Quality detection method of swelling and pain relieving preparation

Abstract

The invention relates to the technical field of medicine detection, in particular to a quality detection method of a swelling and pain relieving preparation. The quality detection method comprises content determination of lupenone and curcumin, and specifically comprises the following steps: preparation of a test solution, preparation of a reference solution and HPLC determination. The quality detection method of the swelling and pain relieving preparation is established, methodological experiments prove that the method is high in accuracy, good in stability and easy to operate, the quality standard of the swelling and pain relieving preparation is further improved, and the medicine quality is better controlled.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a quality detection method for a Zhongtongshu preparation. Background Art

[0002] The Zhongtongshu preparation is a Chinese patent medicine made from banana root, Oxalis and turmeric as the main raw materials. It has the effects of promoting blood circulation, removing blood stasis, reducing swelling and relieving pain. It is often used clinically to treat bruises, blood stasis, swelling and pain, and soft tissue contusion, and has a good therapeutic effect.

[0003] The current quality standard of Zhongtongshu preparation (standard number WS-10540 (ZD-0540) -2002-2012Z) includes thin layer identification of banana root, thin layer identification of turmeric and determination of curcumin content. Among them, the preparation method of the test solution in the determination of curcumin content is: accurately measure 5mL of this product, evaporate to near dryness, mix the sample with a small amount of macroporous resin D101 and place it on a macroporous adsorption resin column D101 (inner diameter 1.5cm, column height 15cm), elute with 80mL of water, discard the water, continue to elute with 100mL of ethanol, collect the eluate, evaporate to dryness, dissolve with acetonitrile and transfer to a 5mL volumetric flask, add acetonitrile to the scale, shake well, filter, and take the filtrate. The preparation method of the above test solution is relatively complicated, the experimental cost is high, and the technical requirements for the experimenter are high, and the human error is large. The content of curcumin was determined according to the method in this standard. The chromatographic peak of curcumin in the obtained HPLC spectrum was asymmetric and had a tailing phenomenon. In addition, this content determination method was only for the turmeric in the preparation and could not achieve a more comprehensive control of the drug quality.

[0004] Based on the above problems, the present invention uses the content of lupetone and curcumin as indicators and establishes a quality detection method for the Zhongtongshu preparation based on high performance liquid chromatography, aiming to further improve the quality standard of the Zhongtongshu preparation, improve the detection efficiency of experimenters, reduce the detection cost, and achieve better control of drug quality. Summary of the invention

[0005] The purpose of the present invention is to provide a quality detection method for a zhongtongshu preparation.

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

[0007] The swollen and pain-relieving preparation of the present invention is calculated based on the preparation of 1000mL of liquid medicine, and the dosage of its effective components is: 63g of banana root, 17g of oxalis, and 20g of turmeric; the preparation process of the swollen and pain-relieving preparation is: the above three flavors are crushed into coarse powder, and according to the percolation method under the fluid extract and extract of the "Chinese Pharmacopoeia", 45% ethanol is used as a solvent, and after immersion for 48 hours, the percolation is slowly performed, and the percolation liquid is collected to 1000mL at a speed of 1-2mL per minute, and the percolation liquid is allowed to stand and filtered to obtain the swollen and pain-relieving preparation.

[0008] The quality detection method of the Zhongtongshu preparation of the present invention includes the determination of the content of lupine ketone and curcumin, which is specifically as follows:

[0009] 1) Preparation of test solution: accurately weigh 25-35g of the drug solution, evaporate the solvent at 50℃, dissolve the residue in appropriate amount of ethanol and transfer to a 10mL volumetric flask, make up to volume, shake well, filter, and obtain the solution;

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

[0011] 3)HPLC determination:

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

[0013] 0-8min, the volume ratio of mobile phase A to mobile phase B was 45:55;

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

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

[0016] 16-25 min, the volume ratio of mobile phase A to mobile phase B was changed from 100:0 to 70:30;

[0017] 25-30 min, the volume ratio of mobile phase A to mobile phase B was changed from 70:30 to 45:55;

[0018] Determination method: Accurately pipette 10 μL of reference solution and test solution respectively, inject into high performance liquid chromatography, and determine.

[0019] Preferably, in the quality detection method of the Zhongtongshu preparation of the present invention, the mass of the accurately weighed medicinal solution in step 1) is 30-35 g.

[0020] Further preferably, in the quality detection method of the Zhongtongshu preparation of the present invention, the mass of the accurately weighed medicinal solution in step 1) is 30 g.

