A method for determining the contents of five components in Fuzhiqing ointment based on one-test-multiple-evaluation method

Abstract

The present invention relates to a method for determining the contents of five components in Fuzhiqing ointment based on a one-measurement-multiple-evaluation method, wherein the determination method comprises injecting a gallic acid reference solution and a test solution into a high performance liquid chromatograph for determination, and then calculating the contents of gallic acid, phellodendronine hydrochloride, berberine hydrochloride and rheum officinale in Fuzhiqing ointment respectively according to the relative correction factors of gallic acid and aesculetin, phellodendronine hydrochloride, berberine hydrochloride and rheum officinale. The determination method is highly efficient, can synchronously calculate the contents of multiple components by determining one component, saves the detection cost of the quality control of Fuzhiqing ointment, is conducive to comprehensively monitoring the product quality, avoids the singleness and one-sidedness of the quality control, and is of great significance to strengthening the overall quality control of Fuzhiqing ointment and ensuring the clinical efficacy.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to a method for determining the contents of five components in Fuzhiqing ointment based on a one-test-multiple-evaluation method. Background Art

[0002] Fuzhiqing Ointment is a product of Guizhou Green Sun Pharmaceutical Co., Ltd. (Drug Approval Number: National Medicine Standard Z20025745), and is also one of the representative products of Guizhou Miao medicine brand. It has the effects of clearing away heat and detoxifying, removing blood stasis and swelling, removing dampness and relieving itching. It is used for tinea pedis, tinea corporis, tinea cruris, maceration, internal hemorrhoids, external hemorrhoids, swelling, pain, bleeding and leucorrhea caused by damp-heat accumulation. Due to its significant clinical efficacy, this product is favored by patients and doctors.

[0003] The prescription of Fuzhiqing ointment is: 50g of Jin Guolan, 50g of Rheum officinale, 20g of Phellodendron, 30g of Cinnabar Root, 30g of Chrysanthemum, 40g of Viola yedoensis, 20g of Snow Gallbladder, 50g of Sophora flavescens, 20g of Paris polyphylla, 20g of Dioscorea bulbifera, 20g of Curcuma longa, 50g of Sanguisorba officinalis, 20g of Kudingcha, 16g of Borneolum, and 3g of menthol. The preparation method of the Fuzhiqing ointment is as follows: the above 15 ingredients, except Borneolum and menthol, and the remaining 13 ingredients such as Phellodendron are decocted twice with water, the first time for 2 hours and the second time for 1.5 hours, the decoctions are combined, filtered, and the filtrate is concentrated to a relative density of 60°C. A clear paste with a concentration of about 1.05; add 60g of sorbitol, 4.2g of guar gum, and 30g of beta-cyclodextrin, heat to 80℃ to dissolve, and set aside; separately take 10g of stearic acid, 80g of monoglyceride, 80g of octadecyl alcohol, 20g of polysorbate 80, 20g of paraffin oil, and 40g of Span 80, heat to 80℃ to melt, add to the above solution, mix, emulsify for 15 minutes, keep warm at 80℃ for 30 minutes, when the temperature drops to 40℃, add borneol, menthol and 10g of laurocapram, 6g of benzoic acid, and 1.5g of ethylparaben, mix well, and make 1000g.

[0004] The current quality standard of Fuzhiqing ointment only detects the content of berberine hydrochloride in Phellodendron chinense, which cannot reflect the quality of Fuzhiqing ointment more comprehensively. In order to improve the quality inspection standard of Fuzhiqing ointment, the invention team conducted in-depth research and development and investigation on the original detection method of Fuzhiqing ointment, and established a method for determining the content of 5 components in Fuzhiqing ointment by a one-test-multiple-evaluation method. This method realizes the simultaneous analysis of the content of multiple components, reflects the product quality through multi-index synchronous quality control, and provides a reference for further promoting the improvement of the quality control level of Fuzhiqing ointment and the improvement of related evaluation systems. The method is efficient, objective, comprehensive, accurate and repeatable, which saves the economic cost of the detection of Fuzhiqing ointment quality control, and the test results are more stable and controllable. It is conducive to comprehensive monitoring of product quality, avoiding the singleness and one-sidedness of quality control, and is of great significance to strengthening the overall quality control of Fuzhiqing ointment and ensuring clinical efficacy. Summary of the invention

[0005] The purpose of the present invention is to provide a method for determining the contents of five components in Fuzhiqing ointment based on a one-test-multiple-evaluation method.

[0006] The prescription of the Fuzhiqing ointment of the present invention is: 50g of Olea europaea, 50g of Rheum officinale, 20g of Phellodendron chinense, 30g of Cinnabar Root, 30g of Chrysanthemum indica, 40g of Viola yedoensis, 20g of Radix Sophorae Flavescentis, 20g of Paris polyphylla, 20g of Dioscorea bulbifera, 20g of Curcuma longa, 50g of Radix Sanguisorbae Officinalis, 20g of Kudingcha, 16g of Borneolum, and 3g of Menthol; the preparation method of the Fuzhiqing ointment is as follows: the above 15 ingredients, except Borneolum and Menthol, the remaining 13 ingredients such as Phellodendron chinense are decocted twice with water, the first time for 2 hours and the second time for 1.5 hours, the decoctions are combined, filtered, and the filtrate is concentrated to 60°C For the clear paste with a density of about 1.05, add 60g of sorbitol, 4.2g of guar gum, and 30g of beta-cyclodextrin, heat to 80℃ to dissolve, and set aside; take another 10g of stearic acid, 80g of monoglyceride, 80g of octadecyl alcohol, 20g of polysorbate 80, 20g of paraffin oil, and 40g of Span 80, heat to 80℃ to melt, add to the above solution, mix, emulsify for 15 minutes, keep warm at 80℃ for 30 minutes, when the temperature drops to 40℃, add borneol, menthol and 10g of laurocapram, 6g of benzoic acid, and 1.5g of ethylparaben, mix well, and make 1000g.

