigh 0.02 g of lupeone reference standard, place it in a 100 mL volumetric flask, dissolve it with ethanol and dilute to the mark to prepare a lupeone reference standard stock solution with a concentration of 0.2 mg / mL; accurately weigh 0.03 g of curcumin reference standard, accurately transfer 1.0 mL of the lupeone reference standard stock solution prepared above into a 10 mL volumetric flask, dissolve it with ethanol and dilute to the mark, shake well to obtain a mixed reference solution;

[0011] 3) HPLC determination:

[0012] Chromatographic conditions: Column type C 18 The column was 250 mm × 4.6 mm, 5 μm in diameter; the column temperature was 30-40℃; the flow rate was 0.8-1.2 mL / min; the detection wavelength was 220 nm; a gradient elution was performed using a methanol-acetonitrile mixture (volume ratio 15-25:75-85) as mobile phase A and a 0.1%-0.3% ammonium acetate aqueous solution as mobile phase B. The gradient elution program is as follows:

[0013] From 0 to 8 minutes, the volume ratio of mobile phase A to mobile phase B was 45:55.

[0014] In 8-9 minutes, the volume ratio of mobile phase A to mobile phase B changed from 45:55 to 100:0;

[0015] 9-16 min, the volume ratio of mobile phase A to mobile phase B is 100:0;

[0016] Over 16-25 minutes, the volume ratio of mobile phase A to mobile phase B changed from 100:0 to 70:30.

[0017] Over 25-30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 70:30 to 45:55.

[0018] Assay: Accurately pipette 10 μL each of the reference solution and the test solution into the high-performance liquid chromatograph and determine the result.

[0019] Preferably, in the quality testing method of the swelling and pain relief preparation of the present invention, the mass of the accurately weighed medicine solution in step 1) is 30-35g.

[0020] In a further preferred embodiment, in the quality testing method of the swelling and pain relief preparation of the present invention, the mass of the accurately weighed medicine solution in step 1) is 30g.

[0021] Preferably, in the quality testing method of the swelling and pain relief preparation of the present invention, the filtration in step 1) is through a 0.22μm microporous membrane.

[0022] Preferably, in the quality testing method of the swelling and pain relief preparation of the present invention, the column temperature in step 3) is 35-40℃.

[0023] In a further preferred embodiment, in the quality testing method for the swelling and pain relief preparation of the present invention, the column temperature in step 3) is 35°C.

[0024] Preferably, in the quality testing method of the swelling and pain relief preparation of the present invention, the flow rate in step 3) is 1.0-1.2 mL / min.

[0025] In a further preferred embodiment, in the quality testing method of the swelling and pain relief preparation of the present invention, the flow rate in step 3) is 1.0 mL / min.

[0026] Preferably, in the quality testing method of the swelling and pain relief preparation of the present invention, in step 3), the volume ratio of methanol to acetonitrile in the mobile phase A is 20-25:75-80; and the mobile phase B is a 0.1%-0.2% aqueous solution of ammonium acetate.

[0027] In a further preferred embodiment, in the quality testing method of the swelling and pain relief preparation of the present invention, the volume ratio of methanol to acetonitrile in the mobile phase A in step 3) is 20:80; and the mobile phase B is a 0.1% aqueous solution of ammonium acetate.

[0028] The beneficial effects of this invention are:

[0029] This invention establishes a quality detection method for a swelling and pain relief preparation, and extensively investigated the chromatographic conditions of this method. The optimal chromatographic conditions were determined to be: column temperature 35℃; mobile phase flow rate 1.0 mL / min; a methanol-acetonitrile ratio of 20:80 in mobile phase A; and mobile phase B being a 0.1% ammonium acetate aqueous solution. Under these optimal chromatographic conditions, HPLC analysis of the swelling and pain relief preparation showed symmetrical peaks of lupinone and curcumin in the obtained chromatograms, without tailing, and the resolution met the requirements. Methodological validation experiments confirmed that this method has high accuracy and good stability, further improving the quality standard of the swelling and pain relief preparation and enabling better control of drug quality. Attached Figure Description

