CN115541773A - Detection method for 5 rhubarb anthraquinone components in traditional Chinese medicine composition for treating nephropathy - Google Patents

Abstract

The invention relates to a detection method of 5 rhubarb anthraquinone components in a traditional Chinese medicine composition for treating nephropathy, which can simultaneously determine the contents of aloe-emodin, rhein, emodin, chrysophanol and physcion in the traditional Chinese medicine composition for treating nephropathy by high performance liquid chromatography. The detection method can simply, conveniently and efficiently quantitatively detect the content of the effective components of the traditional Chinese medicine composition for treating the nephropathy, thereby realizing objective, comprehensive and accurate evaluation of the quality of the traditional Chinese medicine composition for treating the nephropathy, and having important significance for controlling the quality of the traditional Chinese medicine composition for treating the nephropathy and ensuring the curative effect.

Description

Detection method for 5 rhubarb anthraquinone components in traditional Chinese medicine composition for treating nephropathy

The application is a divisional application of an invention patent application with the application date of 31/12/2019 and the application number of 201911406208.2, and the invention name of a method for detecting 5 rhubarb anthraquinone components in a traditional Chinese medicine composition for treating nephropathy.

Technical Field

The invention relates to the field of drug detection, in particular to a method for detecting the content of 5 rhubarb anthraquinone components in a traditional Chinese medicine composition for treating nephropathy.

Background

Renal diseases such as renal failure are common serious diseases in clinic, dialysis and kidney transplantation are generally adopted for treatment, but the medical cost is too high, the general family economy cannot bear the treatment, and complications are easily caused in the dialysis treatment process. Rejection reactions also commonly occur after kidney transplantation surgery, and for this reason, patients and their families are generally difficult to bear. The traditional Chinese medicine such as Shenshuaining capsules and the like can be applied to effectively avoid the occurrence of the above situations, can effectively relieve symptoms and reduce the pain of patients, but needs to be improved in order to ensure more remarkable clinical curative effect. In order to ensure that the high-quality traditional Chinese medicines for treating the kidney diseases, such as Shenshuaining capsules, can be used for treating the kidney diseases, and can fully exert the effects in clinic, so that patients can recover the health as soon as possible, the quality control of the medicines is very important, but the quality control method of the traditional medicine, shenshuaining capsules for treating the kidney diseases does not include the quantitative detection of the active ingredients of the monarch drug rhubarb, can not well reflect the quality of a preparation, and thus the active ingredients of the rhubarb are not fully utilized and monitored. Therefore, in order to maintain the benefit of patients, it is necessary to research and design a detection method capable of simultaneously determining the contents of the effective components of aloe-emodin, rhein, emodin, chrysophanol and physcion in rhubarb on the basis of the prior art.

In the aspect of preventing and treating diseases, the traditional Chinese medicine and the extracted preparation thereof occupy important positions, and the quality control method mainly comprises microscopic identification, thin-layer identification, content measurement and the like. In order to eliminate interference, the pretreatment procedure of the sample is complex and complicated, a large amount of organic reagents are needed for repeated purification treatment, the method is labor-consuming, time-consuming, reagent-consuming, environment-polluting, health-endangering and long in detection period. The quality standard detection comprising 5 to 6 items of identification and content measurement is finished, generally, the time is 4 to 5 days, if the time is longer in the case of repeated tests, the detection speed seriously restricts the modernized production speed of the traditional Chinese medicine. Therefore, finding a simple, fast and comprehensive content detection method for controlling quality becomes a necessary breakthrough in traditional Chinese medicine production.

Disclosure of Invention

In order to solve the above problems, the present invention provides a detection method for simultaneously determining the content of aloe-emodin, rhein, emodin, chrysophanol and physcion, which are traditional Chinese medicine compositions for treating nephropathy, wherein the detection method is performed by high performance liquid chromatography, and the conditions of the high performance liquid chromatography comprise: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile is used as a mobile phase A, phosphoric acid aqueous solution with the volume ratio concentration of 0.1% is used as a mobile phase B, and elution is carried out according to the gradient that the volume ratio of the mobile phase A to the mobile phase B is changed from 35:65 to 80:20 within 0-40 minutes.

Preferably, the detection method comprises the following steps:

(1) Preparation of control solutions: mixing appropriate amount of aloe-emodin reference substance, rhein reference substance, emodin reference substance, chrysophanol reference substance, and physcion reference substance, and adding methanol to obtain reference substance solution;

(2) Preparation of a test solution: adding methanol into the Chinese medicinal composition, ultrasonic treating, filtering, collecting filtrate, evaporating to dryness under reduced pressure, adding 4% hydrochloric acid, heating in water bath under reflux, extracting with diethyl ether, evaporating to dryness under reduced pressure, dissolving the residue with methanol, filtering, and collecting filtrate to obtain total anthraquinone sample solution;

(3) The measuring method comprises the following steps: precisely sucking the reference substance solution prepared in the step (1) and the test solution prepared in the step (2) respectively, injecting the reference substance solution and the test solution into a high performance liquid chromatograph, and measuring the chromatograms of the test solution and the reference substance solution according to the conditions of the high performance liquid chromatography, wherein the conditions of the high performance liquid chromatography comprise: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile is used as a mobile phase A, phosphoric acid aqueous solution with the volume ratio concentration of 0.1 percent is used as a mobile phase B, and elution is carried out according to the gradient that the volume ratio of the mobile phase A to the mobile phase B is changed from 35:65 to 80:20 at a constant speed within 0-40 minutes.

More preferably, the detection method comprises the following steps:

(1) Preparation of control solutions: respectively taking appropriate amount of aloe-emodin reference substance, rhein reference substance, emodin reference substance, chrysophanol reference substance, and physcion reference substance, and adding methanol to obtain reference substance solution;

(2) Preparing a test solution: precisely weighing the traditional Chinese medicine composition, precisely adding methanol, weighing, ultrasonically treating for 60 minutes, cooling, supplementing the lost weight with methanol, shaking up, filtering, taking the subsequent filtrate, evaporating to dryness under reduced pressure, adding hydrochloric acid with a volume ratio of 4%, heating and refluxing in a water bath for 30-60 minutes, immediately cooling, adding diethyl ether for extraction, evaporating to dryness under reduced pressure, dissolving the residue with methanol, transferring into a measuring flask, fixing the volume to the scale with methanol, shaking up, filtering, taking the subsequent filtrate, and obtaining the total anthraquinone sample solution; preferably, the water bath is heated to reflux for 60 minutes;

(3) The measuring method comprises the following steps: and (3) precisely sucking the reference substance solution prepared in the step (1) and the test substance solution prepared in the step (2) respectively, injecting the reference substance solution and the test substance solution into a high performance liquid chromatograph, and measuring the chromatograms of the test substance solution and the reference substance solution according to the conditions of the high performance liquid chromatography.

