A method for determining the contents of five components in Baiyu ointment based on quantitative analysis of multi-components by single marker

Abstract

The application discloses a method for determining the content of five components in Baiyu ointment based on a quantitative analysis of multiple components, and relates to the technical field of drug detection. ′ The method uses tanshinone II A as an internal standard for quantitative analysis of multiple components, establishes the relative correction factors of 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malonic acid ester, tanshinone I, 6-gingerol, beta, beta The content of each component in the Baiyu ointment is calculated through the relative correction factors, and the specific steps include: preparation of a test sample solution; preparation of a reference sample solution; chromatographic conditions and system suitability test; determination and calculation. The application overcomes the single and one-sided quality control of the Baiyu ointment, and has important significance for further improving the quality control level of the Baiyu ointment, guaranteeing the clinical curative effect and improving the related evaluation system.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, specifically to a method for determining the content of five components in Baiyu Ointment based on a one-test-multiple-evaluation method. Background Technology

[0002] Baiyu Ointment is a product of Guizhou Lvtaiyang Pharmaceutical Co., Ltd. It has the effects of promoting blood circulation, dispelling wind, nourishing blood and moisturizing the skin. It is used for skin cracking caused by blood deficiency and wind dryness. Its clinical treatment effect is significant and it is highly favored by patients.

[0003] The formula for Baiyu Ointment consists of nine herbs: Bletilla striata, Polygonatum odoratum, Ligusticum striatum, Polygonum hydropiper, Geranium wilfordii, Salvia miltiorrhiza, Lithospermum erythrorhizon, Zingiber officinale, and Angelica sinensis. The preparation method is as follows: For the above nine herbs, take Salvia miltiorrhiza, Angelica sinensis, and Zingiber officinale, and use 80% ethanol as a solvent. Soak for 12 hours, then slowly percolate at a rate of 1-3 mL / min. Collect 1200 mL of the percolate, recover the ethanol under reduced pressure, and concentrate to a clear extract with a relative density of 1.15 (50℃). Set aside. The remaining six herbs, including Bletilla striata, are decocted twice with water, the first time for 2 hours and the second time for 1.5 hours. Combine the decoctions, filter, and concentrate the filtrate to a clear extract with a relative density of 1.05 (60℃). Combine this with the above clear extract. Add 60 g of sorbitol, 60 g of beta-cyclodextrin, and 60 g of glycerin. 20g of titanium dioxide (titanium white powder) and 3g of sodium hyaluronate are dissolved by heating (80℃) and set aside. Separately, 10g of mink oil, 30g of lanolin, 60g of white petrolatum, 12g of mono- and di-stearate glyceryl monostearate, 10g of sucrose stearate, 100g of liquid paraffin (paraffin oil), 50g of polysorbate 80, 50g of sorbitan oleate (Span 80), and 20g of emulsified silicone oil are melted by heating (80℃) and mixed with the above-mentioned ointment. The mixture is emulsified for 15 minutes, kept at 80℃ for 30 minutes, and then cooled to 40℃. 2g of elastase, 6g of benzoic acid, and 1.5g of ethylparaben are then added and mixed thoroughly to produce 1000g of the final product. The current quality standard for Baiyu Ointment only uses the content of tanshinone IIA as the detection standard, which cannot comprehensively reflect the quality of Baiyu Ointment.

[0004] Based on this, the research team, after in-depth investigation of the quality testing methods for Baiyu Ointment, established a method for determining the content of five components in Baiyu Ointment using a single-analysis-multiple-evaluation approach. This method uses tanshinone IIA as an internal standard for single-analysis-multiple-evaluation and establishes the content of 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutyl malate, tanshinone I, 6-gingerol, and β,β-glucanol. ´ The relative correction factor of dimethacryloyl argentine was used to calculate the content of each component in Baiyu ointment. The present invention aims to achieve more comprehensive quality control of Baiyu ointment. Summary of the Invention

[0005] The purpose of this invention is to provide a method for determining the content of five components in Baiyu Ointment based on a one-test-multiple-evaluation method.

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

[0007] The method for determining the content of five components in Baiyu Ointment based on a single-test, multiple-evaluation approach described in this invention uses tanshinone IIA as an internal standard to establish a method for determining the content of 1,4-bis[4-(glucosinolate)benzyl]-2-isobutyl malate, tanshinone I, 6-gingerol, and β,β-glucanol. ´ The relative correction factor for dimethacryloyl argentine was determined, and the content of each component was calculated using the relative correction factor. The specific steps included: preparation of the test solution; preparation of the reference solution; chromatographic conditions and system suitability test; determination and calculation.

[0008] Preferably, in the method for determining the content of five components in Baiyu Ointment based on the one-test-multiple-evaluation method of the present invention, the preparation of the test solution is as follows: take 1.8-4.2g of Baiyu Ointment, accurately weigh it, accurately add 15-35mL of 70%-100% methanol solution or 60%-75% ethanol solution, and weigh it; sonicate for 30-60min, cool, replenish the lost weight with the above methanol solution or ethanol solution, shake well, and filter; extract the filtrate with chloroform 1-4 times by shaking, 20-40mL each time; combine the extracts, evaporate to near dryness, add an appropriate amount of methanol to dissolve and transfer to a 5mL volumetric flask, make up to volume, shake well, and filter to obtain the final product.

[0009] In a further preferred embodiment, the method for determining the content of five components in Baiyu Ointment based on the one-test-multiple-evaluation method of the present invention comprises the following steps for preparing the test solution: accurately weigh 2.4-4.2 g of Baiyu Ointment, accurately add 20-35 mL of 60%-75% ethanol solution, and weigh again; sonicate for 30-45 min, cool, replenish the lost weight with the above-mentioned ethanol solution, shake well, and filter; extract the filtrate with chloroform 3-4 times by shaking, 30-40 mL each time; combine the extracts, evaporate to near dryness, dissolve in an appropriate amount of methanol and transfer to a 5 mL volumetric flask, make up to volume, shake well, and filter through a 0.45 μm microporous membrane to obtain the final product.