[0030] Figure 1 The spectrum of curcumin reference standard;

[0031] Figure 2 The chromatogram of lupinone reference standard;

[0032] Figure 3 The chromatogram is for a mixture of reference standards;

[0033] Figure 4 The chromatogram of the test sample for the swelling and pain relief preparation. Detailed Implementation

[0034] The technical solution of the present invention will be described in detail below with reference to specific embodiments. The following embodiments are for explanation and illustration only, and do not constitute a limitation on the technical solution of the present invention.

[0035] Example 1

[0036] The quality testing methods for swelling and pain relief preparations, including the determination of lupinone and curcumin content, are as follows:

[0037] 1) Preparation of test solution: Accurately weigh 30g of drug solution, evaporate the solvent by rotary evaporation at 50℃, dissolve the residue in an appropriate amount of ethanol and transfer it to a 10mL volumetric flask, make up to volume, shake well, and filter through a 0.22μm microporous membrane to obtain the test solution;

[0038] 2) Preparation of reference solution: Accurately weigh 0.02 g of lupeone reference standard, place it in a 100 mL volumetric flask, dissolve it with ethanol and dilute to the mark to prepare a lupeone reference standard stock solution with a concentration of 0.2 mg / mL; accurately weigh 0.03 g of curcumin reference standard, accurately transfer 1.0 mL of the lupeone reference standard stock solution prepared above into a 10 mL volumetric flask, dissolve it with ethanol and dilute to the mark, shake well to obtain a mixed reference solution;

[0039] 3) HPLC determination:

[0040] Chromatographic conditions: Column type C 18The column, with dimensions of 250 mm × 4.6 mm and a diameter of 5 μm, was used. The column temperature was 35 °C, the flow rate was 1.0 mL / min, and the detection wavelength was 220 nm. Gradient elution was performed using a 20:80 methanol-acetonitrile mixture as mobile phase A and a 0.1% ammonium acetate aqueous solution as mobile phase B. The gradient elution program is as follows:

[0041] From 0 to 8 minutes, the volume ratio of mobile phase A to mobile phase B was 45:55.

[0042] In 8-9 minutes, the volume ratio of mobile phase A to mobile phase B changed from 45:55 to 100:0;

[0043] 9-16 min, the volume ratio of mobile phase A to mobile phase B is 100:0;

[0044] Over 16-25 minutes, the volume ratio of mobile phase A to mobile phase B changed from 100:0 to 70:30.

[0045] Over 25-30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 70:30 to 45:55.

[0046] Assay: Accurately pipette 10 μL each of the reference solution and the test solution into the high-performance liquid chromatograph and determine the result.

[0047] Example 2

[0048] The quality testing methods for swelling and pain relief preparations, including the determination of lupinone and curcumin content, are as follows:

[0049] 1) Preparation of test solution: Accurately weigh 25g of drug solution, evaporate the solvent by rotary evaporation at 50℃, dissolve the residue in an appropriate amount of ethanol and transfer it to a 10mL volumetric flask, make up to volume, shake well, and filter through a 0.22μm microporous membrane to obtain the test solution;

[0050] 2) Preparation of reference solution: Accurately weigh 0.02 g of lupeone reference standard, place it in a 100 mL volumetric flask, dissolve it with ethanol and dilute to the mark to prepare a lupeone reference standard stock solution with a concentration of 0.2 mg / mL; accurately weigh 0.03 g of curcumin reference standard, accurately transfer 1.0 mL of the lupeone reference standard stock solution prepared above into a 10 mL volumetric flask, dissolve it with ethanol and dilute to the mark, shake well to obtain a mixed reference solution;

[0051] 3) HPLC determination:

[0052] Chromatographic conditions: Column type C 18The column was 250 mm × 4.6 mm, 5 μm in diameter; the column temperature was 30 °C; the flow rate was 0.8 mL / min; the detection wavelength was 220 nm; gradient elution was performed using a 15:85 methanol and acetonitrile mixture as mobile phase A and a 0.1% ammonium acetate aqueous solution as mobile phase B. The gradient elution program is as follows:

[0053] From 0 to 8 minutes, the volume ratio of mobile phase A to mobile phase B was 45:55.