According to one embodiment of the present invention, the Chinese medicinal composition comprises salvia miltiorrhiza, rhubarb, radix pseudostellariae, coptis chinensis, achyranthes bidentata, pinellia ternate, safflower, poria cocos, dried orange peel and liquorice.

According to a preferred embodiment of the present invention, wherein the Chinese medicinal composition comprises, in parts by weight: 20-30 parts of radix pseudostellariae, 20-30 parts of pinellia ternate, 16-24 parts of poria cocos, 56-84 parts of salvia miltiorrhiza, 8-12 parts of safflower, 8-12 parts of coptis chinensis, 8-12 parts of pericarpium citri reticulatae, 32-48 parts of rheum officinale, 16-24 parts of achyranthes bidentata and 8-12 parts of liquorice.

According to a more preferred embodiment of the present invention, said pinellia ternata is rhizoma pinellinae praeparata.

According to a most preferred embodiment of the present invention, the Chinese medicinal composition is shenshuaining capsule.

Preferably, in the detection method, the high performance liquid chromatography conditions further include: the column temperature is 20-40 ℃; the flow rate is 0.95-1.05ml per minute; the ultraviolet detection wavelength is 250-260nm; the number of theoretical plates is not less than 4000 calculated according to emodin peak.

Further preferably, in the detection method, the high performance liquid chromatography conditions further include a column temperature of 40 ℃; the flow rate was 1ml per minute; ultraviolet detection wavelength: 254nm.

According to a specific embodiment of the present invention, the detection method comprises the steps of:

(1) Preparation of control solutions: respectively taking appropriate amount of aloe-emodin reference substance, rhein reference substance, emodin reference substance, chrysophanol reference substance, and physcion reference substance, precisely weighing, and adding methanol to obtain solutions containing 100 μ g of aloe-emodin, rhein, emodin or chrysophanol and 50 μ g of physcion per 1ml; respectively precisely measuring 10ml of each reference substance solution, placing in a 100ml measuring flask, adding methanol to scale, and shaking to obtain reference substance solution containing aloe-emodin, rhein, emodin or chrysophanol 10 μ g and physcion 5 μ g per 1ml;

(2) Preparing a test solution: precisely weighing 0.5g of the Chinese medicinal composition, precisely adding 25ml of methanol, weighing, ultrasonically treating at the power of 250W and the frequency of 50kHz for 60 minutes, cooling, supplementing the lost weight with methanol, shaking up, filtering, and taking the subsequent filtrate to obtain a free anthraquinone sample solution; and

taking 0.5g of the traditional Chinese medicine composition, precisely weighing, precisely adding 50ml of methanol, weighing the weight, carrying out ultrasonic treatment with the power of 250W and the frequency of 50kHz for 60 minutes, cooling, complementing the weight loss by using the methanol, shaking up, filtering, taking 25ml of subsequent filtrate, carrying out reduced pressure evaporation to dryness, adding 50ml of 4% (volume ratio) hydrochloric acid, heating and refluxing in a water bath for 1 hour, immediately cooling, adding diethyl ether for extraction for 3 times, 50ml each time, combining diethyl ether layers, carrying out reduced pressure evaporation to dryness, adding 20ml of methanol to residues for dissolution, transferring into a 25ml measuring flask, fixing the volume to a scale by using the methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the total anthraquinone sample solution.

(3) High performance liquid chromatography conditions: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile is used as a mobile phase A, 0.1 percent (volume ratio) phosphoric acid aqueous solution is used as a mobile phase B, and uniform-speed elution is carried out according to the specified gradient in the following table, wherein the column temperature is 40 ℃, the flow rate is 1ml per minute, the ultraviolet detection wavelength is 254nm, and the number of theoretical plates is not less than 4000 according to the emodin peak.

(4) The determination method comprises the following steps: and (3) precisely weighing and sucking 10 mu l of each of the reference solution prepared in the step (1) and the test solution prepared in the step (2), injecting into a liquid chromatograph, and measuring the chromatograms of the test solution and the reference solution.

According to the invention, according to the policy of ensuring the safety and the effectiveness of the preparation in China, the high performance liquid chromatography technology is utilized to establish the method for detecting the effective components of the traditional Chinese medicine composition (such as Shenshuaining capsules) for treating the nephropathy, so that the quantitative determination of the effective components of the monarch drug rhubarb in the traditional Chinese medicine prescription is realized, and the safety and the effectiveness of the preparation are ensured. Compared with the conventional method, the detection method does not need concentration, extraction and evaporation steps, is simple, convenient, rapid, high in efficiency, low in cost and free of pollution, effectively improves the accuracy and reproducibility of results, and can be used for controlling the quality of the traditional Chinese medicine composition for treating the nephropathy.

