Method for measuring content of rhizoma alismatis

Abstract

The invention relates to the technical field of drug analysis and detection, in particular to a rhizoma alismatis content determination method which comprises the following steps: preparing a clobetasol propionate reference substance solution; preparing a mixed reference substance solution, namely mixing the 23-acetyl alisol C reference substance solution, the alisol B reference substance solution, the 23-acetyl alisol B reference substance solution and the clobetasol propionate reference substance solution; preparing a test solution: mixing a rhizoma alismatis sample with the clobetasol propionate reference substance solution; and quantitative determination: injecting the test solution and the mixed reference solution into a high performance liquid chromatograph, determining the nature by adopting an ultraviolet spectrogram, and determining and calculating the contents of 23-acetyl alisol C, alisol B and 23-acetyl alisol B in the test solution. According to the present invention, the chemical drug clobetasol propionate with characteristics of low price, easy obtaining and stable property is adopted as the alternative reference substance, such that the problems of transportation, storage, use and low cost of the existing determination method using 23-acetyl alisol C, 23-acetyl alisol B and alisol B as the reference substances are avoided.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical analysis and detection, in particular to a determination method of alisma orientalis content. BACKGROUND

[0002] Modern research shows that alisma orientalis has multiple effects such as blood lipid regulation, blood sugar reduction, anti-tumor and anti-virus in addition to diuretic effect. Alisma orientalis is a large-scale medicinal material, and the demand for alisma orientalis is large whether in traditional Chinese medicine decoction pieces or in Chinese patent medicines. Alisma orientalis is used in combination in classic famous prescriptions such as Liuwei Dihuang Wan, Longdan Xiegan Wan and Wuling San.

[0003] The existing standards all use HPLC external standard method to calculate the content, and 23-acetyl alismoxide C, 23-acetyl alismoxide B and alismoxide B are commonly used as reference substances, but there are two problems: first, the 23-acetyl alismoxide C, 23-acetyl alismoxide B and alismoxide B reference substances required by the existing standards need cold chain transportation and storage at 2-8 DEG C, which is harsh and difficult for transportation, storage and use, and there is a certain risk; second, the 23-acetyl alismoxide C, 23-acetyl alismoxide B and alismoxide B reference substances are extracted from alisma orientalis medicinal materials, and the extraction efficiency is low, the source is difficult, and the price is expensive. SUMMARY

[0004] In order to solve the defects in the prior art, the present application provides a determination method of alisma orientalis content. The present application uses cheap and stable chemical drug chlortetracycline propionic acid as a substitute reference substance to determine the contents of 23-acetyl alismoxide C, 23-acetyl alismoxide B and alismoxide B in alisma orientalis, and the method is stable and reliable.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0006] The present application provides a determination method of alisma orientalis content, which comprises the following steps:

[0007] Preparation of chlortetracycline propionic acid reference substance solution;

[0008] Preparation of mixed reference substance solution: mixing 23-acetyl alismoxide C reference substance solution, alismoxide B reference substance solution, 23-acetyl alismoxide B reference substance solution and the chlortetracycline propionic acid reference substance solution;

[0009] Preparation of test sample solution: mixing alisma orientalis sample and the chlortetracycline propionic acid reference substance solution;

[0010] Quantitative determination: injecting the test sample solution and the mixed reference substance solution into a high performance liquid chromatograph, using ultraviolet spectrum for qualitative determination, and determining and calculating the contents of 23-acetyl alismoxide C, alismoxide B and 23-acetyl alismoxide B in the test sample solution.

[0011] Preferably, the chromatographic conditions of the high performance liquid chromatograph for determination are as follows: octadecylsilane-bonded silica gel as the filler; mobile phase: water-acetonitrile; detection wavelength of 23-acetylrhizoxinol B and rhizoxinol B: 207.8-208.2 nm, detection wavelength of 23-acetylrhizoxinol C: 245.8-246.2 nm, detection wavelength of chlobetasol propionate: 238.8-239.2 nm; column temperature: 28-32℃; flow rate: 0.98-1.02 ml / min.

[0012] Preferably, the chromatographic conditions of the high performance liquid chromatograph for determination are as follows: octadecylsilane-bonded silica gel as the filler; mobile phase: water-acetonitrile; detection wavelength of 23-acetylrhizoxinol B and rhizoxinol B: 208 nm, detection wavelength of 23-acetylrhizoxinol C: 246 nm, detection wavelength of chlobetasol propionate: 239 nm; column temperature: 30℃; flow rate: 1.0 ml / min.