[0021] Preferably, in the quality inspection method of the Zhongtongshu preparation of the present invention, the filtration in step 1) is through a 0.22 μm microporous filter membrane.

[0022] Preferably, in the quality detection method of the Zhongtongshu preparation of the present invention, the column temperature in step 3) is 35-40°C.

[0023] Further preferably, in the quality detection method of the Zhongtongshu preparation of the present invention, the column temperature in step 3) is 35°C.

[0024] Preferably, in the quality detection method of the Zhongtongshu preparation of the present invention, the flow rate in step 3) is 1.0-1.2 mL / min.

[0025] Further preferably, in the quality detection method of the Zhongtongshu preparation of the present invention, the flow rate in step 3) is 1.0 mL / min.

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

[0027] Further preferably, in the quality detection method of the Zhongtongshu 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% ammonium acetate aqueous solution.

[0028] Beneficial effects of the present invention:

[0029] The present invention establishes a quality detection method for a swollen and tongshu preparation, and conducts a large number of investigations on the chromatographic conditions in the detection method. As a result, the optimal chromatographic conditions are as follows: the column temperature is 35°C; the mobile phase flow rate is 1.0 mL / min; the ratio of methanol to acetonitrile in mobile phase A is 20:80; and the mobile phase B is a 0.1% ammonium acetate aqueous solution. Under the above-mentioned optimal chromatographic conditions, HPLC analysis is performed on the swollen and tongshu preparation, and the chromatographic peaks of lupetone and curcumin in the obtained spectrum are symmetrical and tail-free, and the separation degree meets the requirements. The methodological investigation experiment confirms that the method has high accuracy and good stability, further improves the quality standard of the swollen and tongshu preparation, and enables the quality of the drug to be better controlled. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the reference substance spectrum of curcumin;

[0031] Figure 2 This is the spectrum of lupine ketone reference substance;

[0032] Figure 3 It is the mixed reference sample spectrum;

[0033] Figure 4 This is the test sample spectrum of Zhongtongshu preparation. DETAILED DESCRIPTION

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

[0035] Example 1

[0036] The quality test method of the Zhongtongshu preparation, including the determination of lupine ketone and curcumin, is as follows:

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

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

[0039] 3)HPLC determination:

[0040] Chromatographic conditions: Chromatographic column is C 18Column, specification: 250mm×4.6mm, 5μm; column temperature: 35℃; flow rate: 1.0mL / min; detection wavelength: 220nm; mobile phase A: a mixture of methanol and acetonitrile in a volume ratio of 20:80, mobile phase B: 0.1% ammonium acetate aqueous solution, gradient elution, the gradient elution program is as follows:

[0041] 0-8min, the volume ratio of mobile phase A to mobile phase B was 45:55;

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

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

[0044] 16-25 min, the volume ratio of mobile phase A to mobile phase B was changed from 100:0 to 70:30;

[0045] 25-30 min, the volume ratio of mobile phase A to mobile phase B was changed from 70:30 to 45:55;

[0046] Determination method: Accurately pipette 10 μL of reference solution and test solution respectively, inject into high performance liquid chromatography, and determine.

[0047] Example 2

[0048] The quality test method of the Zhongtongshu preparation, including the determination of lupine ketone and curcumin, is as follows:

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

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

[0051] 3)HPLC determination:

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

[0053] 0-8min, the volume ratio of mobile phase A to mobile phase B was 45:55;

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

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

[0056] 16-25 min, the volume ratio of mobile phase A to mobile phase B was changed from 100:0 to 70:30;

[0057] 25-30 min, the volume ratio of mobile phase A to mobile phase B was changed from 70:30 to 45:55;

[0058] Determination method: Accurately pipette 10 μL of reference solution and test solution respectively, inject into high performance liquid chromatography, and determine.

[0059] Example 3

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

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

[0062] 3)HPLC determination:

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

[0064] 0-8min, the volume ratio of mobile phase A to mobile phase B was 45:55;

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

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

[0067] 16-25 min, the volume ratio of mobile phase A to mobile phase B was changed from 100:0 to 70:30;

[0068] 25-30 min, the volume ratio of mobile phase A to mobile phase B was changed from 70:30 to 45:55;

[0069] Determination method: Accurately pipette 10 μL of reference solution and test solution respectively, inject into high performance liquid chromatography, and determine.