Specifically, the method for detecting the content of 5 free anthraquinone and total anthraquinone components in the traditional Chinese medicine composition for treating nephropathy (such as Shenshuaining capsules) by using the high performance liquid chromatography established by the invention can realize one-time sample injection, simultaneously separate 5 components and accurately quantify the components. The method has high specificity and accuracy, and can be used for controlling quality of Chinese medicinal materials. In the preparation process of the total anthraquinone sample, trichloromethane and other reagents with high toxicity are not used, thus meeting the requirements of environmental protection and safety. Moreover, the detection method provided by the invention is designed by combining years of medicine detection research and clinical experience, has good linear relation, precision, repeatability, stability, accuracy and specificity, is stable and reliable, can objectively, comprehensively and accurately evaluate the quality of the traditional Chinese medicine composition for treating the nephropathy, and provides guarantee for controlling the quality of the traditional Chinese medicine composition (such as Shenshuaining capsules) and ensuring the clinical curative effect.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a graph of the UV absorption of aloe-emodin;

FIG. 2 is a graph showing the UV absorption of rhein;

FIG. 3 is a graph of the ultraviolet absorption of emodin;

fig. 4 is a graph of the ultraviolet absorption of chrysophanol;

FIG. 5 is a diagram showing the ultraviolet absorption of physcion;

FIG. 6 is an HPLC chromatogram of 5 anthraquinone controls in rhubarb;

FIG. 7 is an HPLC chromatogram for the determination of 5 free anthraquinone samples;

FIG. 8 is an HPLC chromatogram for measuring total anthraquinone sample;

FIG. 9 is an HPLC chromatogram of a blank sample lacking rhubarb free anthraquinone;

FIG. 10 is an HPLC chromatogram of a blank sample lacking total anthraquinone in rhubarb;

FIG. 11 is a standard graph of aloe-emodin;

FIG. 12 is a standard graph of rhein;

FIG. 13 is a standard graph of emodin;

fig. 14 is a standard graph of chrysophanol;

FIG. 15 is a graph of physcion standard curve;

FIG. 16 is the HPLC chromatogram of the second method for hydrolyzing total anthraquinone in rhubarb;

FIG. 17 is an HPLC chromatogram of the first method for hydrolyzing total anthraquinone in rhubarb.

FIG. 18 is an HPLC chromatogram of 5 anthraquinone controls in rhubarb of comparative example 1;

FIG. 19 is an HPLC chromatogram of 5 free anthraquinone samples tested for sample 1 in comparative example 1;

FIG. 20 is an HPLC chromatogram of the total anthraquinone test sample of sample 1 determined in comparative example 1;

FIG. 21 is an HPLC chromatogram of 5 anthraquinone controls in rhubarb of comparative example 2;

FIG. 22 is an HPLC chromatogram of 5 free anthraquinone samples tested for sample 1 in comparative example 2;

FIG. 23 is an HPLC chromatogram of 5 total anthraquinone samples tested for sample 1 in comparative example 2;

FIG. 24 is an HPLC chromatogram of 5 anthraquinone controls in rhubarb of comparative example 3;

FIG. 25 is an HPLC chromatogram of 5 free anthraquinone samples tested in sample 1 of comparative example 3;

FIG. 26 is an HPLC chromatogram of the total anthraquinone test sample of sample 1 determined in comparative example 3.

Detailed Description

The present invention will be described in further detail with reference to specific embodiments thereof, which will be better understood from the following examples. However, it should be readily understood by those skilled in the art that the following examples are illustrative only and are not intended to limit the present invention to these specific embodiments. It will be appreciated by those skilled in the art that the present invention encompasses all modifications, alternatives, and equivalents as may be included within the scope of the claims.

EXAMPLE 1 content determination of free anthraquinone and Total anthraquinone in Rheum officinale

1.1 instruments and reagents

1.1.1 Instrument and test conditions

Agilent-1260 liquid chromatograph (HP-1260 series quaternary pump, DAD detector, autosampler)

Eilide ODS-BP column (4.6X 250mm,5 μm)

Agilent EC-C18(4.6×250mm，4μm)

HP-1260 liquid chromatograph for ultraviolet spectrum determination

Acetonitrile (chromatographically pure, merck), methanol (chromatographically pure, merck), water is ultrapure water, and other reagents are analytically pure.

Balance: OHAUS AR224-N electronic balance (ten thousandths), EX125ZH electronic analytical balance (one hundred thousand), aohaus instruments (Changzhou), inc.;

the instrument comprises the following steps: DK-98II A electric heating constant temperature water bath, tester instruments ltd, tianjin;

an ultrasonic instrument: SB25-12DTD ultrasonic instrument, ningbo Xinzhi Biotech GmbH;

1.1.2 reagents and samples

Aloe-emodin reference substance (number: 110795-201710, provided by China institute for food and drug testing, for content determination, 98.3%)

Rhein reference substance (number: 110757-201607, provided by China institute for testing food and drug, for content determination, 99.3%)

Emodin reference substance (number: 110796-2011512, provided by China food and drug testing research institute for content determination, 98.7%)

Chrysophanol reference substance (number: 110756-201621, provided by China institute for testing food and drug, 99.2%)

Physcion reference substance (number: 110758-201616, provided by China institute for testing food and drug, for content determination, 99.0%)

Shenshuaining capsule: are all provided by Yunan Lei Yi ideal pharmaceutical Co., ltd (batch No.: 20190303, 20190403, 20190404, 20190507, 20170703, 20190520, 20190523, 20190524, 20190605, 20190615, 20190812, 20190414, 20190419, 20190421, 20190705, 20190708, 20190711, 20190714, 20190716 and 20190720)

Preparing negative control medicinal materials: the used medicinal materials are purchased from Kunming Jingtian pharmaceutical industry Co Ltd, and prepared according to the preparation method.

1.2 determination method:

chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile is taken as a mobile phase A, 0.1 percent (volume ratio) phosphoric acid aqueous solution is taken as a mobile phase B, and uniform elution is carried out according to the gradient specified in the following table; column temperature: 40 ℃; the flow rate was 1ml per minute; detection wavelength: 254nm. The number of theoretical plates is not less than 4000 calculated according to emodin peak.

Preparation of control solutions: taking appropriate amount of aloe-emodin reference substance, rhein reference substance, emodin reference substance, chrysophanol reference substance, and physcion reference substance, precisely weighing, and adding methanol to obtain solution containing 100 μ g of aloe-emodin, rhein, emodin, and chrysophanol and 50 μ g of physcion per 1ml; respectively precisely measuring 10ml of each of the above reference solutions, placing in a 100ml measuring flask, adding methanol to scale, and shaking to obtain the final product (each 1ml contains 10 μ g of aloe-emodin, rhein, emodin, and chrysophanol, and 5 μ g of physcion).

Preparation of a test solution:

free anthraquinone: precisely weighing 0.5g of the content, precisely adding 25ml of methanol, weighing, ultrasonically treating for 60 minutes (power 250W and frequency 50 kHz), cooling, supplementing the lost weight with methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the free anthraquinone sample solution.