[0013] Preferably, the volume ratio of water to acetonitrile in the mobile phase is (36-38):(62-64).

[0014] Preferably, the concentration of chlobetasol propionate in the chlobetasol propionate control solution is 0.8-0.9 mg / ml.

[0015] Preferably, the concentration of 23-acetylrhizoxinol C in the mixed control solution is 4-5 μg / ml, the concentration of rhizoxinol B is 117-118 μg / ml, the concentration of 23-acetylrhizoxinol B is 61-62 μg / ml, and the concentration of chlobetasol propionate is 34-35 μg / ml.

[0016] Preferably, the process for preparing the chlobetasol propionate control solution comprises the following steps: weighing chlobetasol propionate control, dissolving and diluting with acetonitrile, and obtaining the solution.

[0017] Preferably, the process for preparing the mixed control solution comprises the following steps: dissolving 23-acetylrhizoxinol C control, rhizoxinol B control and 23-acetylrhizoxinol B control with acetonitrile respectively, mixing, adding the chlobetasol propionate control solution, diluting with acetonitrile, and shaking to obtain the solution.

[0018] Preferably, the process for preparing the sample solution comprises the following steps: taking a rhizoma alismatis sample, adding the chlobetasol propionate control solution, ultrasonic treatment after adding acetonitrile, filtering, and obtaining the filtrate.

[0019] Preferably, the quantitative determination specifically comprises:

[0020] (1) injecting the mixed control solution and the sample solution into the high performance liquid chromatograph respectively, and determining;

[0021] (2) based on the high performance liquid chromatography determination results obtained from the mixed control solution, a relative correction factor is calculated;

[0022] (3) according to the relative correction factor and the high performance liquid chromatography determination results obtained from the test sample solution, the contents of 23-acetyloalanginol C, alanginol B and 23-acetyloalanginol B in the test sample solution are calculated.

[0023] Preferably, the step (2) specifically comprises: based on the high performance liquid chromatograms obtained from the mixed control solutions with different volumes and same concentration, a relative correction factor is calculated;

[0024] The correction calculation formula is f rt =(C r ×A t ) / (A r ×C t );

[0025] Wherein, C t and A t are the concentration and peak area of the component to be measured, and C r and A r are the concentration and peak area of the propionate.

[0026] Preferably, the relative correction factor of 23-acetyloalanginol C is 0.8438, the relative correction factor of alanginol B is 0.5115, and the relative correction factor of 23-acetyloalanginol B is 0.4677.

[0027] The beneficial effects of the present application are:

[0028] In the determination method of the present application, the cheap and easily obtained chemical drug propionate is used as the substitute control sample, and the contents of 23-acetyloalanginol C, 23-acetyloalanginol B and alanginol B in alangium can be determined at the same time. The problem that the control samples of 23-acetyloalanginol C, 23-acetyloalanginol B and alanginol B need to be transported and stored at 2-8℃ cold chain and have high cost can be solved. In addition, the propionate can be synthesized on a large scale and is easy to obtain and has low cost, and the problem that the alangium control sample can only rely on plant extraction and is expensive can be solved. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The liquid chromatogram of the mixed control sample solution.

[0030] Figure 2 The liquid chromatogram of the test sample solution.

[0031] Figure 3 The liquid chromatogram of the solvent blank.

[0032] Figure 4Liquid chromatograms under different proportions of mobile phase.

[0033] Figure 5 UV spectra of propionylclobetasol, 23-acetylrhizanol C, 23-acetylrhizanol B and rhizanol B. DETAILED DESCRIPTION

[0034] In order to make the technical solution of the application better understood by the skilled in the art, the application will be further described in detail below in combination with specific embodiments.

[0035] In order to solve the problems of transportation, storage, use and cost of the current determination method using 23-acetylrhizanol C, 23-acetylrhizanol B and rhizanol B as the control, the application uses propionylclobetasol as a substitute control to determine the content of rhizoma alismatis.

