A method for simultaneously determining contents of various saponin components in Huoxue Jiegu Zhitong Plaster by HPLC

Abstract

The application discloses a method for simultaneously determining the content of multiple saponin components in Huoxue Jiegushangtong Plaster by HPLC, relates to the technical field of traditional Chinese medicines, and comprises notoginsenoside R1, ginsenoside Rg1, Asiasarum saponin VI, ginsenoside Rb1 and ginsenoside Rd. The method is high in specificity, precision, repeatability and recovery rate, can be used for simultaneously determining the content of the five saponin components in the Huoxue Jiegushangtong Plaster, effectively solves the technical problem of determining the content of the big compound black plaster dosage form, and can be used for the product quality control of the Huoxue Jiegushangtong Plaster.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and in particular to a method for simultaneously determining the content of multiple saponin components in a blood-activating, bone-setting, and pain-relieving plaster using HPLC. Background Technology

[0002] Black plaster is one of the oldest dosage forms in traditional Chinese medicine. It has a large drug loading capacity, long-lasting effect, and acts directly on the affected area. Its thousands of years of development have proven its important role in treating human diseases, making it a valuable part of my country's cultural heritage that needs protection and promotion. For a long time, due to the often multiple medicinal ingredients and special processing methods of black plaster, quality control has largely remained at the experiential stage. For example, the ideal quality is described as "clear as a mirror, black as lacquer," and there is the view that "the effect of black plaster lies in stirring, and the effect of bright plaster lies in fanning." The General Rules for Preparations in the Chinese Pharmacopoeia only specifies the properties, softening point, and weight variation tests for plasters. The quality standards for varieties such as "Yanghe Jiening Plaster," "Nuanqi Plaster," and "Goupi Plaster" lack other quality control indicators. Previously, the Pharmacopoeia only specified the gas chromatographic identification of camphor for "Badu Plaster," and only specified the thin-layer chromatography identification of cinnamon and angelica and the determination of musk content for "Wanling Wuxiang Plaster." However, these ingredients are separately packaged powders and are not mixed into the plaster. Compared to most traditional Chinese medicine preparations, the quality control of black plaster has always been a challenge. This is due to the numerous medicinal ingredients and unique processing methods involved, making it difficult to separate the components and causing numerous interferences. The highly viscous and absorbent matrix further complicates sample processing during analysis, posing significant difficulties for the identification of black plaster, especially in determining the content of its active ingredients. In recent years, although gas chromatography and liquid chromatography have been used for the content determination of black plaster, these methods have largely remained at the research stage for individual products and have not been comprehensively and specifically applied to actual product testing. Further practical verification is needed.

[0003] The Blood-Activating and Bone-Setting Pain-Relieving Plaster is made from 22 kinds of traditional Chinese medicine, including Panax notoginseng, Dipsacus asper, Frankincense, Myrrh, Angelica sinensis, Rehmannia glutinosa, Rheum palmatum, Angelica pubescens, Notopterygium incisum, Forsythia suspensa, Angelica dahurica, and Paeonia lactiflora. It comes in three sizes: large, medium, and small. The drug approval numbers are Yu Yao Zhi Zi Z20120261 (Luoyang), Yu Yao Zhi Zi Z20120262 (Luoyang), and Yu Yao Zhi Zi Z20120263 (Luoyang). It has the effects of activating blood circulation and removing blood stasis, reducing swelling and relieving pain, promoting bone and tendon healing, and dispelling wind and dampness. It is used to treat various pain syndromes such as traumatic fractures, soft tissue injuries, strain-induced lower back and leg pain, and neck and shoulder pain.