[0010] In a further preferred embodiment of the present invention, the method for determining the content of five components in Baiyu Ointment based on a single-test-multiple-evaluation method comprises the following steps for preparing the test solution: 3g of Baiyu Ointment is accurately weighed, 25mL of 75% ethanol solution is accurately added, and the weight is weighed; the solution is sonicated for 30min, cooled, and the weight loss is replenished with the above-mentioned ethanol solution, shaken well, and filtered; the filtrate is extracted three times with 30mL of chloroform each time; the extracts are combined, evaporated to near dryness, dissolved in an appropriate amount of methanol, transferred to a 5mL volumetric flask, diluted to volume, shaken well, and filtered through a 0.45μm microporous membrane to obtain the final product.

[0011] Preferably, in the method for determining the content of five components in Baiyu Ointment based on the one-test-multiple-evaluation method of the present invention, the preparation of the reference solution is specifically as follows: take an appropriate amount of tanshinone IIA reference standard, accurately weigh it and place it in a brown volumetric flask, add methanol to prepare a reference solution containing 0.02 mg per 1 mL, and that is obtained.

[0012] Preferably, in the method for determining the content of five components in Baiyu Ointment based on a one-test-multiple-evaluation method according to the present invention, the chromatographic conditions and system suitability test specifically includes:

[0013] The chromatographic column was packed with octadecylsilane-bonded silica gel; the column temperature was 25-35℃; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution containing 0.1% sodium dodecyl sulfonate was used as mobile phase B for gradient elution; the mobile phase flow rate was 0.9-1.1 mL / min; the injection volume was 10 μL; the detection wavelength was 270 nm; the theoretical plate number, calculated based on the tanshinone IIA peak, should not be less than 4000.

[0014] The gradient elution procedure is as follows:

[0015] .

[0016] In a further preferred embodiment, in the method for determining the content of five components in Baiyu Ointment based on the one-test-multiple-evaluation method of the present invention, the column temperature in the chromatographic conditions and system adaptability test is 30°C, and the mobile phase flow rate is 1.0 mL / min.

[0017] Preferably, in the method for determining the content of five components in Baiyu Ointment based on the one-test-multiple-evaluation method of the present invention, the determination and calculation specifically involve: injecting the test solution and the reference solution into a high-performance liquid chromatograph for determination, and determining the content of five components based on tanshinone IIA and 1,4-bis[4-(glucosoxy)benzyl]-2-isobutyl malate, tanshinone I, 6-gingerol, β,β ´ The relative correction factor for dimethacryloyl argentine was used to calculate the content of each component.

[0018] Further preferably, in the method for determining the content of five components in Baiyu Ointment based on a single-measurement-multiple-evaluation method according to the present invention, the determination and calculation of tanshinone IIA, 1,4-bis[4-(glucosoxy)benzyl]-2-isobutyl malate, tanshinone I, 6-gingerol, and β,β ´ The relative retention times of 1,2-dimethacryloyl argentine were 0.33, 0.44, 0.83, and 1.53, respectively, which were within ±10% of the specified values.

[0019] Further preferably, in the method for determining the content of five components in Baiyu Ointment based on a single-measurement-multiple-evaluation method according to the present invention, the determination and calculation of tanshinone IIA, 1,4-bis[4-(glucosoxy)benzyl]-2-isobutyl malate, tanshinone I, 6-gingerol, and β,β ´ The relative correction factors for α-dimethylacryloyl argentine were 2.65, 1.37, 0.68, and 1.12, respectively.

[0020] The beneficial effects of this invention are:

[0021] 1. This invention establishes for the first time a method for determining the content of five components in Baiyu Ointment based on a single-measurement, multi-evaluation approach. Only the content of tanshinone IIA needs to be measured to determine the content of 1,4-bis[4-(glucosoxy)benzyl]-2-isobutyl malate, tanshinone I, 6-gingerol, and β,β-glucanol. ´ The simultaneous analysis of dimethacryloyl argentine content reflects the overall product quality through simultaneous quality control of the above five components. This invention overcomes the limitations and one-sidedness of the previous quality control methods for Baiyu Ointment, and is of great significance for further improving the quality control level of Baiyu Ointment, ensuring clinical efficacy, and enhancing the relevant evaluation system.

[0022] 2. This invention investigated the factors affecting the extraction of five active ingredients in Baiyu Ointment, and finally obtained the optimal extraction scheme for the five active ingredients in Baiyu Ointment as follows: the extraction solvent is 75% ethanol solution; the ultrasonic extraction time is 30 min; the number of chloroform extractions is 3; and the amount of chloroform used is 30 mL each time.

[0023] 3. Methodological verification experiments have confirmed that the method for determining the content of five components in Baiyu Ointment based on the one-test-multiple-evaluation method established in this invention has the advantages of high accuracy, good repeatability, strong stability and good durability. It greatly improves the detection efficiency while enhancing the overall quality control standards of Baiyu Ointment, and saves the detection cost of Baiyu Ointment quality control. Attached Figure Description

[0024] Figure 1 To examine the chromatogram of the mobile phase (elution was performed according to gradient elution program 1);

[0025] Figure 2 To examine the chromatogram of the mobile phase (elution was performed according to gradient elution program 2);

[0026] Figure 3 To examine the chromatogram of the mobile phase (elution was performed according to gradient elution program 3);

[0027] Figure 4 Chromatograms were examined to determine the extraction method (active ingredient extraction was performed according to Method 1).