[0036] The application provides a determination method of the content of rhizoma alismatis, comprising:

[0037] Preparation of propionylclobetasol control solution;

[0038] Preparation of mixed control solution: mixing 23-acetylrhizanol C control solution, rhizanol B control solution, 23-acetylrhizanol B control solution and the propionylclobetasol control solution;

[0039] Preparation of test sample solution: mixing rhizoma alismatis sample and the propionylclobetasol control solution;

[0040] Quantitative determination: injecting the test sample solution and the mixed control solution into a high performance liquid chromatograph, qualitatively determining by using UV spectrum, and determining and calculating the content of 23-acetylrhizanol C, rhizanol B and 23-acetylrhizanol B in the test sample solution.

[0041] The application uses the cheap and easy-to-obtain chemical drug propionylclobetasol with stable properties as a substitute control to determine the content of the components of rhizoma alismatis, can simultaneously determine the content of 23-acetylrhizanol C, 23-acetylrhizanol B and rhizanol B in rhizoma alismatis, can solve the problems of 2-8℃ cold chain transportation, storage and high cost of 23-acetylrhizanol C, 23-acetylrhizanol B and rhizanol B control, in addition, propionylclobetasol can be synthesized on a large scale, is easy to obtain and has low cost, and solves the problem of expensive rhizoma alismatis control relying on plant extraction.

[0042] The chromatograph conditions for the high performance liquid chromatograph in the present application are as follows: octadecylsilane-bonded silica gel as the filler; mobile phase: water-acetonitrile; detection wavelength of 23-acetylrhizoxinol B and rhizoxinol B: 207.8-208.2 nm, detection wavelength of 23-acetylrhizoxinol C: 245.8-246.2 nm, detection wavelength of chlobetasol propionate: 238.8-239.2 nm; column temperature: 28-32℃; flow rate: 0.98-1.02 ml / min. Preferably, the chromatograph conditions for the high performance liquid chromatograph in the present application are as follows: octadecylsilane-bonded silica gel as the filler; mobile phase: water-acetonitrile; detection wavelength of 23-acetylrhizoxinol B and rhizoxinol B: 208 nm, detection wavelength of 23-acetylrhizoxinol C: 246 nm, detection wavelength of chlobetasol propionate: 239 nm; column temperature: 30℃; flow rate: 1.0 ml / min.

[0043] The volume ratio of water to acetonitrile in the mobile phase in the present application is (36-38):(62-64). Preferably, the volume ratio of water to acetonitrile in the mobile phase is 37:63, at which the separation degree of 23-acetylrhizoxinol C peak and chlobetasol propionate peak reaches 2.7, the separation degree of each target peak and adjacent impurity peak meets the requirements, and the retention time of each target peak is suitable.

[0044] The concentration of chlobetasol propionate in the chlobetasol propionate reference solution in the present application is 0.8-0.9 mg / ml. In some embodiments of the present application, the concentration of chlobetasol propionate is about 0.8533 mg / ml.

[0045] The concentration of 23-acetylrhizoxinol C in the mixed reference in the present application is 4-5 μg / ml, the concentration of rhizoxinol B is 117-118 μg / ml, the concentration of 23-acetylrhizoxinol B is 61-62 μg / ml, and the concentration of chlobetasol propionate is 34-35 μg / ml. In some embodiments of the present application, the concentrations of 23-acetylrhizoxinol C, rhizoxinol B and 23-acetylrhizoxinol B are 4.73 μg / ml, 117.07 μg / ml and 61.82 μg / ml, respectively, and the concentration of chlobetasol propionate is about 34.13 μg / ml.

[0046] The process for preparing the chlobetasol propionate reference solution in the present application comprises: weighing chlobetasol propionate reference, dissolving and diluting with acetonitrile, and obtaining the solution.

[0047] The process for preparing the mixed reference solution in the present application comprises: dissolving 23-acetylrhizoxinol C reference, rhizoxinol B reference and 23-acetylrhizoxinol B reference with acetonitrile respectively, mixing, adding the chlobetasol propionate reference solution, diluting with acetonitrile, and shaking to obtain the solution.

[0048] The process for preparing the test solution according to the present invention includes: taking a sample of Alisma plantago-aquatica, adding clobetasol propionate reference solution, adding acetonitrile, sonicating, filtering, and taking the filtrate to obtain the test solution.

[0049] The quantitative determination described in this invention specifically includes:

[0050] (1) Inject the mixed reference solution and the test solution into a high-performance liquid chromatograph for determination;

[0051] (2) The relative correction factor is calculated based on the high performance liquid chromatography results obtained from the mixed reference solution;

[0052] (3) Based on the relative correction factor and the high performance liquid chromatography determination results obtained based on the test solution, the contents of 23-acetylalizool C, alizool B and 23-acetylalizool B in the test solution are calculated.