[0054] In 8-9 minutes, the volume ratio of mobile phase A to mobile phase B changed from 45:55 to 100:0;

[0055] 9-16 min, the volume ratio of mobile phase A to mobile phase B is 100:0;

[0056] Over 16-25 minutes, the volume ratio of mobile phase A to mobile phase B changed from 100:0 to 70:30.

[0057] Over 25-30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 70:30 to 45:55.

[0058] Assay: Accurately pipette 10 μL each of the reference solution and the test solution into the high-performance liquid chromatograph and determine the result.

[0059] Example 3

[0060] 1) Preparation of test solution: Accurately weigh 35g of drug solution, evaporate the solvent by rotary evaporation at 50℃, dissolve the residue in an appropriate amount of ethanol and transfer it to a 10mL volumetric flask, make up to volume, shake well, and filter through a 0.22μm microporous membrane to obtain the test solution;

[0061] 2) Preparation of reference solution: Accurately weigh 0.02 g of lupeone reference standard, place it in a 100 mL volumetric flask, dissolve it with ethanol and dilute to the mark to prepare a lupeone reference standard stock solution with a concentration of 0.2 mg / mL; accurately weigh 0.03 g of curcumin reference standard, accurately transfer 1.0 mL of the lupeone reference standard stock solution prepared above into a 10 mL volumetric flask, dissolve it with ethanol and dilute to the mark, shake well to obtain a mixed reference solution;

[0062] 3) HPLC determination:

[0063] Chromatographic conditions: Column type C 18 The column was 250 mm × 4.6 mm, 5 μm in diameter; the column temperature was 40 °C; the flow rate was 1.2 mL / min; the detection wavelength was 220 nm; gradient elution was performed using a 25:75 methanol and acetonitrile mixture as mobile phase A and a 0.3% ammonium acetate aqueous solution as mobile phase B. The gradient elution program is as follows:

[0064] From 0 to 8 minutes, the volume ratio of mobile phase A to mobile phase B was 45:55.

[0065] In 8-9 minutes, the volume ratio of mobile phase A to mobile phase B changed from 45:55 to 100:0;

[0066] 9-16 min, the volume ratio of mobile phase A to mobile phase B is 100:0;

[0067] Over 16-25 minutes, the volume ratio of mobile phase A to mobile phase B changed from 100:0 to 70:30.

[0068] Over 25-30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 70:30 to 45:55.

[0069] Assay: Accurately pipette 10 μL each of the reference solution and the test solution into the high-performance liquid chromatograph and determine the result.

[0070] Example 4

[0071] The quality testing methods for swelling and pain relief preparations, including the determination of lupinone and curcumin content, are as follows:

[0072] 1) Preparation of test solution: Accurately weigh 25g of drug solution, evaporate the solvent by rotary evaporation at 50℃, dissolve the residue in an appropriate amount of ethanol and transfer it to a 10mL volumetric flask, make up to volume, shake well, and filter through a 0.22μm microporous membrane to obtain the test solution;

[0073] 2) Preparation of reference solution: Accurately weigh 0.02 g of lupeone reference standard, place it in a 100 mL volumetric flask, dissolve it with ethanol and dilute to the mark to prepare a lupeone reference standard stock solution with a concentration of 0.2 mg / mL; accurately weigh 0.03 g of curcumin reference standard, accurately transfer 1.0 mL of the lupeone reference standard stock solution prepared above into a 10 mL volumetric flask, dissolve it with ethanol and dilute to the mark, shake well to obtain a mixed reference solution;