Total anthraquinone: taking 0.5g of contents, accurately weighing, accurately adding 50ml of methanol, weighing the weight, carrying out ultrasonic treatment for 60 minutes (power 250W and frequency 50 kHz), cooling, complementing the lost weight with methanol, shaking up, filtering, taking 25ml of subsequent filtrate, carrying out reduced pressure evaporation to dryness, adding 50ml of 4% (v/v) hydrochloric acid, heating and refluxing in a water bath for 1 hour, cooling immediately, adding diethyl ether for extraction for 50ml each time, combining diethyl ether layers, carrying out reduced pressure evaporation to dryness, adding 20ml of methanol into residues for dissolution, transferring into a 25ml measuring flask, carrying out constant volume metering to the scale with methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the total anthraquinone sample solution.

The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

1.3 chromatographic conditions and System suitability test

1.3.1 chromatographic Condition selection

1.3.1.1 Mobile phase selection

The method for measuring the free anthraquinone components of rhubarb by HPLC (high performance liquid chromatography) adopts a common mobile phase which is a methanol-buffer salt solution system and a methanol-phosphoric acid aqueous solution system, and the methanol-phosphoric acid aqueous solution system is selected as the mobile phase in the experiment. The flow phase ratio was influenced by the brand and length of the column, and this experiment examined methanol-phosphoric acid aqueous solution systems and acetonitrile-phosphoric acid aqueous solution systems, in which methanol-phosphoric acid aqueous solution interfered with aloe-emodin at 75: 25 (by volume) to 85: 15 (by volume) and the tail factor T value deviated by 1.0 more. When the acetonitrile-phosphoric acid aqueous solution is subjected to gradient elution at the volume ratio of 35:65 to 80:20, 5 rhubarb anthraquinone components achieve good separation effect, and the tail factor T value deviates less than 1.0 (see table 1).

TABLE 1 comparison of the separation efficiency of different mobile phase ratios

1.3.1.2 selection of detection wavelength

The components to be detected in the test solution and the reference solution are scanned at the wavelength of 190-400nm, and as a result, 5 anthraquinone components have obvious absorption peaks near the wavelength of 254nm, and under the condition of the wavelength, the chromatographic peak separation degree of each component to be detected is good (see fig. 1-5), so 254nm is selected as the detection wavelength.

The results of the measurement of the rhubarb anthraquinone reference substance solution and the test solution are ideal under the chromatographic condition of 1.2, the measurement result of the negative solution which is prepared by the same method and lacks rhubarb is negative, and the measurement of the sample is not interfered (see figures 6 to 10).

1.3.2 System suitability test

The test was carried out according to the relevant regulations in the general rules 0512 of high performance liquid chromatography in the four ministry of the ministry of record of China pharmacopoeia 2015 edition.

Number of theoretical plates of 1.3.2.1 column

When the chromatographic columns of different brands and different column lengths are adopted to measure the test solution, the number of theoretical plates is more than 80000, and the number of theoretical plates is not less than 4000 according to the calculation of emodin peak on the premise of ensuring the separation effect by considering the influence of the new and old degrees of the columns.

TABLE 2 column efficiency of different brands of chromatographic columns for detecting emodin

The results in the table show that when the emodin is measured under the chromatographic conditions of the invention, the column effects of three different chromatographic columns are basically consistent, which indicates that the emodin has good applicability to the chromatographic columns.

1.3.2.2 reproducibility

The control solution was injected for 5 times, and the RSD (n = 5) of the peak areas of aloe-emodin, rhein, emodin, chrysophanol and physcion were all no more than 2.0% (see table 3), indicating good precision of the instrument.

TABLE 3 repeatability test results for control solutions

1.4 method for preparing test solution

1.4.1 examination of extraction methods

The experiments were performed using reflux extraction for 60 minutes and ultrasonic extraction for 60 minutes.

Method one (see "Chinese pharmacopoeia" 2015 edition one part):

weighing 0.5g of Shenshuaining capsule content (batch number 20190108), accurately weighing, placing in a conical flask with a plug, accurately adding 25ml of methanol, weighing, heating and refluxing for 1 hour, cooling, weighing again, supplementing the lost weight with methanol, shaking up, and filtering.

The second method comprises the following steps:

precisely weighing O.5g of Shenshuaining capsule content (batch number 20190108), precisely adding 25ml of methanol, weighing, carrying out ultrasonic treatment (power of 250W and frequency of 50 kHz) for 1 hour, cooling, complementing the lost weight with methanol, filtering, and taking the subsequent filtrate to obtain the Shenshuaining capsule. The results, as determined by the liquid chromatography conditions of the present invention, are shown in Table 4 below.

TABLE 4 extraction methods test results

As can be seen from Table 4, the test results of the reflux extraction and the ultrasonic extraction are not very different, so we choose to prepare the test sample by ultrasonic extraction.

1.4.2 determination of extraction time

Taking 5 parts of Shenshuaining capsule contents (batch number 20190108), each part is 0.2g, precisely weighing, precisely adding 25ml of methanol, weighing, extracting for different times of 10 minutes, 20 minutes, 30 minutes, 40 minutes and 60 minutes by ultrasonic treatment (power 250W and frequency 50 kHz), cooling, complementing lost weight by methanol, filtering, taking subsequent filtrate, and determining according to the liquid chromatography conditions of the invention, wherein the results are shown in Table 5 below.

TABLE 5 extraction time test results

The preparation method of the rhubarb free anthraquinone test solution is determined by the analysis of experimental results: precisely weighing 0.5g of the content, precisely adding 25ml of methanol, weighing, ultrasonically treating for 60 minutes (power 250W and frequency 50 kHz), cooling, supplementing the lost weight with methanol, shaking up, filtering, and collecting the subsequent filtrate.

1.4.3 hydrolysis method selection of total anthraquinone in Rheum officinale

The method comprises the following steps: taking 2 parts of Shenshuaining capsule content (batch number 20190108), each part is 0.2g, precisely weighing, precisely adding 25ml of methanol, weighing, carrying out ultrasonic treatment for 60 minutes (power is 250W, frequency is 50 kHz), cooling, complementing lost weight with methanol, filtering, taking 25ml of subsequent filtrate, adding 5ml of 36% (v/v) hydrochloric acid, heating and refluxing in water bath for 60 minutes, immediately cooling, transferring into a 25ml measuring flask, fixing volume to scale with methanol, shaking up, filtering, taking subsequent filtrate, and determining according to the liquid chromatography condition of the invention.