[0004] Huoxue Jiegu Zhitong Plaster is a traditional Chinese medicine external black plaster preparation, consisting of two parts: a matrix and a bone-setting powder. The basic process is: 9 traditional Chinese medicines including Angelica sinensis and Rehmannia glutinosa are subjected to frying in oil, refining the oil, adding the powder, and removing the toxic effects of fire to form a plaster matrix. After the matrix is heated, a bone-setting powder composed of 13 traditional Chinese medicines including Panax notoginseng and Dipsacus asperoides is added, and the mixture is spread and coated to form the final product. In addition to the properties, softening point and weight difference specified in the General Rules for Preparations of the Chinese Pharmacopoeia, its quality control indicators only include thin-layer identification of two medicines, Panax notoginseng and borneol, in the preparation standard, lacking content determination of active ingredients, which is far from sufficient for controlling product quality. Like other black plasters on the market, it has many medicinal flavors, and the relative content of active ingredients is low. After frying in oil, some active ingredients undergo unknown changes or transformations. The mixture of medicinal ingredients and the plaster matrix causes difficulties in extraction and separation, which all create difficulties in the analysis and determination of active ingredients, making it very difficult to detect the content of active ingredients using conventional methods. Panax notoginseng and Dipsacus asperoides are the main medicines in the formula, and saponin components such as notoginsenoside R1, ginsenoside Rg1, asperosaponin Ⅵ, ginsenoside Rb1, and ginsenoside Rd are their active ingredients. At present, there is no published literature disclosing a method for determining the content of active ingredients in Huoxue Jiegu Zhitong Plaster, nor a method for simultaneously determining the content of multiple saponin components in black plasters using the HPLC method. Contents of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for simultaneously determining the content of multiple saponin components in Huoxue Jiegu Zhitong Plaster by the HPLC method, so as to solve the technical problems in the prior art that the large-compound black plaster preparation has high matrix viscosity, difficult-to-separate components, and many interferences, which lead to difficulty in accurately determining the content of active ingredients and a low product quality control level, and to better control product quality.

[0006] The technical solution adopted by the present invention to solve the above technical problems is as follows: In a first aspect, the present invention provides a method for simultaneously determining the content of multiple saponin components in Huoxue Jiegu Zhitong Plaster by a liquid chromatography method, wherein the multiple saponin components include notoginsenoside R1, ginsenoside Rg1, asperosaponin Ⅵ, ginsenoside Rb1, and ginsenoside Rd; and the method includes the following steps: S1, Chromatographic conditions: Using octadecylsilane-bonded silica gel as the stationary phase, acetonitrile as mobile phase A, and water as mobile phase B, gradient elution was performed according to the following program: 0–10 min, mobile phase A 20%, mobile phase B 80%; 10–40 min, mobile phase A linearly changed from 20% to 30%, mobile phase B linearly changed from 80% to 70%; 40–60 min, mobile phase A linearly changed from 30% to 31%, mobile phase B linearly changed from 70% to 69%; 60–61 min, mobile phase A linearly changed from 31% to 36%, mobile phase B linearly changed from 69% to 64%; 61–70 min, mobile phase A linearly changed from 36% to 42%, mobile phase B linearly changed from 64% to 58%; 70–72 min, mobile phase A linearly changed from 42% to 90%, mobile phase B linearly changed from 58% to 10%; flow rate was 1 ml / min, detection wavelength was 203 nm; S2, Preparation of reference solutions: Accurately weigh the reference standards of Panax notoginseng saponin R1, Ginsenoside Rg1, Dipsacus asperoidin VI, Ginsenoside Rb1 and Ginsenoside Rd respectively, and add methanol to prepare a mixed solution. S3, Preparation of the test solution: Remove the blood-activating and bone-setting pain-relieving plaster, cut it into small pieces, weigh it accurately, and place it in a stoppered conical flask. According to the ratio of sample weight to the amount of chloroform, ethanol and water, 1g:2ml:2ml:5ml, add chloroform, ethanol and water in sequence, shake well, weigh, place on a water bath shaker for 1 hour, take it out, cool it, make up the lost weight with water, filter, wash the filtrate with ether, discard the ether, evaporate the water layer to dryness, add methanol to the residue to make up to volume, shake well, filter, and take the filtrate to obtain the test solution. S4, Determination: Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatograph, and determine the result.

[0007] As a preferred embodiment, in step S2, the concentrations of notoginsenoside R1, ginsenoside Rg1, ginsenoside VI, ginsenoside Rb1, and ginsenoside Rd in the mixed solution are 10 μg / ml, 40 μg / ml, 200 μg / ml, 40 μg / ml, and 10 μg / ml, respectively.

[0008] As a preferred embodiment, in step S3, the sample weight is 10g; the amounts of chloroform, ethanol, and water are 20ml, 20ml, and 50ml, respectively; the temperature of the water bath shaker is 60℃, and the shaking frequency is 150r / min; the temperature at which the water layer is evaporated is 85℃; and the residue is diluted to 5ml with methanol.