[0074] 3) HPLC determination:

[0075] Chromatographic conditions: Column type C 18 The column, with dimensions of 250 mm × 4.6 mm and a diameter of 5 μm, was used. The column temperature was 35 °C, the flow rate was 1.0 mL / min, and the detection wavelength was 220 nm. Gradient elution was performed using a 20:80 methanol-acetonitrile mixture as mobile phase A and a 0.1% ammonium acetate aqueous solution as mobile phase B. The gradient elution program is as follows:

[0076] From 0 to 8 minutes, the volume ratio of mobile phase A to mobile phase B was 45:55.

[0077] In 8-9 minutes, the volume ratio of mobile phase A to mobile phase B changed from 45:55 to 100:0;

[0078] 9-16 min, the volume ratio of mobile phase A to mobile phase B is 100:0;

[0079] Over 16-25 minutes, the volume ratio of mobile phase A to mobile phase B changed from 100:0 to 70:30.

[0080] Over 25-30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 70:30 to 45:55.

[0081] Assay: Accurately pipette 10 μL each of the reference solution and the test solution into the high-performance liquid chromatograph and determine the result.

[0082] Example 5

[0083] The quality testing methods for swelling and pain relief preparations, including the determination of lupinone and curcumin content, are as follows:

[0084] 1) Preparation of test solution: Accurately weigh 35g of drug solution, evaporate the solvent by rotary evaporation at 50℃, dissolve the residue in an appropriate amount of ethanol and transfer it to a 10mL volumetric flask, make up to volume, shake well, and filter through a 0.22μm microporous membrane to obtain the test solution;

[0085] 2) Preparation of reference solution: Accurately weigh 0.02 g of lupeone reference standard, place it in a 100 mL volumetric flask, dissolve it with ethanol and dilute to the mark to prepare a lupeone reference standard stock solution with a concentration of 0.2 mg / mL; accurately weigh 0.03 g of curcumin reference standard, accurately transfer 1.0 mL of the lupeone reference standard stock solution prepared above into a 10 mL volumetric flask, dissolve it with ethanol and dilute to the mark, shake well to obtain a mixed reference solution;

[0086] 3) HPLC determination:

[0087] Chromatographic conditions: Column type C 18 The column, with dimensions of 250 mm × 4.6 mm and a diameter of 5 μm, was used. The column temperature was 35 °C, the flow rate was 1.0 mL / min, and the detection wavelength was 220 nm. Gradient elution was performed using a 20:80 methanol-acetonitrile mixture as mobile phase A and a 0.1% ammonium acetate aqueous solution as mobile phase B. The gradient elution program is as follows:

[0088] From 0 to 8 minutes, the volume ratio of mobile phase A to mobile phase B was 45:55.

[0089] In 8-9 minutes, the volume ratio of mobile phase A to mobile phase B changed from 45:55 to 100:0;

[0090] 9-16 min, the volume ratio of mobile phase A to mobile phase B is 100:0;

[0091] Over 16-25 minutes, the volume ratio of mobile phase A to mobile phase B changed from 100:0 to 70:30.

[0092] Over 25-30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 70:30 to 45:55.

[0093] Assay: Accurately pipette 10 μL each of the reference solution and the test solution into the high-performance liquid chromatograph and determine the result.