[0007] The determination method of the present invention comprises: (1) preparation of a test solution; (2) preparation of a reference solution; (3) chromatographic conditions and system suitability test; and (4) determination and calculation.

[0008] The test solution (1) of the present invention is prepared as follows: take the contents of Fuzhiqing ointment, mix well, accurately weigh 5-10 g, place in a conical flask, add 50-150 mL of methanol, ultrasonically treat at a power of 250 W and a frequency of 40 kHz for 30-60 min, filter, concentrate the filtrate and make it 5-15 mL, filter with a 0.45 μm microporous filter membrane, and obtain the solution.

[0009] Preferably, the test solution (1) of the present invention is prepared as follows: take the contents of Fuzhiqing ointment, mix well, accurately weigh 8-10 g, place in a conical flask, add 100-150 mL of methanol, ultrasonically treat at a power of 250 W and a frequency of 40 kHz for 30-40 min, filter, concentrate the filtrate and make it 5-10 mL, filter with a 0.45 μm microporous membrane, and obtain.

[0010] Further preferably, the test solution (1) of the present invention is prepared as follows: take the contents of Fuzhiqing ointment, mix well, accurately weigh 10 g, place in a conical flask, add 100 mL of methanol, ultrasonically treat for 30 min, filter, concentrate the filtrate and make the volume to 10 mL, filter with a 0.45 μm microporous filter membrane, and obtain.

[0011] The preparation method of the reference substance solution (2) of the present invention is as follows: take an appropriate amount of gallic acid reference substance, accurately weigh it, and add methanol to prepare a solution containing 0.50 mg of the reference substance per 1 mL.

[0012] The chromatographic conditions and system suitability test of (3) the present invention are as follows: octadecylsilane bonded silica gel is used as filler; acetonitrile is used as mobile phase A, 0.05-0.12% phosphoric acid solution is used as mobile phase B; gradient elution is performed according to the provisions in the following table; the injection volume is 10 μL; the detection wavelength is 254 nm; the flow rate is 1.0 mL / min, and the theoretical plate number calculated based on the esculetin peak should be not less than 2000;

[0013]

[0014]

[0015] Preferably, the chromatographic conditions and system suitability test (3) of the present invention are as follows: octadecylsilane bonded silica gel is used as filler; acetonitrile is used as mobile phase A, and 0.10% phosphoric acid solution is used as mobile phase B; gradient elution is performed according to the provisions in the following table; the injection volume is 10 μL; the detection wavelength is 254 nm; the flow rate is 1.0 mL / min, and the theoretical plate number calculated based on the esculetin peak should be not less than 2000;

[0016]

[0017] The determination and calculation (4) of the present invention are as follows: 10 μl of each of the gallic acid reference solution and the test solution are injected into a high performance liquid chromatograph for determination; then, based on the relative correction factors of gallic acid and aesculetin, phellodendronine hydrochloride, berberine hydrochloride and rhamnolide, the contents of gallic acid, phellodendronine hydrochloride, berberine hydrochloride and rhamnolide in the Fuzhiqing ointment are calculated respectively.

[0018] The relative correction factors of the gallic acid of the present invention with aesculetin, phellodendrine hydrochloride, berberine hydrochloride and emodin are 0.46, 0.36, 0.52 and 0.40 respectively.

[0019] The relative retention time values ​​of the gallic acid of the present invention with aesculetin, phellodendrine hydrochloride, berberine hydrochloride and emodin are 1.44, 2.58, 3.28 and 3.98 respectively, and the relative retention time is within ±10% of the specified value.

[0020] Beneficial Effects

[0021] 1. The present invention uses gallic acid as an internal standard, determines the relative correction factors of gallic acid and aesculetin, phellodendron chinense hydrochloride, berberine hydrochloride and rhamnolide, and establishes a one-measurement-multiple-evaluation method for the contents of five components in Fuzhiqing ointment. The determination method synchronously calculates the other four components by detecting one component, has high detection efficiency, can realize synchronous analysis of the contents of multiple components, saves the amount of reference substances used in the quality control of Fuzhiqing ointment, reduces the detection cost, is conducive to comprehensive monitoring of product quality, avoids the singleness and one-sidedness of quality control, and is of great significance to strengthening the overall quality control of Fuzhiqing ointment and ensuring clinical efficacy.

[0022] 2. The determination method of the present invention has passed the extraction method investigation, and the results show that the contents of the components to be measured extracted by ultrasonic and reflux extraction methods are not much different. In order to simplify the experimental operation, ultrasonic extraction is selected as the extraction method.

[0023] 3. The determination method of the present invention has passed the extraction time test, and the results show that ultrasonic extraction for 30 minutes, 45 minutes and 65 minutes has little effect on the content of each component to be measured. From the perspective of energy conservation and emission reduction and simplified processing methods, the ultrasonic time is selected as 30 minutes.

[0024] 4. The mobile phase of the determination method of the present invention was investigated. The results showed that the gradient elution was performed according to the conditions of the gradient elution program 1. The chromatographic peak had a good peak shape, good separation, and a stable baseline. The gradient elution was performed according to the conditions of the gradient elution program 2. The peak to be measured was poorly separated from the impurity peak, and the peak shape was severely tailed. The gradient elution was performed according to the conditions of the gradient elution program 3. The chromatographic peak shape was poor, and the peaks of aesculetin and phellodendron hydrochloride were severely tailed. The gradient elution was performed according to the conditions of the gradient elution program 4. The gallic acid peak partially overlapped with the aesculetin peak and could not be separated. Therefore, the parameters of the gradient elution program 1 were selected as the mobile phase.