[0028] Figure 5 Chromatograms were examined to determine the extraction method (active ingredient extraction was performed according to Method 2).

[0029] Figure 6 Chromatograms were examined to determine the extraction method (active ingredient extraction was performed according to Method 3).

[0030] Figure 7 Chromatograms were examined to determine the extraction method (active ingredient extraction was performed according to Method 4).

[0031] Figure 8 The chromatograms of the reference standards for each component are shown below (in the figure: 1 is 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutylmalate; 2 is tanshinone I; 3 is 6-gingerol; 4 is tanshinone IIA; 5 is β,β ´ (-Dimethylacryloyl acrylin)

[0032] Figure 9 The figures show the chromatograms of the sample and the mixed reference standard (in the figure: A is the chromatogram of the sample; B is the chromatogram of the mixed reference standard). Detailed Implementation

[0033] The technical solution of the present invention will be described in detail below with reference to specific embodiments. These embodiments are for explanation and illustration only and do not constitute a limitation on the technical solution of the present invention.

[0034] Example 1

[0035] The method for determining the content of five components in Baiyu Ointment based on the one-test-multiple-evaluation approach is as follows:

[0036] (1) Preparation of the test solution

[0037] Accurately weigh 3 g of white jade ointment, add 25 mL of 75% ethanol solution, and weigh again. Sonicate for 30 min, cool, replenish the lost weight with the ethanol solution, shake well, and filter. Extract the filtrate three times with 30 mL of chloroform each time. Combine the extracts, evaporate to near dryness, dissolve in an appropriate amount of methanol, transfer to a 5 mL volumetric flask, make up to volume, shake well, and filter through a 0.45 μm microporous membrane to obtain the final product.

[0038] (2) Preparation of reference solution

[0039] Take an appropriate amount of tanshinone IIA reference standard, accurately weigh it and place it in a brown volumetric flask, add methanol to prepare a reference standard solution containing 0.02 mg per 1 mL.

[0040] (3) Chromatographic conditions and system suitability test

[0041] The chromatographic column was packed with octadecylsilane-bonded silica gel; the column temperature was 30℃; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution containing 0.1% sodium dodecyl sulfonate was used as mobile phase B for gradient elution; the mobile phase flow rate was 1.0 mL / min; the injection volume was 10 μL; the detection wavelength was 270 nm; the theoretical plate number, calculated based on the tanshinone IIA peak, should not be less than 4000.

[0042] The gradient elution procedure is as follows:

[0043] .

[0044] (4) Measurement and Calculation

[0045] The test solution and reference solution were injected into a high-performance liquid chromatograph for determination. The relative correction factors were 2.65 for tanshinone IIA with 1,4-bis[4-(glucosoxy)benzyl]-2-isobutylmalate, 1.37 for tanshinone I, 0.68 for 6-gingerol, and β,β... ´ The relative correction factor for dimethacryloyl argentine was 1.12, and the content of each component was calculated separately.

[0046] Example 2

[0047] The method for determining the content of five components in Baiyu Ointment based on the one-test-multiple-evaluation approach is as follows:

[0048] (1) Preparation of the test solution

[0049] Accurately weigh 1.8 g of white jade ointment, add 15 mL of 75% ethanol solution, and weigh again. Sonicate for 30 min, cool, replenish the lost weight with the above ethanol solution, shake well, and filter. Extract the filtrate three times with 30 mL of chloroform each time. Combine the extracts, evaporate to near dryness, dissolve in an appropriate amount of methanol, transfer to a 5 mL volumetric flask, make up to volume, shake well, and filter through a 0.45 μm microporous membrane to obtain the final product.

[0050] (2) Preparation of reference solution

[0051] Take an appropriate amount of tanshinone IIA reference standard, accurately weigh it and place it in a brown volumetric flask, add methanol to prepare a reference standard solution containing 0.02 mg per 1 mL.

[0052] (3) Chromatographic conditions and system suitability test

[0053] The chromatographic column was packed with octadecylsilane-bonded silica gel; the column temperature was 25℃; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution containing 0.1% sodium dodecyl sulfonate was used as mobile phase B for gradient elution; the mobile phase flow rate was 0.9 mL / min; the injection volume was 10 μL; the detection wavelength was 270 nm; the theoretical plate number, calculated based on the tanshinone IIA peak, should not be less than 4000.

[0054] The gradient elution procedure is as follows:

[0055] .

[0056] (4) Measurement and Calculation

[0057] The test solution and reference solution were injected into a high-performance liquid chromatograph for determination. The relative correction factors were 2.65 for tanshinone IIA with 1,4-bis[4-(glucosoxy)benzyl]-2-isobutylmalate, 1.37 for tanshinone I, 0.68 for 6-gingerol, and β,β... ´ The relative correction factor for dimethacryloyl argentine was 1.12, and the content of each component was calculated separately.

[0058] Example 3

[0059] The method for determining the content of five components in Baiyu Ointment based on the one-test-multiple-evaluation approach is as follows:

[0060] (1) Preparation of the test solution

[0061] Accurately weigh 2.4 g of white jade ointment, add 20 mL of 75% ethanol solution, and weigh again. Sonicate for 30 min, cool, replenish the lost weight with the above ethanol solution, shake well, and filter. Extract the filtrate three times with 30 mL of chloroform each time. Combine the extracts, evaporate to near dryness, dissolve in an appropriate amount of methanol, transfer to a 5 mL volumetric flask, make up to volume, shake well, and filter through a 0.45 μm microporous membrane to obtain the final product.

[0062] (2) Preparation of reference solution

[0063] Take an appropriate amount of tanshinone IIA reference standard, accurately weigh it and place it in a brown volumetric flask, add methanol to prepare a reference standard solution containing 0.02 mg per 1 mL.