[0094] Example 6

[0095] The quality testing methods for swelling and pain relief preparations, including the determination of lupinone and curcumin content, are as follows:

[0096] 1) Preparation of test solution: Accurately weigh 30g of drug solution, evaporate the solvent by rotary evaporation at 50℃, dissolve the residue in an appropriate amount of ethanol and transfer it to a 10mL volumetric flask, make up to volume, shake well, and filter through a 0.22μm microporous membrane to obtain the test solution;

[0097] 2) Preparation of reference solution: Accurately weigh 0.02 g of lupeone reference standard, place it in a 100 mL volumetric flask, dissolve it with ethanol and dilute to the mark to prepare a lupeone reference standard stock solution with a concentration of 0.2 mg / mL; accurately weigh 0.03 g of curcumin reference standard, accurately transfer 1.0 mL of the lupeone reference standard stock solution prepared above into a 10 mL volumetric flask, dissolve it with ethanol and dilute to the mark, shake well to obtain a mixed reference solution;

[0098] 3) HPLC determination:

[0099] Chromatographic conditions: Column type C 18 The column was 250 mm × 4.6 mm, 5 μm in diameter; the column temperature was 30 °C; the flow rate was 0.8 mL / min; the detection wavelength was 220 nm; gradient elution was performed using a 15:85 methanol and acetonitrile mixture as mobile phase A and a 0.1% ammonium acetate aqueous solution as mobile phase B. The gradient elution program is as follows:

[0100] From 0 to 8 minutes, the volume ratio of mobile phase A to mobile phase B was 45:55.

[0101] In 8-9 minutes, the volume ratio of mobile phase A to mobile phase B changed from 45:55 to 100:0;

[0102] 9-16 min, the volume ratio of mobile phase A to mobile phase B is 100:0;

[0103] Over 16-25 minutes, the volume ratio of mobile phase A to mobile phase B changed from 100:0 to 70:30.

[0104] Over 25-30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 70:30 to 45:55.

[0105] Assay: Accurately pipette 10 μL each of the reference solution and the test solution into the high-performance liquid chromatograph and determine the result.

[0106] Example 7

[0107] The quality testing methods for swelling and pain relief preparations, including the determination of lupinone and curcumin content, are as follows:

[0108] 1) Preparation of test solution: Accurately weigh 30g of drug solution, evaporate the solvent by rotary evaporation at 50℃, dissolve the residue in an appropriate amount of ethanol and transfer it to a 10mL volumetric flask, make up to volume, shake well, and filter through a 0.22μm microporous membrane to obtain the test solution;

[0109] 2) Preparation of reference solution: Accurately weigh 0.02 g of lupeone reference standard, place it in a 100 mL volumetric flask, dissolve it with ethanol and dilute to the mark to prepare a lupeone reference standard stock solution with a concentration of 0.2 mg / mL; accurately weigh 0.03 g of curcumin reference standard, accurately transfer 1.0 mL of the lupeone reference standard stock solution prepared above into a 10 mL volumetric flask, dissolve it with ethanol and dilute to the mark, shake well to obtain a mixed reference solution;

[0110] 3) HPLC determination:

[0111] Chromatographic conditions: Column type C 18 The column was 250 mm × 4.6 mm, 5 μm in diameter; the column temperature was 40 °C; the flow rate was 1.2 mL / min; the detection wavelength was 220 nm; gradient elution was performed using a 25:75 methanol and acetonitrile mixture as mobile phase A and a 0.3% ammonium acetate aqueous solution as mobile phase B. The gradient elution program is as follows:

[0112] From 0 to 8 minutes, the volume ratio of mobile phase A to mobile phase B was 45:55.

[0113] In 8-9 minutes, the volume ratio of mobile phase A to mobile phase B changed from 45:55 to 100:0;

[0114] 9-16 min, the volume ratio of mobile phase A to mobile phase B is 100:0;

[0115] Over 16-25 minutes, the volume ratio of mobile phase A to mobile phase B changed from 100:0 to 70:30.

[0116] Over 25-30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 70:30 to 45:55.

[0117] Assay: Accurately pipette 10 μL each of the reference solution and the test solution into the high-performance liquid chromatograph and determine the result.

[0118] To further verify the reliability of the present invention and select the optimal solution, the inventors conducted a series of experiments, as follows:

[0119] 1. Experimental Materials

[0120] 1.1 Main Reagents

[0121] Methanol, chromatographic grade; acetonitrile, chromatographic grade; ammonium acetate, analytical grade; water was laboratory-prepared ultrapure water; lupinone reference standard; curcumin reference standard.