[0025] 5. The determination method of the present invention has passed the methodological verification, and the results show that the linear relationship is good; the precision investigation shows that the RSD of each component to be measured is in the range of 0.82-2.07%, indicating that the precision of the instrument is good; the repeatability investigation shows that the RSD of each component to be measured in the six repeatability experimental samples is in the range of 1.04-2.07%, indicating that the repeatability is good; the stability investigation results show that the RSD values ​​of esculetin, berberine hydrochloride, phellodendron hydrochloride, gallic acid and rhamnolide are 0.82%, 1 .32%, 1.47%, 1.14% and 2.07%, indicating that the test solution had good stability within 12 h; the spiked recovery test showed that the average recoveries of quinoline, berberine hydrochloride, phellodendron chinense hydrochloride, rhamnolide and gallic acid were 99.52%, 99.74%, 98.99%, 98.98% and 100.01%, respectively, and the RSD values ​​were 0.49%, 0.28%, 0.48%, 0.45% and 0.21%, respectively, indicating that the recovery rate of this method was good.

[0026] 6. The present invention conducted experiments on relative correction factors, investigated different instruments and different chromatographic columns, investigated different flow rates, investigated different column temperatures, and located the chromatographic peaks of the components to be measured. The results showed that the relative retention time values ​​corresponding to the relative correction factors of gallic acid and aesculetin, phellodendronine hydrochloride, berberine hydrochloride and rhamnolide were 1.44, 2.58, 3.28, and 3.98, respectively, and the relative retention time was within ±10% of the specified value; the relative correction factors of gallic acid and aesculetin, phellodendronine hydrochloride, berberine hydrochloride and rhamnolide were 0.46, 0.36, 0.52, and 0.40, respectively.

[0027] 7. The present invention takes 3 batches of Fuzhiqing ointment respectively, and prepares the test solution according to the above method. The contents of gallic acid, berberine hydrochloride, phellodendron hydrochloride, rhamnolide, and fraxin are calculated by the one-measurement multiple evaluation method (QASM method) and the external standard method (ESM method). The contents calculated by the external standard method and the one-measurement multiple evaluation method are compared by t test, and P is much greater than 0.05, indicating that there is no significant difference in the contents measured by the two methods, and the relative error between the two groups of contents is <3%, indicating that the established one-measurement multiple evaluation method has good accuracy and can be used for the content determination of Fuzhiqing ointment. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Extraction method investigation chart (extraction method is ultrasonic extraction);

[0029] Figure 2 Extraction method investigation spectrum (extraction method is reflux extraction);

[0030] Figure 3 Extraction solvent investigation spectrum (extraction solvent is methanol);

[0031] Figure 4 Extraction solvent investigation spectrum (extraction solvent is 30% methanol);

[0032] Figure 5 Extraction solvent investigation spectrum (extraction solvent is 50% methanol);

[0033] Figure 6 Extraction solvent investigation spectrum (extraction solvent is 80% methanol);

[0034] Figure 7 Extraction time investigation spectrum (ultrasonic treatment for 30 min);

[0035] Figure 8 Extraction time investigation spectrum (ultrasonic treatment 45min);

[0036] Fig. 9 Extraction time investigation spectrum (ultrasonic treatment for 60 min);

[0037] Fig.10 Mobile phase investigation spectrum (elution according to gradient elution program 1);

[0038] Fig.11 Mobile phase investigation spectrum (eluted according to gradient elution program 2);

[0039] Fig.12 Mobile phase investigation spectrum (eluted according to gradient elution program 3);

[0040] Fig.13 Mobile phase investigation spectrum (elution according to gradient elution program 4). DETAILED DESCRIPTION

[0041] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0042] Example 1

[0043] (1) Preparation of test solution: Take the contents of Fuzhiqing ointment, mix well, accurately weigh 10 g, place in a conical flask, add 100 mL of methanol, ultrasonically treat at a power of 250 W and a frequency of 40 kHz for 30 min, filter, concentrate the filtrate and make it up to 10 mL, filter with a 0.45 μm microporous membrane, and obtain the solution;

[0044] (2) Preparation of reference solution: Take an appropriate amount of gallic acid reference, weigh accurately, and add methanol to make a solution containing 0.50 mg of reference substance per 1 mL.

[0045] (3) Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A and 0.1% phosphoric acid solution was used as mobile phase B; gradient elution was performed according to the provisions in the following table; injection volume was 10 μL; detection wavelength was 254 nm; flow rate was 1.0 mL / min, and the number of theoretical plates calculated based on the esculetin peak should be no less than 2000;

[0046]

[0047]

[0048] (4) Determination and calculation: 10 μl of each of the gallic acid reference solution and the test solution were injected into a high performance liquid chromatograph for determination; then, the contents of gallic acid, phellodendronine hydrochloride, berberine hydrochloride and rhein in the Fuzhiqing ointment were calculated based on the relative correction factors of gallic acid and aesculetin (0.46), phellodendronine hydrochloride (0.36), berberine hydrochloride (0.52) and rhein (0.40).

[0049] Example 2

[0050] (1) Preparation of test solution: Take the contents of Fuzhiqing ointment, mix well, accurately weigh 5 g, place in a conical flask, add 50 mL of methanol, ultrasonically treat at a power of 250 W and a frequency of 40 kHz for 30 min, filter, concentrate the filtrate and make it up to 5 mL, filter with a 0.45 μm microporous membrane, and obtain the solution;

[0051] (2) Preparation of reference solution: Take an appropriate amount of gallic acid reference, weigh accurately, and add methanol to make a solution containing 0.50 mg of reference substance per 1 mL.