[0053] Preferably, step (2) specifically includes: obtaining high performance liquid chromatograms based on mixed reference solutions of the same concentration but different volumes, and calculating relative correction factors;

[0054] Correction calculation formula: f rt =(C r ×A t ) / (A r ×C t );

[0055] Among them, C t and A t C represents the concentration and peak area of ​​the analyte. r and A r The values ​​represent the concentration and peak area of ​​clobetasol propionate.

[0056] The relative correction factor for 23-acetylalizool C in this invention is 0.8438, the relative correction factor for alismool B is 0.5115, and the relative correction factor for 23-acetylalizool B is 0.4677.

[0057] The relative correction factor of this invention has been subjected to tolerance tests, which show that different liquid chromatographs, different brands of C18 columns, different batches of the same brand of C18 columns, wavelength, column temperature, mobile phase ratio, and flow rate have no significant effect on the relative correction factor (f).

[0058] The above is a detailed description of the technical solution of the present invention. The following are embodiments of the present invention.

[0059] Example 1

[0060] 1. Experimental instruments and reagents

[0061] 1.1 Instruments

[0062] Waters 2695-2998 liquid chromatograph: Waters Corporation, PDA, S / N: M21SM7923A;

[0063] Waters 2695-2998 liquid chromatograph: Waters Corporation, PDA, S / N: E22SM4041A;

[0064] SHIMADZU LC-2050C 3D liquid chromatograph: PDA, Shimadzu International Trading (Shanghai) Co., Ltd.;

[0065] XSE205 DualRange electronic balance: Mettler-Toledo;

[0066] 3 different brands and batch numbers C 18 Chromatographic column: Waters Symmetry C18 (250 mm x 4.6 mm, 5 μm), S / N: 03413222212407; Waters Symmetry C18 (250 mm x 4.6 mm, 5 μm), S / N: 03413222212469; Waters Symmetry C18 (250 mm x 4.6 mm, 5 μm), S / N: 03413222212463; Kromasil 100-5-C18 (250 mm x 4.6 mm, 5 μm), S / N: M05CLA25 / H229318; Perkin Elmer Analytical C18 (250 mm x 4.6 mm, 5 μm), S / N: 16020547S;

[0067] ULUP-II-10T ultra-pure water generator: Chengdu Superpure Technology Co., Ltd.

[0068] 1.2 reagents

[0069] Clobetasol propionate reference substance (100302-202405, content 99.2%) from China National Research Center for Clinical Medicine;

[0070] Reference substances 23-acetylrhizoxinol C (CAS: 26575-93-9, content 98.80%), 23-acetylrhizoxinol B (CAS: 26575-95-1, content 98.50%), and rhizoxinol B (CAS: 18649-93-9, content 98.94%) were purchased from Zhuhai Anzhe Biological Technology Co., Ltd.

[0071] Rhizoma Alismatis 3 batches were identified by Leshan Food and Drug Inspection and Testing Center, K1 from Caijin Town, Wutongqiao District, Leshan City, K2 from Xiejia Street, Hansan Village, Pengshan District, Meishan City, K3 from Sisheng Village, Xuzhou Town, Zitong County, Mianyang City; Sal Rhizoma Alismatis 3 batches, commercially available, K4 and K5 from Sichuan Guoqiang Traditional Chinese Medicine Herbal Pieces Co., Ltd., batch numbers are 2411109 and 2412186 respectively; K6 from Anhui Tonghuatang Traditional Chinese Medicine Herbal Pieces Technology Co., Ltd., batch number 20231001.

[0072] Acetonitrile (chromatographically pure, Beijing Biotopped Science and Technology Co., Ltd.), acetonitrile (analytically pure, Beijing Biotopped Science and Technology Co., Ltd.), and test water were purified water.

[0073] 2. Methods and Results

[0074] 2.1 Chromatographic conditions

[0075] Chromatographic column: octadecylsilane-bonded silica gel as the filler; mobile phase: water-acetonitrile (volume ratio 37:63); detection wavelength: 208 nm (23-acetylrhizalol B, rhizalol B), 246 nm (23-acetylrhizalol C), 239 nm (clotbesol propionate); column temperature: 30℃; flow rate: 1.0 ml / min; injection volume: 20 μl.