[0122] 1.2 Main Instruments

[0123] Electronic balance (Shanghai Mettler Toledo Instruments Co., Ltd.); High performance liquid chromatograph (Agilent Technologies, Inc., USA); Rotary evaporator (Henan Yiyuan Instruments Co., Ltd.).

[0124] 2. Determination of lupinone and curcumin content

[0125] 2.1 Preparation of the test solution

[0126] Accurately weigh 30g of the drug solution, evaporate the solvent by rotary evaporation at 50℃, dissolve the residue in an appropriate amount of ethanol and transfer it to a 10mL volumetric flask, make up to volume, shake well, and filter through a 0.22μm microporous membrane to obtain the final product.

[0127] 2.2 Preparation of reference solution

[0128] Accurately weigh 0.02 g of lupeone reference standard, place it in a 100 mL volumetric flask, dissolve it in ethanol and dilute to the mark to prepare a lupeone reference standard stock solution with a concentration of 0.2 mg / mL.

[0129] Accurately weigh 0.03 g of curcumin reference standard, precisely transfer 1.0 mL of the lupin ketone reference standard stock solution prepared above into a 10 mL volumetric flask, dissolve in ethanol and dilute to the mark, shake well to obtain the mixed reference standard solution.

[0130] Accurately weigh appropriate amounts of lupinone and curcumin reference standards, dissolve them in ethanol, and prepare lupinone and curcumin reference standard solutions respectively.

[0131] 2.3 Chromatographic conditions

[0132] The chromatographic column was a Shim-pack GIST C10. 18The column was 250 mm × 4.6 mm, 5 μm in diameter; column temperature: 35 °C; flow rate: 1.0 mL / min; detection wavelength: 220 nm; gradient elution was performed using a 20:80 methanol and acetonitrile mixture as mobile phase A and 0.1% ammonium acetate aqueous solution as mobile phase B. The gradient elution program is as follows:

[0133] From 0 to 8 minutes, the volume ratio of mobile phase A to mobile phase B was 45:55.

[0134] In 8-9 minutes, the volume ratio of mobile phase A to mobile phase B changed from 45:55 to 100:0;

[0135] 9-16 min, the volume ratio of mobile phase A to mobile phase B is 100:0;

[0136] Over 16-25 minutes, the volume ratio of mobile phase A to mobile phase B changed from 100:0 to 70:30.

[0137] Over 25-30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 70:30 to 45:55.

[0138] 2.4 Determination Method

[0139] Accurately pipette 10 μL each of the mixed reference solution and the test solution into the high-performance liquid chromatograph and determine the result.

[0140] 2.5 Results and Analysis

[0141] See results Figure 1-4 (in Figure 1 The spectrum of curcumin reference standard; Figure 2 The chromatogram of lupinone reference standard; Figure 3 The chromatogram is for a mixture of reference standards; Figure 4 (The HPLC chromatogram of the test sample of the swelling and pain relief preparation is shown). The results show that the swelling and pain relief preparation exhibits two characteristic peaks in its HPLC chromatogram, with peak 1 being curcumin and peak 2 being lupinone.

[0142] 3. Selection of chromatographic conditions

[0143] 3.1 Column Temperature Investigation

[0144] Accurately weigh 30g of the swelling and pain relief preparation and prepare the test solution according to the method in section "2.1". Use the mixed reference solution prepared in section "2.2" as a control to investigate the effect of column temperatures of 30℃, 35℃, and 40℃ on the chromatographic separation effect. The results showed that the chromatographic peak shapes of lupinone and curcumin were better when the column temperature was 35-40℃. Considering all factors, a column temperature of 35℃ was selected.