[0052] (3) Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A and 0.05% phosphoric acid solution was used as mobile phase B; gradient elution was performed according to the provisions in the following table; injection volume was 10 μL; detection wavelength was 254 nm; flow rate was 1.0 mL / min, and the number of theoretical plates calculated based on the esculetin peak should be no less than 2000;

[0053]

[0054] Determination and calculation: 10 μl of gallic acid reference solution and test solution were injected into high performance liquid chromatograph for determination; then the contents of gallic acid, phellodendron HCl, berberine hydrochloride and rhein in Fuzhiqing ointment were calculated based on the relative correction factors of gallic acid and fraxinus 0.46, phellodendron HCl relative correction factor 0.36, berberine hydrochloride relative correction factor 0.52 and rhein relative correction factor 0.40.

[0055] Example 3

[0056] (1) Preparation of test solution: Take the contents of Fuzhiqing ointment, mix well, accurately weigh 10 g, place in a conical flask, add 150 mL of methanol, ultrasonically treat at a power of 250 W and a frequency of 40 kHz for 60 min, filter, concentrate the filtrate and make it to 15 mL, filter with a 0.45 μm microporous membrane, and obtain the solution;

[0057] (2) Preparation of reference solution: Take an appropriate amount of gallic acid reference, weigh accurately, and add methanol to make a solution containing 0.50 mg of reference substance per 1 mL.

[0058] (3) Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A and 0.12% phosphoric acid solution was used as mobile phase B; gradient elution was performed according to the provisions in the following table; injection volume was 10 μL; detection wavelength was 254 nm; flow rate was 1.0 mL / min, and the number of theoretical plates calculated based on the esculetin peak should be no less than 2000;

[0059]

[0060] (4) Determination and calculation: 10 μl of each of the gallic acid reference solution and the test solution were injected into a high performance liquid chromatograph for determination; then, the contents of gallic acid, phellodendronine hydrochloride, berberine hydrochloride and rhein in the Fuzhiqing ointment were calculated based on the relative correction factors of gallic acid and aesculetin (0.46), phellodendronine hydrochloride (0.36), berberine hydrochloride (0.52) and rhein (0.40).

[0061] Example 4

[0062] (1) Preparation of test solution: Take the contents of Fuzhiqing ointment, mix well, accurately weigh 6 g, place in a conical flask, add 60 mL of methanol, ultrasonically treat at a power of 250 W and a frequency of 40 kHz for 35 min, filter, concentrate the filtrate and make it up to 6 mL, filter through a 0.45 μm microporous membrane, and obtain the solution;

[0063] (2) Preparation of reference solution: Take an appropriate amount of gallic acid reference, weigh accurately, and add methanol to make a solution containing 0.50 mg of reference substance per 1 mL.

[0064] (3) Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A and 0.06% phosphoric acid solution was used as mobile phase B; gradient elution was performed according to the provisions in the following table; injection volume was 10 μL; detection wavelength was 254 nm; flow rate was 1.0 mL / min, and the number of theoretical plates calculated based on the esculetin peak should be no less than 2000;

[0065]

[0066] (4) Determination and calculation: 10 μl of each of the gallic acid reference solution and the test solution were injected into a high performance liquid chromatograph for determination; then, the contents of gallic acid, phellodendronine hydrochloride, berberine hydrochloride and rhein in the Fuzhiqing ointment were calculated based on the relative correction factors of gallic acid and aesculetin (0.46), phellodendronine hydrochloride (0.36), berberine hydrochloride (0.52) and rhein (0.40).

[0067] Example 5

[0068] (1) Preparation of test solution: Take the contents of Fuzhiqing ointment, mix well, accurately weigh 8 g, place in a conical flask, add 80 mL of methanol, ultrasonically treat at a power of 250 W and a frequency of 40 kHz for 40 min, filter, concentrate the filtrate and make it 8 mL, filter through a 0.45 μm microporous membrane, and obtain the solution;

[0069] (2) Preparation of reference solution: Take an appropriate amount of gallic acid reference, weigh accurately, and add methanol to make a solution containing 0.50 mg of reference substance per 1 mL.

[0070] (3) Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A and 0.07% phosphoric acid solution was used as mobile phase B; gradient elution was performed according to the provisions in the following table; injection volume was 10 μL; detection wavelength was 254 nm; flow rate was 1.0 mL / min, and the number of theoretical plates calculated based on the esculetin peak should be no less than 2000;

[0071]

[0072]

[0073] (4) Determination and calculation: 10 μl of each of the gallic acid reference solution and the test solution were injected into a high performance liquid chromatograph for determination; then, the contents of gallic acid, phellodendronine hydrochloride, berberine hydrochloride and rhein in the Fuzhiqing ointment were calculated based on the relative correction factors of gallic acid and aesculetin (0.46), phellodendronine hydrochloride (0.36), berberine hydrochloride (0.52) and rhein (0.40).

[0074] Example 6

[0075] (1) Preparation of test solution: Take the contents of Fuzhiqing ointment, mix well, accurately weigh 8 g, place in a conical flask, add 100 mL of methanol, ultrasonically treat at a power of 250 W and a frequency of 40 kHz for 45 min, filter, concentrate the filtrate and make it up to 10 mL, filter through a 0.45 μm microporous membrane, and obtain the solution;

[0076] (2) Preparation of reference solution: Take an appropriate amount of gallic acid reference, weigh accurately, and add methanol to make a solution containing 0.50 mg of reference substance per 1 mL.