[0070] Example 4

[0071] The quality test method of the Zhongtongshu preparation, including the determination of lupine ketone and curcumin, is as follows:

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

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

[0074] 3)HPLC determination:

[0075] Chromatographic conditions: Chromatographic column is C 18 Column, specification: 250mm×4.6mm, 5μm; column temperature: 35℃; flow rate: 1.0mL / min; detection wavelength: 220nm; mobile phase A: a mixture of methanol and acetonitrile in a volume ratio of 20:80, mobile phase B: 0.1% ammonium acetate aqueous solution, gradient elution, the gradient elution program is as follows:

[0076] 0-8min, the volume ratio of mobile phase A to mobile phase B was 45:55;

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

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

[0079] 16-25 min, the volume ratio of mobile phase A to mobile phase B was changed from 100:0 to 70:30;

[0080] 25-30 min, the volume ratio of mobile phase A to mobile phase B was changed from 70:30 to 45:55;

[0081] Determination method: Accurately pipette 10 μL of reference solution and test solution respectively, inject into high performance liquid chromatography, and determine.

[0082] Example 5

[0083] The quality test method of the Zhongtongshu preparation, including the determination of lupine ketone and curcumin, is as follows:

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

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

[0086] 3)HPLC determination:

[0087] Chromatographic conditions: Chromatographic column is C 18 Column, specification: 250mm×4.6mm, 5μm; column temperature: 35℃; flow rate: 1.0mL / min; detection wavelength: 220nm; mobile phase A: a mixture of methanol and acetonitrile in a volume ratio of 20:80, mobile phase B: 0.1% ammonium acetate aqueous solution, gradient elution, the gradient elution program is as follows:

[0088] 0-8min, the volume ratio of mobile phase A to mobile phase B was 45:55;

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

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

[0091] 16-25 min, the volume ratio of mobile phase A to mobile phase B was changed from 100:0 to 70:30;

[0092] 25-30 min, the volume ratio of mobile phase A to mobile phase B was changed from 70:30 to 45:55;

[0093] Determination method: Accurately pipette 10 μL of reference solution and test solution respectively, inject into high performance liquid chromatography, and determine.

[0094] Example 6

[0095] The quality test method of the Zhongtongshu preparation, including the determination of lupine ketone and curcumin, is as follows:

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

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

[0098] 3)HPLC determination:

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

[0100] 0-8min, the volume ratio of mobile phase A to mobile phase B was 45:55;

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

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

[0103] 16-25 min, the volume ratio of mobile phase A to mobile phase B was changed from 100:0 to 70:30;

[0104] 25-30 min, the volume ratio of mobile phase A to mobile phase B was changed from 70:30 to 45:55;

[0105] Determination method: Accurately pipette 10 μL of reference solution and test solution respectively, inject into high performance liquid chromatography, and determine.

[0106] Example 7

[0107] The quality test method of the Zhongtongshu preparation, including the determination of lupine ketone and curcumin, is as follows:

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

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

[0110] 3)HPLC determination:

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

[0112] 0-8min, the volume ratio of mobile phase A to mobile phase B was 45:55;

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

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

[0115] 16-25 min, the volume ratio of mobile phase A to mobile phase B was changed from 100:0 to 70:30;

[0116] 25-30 min, the volume ratio of mobile phase A to mobile phase B was changed from 70:30 to 45:55;

[0117] Determination method: Accurately pipette 10 μL of reference solution and test solution respectively, inject into high performance liquid chromatography, and determine.

[0118] In order to further verify the reliability of the present invention and screen out the best solution, the inventor 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 is laboratory-made ultrapure water; lupine ketone reference substance; curcumin reference substance.

[0122] 1.2 Main instruments

[0123] Electronic balance (Shanghai Mettler-Toledo Instrument Co., Ltd.); high performance liquid chromatograph (Agilent Technologies, USA); rotary evaporator (Henan Yiyuan Instrument Co., Ltd.).

[0124] 2. Determination of lupine ketone and curcumin content

[0125] 2.1 Preparation of test solution

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

[0127] 2.2 Preparation of reference solution

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

[0129] Accurately weigh 0.03 g of curcumin reference substance, accurately pipette 1.0 mL of the lupine ketone reference substance mother solution prepared above, place it in a 10 mL volumetric flask, dissolve it with ethanol and make up to the scale, shake well, and obtain a mixed reference substance solution.

[0130] Accurately weigh appropriate amounts of lupine ketone reference substance and curcumin reference substance, add ethanol to dissolve, and prepare lupine ketone reference substance solution and curcumin reference substance solution, respectively.