[0009] As a preferred embodiment, in step S3, the amount of ether used for washing is 30 ml each time, and the washing is performed twice.

[0010] As a preferred embodiment, in step S4, the injection volumes of the reference solution and the test solution are 10 μl each.

[0011] Secondly, the present invention provides the application of the above method in the quality control of the blood-activating, bone-setting, and pain-relieving plaster product.

[0012] According to the above technical solution, the beneficial effects of the present invention are: 1. This invention employs liquid chromatography to achieve, for the first time, the simultaneous determination of five saponin components—Panax notoginsenoside R1, Ginsenoside Rg1, Dipsacus asperoidin VI, Ginsenoside Rb1, and Ginsenoside Rd—in Huoxue Jiegu Zhitong Gao (a traditional Chinese medicine plaster for promoting blood circulation, bone setting, and relieving pain). This method overcomes the limitations of traditional black plaster quality control, which relies solely on routine examinations such as appearance, softening point, weight variation, and thin-layer chromatography. It also overcomes the shortcomings of conventional methods that only control a single active ingredient. The method is simple, accurate, and effectively solves the technical challenge of content determination in large-component black plaster dosage forms, providing a comprehensive and reliable quality control method for Huoxue Jiegu Zhitong Gao.

[0013] 2. Addressing the challenges of high viscosity, strong adsorption, and difficulty in extracting active ingredients from black plaster matrices, this invention employs a ternary mixed solvent system of chloroform, ethanol, and water. Extraction is carried out through stepwise addition. Chloroform effectively dissolves the lipid-soluble matrix of the black plaster, ethanol acts as a bridging solvent miscible with both chloroform and water, and water, as the extraction solvent, fully extracts saponins. The synergistic effect of these three solvents achieves highly efficient extraction of saponins from black plasters. Furthermore, this invention utilizes a 60°C water bath oscillation extraction method, which avoids the difficulty in controlling the temperature of ultrasonic extraction and the damage to active ingredients caused by excessively high reflux extraction temperatures. It also ensures sufficient contact between the extraction solvent and the drug, guaranteeing the thoroughness of the extraction process and the reliability of the results.

[0014] 3. The content determination method provided by this invention is simple to operate. The preparation process of the reference solution and the test solution does not require complicated purification steps. Liquid chromatography analysis can complete the simultaneous determination of five components in a short time. It is suitable for daily quality inspection and quality control of Huoxue Jiegu Zhitong Ointment and has good prospects for promotion and application. Attached Figure Description

[0015] Figure 1 The HPLC chromatogram of the reference standard shows that: peak 1: Panax notoginseng saponin R1, peak 2: Ginsenoside Rg1, peak 3: Dipsacus saponin VI, peak 4: Ginsenoside Rb1, and peak 5: Ginsenoside Rd.

[0016] Figure 2 The HPLC chromatogram of the blood-activating, bone-setting, and pain-relieving plaster sample is shown below. Peak 1: Panax notoginseng saponin R1, Peak 2: Ginsenoside Rg1, Peak 3: Dipsacus saponin VI, Peak 4: Ginsenoside Rb1, Peak 5: Ginsenoside Rd.

[0017] Figure 3 The image shows the HPLC chromatogram of a negative sample of the blood-activating, bone-setting, and pain-relieving plaster.

[0018] Figure 4 The linear relationship of Panax notoginseng saponin R1 is shown in the graph.

[0019] Figure 5 Linear relationship diagram of ginsenoside Rg1.

[0020] Figure 6 Linear relationship diagram of Dipsacus saponin VI.

[0021] Figure 7 Linear relationship diagram of ginsenoside Rb1.

[0022] Figure 8 Linear relationship graph of ginsenoside Rd. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1

[0025] This embodiment provides a method for simultaneously determining the content of multiple saponin components in a blood-activating and bone-setting analgesic plaster using HPLC, including the following steps.

[0026] 1. Instruments and reagents 1.1 Instruments LC-20AD High Performance Liquid Chromatograph (Shimadzu, Japan): SPD-M20A diode array detector, LC-20ADXR pump, SIL-20AXR autosampler, CTO-20AC column oven, LabSolutions chromatography workstation; nacalai tesque COSMOSIL 5 C 18 -MS-II column (4.6ID×250mm); Shimadzu Wondasil C 18 Superb column 5 μm (4.6ID×250mm); AB135-S electronic analytical balance (METTLER, Switzerland); MS204S electronic analytical balance (METTLER, Switzerland). SHA-B Dual-Function Water Bath Thermostatic Oscillator (Hangzhou Jingfei Instrument Technology Co., Ltd.)