[0077] (3) Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A and 0.09% phosphoric acid solution was used as mobile phase B; gradient elution was performed according to the provisions in the following table; injection volume was 10 μL; detection wavelength was 254 nm; flow rate was 1.0 mL / min, and the number of theoretical plates calculated based on the esculetin peak should be no less than 2000;

[0078]

[0079]

[0080] (4) Determination and calculation: 10 μl of each of the gallic acid reference solution and the test solution were injected into a high performance liquid chromatograph for determination; then, the contents of gallic acid, phellodendronine hydrochloride, berberine hydrochloride and rhein in the Fuzhiqing ointment were calculated based on the relative correction factors of gallic acid and aesculetin (0.46), phellodendronine hydrochloride (0.36), berberine hydrochloride (0.52) and rhein (0.40).

[0081] Example 7

[0082] (1) Preparation of test solution: Take the contents of Fuzhiqing ointment, mix well, accurately weigh 10 g, place in a conical flask, add 120 mL of methanol, ultrasonically treat at a power of 250 W and a frequency of 40 kHz for 50 min, filter, concentrate the filtrate and make it up to 12 mL, filter with a 0.45 μm microporous membrane, and obtain the solution;

[0083] (2) Preparation of reference solution: Take an appropriate amount of gallic acid reference, weigh accurately, and add methanol to make a solution containing 0.50 mg of reference substance per 1 mL.

[0084] (3) Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A and 0.11% phosphoric acid solution was used as mobile phase B; gradient elution was performed according to the provisions in the following table; injection volume was 10 μL; detection wavelength was 254 nm; flow rate was 1.0 mL / min, and the number of theoretical plates calculated based on the esculetin peak should be no less than 2000;

[0085]

[0086] (4) Determination and calculation: 10 μl of each of the gallic acid reference solution and the test solution were injected into a high performance liquid chromatograph for determination; then, the contents of gallic acid, phellodendronine hydrochloride, berberine hydrochloride and rhein in the Fuzhiqing ointment were calculated based on the relative correction factors of gallic acid and aesculetin (0.46), phellodendronine hydrochloride (0.36), berberine hydrochloride (0.52) and rhein (0.40).

[0087] Example 8

[0088] (1) Preparation of test solution: Take the contents of Fuzhiqing ointment, mix well, accurately weigh 5 g, place in a conical flask, add 60 mL of methanol, ultrasonically treat at a power of 250 W and a frequency of 40 kHz for 45 min, filter, concentrate the filtrate and make it up to 12 mL, filter through a 0.45 μm microporous membrane, and obtain the solution;

[0089] (2) Preparation of reference solution: Take an appropriate amount of gallic acid reference, weigh accurately, and add methanol to make a solution containing 0.50 mg of reference substance per 1 mL.

[0090] (3) Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A and 0.10% phosphoric acid solution was used as mobile phase B; gradient elution was performed according to the provisions in the following table; injection volume was 10 μL; detection wavelength was 254 nm; flow rate was 1.0 mL / min, and the number of theoretical plates calculated based on the esculetin peak should be no less than 2000;

[0091]

[0092] (4) Determination and calculation: 10 μl of each of the gallic acid reference solution and the test solution were injected into a high performance liquid chromatograph for determination; then, the contents of gallic acid, phellodendronine hydrochloride, berberine hydrochloride and rhein in the Fuzhiqing ointment were calculated based on the relative correction factors of gallic acid and aesculetin (0.46), phellodendronine hydrochloride (0.36), berberine hydrochloride (0.52) and rhein (0.40).

[0093] In order to further verify the effectiveness of the present invention, the inventor conducted a series of verification experiments, the excerpts of which are as follows:

[0094] Experimental example

[0095] 1. Reagents and test drugs

[0096] Berberine hydrochloride reference substance (batch number: 110713-202109, source: China Food and Drug Inspection Institute); phellodendron chinense hydrochloride (batch number: 111895-201805, source: China Food and Drug Inspection Institute); rhubarb reference substance (batch number: 110756-201913, source: China Food and Drug Inspection Institute); quercetin reference substance (batch number: 110741-202310, source: China Food and Drug Inspection Institute); gallic acid reference substance (batch number: 110756-201913, source: China Food and Drug Inspection Institute); Fuzhiqing Ointment (Guizhou Green Sun Pharmaceutical Co., Ltd., batch numbers: 20230316, 20230418, 20230427). Water was Wahaha purified water, acetonitrile was chromatographically pure (Tian Di Co., Ltd., USA), and phosphoric acid was analytically pure (Chongqing Wansheng Chuandong Chemical Co., Ltd.).

[0097] 2 Instruments

[0098] Electronic balance (AUW-220D, Shimadzu, Japan); CNC ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd., Jiangsu Province); Electronic balance (Shimadzu Libror AEL 40SM, Sartorius); High performance liquid chromatograph (LC-2010AHT, Shimadzu, Japan). Chromatographic columns: ZORBAX Eclipse XDB-C18 (4.6×250mm, 5μm); Agilent5TC-C18 (4.6×250mm, 5μm); XTERRA MS C18 (4.6×250mm, 5μm).

[0099] 3. Detection method research

[0100] 3.1 Investigation of extraction methods

[0101] Take about 10g of Fuzhiqing ointment (20230316), accurately weigh two portions, put them in a stoppered conical flask, accurately add 100ml of methanol, weigh the weight, seal it, heat reflux and ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes respectively, filter, concentrate the filtrate and make the volume to 10mL, filter, and obtain the test solution; take appropriate amounts of gallic acid, esculetin, phellodendrin hydrochloride, berberine hydrochloride and rhamnosine reference substances, accurately weigh them, add methanol to prepare a mixed reference substance solution containing 0.50mg, 0.050mg, 0.020mg, 0.10mg and 0.020mg per 1mL; take 10μl of the test solution and the mixed reference substance solution, inject them into the liquid chromatograph for determination, and calculate the contents of berberine hydrochloride, phellodendrin hydrochloride, esculetin, gallic acid and rhamnosine. The results are shown in Table 1, Figure 1 , Figure 2 .