[0064] (3) Chromatographic conditions and system suitability test

[0065] The chromatographic column was packed with octadecylsilane-bonded silica gel; the column temperature was 35℃; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution containing 0.1% sodium dodecyl sulfonate was used as mobile phase B for gradient elution; the mobile phase flow rate was 1.1 mL / min; the injection volume was 10 μL; the detection wavelength was 270 nm; the theoretical plate number, calculated based on the tanshinone IIA peak, should not be less than 4000.

[0066] The gradient elution procedure is as follows:

[0067] .

[0068] (4) Measurement and Calculation

[0069] The test solution and reference solution were injected into a high-performance liquid chromatograph for determination. The relative correction factors were 2.65 for tanshinone IIA with 1,4-bis[4-(glucosoxy)benzyl]-2-isobutylmalate, 1.37 for tanshinone I, 0.68 for 6-gingerol, and β,β... ´ The relative correction factor for dimethacryloyl argentine was 1.12, and the content of each component was calculated separately.

[0070] Example 4

[0071] The method for determining the content of five components in Baiyu Ointment based on the one-test-multiple-evaluation approach is as follows:

[0072] (1) Preparation of the test solution

[0073] Accurately weigh 4.2 g of white jade ointment, accurately add 35 mL of 75% ethanol solution, and weigh again; sonicate for 30 min, cool, replenish the lost weight with the above ethanol solution, shake well, and filter; extract the filtrate three times with 30 mL of chloroform each time; combine the extracts, evaporate to near dryness, dissolve in an appropriate amount of methanol, transfer to a 5 mL volumetric flask, make up to volume, shake well, and filter through a 0.45 μm microporous membrane to obtain the final product.

[0074] (2) Preparation of reference solution

[0075] Take an appropriate amount of tanshinone IIA reference standard, accurately weigh it and place it in a brown volumetric flask, add methanol to prepare a reference standard solution containing 0.02 mg per 1 mL.

[0076] (3) Chromatographic conditions and system suitability test

[0077] The chromatographic column was packed with octadecylsilane-bonded silica gel; the column temperature was 30℃; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution containing 0.1% sodium dodecyl sulfonate was used as mobile phase B for gradient elution; the mobile phase flow rate was 1.0 mL / min; the injection volume was 10 μL; the detection wavelength was 270 nm; the theoretical plate number, calculated based on the tanshinone IIA peak, should not be less than 4000.

[0078] The gradient elution procedure is as follows:

[0079] .

[0080] (4) Measurement and Calculation

[0081] The test solution and reference solution were injected into a high-performance liquid chromatograph for determination. The relative correction factors were 2.65 for tanshinone IIA with 1,4-bis[4-(glucosoxy)benzyl]-2-isobutylmalate, 1.37 for tanshinone I, 0.68 for 6-gingerol, and β,β... ´ The relative correction factor for dimethacryloyl argentine was 1.12, and the content of each component was calculated separately.

[0082] Example 5

[0083] The method for determining the content of five components in Baiyu Ointment based on the one-test-multiple-evaluation approach is as follows:

[0084] (1) Preparation of the test solution

[0085] Take 3 g of white jade ointment, accurately weigh it, add 25 mL of 60% ethanol solution, and weigh it again; sonicate for 60 min, cool it, replenish the lost weight with the above ethanol solution, shake well, and filter it; extract the filtrate with 40 mL of chloroform by shaking; evaporate the extract to near dryness, add an appropriate amount of methanol to dissolve it and transfer it to a 5 mL volumetric flask, make up to volume, shake well, and filter through a 0.45 μm microporous membrane to obtain the final product.

[0086] (2) Preparation of reference solution

[0087] Take an appropriate amount of tanshinone IIA reference standard, accurately weigh it and place it in a brown volumetric flask, add methanol to prepare a reference standard solution containing 0.02 mg per 1 mL.

[0088] (3) Chromatographic conditions and system suitability test

[0089] The chromatographic column was packed with octadecylsilane-bonded silica gel; the column temperature was 30℃; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution containing 0.1% sodium dodecyl sulfonate was used as mobile phase B for gradient elution; the mobile phase flow rate was 1.0 mL / min; the injection volume was 10 μL; the detection wavelength was 270 nm; the theoretical plate number, calculated based on the tanshinone IIA peak, should not be less than 4000.

[0090] The gradient elution procedure is as follows:

[0091] .

[0092] (4) Measurement and Calculation

[0093] The test solution and reference solution were injected into a high-performance liquid chromatograph for determination. The relative correction factors were 2.65 for tanshinone IIA with 1,4-bis[4-(glucosoxy)benzyl]-2-isobutylmalate, 1.37 for tanshinone I, 0.68 for 6-gingerol, and β,β... ´ The relative correction factor for dimethacryloyl argentine was 1.12, and the content of each component was calculated separately.

[0094] Example 6

[0095] The method for determining the content of five components in Baiyu Ointment based on the one-test-multiple-evaluation approach is as follows:

[0096] (1) Preparation of the test solution

[0097] Accurately weigh 3 g of white jade ointment, add 25 mL of 70% methanol solution, and weigh again. Sonicate for 45 min, cool, replenish the lost weight with the methanol solution, shake well, and filter. Extract the filtrate three times with 30 mL of chloroform each time. Combine the extracts, evaporate to near dryness, dissolve in an appropriate amount of methanol, transfer to a 5 mL volumetric flask, make up to volume, shake well, and filter through a 0.45 μm microporous membrane to obtain the final product.

[0098] (2) Preparation of reference solution

[0099] Take an appropriate amount of tanshinone IIA reference standard, accurately weigh it and place it in a brown volumetric flask, add methanol to prepare a reference standard solution containing 0.02 mg per 1 mL.