[0027] 1.2 Drug Testing Blood-activating and bone-setting analgesic plaster (batch numbers: 20260101, 20260201, 20260202, 20260203, 20260204, 20260205, 20260206), provided by the Pharmaceutical Preparation Department of Luoyang Orthopedic Hospital (Henan Provincial Orthopedic Hospital); The following reference standards were purchased from Shanghai Yuanye Biotechnology Co., Ltd.: Panax notoginseng saponin R1 (LOT: JV335638), Ginseng saponin Rg1 (LOT: LS447552), Dipsacus saponin VI (LOT: Z19J11L108584), Ginseng saponin Rb1 (LOT: KB419866), and Ginseng saponin Rd (LOT: KB409084). Acetonitrile was of chromatographic grade, and all other grades were of analytical grade. Pure water, Wahaha Group.

[0028] 2. Methods and Results 2.1 Chromatographic conditions Using octadecylsilane-bonded silica gel as the packing material, acetonitrile as mobile phase A, and water as mobile phase B, gradient elution was performed according to the specifications in the table below; the flow rate was 1 ml / min; and the detection wavelength was 203 nm.

[0029] Table 1 HPLC gradient elution conditions

[0030] 2.2 Preparation of reference solution Accurately weigh appropriate amounts of notoginsenoside R1 reference standard, ginsenoside Rg1 reference standard, dipsacusin VI reference standard, ginsenoside Rb1 reference standard, and ginsenoside Rd reference standard, and add methanol to prepare a mixed solution containing 10 μg of notoginsenoside R1, 40 μg of ginsenoside Rg1, 200 μg of dipsacusin VI, 40 μg of ginsenoside Rb1, and 10 μg of ginsenoside Rd per ml. (Actual concentrations of the mixed reference solution: notoginsenoside R1 10.1517 μg / ml; ginsenoside Rg1 39.5166 μg / ml; dipsacusin VI 198.5538 μg / ml; ginsenoside Rb1 40.9481 μg / ml; ginsenoside Rd 10.1038 μg / ml).

[0031] 2.3 Preparation of the test solution Take an appropriate amount of the blood-activating, bone-setting, and pain-relieving plaster, cut it into small pieces, accurately weigh 10g, place it in a stoppered conical flask, and accurately add 20ml of chloroform and 20ml of ethanol in sequence, shake well, then add 50ml of water, weigh it, and place it on a 60℃ water bath shaker for 1 hour (150 r / min). Remove it, let it cool, replenish the lost weight with water, filter it, wash the filtrate twice with 30ml of ether each time, discard the ether, evaporate the aqueous layer to dryness at 85℃, dissolve the residue in an appropriate amount of methanol, transfer it to a 5ml volumetric flask, add methanol to the mark, shake well, filter it, and collect the filtrate to obtain the final product.

[0032] 2.4 Preparation of negative sample solution Take the negative preparation of the blood-activating, bone-setting, and pain-relieving plaster lacking Panax notoginseng and Dipsacus asper, and prepare the negative sample solution according to the preparation method of the test sample solution.

[0033] 2.5 Specificity Examination Inject 10 μL each of the reference solution, test solution, and negative sample solution into the liquid chromatograph and analyze according to the chromatographic conditions described in section 2.1. The HPLC chromatograms of the reference solution, test solution, and negative sample are shown below. Figures 1 to 3 .

[0034] The results showed that the HPLC chromatograms of the negative samples showed no interference at the corresponding locations of the reference standards for notoginsenoside R1, ginsenoside Rg1, dipsacusin VI, ginsenoside Rb1, and ginsenoside Rd.

[0035] 2.6 Examination of Linear Relationships Panax notoginseng saponin R1: 2 μl, 4 μl, 10 μl, 16 μl, and 22 μl of the Panax notoginseng saponin R1 reference solution with a concentration of 10.1517 μg / ml were injected into the liquid chromatograph for analysis. The peak area was determined according to the chromatographic conditions in section 2.1. The results are shown in Table 2.