The second method comprises the following steps: taking 2 parts of Shenshuaining capsule content (batch number 20190108), each part being 0.2g, precisely weighing, precisely adding 25ml of methanol, weighing, carrying out ultrasonic treatment for 60 minutes (power is 250W, frequency is 50 kHz), cooling, complementing lost weight with methanol, filtering, taking 25ml of subsequent filtrate, carrying out reduced pressure evaporation to dryness, adding 50ml of 4% (v/v) hydrochloric acid, heating and refluxing in a water bath for 60 minutes, immediately cooling, adding diethyl ether for extraction for 3 times, each time 50ml, combining diethyl ether layers, carrying out reduced pressure evaporation to dryness, adding 20ml of methanol to dissolve residues, transferring the residues into a 25ml measuring flask, carrying out volume fixing to a scale with methanol, shaking up, filtering, taking subsequent filtrate, and determining according to the liquid chromatography conditions of the invention.

The chromatogram of the sample obtained by the first method has more unknown peaks, which indicates that more hydrolysis products are obtained and the peak areas of the 5 rhubarb anthraquinones are all reduced.

The chromatogram obtained by the second method has less interference peaks, which indicates that the hydrolysate is less and the peak areas of 5 rhubarb anthraquinones are increased (see fig. 16 and 17).

1.4.4 hydrolysis time selection of Total anthraquinone in rhubarb

Taking 4 parts of Shenshuaining capsule content (batch number 20190108), each part being 0.2g, precisely weighing, precisely adding 25ml of methanol, weighing, carrying out ultrasonic treatment for 60 minutes (power is 250W, frequency is 50 kHz), cooling, complementing lost weight with methanol, filtering, taking 25ml of subsequent filtrate, carrying out reduced pressure evaporation to dryness, adding 50ml of 4% (v/v) hydrochloric acid, carrying out water bath heating reflux for 30, 40, 50 and 60 minutes, immediately cooling, adding diethyl ether for extraction for 3 times, each time taking 50ml, combining diethyl ether layers, carrying out reduced pressure evaporation to dryness, adding 20ml of methanol into residues for dissolving, transferring into a 25ml measuring flask, carrying out constant volume to scale with methanol, shaking up, filtering, taking subsequent filtrate, and determining according to the liquid chromatography conditions of the invention, wherein the results are shown in Table 6 below.

TABLE 6 hydrolysis time test results for total anthraquinone in rhubarb

The experimental result analysis confirms that the preparation method of the rhubarb total anthraquinone sample solution comprises the following steps: taking 0.2g of Shenshuaining capsule content, accurately weighing, accurately adding 25ml of methanol, weighing, ultrasonically treating for 60 minutes (power 250W and frequency 50 kHz), cooling, supplementing lost weight with methanol, filtering, taking 25ml of subsequent filtrate, evaporating to dryness under reduced pressure, adding 50ml of 4% (v/v) hydrochloric acid, heating and refluxing in a water bath for 60 minutes, immediately cooling, adding diethyl ether for extraction for 3 times, each time extracting for 50ml, combining diethyl ether layers, evaporating to dryness under reduced pressure, adding 20ml of methanol into residues for dissolving, transferring into a 25ml measuring flask, fixing the volume to scale with methanol, shaking uniformly, filtering, and taking subsequent filtrate to obtain the Shenshuaining capsule.

1.5 specificity

According to the prescription and the process method, a rheum officinale-lacking negative sample is prepared, then a negative sample solution is prepared according to the preparation method of the test sample solution, the test is carried out by injecting the test sample solution into a liquid chromatograph under the liquid chromatogram condition of the invention, and the result negative sample is not interfered (see figure 9 and figure 10), which indicates that the method has certain specificity.

1.6 Linear relationship investigation

Accurately weighing 10.47mg of aloe-emodin reference substance, 10.18mg of rhein reference substance, 10.57mg of emodin reference substance and 10.04mg of chrysophanol reference substance respectively in 100ml measuring bottles, putting 9.65mg of physcion reference substance in 250ml measuring bottles, adding methanol to dissolve and dilute to scale, shaking uniformly to prepare aloe-emodin reference substance solution (0.1029 mg/ml), rhein reference substance solution (0.1011 mg/ml), emodin reference substance solution (0.1043 mg/ml), chrysophanol reference substance solution (0.09960 mg/m 1) and physcion reference substance solution (0.007643 mg/ml) as reference substance solutions; accurately weighing the above control solutions, placing into 10ml measuring bottles, and shaking to obtain the final product (each 1ml contains aloe-emodin 0.02058mg, rhein 0.02022mg, emodin 0.02087mg, chrysophanol 0.01992mg, and physcion 0.0077643 mg).

Precisely sucking mixed reference substance solutions 1, 2, 3, 5 and 10ml respectively, placing the mixed reference substance solutions 10 and 15ml respectively in 10ml measuring bottles, adding methanol to dilute to scale, shaking up, injecting into a liquid chromatograph according to the liquid chromatogram condition of the invention, measuring peak area, measuring the sample amount of the measured object according to the peak area integral value, and performing linear regression by using a least square method to obtain regression equations and linear ranges of 5 rhubarb anthraquinone components, wherein the results are shown in Table 7 and figures 11 to 15.

TABLE 7 measurement of the Linear relationship between anthraquinone components of rhubarb

1.7 precision test

1.7.1 repeatability test

1.7.1.1 free anthraquinones

About 0.5g of the same batch of shenshuaining capsules (batch No. 20190108) is taken, 6 parts are weighed precisely, and the operation and the measurement are carried out according to the '1.2 item content measurement method', and the results are shown in Table 8.

TABLE 8 repeatability test results for free anthraquinone test sample solutions

The average contents of aloe-emodin, rhein, emodin, chrysophanol and physcion in the shenshuaining capsule are respectively measured to be 0.035%,0.067%,0.024%,0.056%,0.017% and RSD (n = 6) is respectively 0.77%,0.53%,0.76%,0.23% and 0.48%, which shows that the method has good repeatability.