[0102] Table 1 Results of investigation on extraction methods

[0103]

[0104] The test results show that the contents of the components extracted by the two extraction methods are not much different. In order to simplify the experimental operation, ultrasonic extraction was selected as the extraction method.

[0105] 3.2 Investigation of extraction solvent

[0106] Take about 10g of Fuzhiqing Ointment (20230316), accurately weigh 4 portions, and use methanol, 30% methanol, 50% methanol, and 80% methanol to ultrasonically prepare the test solution, respectively. Determine according to the method under "3.1" and calculate the contents of berberine hydrochloride, phellodendrine hydrochloride, fraxinothrin, gallic acid and rhamnolide. The results are shown in Table 2. Figure 3 , Figure 4 , Figure 5 , Figure 6 .

[0107] Table 2 Extraction solvent investigation results

[0108]

[0109] The test results show that when methanol is used for extraction, the content of each component to be tested is the highest and the extraction is more complete, so methanol is selected as the extraction solvent.

[0110] 3.3 Extraction time investigation

[0111] Take about 10g of Fuzhiqing Ointment (20230316), accurately weigh 3 portions, set the ultrasonic time to 30 minutes, 45 minutes, and 60 minutes respectively, prepare the test solution, determine the peak area of ​​the test solution according to the method under "3.1", and calculate the contents of berberine hydrochloride, phellodendronine hydrochloride, esculetin, gallic acid and rhamnolide. The results are shown in Table 3. Figure 7 , Figure 8 , Fig. 9 .

[0112] Table 3 Extraction time investigation results

[0113]

[0114] The test results show that different ultrasonic times have little effect on the content of each component to be tested. From the perspective of energy saving and emission reduction and simplifying the processing method, the ultrasonic time is selected as 30 minutes.

[0115] 3.4 Detection wavelength selection

[0116] The test solution and the reference solution under item "3.3" were scanned at full wavelength using a UV detector, and the chromatographic information at wavelengths of 254nm, 265nm, 272nm, 284nm, and 344nm was compared. The results showed that the chromatographic peak separation and peak shape at 254nm were better, the baseline was more stable, and it was better than other wavelengths. Therefore, 254nm was selected as the detection wavelength.

[0117] 3.5 Mobile phase investigation

[0118] Take about 10g of Fuzhiqing Ointment (20230316), accurately weigh it, prepare the test solution according to the preferred preparation method of the test solution, select methanol and acetonitrile as the organic phase, select 0.1% phosphoric acid as the aqueous phase, set the gradient according to Table 4, Table 5, Table 6 and Table 7 for elution, and the results are shown in Table Fig.10 , Fig.11 , Fig.12 , Fig.13 .

[0119] Table 4 Gradient elution program 1

[0120]

[0121] Table 5 Gradient elution program 2

[0122]

[0123] Table 6 Gradient elution program 3

[0124]

[0125]

[0126] Table 7 Gradient elution program 4

[0127]

[0128] The test results show that the chromatographic peak shape is good, the separation is good, and the baseline is stable when gradient elution is performed according to the conditions of gradient elution program 1; the peak to be measured and the impurity peak are poorly separated, and the peak shape is seriously tailing when gradient elution is performed according to the conditions of gradient elution program 2; the chromatographic peak shape is poor, and the peaks of aesculetin and phellodendrine hydrochloride are seriously tailing when gradient elution is performed according to the conditions of gradient elution program 3; the gallic acid peak and the aesculetin peak partially overlap and cannot be separated when gradient elution is performed according to the conditions of gradient elution program 4. Therefore, the parameters of gradient elution program 1 are selected as the mobile phase.

[0129] 4 Methodological Validation

[0130] 4.1 Investigation of linear relationship

[0131] 2 μL, 6 μL, 10 μL, 14 μL, 18 μL and 22 μL of the mixed reference solution were accurately aspirated respectively, and the samples were injected and measured according to the above chromatographic conditions. 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 that the linear relationship is good.

[0132] Table 8 Linear relationship investigation results

[0133]

[0134] 4.2 Precision investigation

[0135] Take about 10g of Fuzhiqing Ointment (20230316), weigh accurately, prepare the test solution according to the preparation method of the test solution, measure according to the above chromatographic conditions, inject 6 injections continuously, and measure the precision of the instrument. Calculate the contents of berberine hydrochloride, phellodendrine hydrochloride, fraxinoyl ester, gallic acid and emodin, and calculate the RSD. The results are shown in Table 9.

[0136] Table 9 Precision investigation results

[0137]

[0138] The results of the precision experiment showed that the RSD of each component to be tested was in the range of 0.82-2.07%, indicating that the precision of the instrument was good.

[0139] 4.3 Repeatability study

[0140] Using Shimadzu LC-2010AHT high performance liquid chromatograph, take about 10g of Fuzhiqing ointment (20230316), accurately weigh, take 6 portions in total, prepare the test solution according to the preparation method of the test solution, and determine according to the above chromatographic conditions. Calculate the contents of berberine hydrochloride, phellodendron hydrochloride, fraxin, gallic acid and rhamnolide, and calculate the RSD. The results are shown in Table 10.

[0141] Table 10 Repeatability test results

[0142]

[0143]

[0144] The results of the repeatability experiment showed that the RSD of each component to be tested in the six repeatability experimental samples was in the range of 1.04-2.07%, indicating that the repeatability was good.