[0100] (3) Chromatographic conditions and system suitability test

[0101] The chromatographic column was packed with octadecylsilane-bonded silica gel; the column temperature was 30℃; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution containing 0.1% sodium dodecyl sulfonate was used as mobile phase B for gradient elution; the mobile phase flow rate was 1.0 mL / min; the injection volume was 10 μL; the detection wavelength was 270 nm; the theoretical plate number, calculated based on the tanshinone IIA peak, should not be less than 4000.

[0102] The gradient elution procedure is as follows:

[0103] .

[0104] (4) Measurement and Calculation

[0105] The test solution and reference solution were injected into a high-performance liquid chromatograph for determination. The relative correction factors were 2.65 for tanshinone IIA with 1,4-bis[4-(glucosoxy)benzyl]-2-isobutylmalate, 1.37 for tanshinone I, 0.68 for 6-gingerol, and β,β... ´ The relative correction factor for dimethacryloyl argentine was 1.12, and the content of each component was calculated separately.

[0106] Example 7

[0107] The method for determining the content of five components in Baiyu Ointment based on the one-test-multiple-evaluation approach is as follows:

[0108] (1) Preparation of the test solution

[0109] Take 3 g of white jade ointment, accurately weigh it, add 25 mL of methanol accurately, and weigh it again; sonicate for 30 min, cool it, replenish the lost weight with methanol, shake well, and filter it; extract the filtrate with chloroform 4 times, 20 mL each time; combine the extracts, evaporate to near dryness, add an appropriate amount of methanol to dissolve it and transfer it to a 5 mL volumetric flask, make up to volume, shake well, and filter through a 0.45 μm microporous membrane to obtain the final product.

[0110] (2) Preparation of reference solution

[0111] Take an appropriate amount of tanshinone IIA reference standard, accurately weigh it and place it in a brown volumetric flask, add methanol to prepare a reference standard solution containing 0.02 mg per 1 mL.

[0112] (3) Chromatographic conditions and system suitability test

[0113] The chromatographic column was packed with octadecylsilane-bonded silica gel; the column temperature was 30℃; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution containing 0.1% sodium dodecyl sulfonate was used as mobile phase B for gradient elution; the mobile phase flow rate was 1.0 mL / min; the injection volume was 10 μL; the detection wavelength was 270 nm; the theoretical plate number, calculated based on the tanshinone IIA peak, should not be less than 4000.

[0114] The gradient elution procedure is as follows:

[0115] .

[0116] (4) Measurement and Calculation

[0117] The test solution and reference solution were injected into a high-performance liquid chromatograph for determination. The relative correction factors were 2.65 for tanshinone IIA with 1,4-bis[4-(glucosoxy)benzyl]-2-isobutylmalate, 1.37 for tanshinone I, 0.68 for 6-gingerol, and β,β... ´ The relative correction factor for dimethacryloyl argentine was 1.12, and the content of each component was calculated separately.

[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 apparatus

[0120] Electronic balance (BSM-220.4, Shanghai Zhuojing Electronic Technology Co., Ltd.); Ultrasonic cleaner (MH-0403, Dong'an Meihong Metal Processing Plant); Electronic balance (AB-135-S, Mettler Toledo); High-performance liquid chromatograph (LC-2010AHT, Shimadzu, Japan). Chromatographic column: Welchrom-C 18 (4.6×250 mm, 5 μm); Agilent 5TC-C 18 (4.6×250 mm, 5μm); Shim-pack GIST C 18 (4.6×250 mm, 5 μm).

[0121] 2. Materials and Reagents

[0122] Tanshinone IIA reference standard (batch number: 110766-202323), 1,4-bis[4-(glucosoxy)benzyl]-2-isobutylmalate reference standard (batch number: 112061-202102), Tanshinone I reference standard (batch number: 110867-201607), 6-gingerol reference standard (batch number: 111833-202408), β,β ，- Dimethacryloyl acrylin reference standard (batch number: 111689-201805) were all obtained from the China National Institutes for Food and Drug Control; water was Wahaha purified water; methanol (chromatographic grade, Tiandi Inc., USA); acetonitrile (chromatographic grade, Tiandi Inc., USA); phosphoric acid (analytical grade, Chongqing Wansheng Chuandong Chemical Co., Ltd.); sodium dodecyl sulfonate (chromatographic grade, Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0123] 3. Examination of testing methods

[0124] (1) Investigation of the mobile phase

[0125] Accurately weigh 3 g of white jade ointment, accurately add 25 mL of 60% ethanol solution, and weigh again; sonicate for 30 min, cool, replenish the lost weight with 60% ethanol solution, shake well, and filter; extract the filtrate three times with 30 mL of chloroform each time; combine the extracts, evaporate to near dryness, dissolve in an appropriate amount of methanol, transfer to a 5 mL volumetric flask, make up to volume, shake well, filter through a 0.45 μm microporous membrane to obtain the test solution, and perform gradient elution according to the elution programs in Tables 1-3.

[0126] The HPLC chromatographic conditions and system suitability test specifically includes:

[0127] Column: Shim-pack GIST C 18 (4.6×250 mm, 5 μm); column temperature: 30℃; mobile phase flow rate: 1.0 mL / min; injection volume: 10 μL; detection wavelength: 270 nm; the theoretical plate number calculated based on the tanshinone IIA peak should not be less than 4000.

[0128] ;

[0129] ;

[0130] ;

[0131] The mobile phase was examined using the chromatogram as shown below. Figures 1-3 As shown, the results indicate that gradient elution using the elution program in Table 1 did not achieve the required resolution for each component, and the peak shapes were poor. Gradient elution using the elution program in Table 2 also failed to achieve the required resolution. However, gradient elution using the elution program in Table 3 resulted in good peak shapes and met the required resolution. Therefore, acetonitrile was selected as mobile phase A, and 0.1% phosphoric acid solution (containing 0.1% sodium dodecyl sulfate) was selected as mobile phase B, with the elution program in Table 3 used for subsequent studies.