[0036] Table 2 Linearity and range of Panax notoginsenoside R1

[0037] Plot the peak area integral value on the ordinate and the injection concentration (μg·ml). -1 Using y = 2287.53X + 2690.89 as the x-axis, a standard curve was plotted, yielding the regression equation Y = 2287.53X + 2690.89, with a correlation coefficient r = 0.999. Figure 4 As shown, the results indicate that notoginsenoside R1 exhibits good linearity in the range of 2.0303 μg / ml to 22.3337 μg / ml.

[0038] Ginsenoside Rg1: 2 μl, 4 μl, 10 μl, 16 μl, 22 μl, and 30 μl of ginsenoside Rg1 reference solution with a concentration of 39.5166 μg / ml were injected into the liquid chromatograph for analysis. The peak area was determined according to the chromatographic conditions in section 2.1. The results are shown in Table 3.

[0039] Table 3 Linearity and Range of Ginsenoside Rg1

[0040] Plot the peak area integral value on the ordinate and the injection concentration (μg·ml). -1 Using y = 2897.23X + 6671.56 as the x-axis, a standard curve was plotted, yielding the regression equation Y = 2897.23X + 6671.56, with a correlation coefficient r = 0.999. Figure 5 As shown, ginsenoside Rg1 exhibits good linearity in the range of 7.9033 μg / ml to 118.5498 μg / ml.

[0041] Dipsacus saponin VI: 2 μl, 4 μl, 10 μl, 16 μl, 22 μl and 30 μl of the reference solution with a concentration of 198.5538 μg / ml were injected into the liquid chromatograph for analysis. The peak area was determined according to the chromatographic conditions in section 2.1. The results are shown in Table 4.

[0042] Table 4. Linearity and range of Dipsacus saponin VI

[0043] Plot the peak area integral value on the ordinate and the injection concentration (μg·ml). -1 Using y = 3342.88X + 19805.55 as the x-axis, a standard curve was plotted, and the regression equation was calculated to be Y = 3342.88X + 19805.55, with a correlation coefficient r = 0.999. Figure 6 As shown, the linear relationship of Dipsacus saponin VI is good in the range of 39.7107 μg / ml to 595.6614 μg / ml.

[0044] Ginsenoside Rb1: 2 μl, 4 μl, 10 μl, 16 μl, 22 μl, and 30 μl of the reference solution with a concentration of 40.9481 μg / ml were injected into the liquid chromatograph for analysis. The peak area was determined according to the chromatographic conditions in section 2.1. The results are shown in Table 5.

[0045] Table 5. Linearity and range of ginsenoside Rb1

[0046] Plot the peak area integral value on the ordinate and the injection concentration (μg·ml). -1Using y = 2341.81X + 4409.74 as the x-axis, a standard curve was plotted, and the regression equation was calculated to be Y = 2341.81X + 4409.74, with a correlation coefficient r = 0.999. Figure 7 As shown, ginsenoside Rb1 exhibits good linearity in the range of 8.1896 μg / ml to 122.8443 μg / ml.

[0047] Ginsenoside Rd: 2 μl, 4 μl, 10 μl, 16 μl, 22 μl and 30 μl of the reference solution with a concentration of 10.1038 μg / ml were injected into the liquid chromatograph for analysis. The peak area was determined according to the chromatographic conditions in section 2.1. The results are shown in Table 6.

[0048] Table 6 Linearity and Range of Ginsenoside Rd

[0049] Plot the peak area integral value on the ordinate and the injection concentration (μg·ml). -1 Using y = 3265.82X - 239.69 as the x-axis, a standard curve was plotted, and the regression equation was calculated to be Y = 3265.82X - 239.69, with a correlation coefficient r = 1. Figure 8 As shown, ginsenoside Rd exhibits good linearity in the range of 2.0208 μg / ml to 30.3114 μg / ml.

[0050] 2.7 Stability Test Accurately pipette 10 μl of the same test solution and analyze it at 0 h, 3 h, 9 h, 18 h, 27 h and 36 h according to the chromatographic conditions in section 2.1. The results are shown in Table 7.

[0051] Table 7 Stability Test Results

[0052] The experimental results showed that the relative standard deviation of the peak area of ​​each component in the test sample was less than 2% within 36 hours, indicating that the test sample solution was stable within 36 hours.