[0076] 2.2 Preparation of solutions

[0077] 2.2.1 Preparation of clotbesol propionate reference substance stock solution

[0078] Precisely weigh 86.02 mg of clotbesol propionate reference substance, place it in a 100 ml volumetric flask, dissolve and dilute to the mark with acetonitrile, shake well, and it is ready. (The concentration of clotbesol propionate is about 0.8533 mg / ml).

[0079] 2.2.2 Preparation of mixed reference substance solution

[0080] Preparation of 23-acetylrhizalol C reference substance stock solution: precisely weigh 11.96 mg of 23-acetylrhizalol C reference substance, place it in a 100 ml volumetric flask, dissolve and dilute to the mark with acetonitrile;

[0081] Preparation of rhizalol B and 23-acetylrhizalol B reference substance stock solution: precisely weigh 29.58 mg of rhizalol B reference substance and 15.69 mg of 23-acetylrhizalol B reference substance, respectively, place them in two 50 ml volumetric flasks, dissolve and dilute to the mark with acetonitrile as reference substance stock solution;

[0082] Accurately pipette 1 ml of the chlorbetamide propionic acid reference stock solution into a 25 ml volumetric flask containing 23-acetyl alisolucinol C 1 ml, alisolubol B 5 ml and 23-acetyl alisolubol B 5 ml. Dilute to the mark with acetonitrile, shake well, and you get it. (The concentrations of 23-acetyl alisolucinol C, alisolubol B and 23-acetyl alisolubol B are 4.73 μg / ml, 117.07 μg / ml and 61.82 μg / ml respectively, and the concentration of chlorbetamide propionic acid is about 34.13 μg / ml).

[0083] 2.2.3 Preparation of test solution

[0084] Take about 0.5 g of the fine powder of the product, accurately weigh it, and place it in a conical flask with a stopper. Accurately add 1 ml of the chlorbetamide propionic acid reference stock solution, and then accurately add 24 ml of acetonitrile. Seal tightly, weigh, ultrasonically treat for 30 minutes, cool, re-weigh, make up the weight loss with acetonitrile, shake well, filter, and take the filtrate, and you get it.

[0085] 2.3 Specificity test

[0086] Take the mixed reference solution, the test solution and the solvent blank respectively, and determine them according to the chromatographic conditions in item 2.1, record the chromatograms, Figure 1 which is the liquid chromatogram of the mixed reference solution, Figure 2 which is the liquid chromatogram of the test solution, Figure 3 which is the liquid chromatogram of the solvent blank (1. 23-acetyl alisolucinol C, 2. chlorbetamide propionic acid, 3. alisolubol B, 4. 23-acetyl alisolubol B). The ratio of the retention time of the 23-acetyl alisolucinol C peak to that of the chlorbetamide propionic acid peak is 0.8906; the ratio of the retention time of the alisolubol B peak to that of the chlorbetamide propionic acid peak is 2.6470; and the ratio of the retention time of the 23-acetyl alisolubol B peak to that of the chlorbetamide propionic acid peak is 4.5067. The ultraviolet spectrum collected by the diode array detector also helps in the qualitative analysis.

[0087] 2.4 Determination of the relative correction factor (f) value

[0088] Accurately pipette 20 μL, 16 μL, 12 μL, 8 μL, 4 μL and 2 μL of the mixed reference solution in item 2.2.2 respectively, and determine them according to the chromatographic conditions in item 2.1, record the chromatograms, and calculate the relative correction factor (f) according to f = (C rt × A r ) / (A t × C r ), and then obtain the arithmetic mean value, wherein C t and A t are the concentration and peak area of the component to be determined, and C t and A r are the concentration and peak area of the reference substance. rThe concentration and peak area of clobetasol propionate. The mean values of each factor investigated under 2.5 were taken as the final relative correction factor (f), the values were 0.8438 for 23-acetylrhoifolin C with RSD of 1.32%, 0.5115 for rhoifolin B with RSD of 2.01%, and 0.4677 for 23-acetylrhoifolin B with RSD of 1.21%, respectively.