[0131] 2.3 Chromatographic conditions

[0132] The chromatographic column is Shim-pack GIST C 18Column, specification: 250mm×4.6mm, 5μm; column temperature: 35℃; flow rate: 1.0mL / min; detection wavelength: 220nm; using a mixed solution of methanol and acetonitrile with a volume ratio of 20:80 as mobile phase A and 0.1% ammonium acetate aqueous solution as mobile phase B, gradient elution was performed, and the gradient elution program was as follows:

[0133] 0-8min, the volume ratio of mobile phase A to mobile phase B was 45:55;

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

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

[0136] 16-25 min, the volume ratio of mobile phase A to mobile phase B was changed from 100:0 to 70:30;

[0137] 25-30 min, the volume ratio of mobile phase A to mobile phase B was changed from 70:30 to 45:55;

[0138] 2.4 Determination method

[0139] Accurately pipette 10 μL of the mixed reference solution and test solution respectively, inject into high performance liquid chromatography, and measure to obtain the result.

[0140] 2.5 Results and Analysis

[0141] Results Figure 1-4 (in Figure 1 This is the reference substance spectrum of curcumin; Figure 2 This is the spectrum of lupine ketone reference substance; Figure 3 It is the mixed reference sample spectrum; Figure 4 ) The results show that there are two characteristic peaks in the HPLC spectrum of the Zhongtongshu preparation, of which peak 1 is curcumin and peak 2 is lupeone.

[0142] 3. Selection of chromatographic conditions

[0143] 3.1 Column temperature investigation

[0144] Weigh 30g of the Zhongtongshu preparation accurately, prepare the test solution according to the method under "2.1", and use the mixed reference solution prepared under "2.2" as the control to investigate the effect of column temperature at 30℃, 35℃, and 40℃ on the chromatographic separation effect. The results show that when the column temperature is 35-40℃, the chromatographic peak shape of lupenone and curcumin is better. Considering all factors, the column temperature of 35℃ is selected.

[0145] 3.2 Flow rate investigation

[0146] Accurately weigh 30g of the Zhongtongshu preparation, prepare the test solution according to the method under "2.1", and use the mixed reference solution prepared under "2.2" as the control to investigate the effect of flow rates of 0.8mL / min, 1.0mL / min, and 1.2mL / min on the chromatographic separation effect. The results showed that when the flow rate was 1.0mL / min, the chromatographic peak separation of lupine ketone and curcumin was the best, and the peak shape was more symmetrical, so the flow rate was selected to be 1.0mL / min.

[0147] 3.3 Mobile phase investigation

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

[0149] Accurately weigh 30g of the Zhongtongshu preparation, prepare the test solution according to the method under "2.1", and use the mixed reference solution prepared under "2.2" as the 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, so the ratio of methanol to acetonitrile in mobile phase A is selected to be 20:80.

[0150] 3.3.2 Investigation of mobile phase B concentration

[0151] Accurately weigh 30g of the Zhongtongshu preparation, prepare the test solution according to the method under "2.1", and use the mixed reference solution prepared under "2.2" as the control to investigate the effect of 0.1%, 0.2%, and 0.3% of the concentration of the ammonium acetate aqueous solution in the mobile phase B on the chromatographic separation effect. The results show that when the concentration of the ammonium acetate aqueous solution is 0.1%, the chromatographic peak separation effect is the best, so the concentration of the ammonium acetate aqueous solution in the mobile phase B is selected to be 0.1%.

[0152] 4. Methodological Validation

[0153] 4.1 Precision investigation

[0154] Accurately pipette 10 μL of the mixed reference solution under item "2.2", repeat the injection 6 times, and record the peak areas of lupetone and curcumin respectively. The results are shown in Table 1. It can be seen from the results that the RSD values ​​are 0.004% and 0.001% respectively, indicating that the precision of the instrument is good.

[0155] Table 1 Precision test results

[0156]

[0157]

[0158] 4.2 Stability test

[0159] The test solution prepared under "2.1" was taken and sampled at 0, 2, 4, 8, 12 and 24 h, and the peak areas of lupeone and curcumin were recorded respectively. The results are shown in Table 2. From the results, it can be seen 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 30 g of the same batch of Zhongtongshu preparation liquid, weigh a total of 6 portions, prepare the test solution according to the method under "2.1", and perform HPLC determination, record the peak area, and calculate the content of lupetone and curcumin. The results are shown in Table 3. It can be seen from the results that the RSD values ​​are 0.54% and 0.10%, respectively, indicating that the method has good reproducibility.