[0053] 2.8 Precision Test Accurately pipette 10 μl of the same test solution and inject it 6 times according to the chromatographic conditions in section 2.1. Measure the peak area and the results are shown in Table 8.

[0054] Table 8 Precision Test Results

[0055] The experimental results showed that the relative standard deviation of the peak area of ​​each component was less than 2% in six consecutive analyses of the test solution, indicating that the instrument has good precision.

[0056] 2.9 Repeatability Test Take an appropriate amount of sample (batch number: 20260101), weigh 6 portions, extract the sample according to the test sample preparation method under section 2.3, and analyze it according to the chromatographic conditions under section 2.1. The results are shown in Table 9.

[0057] Table 9 Results of Repeatability Tests

[0058] The experimental results showed that the relative standard deviation of the content of each component in the six test solutions was less than 2%, indicating that the method has good repeatability.

[0059] 2.10 Recovery Test Take an appropriate amount of reference standard and add ethanol to prepare a mixed solution containing notoginsenoside R1 23.36 μg / ml, ginsenoside Rg 172.83 μg / ml, ginsenoside Rb 166.79 μg / ml, and ginsenoside Rd 16.17 μg / ml. Take an appropriate amount of Dipsacus VI and add ethanol to prepare solutions with concentrations of 0.2890 mg / ml, 0.5583 mg / ml, and 0.7869 mg / ml, respectively.

[0060] Take an appropriate amount of repeatable sample (batch number: 20260101), accurately weigh 5g of sample, make a total of 9 portions, place them in stoppered conical flasks, and add 20 ml of chloroform to each. Take three portions, add 1 ml of the above mixed reference solution to each, then add 1 ml of a 0.2890 mg / ml solution of Dipsacus saponin VI to each, then add 18 ml of ethanol to each sample, shake well, and add 50 ml of water; take another three portions, add 1.5 ml of the above mixed reference solution to each, then add 1 ml of a 0.5583 mg / ml solution of Dipsacus saponin VI to each, then add 17.5 ml of ethanol to each sample, shake well, and add 50 ml of water; take yet another three portions, add 2 ml of the above mixed reference solution to each, then add 1 ml of a 0.7869 mg / ml solution of Dipsacus saponin VI to each, then add 17 ml of ethanol to each sample, shake well, and add 50 ml of water; prepare the test solution according to the method under section 2.3 "Preparation Method of Test Sample", starting from "Weighing". Under the chromatographic conditions described in section 2.1, 10 μl of the solution was injected into the liquid chromatograph for analysis. The results are shown in Tables 10 to 14.

[0061] Table 10 Results of the recovery test of Panax notoginsenoside R1

[0062] Table 11 Results of the recovery test of ginsenoside Rg1

[0063] Table 12 Results of the recovery test of Dipsacus saponin VI

[0064] Table 13 Results of the recovery test of ginsenoside Rb1

[0065] Table 14 Results of Ginsenoside Rd Recovery Test

[0066] The experimental results showed that the average recovery rates of the five analytes were all greater than 95%, and the relative standard deviations were all less than 2%, indicating that the method has good recovery rates.

[0067] 2.11 Durability Test The sample analysis method showed good resolution of each component under different column temperatures (20℃, 25℃, 30℃). Different mobile phases (acetonitrile-0.05% phosphoric acid solution, acetonitrile-0.05% formic acid solution, and acetonitrile-water) were used for evaluation. The baseline fluctuations were larger when using acetonitrile-0.05% phosphoric acid solution and acetonitrile-0.05% formic acid solution, while the baseline was relatively stable and the resolution of each component was good when using acetonitrile-water mobile phase. Different chromatographic columns (Shimadzu WONDASIL C) were also tested. 18 Superb column, 5 μm, 4.6 ID × 250 mm; Nacalai tesque COSMOSIL 5 C 18 (MS-II column, 4.6ID×250mm), sample resolution met requirements. This indicates that the method has good robustness.

[0068] 2.12 Determination of Sample Content Six batches of Huoxue Jiegu Zhitong Gao (a traditional Chinese medicine plaster) were collected, and test solutions were prepared according to the method in section 2.3. The sample content was determined according to the chromatographic conditions in section 2.1. The results are shown in Table 15.