[0145] 3.2 Flow velocity investigation

[0146] Accurately weigh 30g of the swelling and pain relief preparation and prepare the test solution according to the method in section "2.1". Use the mixed reference solution prepared in section "2.2" as a control. Investigate the effect of flow rates of 0.8mL / min, 1.0mL / min, and 1.2mL / min on the chromatographic separation effect. The results show that the chromatographic peak resolution of lupinone and curcumin is the best and the peak shape is more symmetrical when the flow rate is 1.0mL / min. Therefore, the flow rate of 1.0mL / min is selected.

[0147] 3.3 Investigation of the mobile phase

[0148] 3.3.1 Investigation of the proportion of mobile phase A

[0149] Accurately weigh 30g of the swelling and pain relief preparation and prepare the test solution according to the method in section "2.1". Use the mixed reference solution prepared in section "2.2" as a control to investigate the effect of the ratio of methanol to acetonitrile in mobile phase A of 15:85, 20:80, and 25:75 on the chromatographic separation effect. The results show that when the ratio of methanol to acetonitrile in mobile phase A is 20:80, the chromatographic peak separation effect is better and there is no tailing. Therefore, the ratio of methanol to acetonitrile in mobile phase A of 20:80 is selected.

[0150] 3.3.2 Investigation of mobile phase B concentration

[0151] Accurately weigh 30g of the swelling and pain relief preparation and prepare the test solution according to the method in section "2.1". Use the mixed reference solution prepared in section "2.2" as a control to investigate the effect of the concentration of ammonium acetate aqueous solution in mobile phase B (0.1%, 0.2%, and 0.3%) on the chromatographic separation effect. The results show that the chromatographic peak separation effect is best when the concentration of ammonium acetate aqueous solution is 0.1%. Therefore, the concentration of ammonium acetate aqueous solution in mobile phase B is selected as 0.1%.

[0152] 4. Methodological Validation

[0153] 4.1 Precision Examination

[0154] Accurately pipette 10 μL of the mixed reference solution under section “2.2”, and repeat the injection 6 times. Record the peak areas of lupinone and curcumin respectively. The results are shown in Table 1. The results show that the RSD values ​​are 0.004% and 0.001% respectively, indicating that the instrument has good precision.

[0155] Table 1 Precision test results

[0156]

[0157]

[0158] 4.2 Stability Test

[0159] The test solution prepared under section “2.1” was injected at 0, 2, 4, 8, 12 and 24 h respectively, and the peak areas of lupinone and curcumin were recorded respectively. The results are shown in Table 2. The results show that the RSD values ​​are 0.004% and 0.004% respectively, indicating that the test solution has good stability within 24 h.

[0160] Table 2 Stability test results

[0161]

[0162] 4.3 Repeatability Test

[0163] Accurately weigh 30g of the same batch of swelling and pain relief preparation solution, and weigh a total of 6 portions. Prepare the test solution according to the method in section "2.1", and perform HPLC determination. Record the peak area and calculate the contents of lupinone and curcumin. The results are shown in Table 3. The results show that the RSD values ​​are 0.54% and 0.10%, respectively, indicating that the method has good reproducibility.

[0164] Table 3 Results of Repeatability Tests

[0165]

[0166]