1.7.1.2 Total anthraquinones

About 0.5g of the same batch of shenshuaining capsules (batch No. 20190108) is weighed in parallel by 6 parts, and the operation and the measurement are carried out according to the '1.2 item content measurement method', and the results are shown in Table 9.

TABLE 9 repeatability test results for total anthraquinone sample solutions

The measured average contents of aloe-emodin, rhein, emodin, chrysophanol and physcion in the Shenshuaining capsule are 1.38mg/g, 2.24mg/g, 0.90mg/g, 2.22mg/g, 0.70mg/g and 7.44mg/g respectively, and RSD (n = 6) is 1.17%, 1.74%, 0.84%, 0.94%, 1.48% and 1.12% respectively, which indicates that the method has good repeatability.

1.7.2 intermediate precision investigation

In the same laboratory, 3 batches of samples were subjected to the content measuring method of the invention and calculated by different operators on different dates and different chromatographic columns (A: 7/1-mesh in 2019, agilent 260 liquid chromatograph, agilent EC-C18 chromatographic column; B: 6/17-mesh in 2019, agilent 260 liquid chromatograph, elite ODS-BP chromatographic column). The results of the experiments show that the method defined by the invention has good intermediate precision (see tables 10 and 11).

TABLE 10 intermediate precision examination of free anthraquinones (n = 3)

TABLE 11 Total anthraquinone intermediate precision study results (n = 3)

1.8 testing of accuracy

1.8.1 rhubarb free anthraquinone

And a sample recovery method is adopted. Precisely measuring aloe-emodin (0.01544 mg/ml), rhein (0.03033 mg/ml), emodin (0.01043 mg/ml), chrysophanol (0.02490 mg/ml) and physcion (0.00764 mg/ml) to obtain 9 parts of a control solution, dividing the solution into three groups by adding the solution in an amount of 0.5, 1.0 and 1.5 times, each group is 3.0ml,5.0ml and 10.0ml, respectively placing the three groups in conical flasks, volatilizing at low temperature, precisely weighing 0.25g of Shenshuaining Capsule (lot No. 20180108) powder with known content, respectively adding the obtained powder into the 9 conical flasks, preparing test solution according to 1.2 test solution preparation methods, measuring the content according to 1.2 chromatographic conditions, and simultaneously taking the aloe-emodin, rhein, emodin, chrysophanol and physcion control solution with concentrations of 0.010109, 0.99433, 0.0036, 0.00821, 300.300 mg/ml, and 300.300.300 mg/ml as an average peak area. The average recovery rates RSD (n = 9) of aloe-emodin, rhein, emodin, chrysophanol and physcion were 0.54%, 1.77%, 0.96%, 1.31%, 1.98%, and the results are shown in table 12. The results show that the method is good in accuracy.

TABLE 12 sample application and recovery rate test of free anthraquinone components in SHENSHUAINING Capsule

1.8.2 Total anthraquinone of radix et rhizoma Rhei

And a sample recovery method is adopted. Precisely measuring aloe-emodin (0.033697 mg/ml), rhein (0.055091 mg/ml), emodin (0.022029 mg/ml), chrysophanol (0.054401 mg/ml) and physcion (0.017026 mg/ml) mixed reference solution 9 parts, dividing into three groups, each group being 5.0ml,10.0ml and 15.0ml, respectively placing in a conical flask, volatilizing at low temperature, precisely weighing 0.25g of shenshuaining capsule (lot No. 20180108) powder with known content, respectively adding into the above 9 conical flasks, preparing a test solution according to the preparation method of the test solution under 1.2, measuring the content under chromatographic conditions, and simultaneously calculating peak areas of the aloe-emodin, rhein, emodin, chrysophanol and physcion reference solutions with the concentrations of 0.010010010109, 0.433, 0.00996 and 0.003477/ml, average peak areas of 0.821, 780.393, 489.816, and 355.132.132. The average recovery rates RSD (n = 9) of aloe-emodin, rhein, emodin, chrysophanol and physcion were 3.16%, 2.81%, 1.90%, 1.84% and 2.94%, and the results are shown in table 13. The results show that the method has good accuracy.

TABLE 13 test of sample application and recovery rate of total anthraquinone components in SHENSHUAINING Capsule

1.9 determination of content Limit

Twenty batches of shenshuaining capsules were tested and the results are shown in table 14.

TABLE 14 results of twenty sample measurements

According to the actual content measurement results of twenty batches of samples, the average values of the contents of free anthraquinone and total anthraquinone are respectively 0.90 mg/grain and 2.38 mg/grain, and the content of rhubarb in the product is temporarily bound to be not less than 0.60 mg/grain based on the total amount of aloe-emodin, rhein, emodin, chrysophanol and physcion in the free anthraquinone; based on the total amount of aloe-emodin, rhein, emodin, chrysophanol and physcion in the total anthraquinone, each granule should not be less than 1.80mg.

1.10 durability test

1.10.1 column temperature investigation

Because the influence of the column temperature on the separation effect is large, in order to test the stability of the method, the temperature of 20 ℃, 25 ℃, 30 ℃, 35 ℃ and 40 ℃ are selected for testing, and the test result shows that the theoretical plate effect and the separation degree of the tower meet the requirements.

TABLE 15 comparison of different column temperatures

1.10.2 investigation of flow Rate

In order to examine the influence degree of different flow rates on the test result, 0.95ml/min, 1.0ml/min and 1.05ml/min are selected for testing, and the test result shows that the theoretical plate number and the separation degree of the mixed gas meet the requirements.

TABLE 16 comparison of different flow rates

1.10.4 examination of the detection wavelength

In order to examine the influence degree of different detection wavelengths on the test result, 250nm, 254nm and 260nm are selected for the test, the test result shows that the theoretical plate number and the separation degree of the catalyst meet the requirements.

Tests show that small changes of the test conditions have little influence on the system applicability test, and the method selected by the user is feasible.

1.10.5 investigation of different columns

TABLE 17 comparison of different chromatography columns

1.10.6 stability Studies

Taking the same sample, preparing a test solution according to a content measurement method, placing the test solution at room temperature under a lightproof condition, carrying out multiple measurements at different time intervals, and calculating according to peak areas, wherein the RSD (n = 12) is 0.72%,0.27%,0.78%,0.19% and 0.24%, and the peak areas of the obtained aloe-emodin, rhein, emodin, chrysophanol and physcion are basically consistent, which indicates that the sample is stable within 42 hours, and the results are shown in Table 18.