[0089] 2.5 Relative correction factor (f) tolerance investigation

[0090] Take the mixed reference solution under 2.2.2, and determine under the chromatographic conditions of 2.1, respectively, using 3 different liquid chromatographs, 3 different brands of C18 chromatographic columns, 3 different batches of C18 chromatographic columns of the same brand, and changing the determination wavelength (207.8 / 245.8 / 238.8, 207.9 / 245.9 / 238.9, 208 / 246 / 239, 208.1 / 246.1 / 239.1, 208.2 / 246.2 / 239.2 nm), column temperature (28, 29, 30, 31 ℃, 32 ℃), proportion of mobile phase (water-acetonitrile) (36.0:64.0, 36.5:63.5, 37:63, 37.5:62.5, 38:62), and flow rate (0.98, 0.99, 1.00, 1.01, 1.02 mL·min-1), calculate the relative correction factor (f). The results are shown in Table 1, which shows that different liquid chromatographs, different brands of C18 chromatographic columns, different batches of C18 chromatographic columns of the same brand, wavelength, column temperature, proportion of mobile phase, and flow rate have no significant effect on the relative correction factor (f). -1 ) of the same brand, wavelength, column temperature, proportion of mobile phase, and flow rate, the relative retention times of the 23-acetylrhoifolin C peak, the rhoifolin B peak, the 23-acetylrhoifolin B peak, and the clobetasol propionate peak are stable and have a small fluctuation, with an RSD of less than 3%. When different brands of chromatographic columns are used, the relative retention times of the 23-acetylrhoifolin C peak and the clobetasol propionate peak have a small fluctuation, with an RSD of less than 3%; however, the relative retention times of the rhoifolin B peak, the 23-acetylrhoifolin B peak, and the clobetasol propionate peak have a large fluctuation, and can be assisted by ultraviolet spectroscopy for qualitative analysis. It is also suggested that when different brands of chromatographic columns have an effect on the relative retention time in the alternative reference substance method, a specific model of chromatographic column needs to be specified.

[0091] Table 1. Relative correction factor (f) tolerance investigation

[0092]

[0093]

[0094] 2.6 Relative retention time investigation

[0095] Under the conditions of 2.5, different liquid chromatographs, different batches of chromatographic columns of the same brand, column temperature, proportion of mobile phase, and flow rate, the relative retention times of the 23-acetylrhoifolin C peak, the rhoifolin B peak, the 23-acetylrhoifolin B peak, and the clobetasol propionate peak are stable and have a small fluctuation, with an RSD of less than 3%. When different brands of chromatographic columns are used, the relative retention times of the 23-acetylrhoifolin C peak and the clobetasol propionate peak have a small fluctuation, with an RSD of less than 3%; however, the relative retention times of the rhoifolin B peak, the 23-acetylrhoifolin B peak, and the clobetasol propionate peak have a large fluctuation, and can be assisted by ultraviolet spectroscopy for qualitative analysis. It is also suggested that when different brands of chromatographic columns have an effect on the relative retention time in the alternative reference substance method, a specific model of chromatographic column needs to be specified.

[0096] Table 2. Relative retention time

[0097]

[0098] 2.7 Linearity and range test

[0099] Accurately pipette 20 μL, 16 μL, 12 μL, 8 μL, 4 μL and 2 μL of the mixed control solution under 2.2.2 respectively, and determine by 2.1 chromatographic condition. Record the chromatogram, and plot the standard curve with peak area (A) and concentration (c, μg·mL -1 ) for linear regression. The 23-acetylrhoifolin C, rhoifolin B, 23-acetylrhoifolin B and clobetasol propionate have good linear relationship with peak area in the concentration range of 0.95-9.45 μg·mL -1 (r=0.9998), 23.41-234.13 μg·mL -1 (r=0.9999), 12.36-123.64 μg·mL -1 (r=0.9999) and 6.83-68.27 μg·mL -1 (r=0.9998) respectively, and the regression equations are A=31450c-1424.2, A=19086c-54617, A=17253c-13328 and A=36834c-12013 respectively.

[0100] 2.8 Precision test

[0101] Accurately pipette the mixed control solution under 2.2.2, and analyze by 2.1 injection. Repeat the injection for 6 times, and record the peak area. The RSD of peak area is 0.50% for 23-acetylrhoifolin C peak, 0.55% for rhoifolin B peak, 1.08% for 23-acetylrhoifolin B peak and 0.39% for clobetasol propionate peak respectively, indicating that the injection precision of the instrument is good.

[0102] 2.9 Reproducibility test

[0103] Take sample K1, and prepare 6 sample solutions in parallel according to 2.2.3. Analyze by 2.1 injection, and record the peak area. Calculate the content with relative correction factor (f). The content (mg / g, calculated as dry product) is 0.3519 for 23-acetylrhoifolin C, RSD is 0.67%, 2.7777 for rhoifolin B, RSD is 0.77%, 1.8536 for 23-acetylrhoifolin B, RSD is 0.35% respectively, indicating that the method has good reproducibility.