[0145] 4.4 Stability investigation

[0146] Take about 10g of Fuzhiqing ointment (20230316), accurately weigh, prepare the test solution according to the preparation method of the test solution, accurately draw 10μL of the test solution, inject it into the liquid chromatograph for detection at 0, 2, 4, 6, 8, and 12h, respectively, and determine it according to the chromatographic conditions of Example 1. Calculate the contents of berberine hydrochloride, phellodendrine hydrochloride, fraxinothrin, gallic acid and rhamnolide, and calculate the RSD. The results are shown in Table 11.

[0147] Table 11 Stability test results

[0148]

[0149] The results of stability study showed that the RSD values ​​of aesculetin, berberine hydrochloride, phellodendronine hydrochloride, gallic acid and rhamnolide were 0.82%, 1.32%, 1.47%, 1.14% and 2.07%, respectively, indicating that the test solution had good stability within 12 hours.

[0150] 4.5 Spike recovery test

[0151] Weigh 6 samples accurately, 5g each, and add each reference substance accurately according to the ratio of 1:1 between the content of each component in the sample and the corresponding reference substance. Prepare the test solution according to the test sample preparation method, inject and analyze, and calculate the average recovery rate and RSD value of each component. The results are shown in Table 12, Table 13, Table 14, Table 15, Table 16. The average recovery rates of esculetin, berberine hydrochloride, phellodendron hydrochloride, rhamnolide and gallic acid were calculated to be 99.52%, 99.93%, 98.99%, 98.98% and 100.01%, respectively, and the RSD values ​​were 0.49%, 0.28%, 0.48%, 0.45% and 0.21%, respectively, indicating that the recovery rate of this method is good.

[0152] Table 12 Fraxinyl recovery test

[0153]

[0154] Table 13 Berberine hydrochloride recovery test

[0155]

[0156] Table 14 Phellodendron hydrochloride recovery test

[0157]

[0158] Table 15 Rhein recovery test

[0159]

[0160] Table 16 Gallic acid recovery test

[0161]

[0162]

[0163] 5Relative correction factor

[0164] 5.1 Calculation of relative correction factor

[0165] The mixed reference solution was injected into a liquid chromatograph for determination. Gallic acid was used as an internal reference to calculate the relative correction factors of aesculetin, phellodendrine hydrochloride, berberine hydrochloride and rhamnolide. The results are shown in Table 17.

[0166]

[0167] Among them, A s is the peak area of ​​the internal reference, W S is the concentration (or mass) of the internal reference, A k is the peak area of ​​the component to be measured, W k is the concentration (or mass) of the component k to be measured.

[0168] Table 17 Relative correction factors

[0169]

[0170] 5.2 Reproducibility study of relative correction factor

[0171] 5.2.1 Investigation of different instruments and different chromatographic columns

[0172] The relative correction factor was measured using different instruments and different chromatographic columns. The results are shown in Table 18. The measured RSD values ​​of the relative correction factors were between 0.64% and 0.95%, indicating that the relative correction factors of the components to be measured had good reproducibility between different instruments and different chromatographic columns.

[0173] Table 18 Effects of different instruments and different chromatographic columns on the relative correction factors of the components to be measured

[0174]

[0175]

[0176] 5.2.2 Investigation of different flow rates

[0177] The relative correction factor was measured at different flow rates. The results are shown in Table 19. The measured RSD values ​​of the relative correction factors were between 1.12% and 1.62%, indicating that the relative correction factors of the components to be measured had good reproducibility at different flow rates.

[0178] Table 19 Effect of different flow rates on relative correction factors of the components to be measured

[0179]

[0180] 5.2.3 Investigation of different column temperatures

[0181] The relative correction factor was measured using different column temperatures. The results are shown in Table 20. The measured RSD values ​​of the relative correction factors were between 1.12% and 1.62%, indicating that the relative correction factors of the components to be measured had good reproducibility between different column temperatures.

[0182] Table 20 Effect of different column temperatures on the relative correction factors of the components to be measured

[0183]

[0184] 6. Chromatographic peak location of the component to be measured

[0185] The relative retention values ​​(r) of each component to be tested and the internal reference gallic acid were calculated respectively, and the reproducibility between different instruments and different chromatographic columns was investigated. The results are shown in Table 21. The relative retention time fluctuated slightly. Finally, the average value of the relative retention time values ​​of each component to be tested in different instruments and different chromatographic columns was selected as the basis for peak positioning. According to the test results, the relative retention time values ​​of gallic acid and aesculetin, phellodendrine hydrochloride, and berberine hydrochloride were determined to be 1.44, 2.58, 3.28, and 3.98, respectively. According to relevant technical principles, the relative retention time should be within ±10% of the specified value.

[0186] Table 21 Relative retention time of each component in different instruments and different chromatographic columns

[0187]

[0188]

[0189] 7 Comparison of the results of the one-measurement-multiple-evaluation method and the external standard method

[0190] Three batches of Fuzhiqing ointment were taken respectively, and the test solution was prepared according to the above method. The contents of gallic acid, berberine hydrochloride, phellodendron hydrochloride, rhamnolide, and fraxin were calculated by the one-measurement multiple evaluation method (QASM method) and the external standard method (ESM method), respectively, and the results are shown in Table 22. The contents calculated by the external standard method and the one-measurement multiple evaluation method were compared by t-test, and P was much greater than 0.05, indicating that there was no significant difference in the contents measured by the two methods, and the relative error between the two groups of contents was <3%, indicating that the established one-measurement multiple evaluation method has good accuracy and can be used for the content determination of Fuzhiqing ointment.