[0164] Table 3 Repeatability test results

[0165]

[0166]

[0167] Although the present invention has been described in detail above by means of general description, specific implementation methods and tests, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A quality inspection method for a swollen and sour preparation, wherein the dosage of the effective components of the swollen and sour preparation for preparing 1000 mL of liquid medicine is: 63 g of banana root, 17 g of oxalis, and 20 g of turmeric; the preparation process of the swollen and sour preparation is: the above three ingredients are crushed into coarse powder, and the 45% ethanol is used as a solvent according to the percolation method under the fluid extract and extract of the "Chinese Pharmacopoeia", and after immersion for 48 hours, the percolation is slowly performed, and the percolation liquid is collected to 1000 mL at a speed of 1-2 mL per minute, and the percolation liquid is allowed to stand and filtered to obtain the swollen and sour preparation; It is characterized in that The quality inspection method of the Zhongtongshu preparation includes the determination of the content of lupine ketone and curcumin, which is as follows: 1) Preparation of test solution: accurately weigh 25-35g of the drug solution, evaporate the solvent at 50℃, dissolve the residue in appropriate amount of ethanol and transfer to a 10mL volumetric flask, make up to volume, shake well, filter, and obtain the solution; 2) Preparation of reference solution: accurately weigh 0.02 g of lupine ketone reference substance, place it in a 100 mL volumetric flask, dissolve it with ethanol and dilute it to the mark, to obtain a lupine ketone reference substance mother solution with a concentration of 0.2 mg / mL; accurately weigh 0.03 g of curcumin reference substance, accurately transfer 1.0 mL of the lupine ketone reference substance mother solution prepared above, place it in a 10 mL volumetric flask, dissolve it with ethanol and dilute it to the mark, shake well, and obtain a mixed reference substance solution; 3)HPLC determination: Chromatographic conditions: Chromatographic column is C 18 Column, specification: 250mm×4.6mm, 5μm; column temperature: 30-40℃; flow rate: 0.8-1.2mL / min; detection wavelength: 220nm; using a mixed solution of methanol and acetonitrile in a volume ratio of 15-25:75-85 as mobile phase A and a 0.1%-0.3% ammonium acetate aqueous solution as mobile phase B, gradient elution was performed, and the gradient elution procedure was as follows: 0-8min, the volume ratio of mobile phase A to mobile phase B was 45:55; At 8-9 min, the volume ratio of mobile phase A to mobile phase B was changed from 45:55 to 100:0; 9-16min, the volume ratio of mobile phase A to mobile phase B was 100:0; 16-25 min, the volume ratio of mobile phase A to mobile phase B was changed from 100:0 to 70:30; 25-30 min, the volume ratio of mobile phase A to mobile phase B was changed from 70:30 to 45:55; Determination method: Accurately pipette 10 μL of the mixed reference solution and test solution respectively, inject into high performance liquid chromatography, and determine.

2. The quality inspection method of the Zhongtongshu preparation according to claim 1, characterized in that: In the determination of the content of lupine ketone and curcumin, the mass of the accurately weighed medicinal solution in step 1) is 30-35 g.

3. The quality inspection method of the Zhongtongshu preparation according to claim 2, characterized in that: In the determination of the content of lupine ketone and curcumin, the mass of the accurately weighed medicinal solution in step 1) is 30 g.

4. The quality inspection method of the Zhongtongshu preparation according to claim 1, characterized in that: In the determination of the content of lupine ketone and curcumin, the filtration in step 1) is through a 0.22 μm microporous filter membrane.

5. The quality inspection method of the Zhongtongshu preparation according to claim 1, characterized in that: In the determination of the content of lupine ketone and curcumin, the column temperature in step 3) is 35-40°C.

6. The quality inspection method of the Zhongtongshu preparation according to claim 5, characterized in that: In the determination of the content of lupine ketone and curcumin, the column temperature in step 3) is 35°C.

7. The quality inspection method of the Zhongtongshu preparation according to claim 1, characterized in that: In the determination of the content of lupine ketone and curcumin, the flow rate in step 3) is 1.0-1.2 mL / min.

8. The quality inspection method of the Zhongtongshu preparation according to claim 7, characterized in that: In the determination of the content of lupine ketone and curcumin, the flow rate in step 3) is 1.0 mL / min.

9. The quality inspection method of the Zhongtongshu preparation according to claim 1, characterized in that: In the determination of the content of lupine ketone and curcumin, the volume ratio of methanol to acetonitrile in the mobile phase A in step 3) is 20-25:75-80; and the mobile phase B is a 0.1%-0.2% ammonium acetate aqueous solution.

10. The quality inspection method of the Zhongtongshu preparation according to claim 9, characterized in that: In the determination of the content of lupine ketone and curcumin, 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% ammonium acetate aqueous solution.

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