[0104] 2.10 Stability test

[0105] Take 2.2.3 under test solution, respectively, at room temperature for 0, 4, 6, 8, 12, 24, 48 h, according to 2.1 chromatographic conditions were measured, record peak area. The results of peak area RSD were 23-acetyl alisol C peak 0.78%, alisol B peak 0.95%, 23-acetyl alisol B peak 0.88% and 1.09% propionic acid chlorobetasol peak, sample solution at room temperature within 48 h stable.

[0106] 2.11 recovery test

[0107] Respectively, precision weighing reference 23-acetyl alisol C, alisol B, 23-acetyl alisol B, dissolved in acetonitrile to make concentration of 0.1786 mg / ml, 0.6798 mg / ml, 0.4858 mg / ml reference solution. Precision weighing K1 fine powder (about 0.25 g), placed with stoppered conical flask, respectively, precision 23-acetyl alisol C reference solution 0.5 ml, alisol B reference solution 1 ml, 23-acetyl alisol B reference solution 1 ml, 1 ml "2.2.1" under propionic acid chlorobetasol control solution, according to 2.2.3 parallel preparation of 6 solutions, according to 2.1 under the conditions of sample analysis, according to C t = A t × C r / (A r × f) calculation, C t and A t for the concentration and peak area of the measured substance, C r and A r for the concentration and peak area of propionic acid chlorobetasol. The results are shown in table 3, indicating that the method has high accuracy.

[0108] Table 3. Recovery results (n = 6)

[0109]

[0110] 2.12 sample content determination

[0111] Take sample K1-K6 fine powder, according to 2.2.3 preparation of test solution, take test solution, according to 2.1 chromatographic conditions were measured, record chromatogram, according to 2.11 method to calculate the content of 23-acetyl alisol C, alisol B, 23-acetyl alisol B in sample (calculated on dry basis), and the results with external standard method determination results using paired sample T test analysis, Sig. > P (0.05), there is no significant difference between the results, see table 4.

[0112] Table 4. Sample content determination results (mg / g, n = 3)

[0113]

[0114]

[0115] 3Discussion

[0116] 3.1 Selection of sample solvent and mobile phase

[0117] The mobile phase proportions of water-acetonitrile (32:68), water-acetonitrile (35:65), and water-acetonitrile (37:63) were investigated. See Table 1. Figure 4 (208 nm chromatogram, 1. 23-acetyloleanol C, 2. clobetasol propionate, 3. oleanol B, 4. 23-acetyloleanol B). The results showed that when water-acetonitrile (37:63) was used, the separation degree of the 23-acetyloleanol C peak and the clobetasol propionate peak reached 2.7, the separation degree of each target peak and the adjacent impurity peak met the requirements, and the retention time of each target peak was suitable.

[0118] 3.2 Selection of determination wavelength

[0119] The determination wavelength was selected according to the ultraviolet spectrum of the clobetasol propionate and the 23-acetyloleanol C, 23-acetyloleanol B, and oleanol B reference substance solutions. See Table 2. Figure 5 23-acetyloleanol B and oleanol B have terminal absorption, and 208 nm was selected; 23-acetyloleanol C has maximum absorption at a wavelength of 246 nm; and clobetasol propionate has maximum absorption at a wavelength of 239 nm. After investigation, the determination wavelengths were determined to be 208 nm (23-acetyloleanol B and oleanol B), 246 nm (23-acetyloleanol C), and 239 nm (clobetasol propionate).

[0120] 3.3 Ultraviolet spectrum-assisted qualitative chromatographic peak

[0121] The effects of different liquid chromatographs, different brands of C18 chromatographic columns, the same brand of different batches of C18 chromatographic columns, column temperature, mobile phase proportion, and flow rate on the relative retention time of the three components were investigated. The results showed that different liquid chromatographs, the same brand of different batches of chromatographic columns, column temperature, mobile phase proportion, and flow rate had little effect, the relative retention time was stable, and the RSD was less than 3%. When different brand chromatographic columns were used, the relative retention time of the 23-acetyloleanol C peak fluctuated little, and the RSD was less than 3%; however, the relative retention time of the oleanol B peak and the 23-acetyloleanol B peak fluctuated greatly, and the RSDs were 6.90% and 12.13%, respectively, which could be assisted by ultraviolet spectrum for qualitative analysis. At the same time, it is suggested that when different brand chromatographic columns have an effect on the relative retention time in the replacement reference substance method, a specific model of chromatographic column can be specified.