[0132] (2) Examination of extraction methods

[0133] Method 1: Take 3 g of white jade ointment, weigh it accurately, add 50 mL of 70% ethanol accurately, weigh it again; sonicate for 30 min, cool it, make up the lost weight with the above ethanol solution, shake well, filter it, and you will get the product.

[0134] Method 2: Take 3 g of white jade ointment, weigh it accurately, add 50 mL of 70% ethanol accurately, weigh it again; heat under reflux for 30 min, cool it, make up the lost weight with the above ethanol solution, shake well, filter it, and you will get the product.

[0135] Method 3: Accurately weigh 3 g of white jade ointment, accurately add 25 mL of 70% ethanol, and weigh again; sonicate for 30 min, cool, replenish the lost weight with the above ethanol solution, shake well, and filter; accurately measure 10 mL of the filtrate, pass it through a D101 macroporous adsorption resin (inner diameter 1.5 cm, column height 13 cm), and elute successively with 50 mL of water and 35 mL of 20% ethanol, discarding the eluent; then elute with 15 mL of 40% ethanol, 15 mL of 60% ethanol, and 50 mL of 75% ethanol; collect the above eluent and concentrate it, make up to 10 mL with 75% ethanol, shake well, and filter to obtain the final product.

[0136] Method 4: Take 3 g of white jade ointment, accurately weigh it, add 25 mL of 60% ethanol accurately, and weigh it again; sonicate for 30 min, cool it, replenish the lost weight with the above ethanol solution, shake well, and filter it; extract the filtrate three times with 30 mL of triple-filtered methane; combine the extracts, evaporate to near dryness, add an appropriate amount of methanol to dissolve it and transfer it to a 5 mL volumetric flask, make up to volume, shake well, and filter to obtain the final product.

[0137] The test solutions prepared by the above four methods were analyzed by HPLC using the chromatographic conditions and optimal gradient elution program under the section "(1) Investigation of Mobile Phase". The results are as follows: Figure 4-7 As shown in the figure. The results indicate that: when preparing the test solution using Method 1 or Method 2, the resulting chromatograms showed significant interference from impurities, and the peak resolution did not meet the requirements; when preparing the test solution using Method 3, the resulting chromatograms also showed significant interference from impurities, and the peak resolution did not meet the requirements, possibly due to adsorption and repeated elution leading to component loss; when preparing the test solution using Method 4, the resulting chromatograms showed no interference from impurities, and the peak shapes were good, with the resolution meeting the requirements. Therefore, Method 4 was selected as the extraction method for the effective components in Baiyu Ointment for subsequent studies. The chromatograms of each component reference standard are shown in the figure. Figure 8 As shown in the figure (1 is 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutylmalate; 2 is tanshinone I; 3 is 6-gingerol; 4 is tanshinone IIA; 5 is β,β ´ -Dimethacryloylarcinin), chromatograms of the sample and mixed reference standard under method 4 are shown below. Figure 9 As shown in the figure (A is the chromatogram of the sample; B is the chromatogram of the mixed reference standard).

[0138] (3) Investigation of extraction solvent

[0139] Take 3 g of white jade ointment and accurately weigh 4 portions. Use methanol, 70% methanol, 60% ethanol and 75% ethanol as extraction solvents respectively. Prepare the test solution according to method 4 under “(2) Investigation of extraction method”. Perform HPLC analysis according to the chromatographic conditions and optimal gradient elution program under “(1) Investigation of mobile phase”. Determine and calculate the content of each component. The results are shown in Table 4.

[0140] ;

[0141] The results show that the content of each target component is relatively high and the extraction is more complete when 75% ethanol is used for extraction. Therefore, 75% ethanol solution was selected as the extraction solvent for subsequent studies.

[0142] (4) Examination of extraction time

[0143] Take 3 g of white jade ointment, accurately weigh 3 portions, accurately add 25 mL of 75% ethanol, weigh, and sonicate for 30 min, 45 min, and 60 min respectively. Prepare the test solution according to method 4 under “(2) Extraction method investigation”, and perform HPLC analysis according to the chromatographic conditions and optimal gradient elution program under “(1) Mobile phase investigation”. Determine and calculate the content of each component. The results are shown in Table 5.

[0144] ;

[0145] The results show that the ultrasonic time has little effect on the content of each component to be tested. From the perspective of energy conservation and emission reduction, an ultrasonic time of 30 min was selected for subsequent research.

[0146] (5) Examination of extraction times

[0147] Take 3 g of white jade ointment and accurately weigh 4 portions. Use 75% ethanol as the extraction solvent and prepare the test solution according to method 4 under “(2) Investigation of extraction method”. Extract with 30 mL of chloroform once, twice, three times and four times respectively. Perform HPLC analysis according to the chromatographic conditions and optimal gradient elution program under “(1) Investigation of mobile phase”. Determine and calculate the content of each component. The results are shown in Table 6.

[0148] ;

[0149] The results show that when the extraction was performed 3 or 4 times, the content of each component was not significantly different, and all of them were higher than those when the extraction was performed 1 or 2 times. Therefore, 3 extractions were chosen for subsequent studies.

[0150] (6) Investigation on the dosage of extractant

[0151] Take 3 g of white jade ointment and accurately weigh 3 portions. Use 75% ethanol as the extraction solvent and prepare the test solution according to the optimal effective component extraction method obtained under "(2) Extraction Method Investigation". Extract with 20 mL, 30 mL and 40 mL of chloroform three times respectively. Perform HPLC analysis according to the chromatographic conditions and optimal gradient elution program under "(1) Mobile Phase Investigation". Determine and calculate the content of each component. The results are shown in Table 7.