TABLE 18 test results for stability of test solutions

Comparative example 1:

chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; methanol-0.1% phosphoric acid water solution (volume ratio is 75: 25) is used as a mobile phase; column temperature: 30 ℃; detection wavelength: 254nm. The number of theoretical plates is not less than 4000 calculated according to emodin peak.

Preparation of control solutions: taking appropriate amount of aloe-emodin reference substance, rhein reference substance, emodin reference substance, chrysophanol reference substance, and physcion reference substance, precisely weighing, and adding methanol to obtain solution containing 100 μ g of aloe-emodin, rhein, emodin, and chrysophanol and 50 μ g of physcion per 1ml; precisely weighing each 2ml of the above reference solution, and mixing well to obtain the final product (each 1ml contains 20 μ g of aloe-emodin, rhein, emodin, and chrysophanol, and 10 μ g of physcion).

Preparation of a test solution: precisely weighing 0.2g of the content, precisely adding 25ml of methanol, weighing, ultrasonically treating for 20 minutes (power 250W and frequency 50 kHz), cooling, supplementing the lost weight with methanol, shaking up, filtering, and collecting the subsequent filtrate.

Total anthraquinone: taking 0.5g of content, accurately weighing, accurately adding 50ml of methanol, weighing, ultrasonically treating for 60 minutes (power 250W and frequency 50 kHz), cooling, complementing the lost weight with methanol, shaking up, filtering, taking 25ml of subsequent filtrate, evaporating to dryness under reduced pressure, adding 50ml of 4% (v/v) hydrochloric acid, heating and refluxing in a water bath for 1 hour, immediately cooling, adding diethyl ether for extraction for 3 times, each time extracting for 50ml, combining diethyl ether layers, evaporating to dryness under reduced pressure, adding 20ml of methanol into residues for dissolving, transferring into a 25ml measuring flask, fixing the volume to a scale with methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the total anthraquinone sample solution.

The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

Five samples of Shenshuaining capsules were tested and the results are shown in Table 19 and FIGS. 18-20.

TABLE 19 results of content measurement of five samples

Comparative example 2:

chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; methanol-0.1% phosphoric acid aqueous solution (volume ratio 80; column temperature: 30 ℃; detection wavelength: 254nm. The number of theoretical plates is not less than 4000 calculated according to emodin peak.

Preparation of control solutions: taking appropriate amount of aloe-emodin reference substance, rhein reference substance, emodin reference substance, chrysophanol reference substance, and physcion reference substance, precisely weighing, and adding methanol to obtain solution containing 100 μ g of aloe-emodin, rhein, emodin, and chrysophanol and 50 μ g of physcion per 1ml; precisely weighing the above control solutions respectively at a volume of 2ml, and mixing well to obtain the final product (each 1ml contains 20 μ g of aloe-emodin, rhein, emodin, and chrysophanol, and 10 μ g of physcion).

Preparation of a test solution: precisely weighing about 0.2g of the content, precisely adding 25ml of methanol, weighing, ultrasonically treating for 20 minutes (power 250W and frequency 50 kHz), cooling, supplementing the lost weight with methanol, shaking up, filtering, and taking the subsequent filtrate.

Total anthraquinone: taking 0.5g of content, accurately weighing, accurately adding 50ml of methanol, weighing, ultrasonically treating for 60 minutes (power 250W and frequency 50 kHz), cooling, complementing the lost weight with methanol, shaking up, filtering, taking 25ml of subsequent filtrate, evaporating to dryness under reduced pressure, adding 50ml of 4% (v/v) hydrochloric acid, heating and refluxing in a water bath for 1 hour, immediately cooling, adding diethyl ether for extraction for 3 times, each time extracting for 50ml, combining diethyl ether layers, evaporating to dryness under reduced pressure, adding 20ml of methanol into residues for dissolving, transferring into a 25ml measuring flask, fixing the volume to a scale with methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the total anthraquinone sample solution.

The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

Five samples of shenshuaining capsules were tested and the results are shown in table 20 and fig. 21 to 23.

TABLE 20 measurement of the contents of five lots of samples

Comparative example 3:

chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; methanol-0.1% phosphoric acid water solution (volume ratio 85: 15) is used as a mobile phase; column temperature: 30 ℃; detection wavelength: 254nm. The number of theoretical plates is not less than 4000 calculated according to emodin peak.

Preparation of control solutions: taking appropriate amount of aloe-emodin reference substance, rhein reference substance, emodin reference substance, chrysophanol reference substance, and physcion reference substance, precisely weighing, and adding methanol to obtain solution containing 100 μ g of aloe-emodin, rhein, emodin, and chrysophanol and 50 μ g of physcion per 1ml; precisely weighing the above control solutions respectively at a volume of 2ml, and mixing well to obtain the final product (each 1ml contains 20 μ g of aloe-emodin, rhein, emodin, and chrysophanol, and 10 μ g of physcion).

Preparing a test solution: precisely weighing about 0.2g of the content, precisely adding 25ml of methanol, weighing, ultrasonically treating for 20 minutes (power 250W and frequency 50 kHz), cooling, supplementing the lost weight with methanol, shaking up, filtering, and taking the subsequent filtrate.

Total anthraquinone: taking 0.5g of content, accurately weighing, accurately adding 50ml of methanol, weighing, ultrasonically treating for 60 minutes (power 250W and frequency 50 kHz), cooling, complementing the lost weight with methanol, shaking up, filtering, taking 25ml of subsequent filtrate, evaporating to dryness under reduced pressure, adding 50ml of 4% (v/v) hydrochloric acid, heating and refluxing in a water bath for 1 hour, immediately cooling, adding diethyl ether for extraction for 3 times, each time extracting for 50ml, combining diethyl ether layers, evaporating to dryness under reduced pressure, adding 20ml of methanol into residues for dissolving, transferring into a 25ml measuring flask, fixing the volume to a scale with methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the total anthraquinone sample solution.

The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

Five samples of shenshuaining capsules were tested and the results are shown in table 21 and fig. 24-26.