[0122] The above merely describes the preferred embodiments of the present application, and it should be pointed out that the above preferred embodiments should not be regarded as a limitation to the present application, and the protection scope of the present application should be defined by the scope of the claims. For those skilled in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. A method for determining the content of Alisma plantago-aquatica, characterized in that, include: Preparation of clobetasol propionate reference solution; Preparation of mixed reference solution: Mix 23-acetylalizool C reference solution, alizool B reference solution, 23-acetylalizool B reference solution and the clobetasol propionate reference solution; Preparation of the test solution: Mix the Alisma plantago-aquatica sample and the clobetasol propionate reference solution; Quantitative determination: The test solution and the mixed reference solution were injected into a high-performance liquid chromatograph, and the ultraviolet spectra were used for qualitative analysis. The contents of 23-acetylalizool C, alizool B, and 23-acetylalizool B in the test solution were determined and calculated.

2. The method for determining the content of Alisma plantago-aquatica according to claim 1, characterized in that, The chromatographic conditions for the determination using the high-performance liquid chromatograph were as follows: octadecylsilane-bonded silica gel as the packing material; mobile phase: water-acetonitrile; detection wavelengths for 2,3-acetylalizool B and alizool B: 207.8–208.2 nm, detection wavelengths for 2,3-acetylalizool C: 245.8–246.2 nm, and detection wavelengths for clobetasol propionate: 238.8–239.2 nm; column temperature: 28–32 °C; flow rate: 0.98–1.02 ml / min.

3. The method for determining the content of Alisma plantago-aquatica according to claim 2, characterized in that, The volume ratio of water to acetonitrile in the mobile phase is (36-38):(62-64).

4. The method for determining the content of Alisma plantago-aquatica according to claim 1, characterized in that, The concentration of clobetasol propionate in the clobetasol propionate reference solution is 0.8–0.9 mg / ml.

5. The method for determining the content of Alisma plantago-aquatica according to claim 1 or 4, characterized in that, The concentration of 23-acetylalizool C in the mixed reference standard was 4–5 μg / ml, the concentration of alismazool B was 117–118 μg / ml, the concentration of 23-acetylalizool B was 61–62 μg / ml, and the concentration of clobetasol propionate was 34–35 μg / ml.

6. The method for determining the content of Alisma plantago-aquatica according to claim 1, characterized in that, The process for preparing the clobetasol propionate reference solution includes: weighing the clobetasol propionate reference standard, dissolving and diluting it with acetonitrile to obtain the solution.

7. The method for determining the content of Alisma plantago-aquatica according to claim 1, characterized in that, The process for preparing the mixed reference solution includes: dissolving 23-acetylalizool C reference standard, alismazone B reference standard and 23-acetylalizool B reference standard in acetonitrile, mixing them, adding the clobetasol propionate reference standard solution, diluting with acetonitrile, and shaking well to obtain the solution.

8. The method for determining the content of Alisma plantago-aquatica according to claim 1, characterized in that, The process of preparing the test solution includes: taking a sample of Alisma plantago-aquatica, adding clobetasol propionate reference solution, adding acetonitrile, sonicating, filtering, and taking the filtrate to obtain the test solution.

9. The method for determining the content of Alisma plantago-aquatica according to claim 1, characterized in that, The quantitative determination specifically includes: (1) Inject the mixed reference solution and the test solution into a high-performance liquid chromatograph for determination; (2) The relative correction factor is calculated based on the high performance liquid chromatography results obtained from the mixed reference solution; (3) Based on the relative correction factor and the high performance liquid chromatography determination results obtained based on the test solution, the contents of 23-acetylalizool C, alizool B and 23-acetylalizool B in the test solution are calculated.

10. The method for determining the content of Alisma plantago-aquatica according to claim 9, characterized in that, The specific steps (2) include: obtaining high performance liquid chromatograms based on mixed reference solutions of the same concentration but different volumes, and calculating relative correction factors; Correction calculation formula: f rt =(C r ×A t ) / (A r ×C t ); Among them, C t and A t C represents the concentration and peak area of ​​the analyte. r and A r The values ​​represent the concentration and peak area of ​​clobetasol propionate.

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