[0069] Table 15. Results of content determination of the blood-activating, bone-setting, and pain-relieving plaster (mg / g)

[0070] 2.13 Methodological Investigation Description In the preparation of the test solution, the extraction effects of various extraction solvents were compared, including single solvents, binary solvents, and multi-component mixed solvents. The ternary solvent of chloroform, ethanol, and water at ratios of 20 ml, 20 ml, and 50 ml showed the best extraction effect. The extraction methods were investigated, including ultrasonication, reflux, and water bath shaking. Extraction at 60℃ for 1 hour (150 rpm) yielded the best results. A sample volume of 10 g was recommended. The aqueous layer should be evaporated to dryness at 85℃ to avoid loss of effective substances. The above method for preparing the test solution is simple to operate, provides thorough extraction, and has significant effects, ensuring the accuracy of the method.

[0071] In the chromatographic conditions study, the detection wavelength, mobile phase composition, and gradient elution conditions were selected and optimized to meet the separation requirements of each component.

[0072] It should be noted that the above embodiments are only used to illustrate the present invention, but the present invention is not limited to the above embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for simultaneously determining the content of multiple saponin components in a blood-activating, bone-setting, and pain-relieving plaster using liquid chromatography, characterized in that: The various saponin components include notoginsenoside R1, ginsenoside Rg1, dipsacusin VI, ginsenoside Rb1, and ginsenoside Rd; the method includes the following steps: S1, Chromatographic conditions: Using octadecylsilane-bonded silica gel as the stationary phase, acetonitrile as mobile phase A, and water as mobile phase B, gradient elution was performed according to the following program: 0–10 min, mobile phase A 20%, mobile phase B 80%; 10–40 min, mobile phase A linearly changed from 20% to 30%, mobile phase B linearly changed from 80% to 70%; 40–60 min, mobile phase A linearly changed from 30% to 31%, mobile phase B linearly changed from 70% to 69%; 60–61 min, mobile phase A linearly changed from 31% to 36%, mobile phase B linearly changed from 69% to 64%; 61–70 min, mobile phase A linearly changed from 36% to 42%, mobile phase B linearly changed from 64% to 58%; 70–72 min, mobile phase A linearly changed from 42% to 90%, mobile phase B linearly changed from 58% to 10%; flow rate was 1 ml / min, detection wavelength was 203 nm; S2, Preparation of reference solutions: Accurately weigh the reference standards of Panax notoginseng saponin R1, Ginsenoside Rg1, Dipsacus asperoidin VI, Ginsenoside Rb1 and Ginsenoside Rd respectively, and add methanol to prepare a mixed solution. S3, Preparation of the test solution: Remove the blood-activating and bone-setting pain-relieving plaster, cut it into small pieces, weigh it accurately, and place it in a stoppered conical flask. According to the ratio of sample weight to the amount of chloroform, ethanol and water, 1g:2ml:2ml:5ml, add chloroform, ethanol and water in sequence, shake well, weigh, place on a water bath shaker for 1 hour, take it out, cool it, make up the lost weight with water, filter, wash the filtrate with ether, discard the ether, evaporate the water layer to dryness, add methanol to the residue to make up to volume, shake well, filter, and take the filtrate to obtain the test solution. S4, Determination: Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatograph, and determine the result.

2. The method according to claim 1, characterized in that: In step S2, the concentrations of notoginsenoside R1, ginsenoside Rg1, ginsenoside VI, ginsenoside Rb1, and ginsenoside Rd in the mixed solution are 10 μg / ml, 40 μg / ml, 200 μg / ml, 40 μg / ml, and 10 μg / ml, respectively.

3. The method according to claim 1, characterized in that: In step S3, the sample weight is 10g; the amounts of chloroform, ethanol, and water used are 20ml, 20ml, and 50ml, respectively; the temperature of the water bath shaker is 60℃, and the shaking frequency is 150r / min; the temperature at which the water layer is evaporated is 85℃; and the residue is diluted to 5ml with methanol.

4. The method according to claim 1, characterized in that: In step S3, the amount of ether used for washing is 30 ml each time, and the washing is performed twice.

5. The method according to claim 1, characterized in that: In step S4, the injection volumes of the reference solution and the test solution are 10 μl each.

6. The application of the method according to any one of claims 1 to 5 in the quality control of the blood-activating, bone-setting, and pain-relieving plaster product.

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