[0152] ;

[0153] The results show that the content of each component is not significantly different when the amount of chloroform used as the extractant is 30 mL and 40 mL. Therefore, 30 mL of chloroform was chosen for extraction.

[0154] (7) Selection of detection wavelength

[0155] A full-wavelength scan was performed using an ultraviolet detector, and chromatographic information was compared at wavelengths of 220 nm, 265 nm, 270 nm, 280 nm, and 330 nm. The results showed that the peak shape and resolution were better at wavelength 270 nm than at other wavelengths, and the baseline was stable. Therefore, 270 nm was selected as the detection wavelength for this study.

[0156] 4. Methodological Validation

[0157] (1) Examination of linear relationships

[0158] Accurately pipette 2 μL, 6 μL, 10 μL, 14 μL, 18 μL, and 22 μL of the mixed reference solution, respectively, and inject them according to the optimal chromatographic separation conditions of this study. Linear regression was performed with concentration as the abscissa (x) and peak area as the ordinate (y). The results are shown in Table 8, indicating a good linear relationship.

[0159] ;

[0160] (2) Precision test

[0161] Take 3 g of white jade ointment, accurately weigh it, prepare the test solution according to the optimal extraction method of this study, inject it into the high performance liquid chromatograph for HPLC analysis, inject it continuously for 6 injections, determine and calculate the content of each component and calculate the RSD, and examine the instrument precision. The results are shown in Table 9.

[0162] ;

[0163] The results show that the RSD of each analyte is within the range of 0.06%-0.81%, indicating that the instrument has good precision.

[0164] (3) Repeated examination

[0165] Take 3 g of white jade ointment and accurately weigh 6 portions. Prepare the test solution according to the optimal extraction method of this study. Inject the solution into a high performance liquid chromatograph for HPLC analysis, determine and calculate the content of each component and calculate the RSD. The results are shown in Table 10.

[0166] ;

[0167] The results show that the RSD of the component content determination results in the six repeatability test samples are within the range of 0.09%-1.19%, indicating good repeatability.

[0168] (4) Stability test

[0169] 3 g of white jade ointment was accurately weighed and the test solution was prepared according to the optimal extraction method of this study. The solution was injected into the high performance liquid chromatograph at 0, 2, 4, 6, 8, 12 and 24 h for HPLC analysis. The content of each component was determined and calculated and the RSD was calculated. The results are shown in Table 11.

[0170] ;

[0171] The results show that tanshinone IIA, 1,4-bis[4-(glucosoxy)benzyl]-2-isobutylmalate, tanshinone I, 6-gingerol, and β,β-glucanol are present. ， The RSD values ​​of 1,2-dimethylacryloyl argentine were 0.28%, 0.08%, 0.70%, 0.31%, and 1.30%, respectively, indicating that the test solution had good stability within 24 hours.

[0172] (5) Spike recovery test

[0173] Six samples, each weighing 1.5 g, were accurately weighed. Reference standards were added to each sample at a 1:1 ratio according to the content of each component in the sample to prepare test solutions. The solutions were then injected into a high-performance liquid chromatograph for HPLC analysis. The average recovery rate and RSD value of each component were calculated. The results are shown in Tables 12-16.

[0174] ;

[0175] ;

[0176] ;

[0177] ;

[0178] ;

[0179] The results show that tanshinone IIA, 1,4-bis[4-(glucosoxy)benzyl]-2-isobutylmalate, tanshinone I, 6-gingerol, and β,β-glucanol are present. ， The average recoveries of α-dimethacryloylcarpine were 99.77%, 99.28%, 99.76%, 98.93%, and 99.07%, respectively, with RSD values ​​of 0.82%, 1.23%, 0.78%, 0.98%, and 0.75%, respectively, indicating that the method has good recovery.

[0180] 5. Relative correction factor

[0181] (1) Calculation of relative correction factor

[0182] Take the mixed reference solution, inject it into the liquid chromatograph, and determine the following: using tanshinone IIA as an internal reference, calculate 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutyl malate, tanshinone I, 6-gingerol, and β,β-glucanol using the following formula. ， The relative correction factors for dimethacryloyl argentine are shown in Table 17.

[0183] ;

[0184] Among them, A s W represents the peak area of ​​the internal reference material. S A represents the concentration (or mass) of the internal reference substance. k W represents the peak area of ​​the analyte. k This represents the concentration (or mass) of the analyte.

[0185] Table 17 Relative Correction Factors

[0186]

[0187] (2) Reproducibility test of relative correction factor

[0188] ① Investigation using different instruments and different chromatographic columns

[0189] The relative correction factor was determined using different instruments and different chromatographic columns. The results are shown in Table 18. The measured relative correction factor RSD values ​​were between 0.44% and 1.38%, indicating that the relative correction factor of each analyte had good reproducibility among different instruments and different chromatographic columns.

[0190] Table 18. Effects of different instruments and columns on the relative correction factors of the analytes.

[0191]

[0192]

[0193] ② Investigation of different flow velocities

[0194] The relative correction factor was measured using different flow rates, and the results are shown in Table 19. The results show that the RSD values ​​of the measured relative correction factors ranged from 0.38% to 2.26%, indicating that the relative correction factors of each analyte exhibited good reproducibility across different flow rates.

[0195] Table 19 Effect of different flow rates on the relative correction factor of the analyte

[0196]

[0197] ③ Investigation at different column temperatures

[0198] The relative correction factor was measured at different column temperatures, and the results are shown in Table 20. The measured relative correction factor RSD values ​​were between 0.38% and 1.52%, indicating that the relative correction factor of each analyte had good reproducibility at different column temperatures.