TABLE 21 measurement of the content of five samples

The results of the comparative examples 1 to 3 are higher than those of the present invention because the aloe-emodin has a relatively fast peak time when measuring free anthraquinone and causes severe interference with other components, while the hydrolysis products are abundant when measuring total anthraquinone due to the participation of methanol in hydrolysis, and the other hydrolysis products cannot be separated by the chromatographic conditions of the comparative examples 1 to 3, resulting in higher measurement results.

No matter free anthraquinone or total anthraquinone, the method can completely separate 5 anthraquinone components from other components or hydrolysate component interference peaks, eliminates the influence of the interference peaks, has a peak-shaped tailing factor T value closer to 1.0, and has good peak shape symmetry and more accurate quantitative result.

Claims (10)

1. A detection method for simultaneously determining the contents of aloe-emodin, rhein, emodin, chrysophanol and physcion in a traditional Chinese medicine composition for treating nephropathy is disclosed, wherein the detection method is used for determining by high performance liquid chromatography, and the conditions of the high performance liquid chromatography comprise: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile is used as a mobile phase A, phosphoric acid aqueous solution with the volume ratio concentration of 0.1% is used as a mobile phase B, and elution is carried out according to the gradient that the volume ratio of the mobile phase A to the mobile phase B is changed from 35:65 to 80:20 within 0-40 minutes.

2. The detection method according to claim 1, comprising the steps of:

(1) Preparation of control solutions: respectively taking appropriate amount of aloe-emodin reference substance, rhein reference substance, emodin reference substance, chrysophanol reference substance, and physcion reference substance, and adding methanol to obtain reference substance solution;

(2) Preparation of a test solution: adding methanol into the Chinese medicinal composition, ultrasonic treating, filtering, collecting filtrate, evaporating to dryness under reduced pressure, adding 4% hydrochloric acid, heating in water bath under reflux, extracting with diethyl ether, evaporating to dryness under reduced pressure, dissolving the residue with methanol, filtering, and collecting filtrate to obtain total anthraquinone sample solution;

(3) The determination method comprises the following steps: precisely sucking the reference substance solution prepared in the step (1) and the test solution prepared in the step (2) respectively, injecting the reference substance solution and the test solution into a high performance liquid chromatograph, and measuring the chromatograms of the test solution and the reference substance solution according to the conditions of the high performance liquid chromatography, wherein the conditions of the high performance liquid chromatography comprise: octadecylsilane chemically bonded silica is used as a filling agent; taking acetonitrile as a mobile phase A, taking a phosphoric acid aqueous solution with the volume ratio concentration of 0.1% as a mobile phase B, and mixing the acetonitrile and the phosphoric acid aqueous solution according to the volume ratio of the mobile phase A to the mobile phase B within 0-40 minutes from 35:65 is changed to 80 at a constant speed: a gradient of 20 was run for elution.

3. The detection method according to claim 2, comprising the steps of:

(1) Preparation of control solutions: respectively taking appropriate amount of aloe-emodin reference substance, rhein reference substance, emodin reference substance, chrysophanol reference substance, and physcion reference substance, and adding methanol to obtain reference substance solution;

(2) Preparation of a test solution: precisely weighing the traditional Chinese medicine composition, precisely adding methanol, weighing, ultrasonically treating for 60 minutes, cooling, supplementing the weight loss by using methanol, shaking up, filtering, taking subsequent filtrate, evaporating to dryness under reduced pressure, adding hydrochloric acid with the volume ratio of 4%, heating and refluxing in a water bath for 30-60 minutes, immediately cooling, adding diethyl ether for extraction, evaporating to dryness under reduced pressure, dissolving residue by adding methanol, transferring into a measuring flask, fixing the volume to a scale by using methanol, shaking up, filtering, and taking subsequent filtrate to obtain a total anthraquinone sample solution; preferably, the water bath is heated to reflux for 60 minutes;

(3) The determination method comprises the following steps: and (3) precisely sucking the reference substance solution prepared in the step (1) and the test substance solution prepared in the step (2) respectively, injecting the reference substance solution and the test substance solution into a high performance liquid chromatograph, and measuring the chromatograms of the test substance solution and the reference substance solution according to the conditions of the high performance liquid chromatography.

4. The detection method according to any one of claims 1 to 3, wherein the Chinese medicinal composition comprises Salvia miltiorrhiza, rheum officinale, pseudostellaria heterophylla, coptis chinensis, achyranthes bidentata, pinellia ternate, safflower, poria cocos, citrus reticulata, and Glycyrrhiza uralensis.

5. The detection method according to claim 4, wherein the Chinese medicinal composition comprises, in parts by weight: 20-30 parts of radix pseudostellariae, 20-30 parts of pinellia ternate, 16-24 parts of poria cocos, 56-84 parts of salvia miltiorrhiza, 8-12 parts of safflower, 8-12 parts of coptis chinensis, 8-12 parts of pericarpium citri reticulatae, 32-48 parts of rheum officinale, 16-24 parts of achyranthes bidentata and 8-12 parts of liquorice.

6. The detection method according to claim 5, wherein the Chinese medicinal composition comprises, in parts by weight: 25 parts of radix pseudostellariae, 25 parts of pinellia ternate, 20 parts of poria cocos, 70 parts of salvia miltiorrhiza, 10 parts of safflower carthamus, 10 parts of coptis chinensis, 10 parts of pericarpium citri reticulatae, 40 parts of rheum officinale, 20 parts of achyranthes bidentata and 10 parts of liquorice.

7. The detection method according to claim 5 or 6, wherein the pinellia ternate is rhizoma pinellinae praeparata.

8. The assay of any one of claims 5 to 7, wherein the traditional Chinese medicine composition is a shenshuaining capsule.

9. The detection method of any one of claims 1 to 8, wherein the high performance liquid chromatography conditions further comprise: the column temperature is 20-40 ℃; the flow rate is 0.95-1.05ml per minute; the ultraviolet detection wavelength is 250-260nm; the number of theoretical plates is not less than 4000 calculated according to emodin peak.

10. The detection method of claim 9, wherein the high performance liquid chromatography conditions further comprise a column temperature of 40 ℃; the flow rate was 1ml per minute; the ultraviolet detection wavelength is 254nm.

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