[0191] Table 22 Results of determination of 5 components by one-test-multiple-evaluation method and external standard method

[0192]

[0193] Although the present invention has been described in detail above using general descriptions, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A method for determining the contents of five ingredients in Fuzhiqing ointment based on a one-test-multiple-evaluation method, wherein the prescription of the Fuzhiqing ointment is: 50g of Jin Guolan, 50g of Rheum officinale, 20g of Phellodendron chinense, 30g of Cinnabar Root, 30g of Chrysanthemum indica, 40g of Viola yedoensis, and 20g of Snow Gallbladder , 50g of Sophora flavescens, 20g of Paris polyphylla, 20g of Dioscorea bulbifera, 20g of Curcuma longa, 50g of Sanguisorba officinalis, 20g of Kudingcha, 16g of Borneolum, and 3g of menthol; the preparation method of the Fuzhiqing ointment is as follows: the above 15 flavors, except Borneolum and menthol, the remaining 13 flavors of Phellodendron chinense, are decocted twice with water, the first time for 2 hours and the second time for 1.5 hours, the decoctions are combined, filtered, and the filtrate is concentrated to a clear paste with a relative density of 1.05 at 60°C; 60g of sorbitol, 4.2g of guar gum, and 30g of beta-cyclodextrin are added, heated to 80°C to dissolve, and set aside; 10g of stearic acid, 80g of monoglyceride, 80g of octadecyl alcohol, 20g of polysorbate 80, 20g of paraffin oil, and 40g of Span 80 are heated to 80°C to melt, added to the reserved clear paste, mixed, and emulsified for 15 minutes, Keep warm at 80°C for 30 minutes, when the temperature drops to 40°C, add borneol, menthol and 10g of laurocapram, 6g of benzoic acid and 1.5g of ethylparaben, mix well and make 1000g, that is, The method for determining the contents of the five components in Fuzhiqing ointment by the one-test-multiple-evaluation method is as follows: (1) preparing the test solution; (2) preparing the reference solution; (3) performing chromatographic conditions and system suitability tests; (4) determining and calculating: The (1) test solution is prepared as follows: take the contents of Fuzhiqing ointment, mix well, accurately weigh 5-10 g, place in a conical flask, add 50-150 mL of methanol, ultrasonically treat at a power of 250 W and a frequency of 40 kHz for 30-60 min, filter, concentrate the filtrate and make it 5-15 mL, filter with a 0.45 μm microporous membrane, and obtain; The preparation of the reference substance solution (2) is as follows: take an appropriate amount of gallic acid reference substance, accurately weigh it, and add methanol to make a solution containing 0.50 mg of reference substance per 1 mL; The (3) chromatographic conditions and system suitability test are as follows: octadecylsilane bonded silica gel is used as filler; acetonitrile is used as mobile phase A, and 0.05-0.12% phosphoric acid solution is used as mobile phase B; gradient elution is performed according to the provisions in the following table; the injection volume is 10 μL; the detection wavelength is 254 nm; the flow rate is 1.0 mL / min, and the theoretical plate number calculated based on the esculetin peak should be no less than 2000; ； The determination and calculation of (4) are as follows: 10 μl of each of the gallic acid reference solution and the test solution are injected into a high performance liquid chromatograph for determination; then, based on the relative correction factors of gallic acid and aesculetin, phellodendronine hydrochloride, berberine hydrochloride and rhamnolide, the contents of gallic acid, phellodendronine hydrochloride, berberine hydrochloride and rhamnolide in the Fuzhiqing ointment are calculated respectively.

2. The method for determining the contents of five components in Fuzhiqing ointment by a one-test-multiple-evaluation method as claimed in claim 1, characterized in that: The (1) test solution is prepared as follows: take the contents of Fuzhiqing ointment, mix well, accurately weigh 8-10 g, place in a conical flask, add 100-150 mL of methanol, ultrasonically treat at a power of 250 W and a frequency of 40 kHz for 30-40 min, filter, concentrate the filtrate and make it 5-10 mL, filter with a 0.45 μm microporous membrane, and obtain the solution.

3. The method for determining the contents of five components in Fuzhiqing ointment by a one-test-multiple-evaluation method as claimed in claim 2, characterized in that: The (1) test solution is prepared as follows: take the contents of Fuzhiqing ointment, mix well, accurately weigh 10 g, place in a conical flask, add 100 mL of methanol, ultrasonically treat at a power of 250 W and a frequency of 40 kHz for 30 min, filter, concentrate the filtrate and make it 10 mL, filter with a 0.45 μm microporous membrane, and obtain the solution.

4. The method for determining the contents of five components in Fuzhiqing ointment by a one-test-multiple-evaluation method as claimed in claim 1, characterized in that: The (3) chromatographic conditions and system suitability test are as follows: octadecylsilane bonded silica gel is used as filler; acetonitrile is used as mobile phase A, and 0.10% phosphoric acid solution is used as mobile phase B; gradient elution is performed according to the provisions in the following table; the injection volume is 10 μL; the detection wavelength is 254 nm; the flow rate is 1.0 mL / min, and the theoretical plate number calculated based on the esculetin peak should be no less than 2000; 。 5. The method for determining the contents of five components in Fuzhiqing ointment by a one-test-multiple-evaluation method as claimed in claim 1, characterized in that: The relative correction factors of gallic acid with aesculetin, phellodendrine hydrochloride, berberine hydrochloride and emodin are 0.46, 0.36, 0.52 and 0.40 respectively.

6. The method for determining the contents of five components in Fuzhiqing ointment by a one-test-multiple-evaluation method as claimed in claim 1, characterized in that: The relative retention time values ​​of the gallic acid with aesculetin, phellodendrine hydrochloride and berberine hydrochloride are 1.44, 2.58, 3.28 and 3.98, respectively, and the relative retention time is within ±10% of the specified value.

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