[0199] Table 20 Effect of different column temperatures on the relative correction factor of the analyte

[0200]

[0201] 6. Peak localization of the analyte

[0202] The relative retention values ​​(r) of each analyte and the internal reference tanshinone IIA were calculated, and their reproducibility across different instruments and columns was examined. The results are shown in Table 21. The relative retention time fluctuations were small. Finally, the average value of the relative retention time of each analyte in different instruments and columns was selected as the peak localization basis.

[0203] Table 21 Relative retention times of analytes under different instruments and columns

[0204]

[0205] 7. Comparison of results from the one-measure-multiple-evaluations method and the external standard method

[0206] Three batches of Baiyu ointment were taken and test solutions were prepared according to the optimal extraction method of this study. The contents of tanshinone IIA, 1,4-bis[4-(glucosoxy)benzyl]-2-isobutyl malate, tanshinone I, 6-gingerol, and β,β'-dimethylacryloylcarboxine were determined and calculated using both the quality-assay-multiple-analytes (QASM) method and the external standard method (ESM) method. The results are shown in Table 22. The results show that the relative error between the two groups of contents is <3%, indicating that there is no significant difference in the contents measured by the two methods. This demonstrates that the QASM method established in this study has good accuracy and can be used for the content determination of Baiyu ointment.

[0207] Table 22 Results of determination of five components using the single-test-multiple-evaluation method and external standard method.

[0208]

[0209] 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 method for determining the content of five components in Baiyu Ointment based on a single-test, multi-evaluation method, characterized in that, The method involves using tanshinone IIA as an internal standard to establish the formulations of 1,4-bis[4-(glucosoxy)benzyl]-2-isobutyl malate, tanshinone I, 6-gingerol, and β,β-glucanol. ´ The relative correction factor for dimethacryloyl argentine was determined, and the content of each component was calculated using the relative correction factor. The specific steps included: preparation of the test solution; preparation of the reference solution; chromatographic conditions and system suitability test; determination and calculation. The preparation of the test solution is as follows: 1.8-4.2 g of white jade ointment is accurately weighed, and 15-35 mL of 70%-100% methanol solution or 60%-75% ethanol solution is accurately added. The solution is then weighed again. The solution is sonicated for 30-60 min, cooled, and the lost weight is replenished with the above-mentioned methanol or ethanol solution. The solution is shaken well and filtered. The filtrate is extracted with chloroform 1-4 times, 20-40 mL each time. The extracts are combined, evaporated to near dryness, dissolved in an appropriate amount of methanol, and transferred to a 5 mL volumetric flask. The solution is then diluted to volume, shaken well, and filtered to obtain the final product. The chromatographic conditions and system suitability test are as follows: the chromatographic column is packed with octadecylsilane-bonded silica gel; the column temperature is 25-35℃; acetonitrile is used as mobile phase A, and 0.1% phosphoric acid solution containing 0.1% sodium dodecyl sulfonate is used as mobile phase B, with gradient elution; the mobile phase flow rate is 0.9-1.1 mL / min; the injection volume is 10 μL; the detection wavelength is 270 nm; the theoretical plate number, calculated based on the tanshinone IIA peak, should not be less than 4000; the specific gradient elution program is as follows: 。 2. The method for determining the content of five components in Baiyu Ointment based on the one-test-multiple-evaluation method according to claim 1, characterized in that, The preparation of the test solution is as follows: Take 2.4-4.2 g of white jade ointment, accurately weigh it, add 20-35 mL of 60%-75% ethanol solution, and weigh it; sonicate for 30-45 min, cool it, replenish the lost weight with the above ethanol solution, shake well, and filter it; extract the filtrate with chloroform 3-4 times by shaking, 30-40 mL each time; combine the extracts, evaporate to near dryness, add an appropriate amount of methanol to dissolve it and transfer it to a 5 mL volumetric flask, make up to volume, shake well, and filter through a 0.45 μm microporous membrane to obtain the test solution.

3. The method for determining the content of five components in Baiyu Ointment based on the one-test-multiple-evaluation method according to claim 2, characterized in that, The preparation of the test solution is as follows: Take 3 g of white jade ointment, accurately weigh it, add 25 mL of 75% ethanol solution, and weigh it; sonicate for 30 min, cool it, replenish the lost weight with the above ethanol solution, shake well, and filter it; extract the filtrate three times with 30 mL of chloroform each time; combine the extracts, evaporate to near dryness, add an appropriate amount of methanol to dissolve it and transfer it to a 5 mL volumetric flask, make up to volume, shake well, and filter through a 0.45 μm microporous membrane to obtain the test solution.

4. The method for determining the content of five components in Baiyu Ointment based on the one-test-multiple-evaluation method according to claim 1, characterized in that, The preparation of the reference solution is as follows: Take an appropriate amount of tanshinone IIA reference standard, accurately weigh it and place it in a brown volumetric flask, add methanol to prepare a reference solution containing 0.02 mg per 1 mL.

5. The method for determining the content of five components in Baiyu Ointment based on the one-test-multiple-evaluation method according to claim 1, characterized in that, The column temperature in the chromatographic conditions and system suitability test was 30°C, and the mobile phase flow rate was 1.0 mL / min.

6. The method for determining the content of five components in Baiyu Ointment based on the one-test-multiple-evaluation method according to claim 1, characterized in that, The determination and calculation are specifically as follows: The test solution and the reference solution are injected into a high-performance liquid chromatograph for determination, based on the relationship between tanshinone IIA and 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutyl malate, tanshinone I, 6-gingerol, β,β ´ The relative correction factor for dimethacryloyl argentine was used to calculate the content of each component.

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