[0167] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A quality testing method for a swelling and pain relief preparation, wherein the effective components of the swelling and pain relief preparation, based on the preparation of 1000 mL of medicinal liquid, are: 63 g of banana root, 17 g of wood sorrel, and 20 g of turmeric; the preparation process of the swelling and pain relief preparation is as follows: the above three ingredients are pulverized into coarse powder, and according to the percolation method under the section on fluid extracts and extracts in the Chinese Pharmacopoeia, 45% ethanol is used as solvent, and after soaking for 48 hours, the percolation is slowly carried out, and the percolate is collected at a rate of 1-2 mL per minute to 1000 mL, allowed to stand, and filtered to obtain the product; Its features are, The quality testing method for the swelling and pain relief preparation includes the determination of the contents of lupinone and curcumin, as detailed below: 1) Preparation of test solution: Accurately weigh 25-35g of drug solution, evaporate the solvent by rotary evaporation at 50℃, dissolve the residue in an appropriate amount of ethanol and transfer it to a 10mL volumetric flask, make up to volume, shake well, filter, and the test solution is obtained. 2) Preparation of reference solution: Accurately weigh 0.02 g of lupeone reference standard, place it in a 100 mL volumetric flask, dissolve it with ethanol and dilute to the mark to prepare a lupeone reference standard stock solution with a concentration of 0.2 mg / mL; accurately weigh 0.03 g of curcumin reference standard, accurately transfer 1.0 mL of the lupeone reference standard stock solution prepared above into a 10 mL volumetric flask, dissolve it with ethanol and dilute to the mark, shake well to obtain a mixed reference solution; 3) HPLC determination: Chromatographic conditions: Column type C 18 The column was 250 mm × 4.6 mm, 5 μm in diameter; the column temperature was 30-40℃; the flow rate was 0.8-1.2 mL / min; the detection wavelength was 220 nm; a gradient elution was performed using a methanol-acetonitrile mixture (volume ratio 15-25:75-85) as mobile phase A and a 0.1%-0.3% ammonium acetate aqueous solution as mobile phase B. The gradient elution program is as follows: From 0 to 8 minutes, the volume ratio of mobile phase A to mobile phase B was 45:

55. In 8-9 minutes, the volume ratio of mobile phase A to mobile phase B changed from 45:55 to 100:0; 9-16 min, the volume ratio of mobile phase A to mobile phase B is 100:0; Over 16-25 minutes, the volume ratio of mobile phase A to mobile phase B changed from 100:0 to 70:

30. Over 25-30 minutes, the volume ratio of mobile phase A to mobile phase B changed from 70:30 to 45:

55. Assay: Accurately pipette 10 μL each of the mixed reference solution and the test solution into the high performance liquid chromatograph and determine the result.

2. The quality testing method for the swelling and pain relief preparation according to claim 1, characterized in that, In the determination of lupinone and curcumin content, the mass of the accurately weighed medicinal solution in step 1) is 30-35g.

3. The quality testing method for the swelling and pain relief preparation according to claim 2, characterized in that, In the determination of the contents of lupinone and curcumin, the mass of the accurately weighed medicinal solution in step 1) is 30g.

4. The quality testing method for the swelling and pain relief preparation according to claim 1, characterized in that, In the determination of the contents of lupinone and curcumin, the filtration in step 1) is through a 0.22 μm microporous membrane.

5. The quality testing method for the swelling and pain relief preparation according to claim 1, characterized in that, In the determination of the content of lupinone and curcumin, the column temperature in step 3) is 35-40℃.

6. The quality testing method for the swelling and pain relief preparation according to claim 5, characterized in that, In the determination of the content of lupinone and curcumin, the column temperature in step 3) is 35°C.

7. The quality testing method for the swelling and pain relief preparation according to claim 1, characterized in that, In the determination of the contents of lupinone and curcumin, the flow rate in step 3) is 1.0-1.2 mL / min.

8. The quality testing method for the swelling and pain relief preparation according to claim 7, characterized in that, In the determination of the contents of lupinone and curcumin, the flow rate in step 3) is 1.0 mL / min.

9. The quality testing method for the swelling and pain relief preparation according to claim 1, characterized in that, In the determination of the contents of lupinone and curcumin, the volume ratio of methanol to acetonitrile in mobile phase A in step 3) is 20-25:75-80; and mobile phase B is a 0.1%-0.2% aqueous solution of ammonium acetate.

10. The quality testing method for the swelling and pain relief preparation according to claim 9, characterized in that, In the determination of the contents of lupinone and curcumin, the volume ratio of methanol to acetonitrile in mobile phase A in step 3) is 20:80; and mobile phase B is a 0.1% aqueous solution of ammonium acetate.

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