CN113588815B - Method for simultaneously detecting content of multiple active ingredients in Rupishu capsule - Google Patents

Abstract

The invention relates to a content determination method for simultaneously detecting various effective components in Rupishu capsules, which adopts a liquid chromatography method to simultaneously determine the contents of an effective component tubeimoside A in the Rupishu capsules, effective components saikoside a and saikoside d in bupleurum, effective components tetrahydropalmatine in corydalis tuber, effective components tanshinone IIA and salvianolic acid B in salvia miltiorrhiza and effective components paeoniflorin in red paeony root; the detection method can make up the defects of the existing method, can simultaneously detect a plurality of effective components in the capsule, effectively controls the quality of the medicine, improves the detection efficiency, reduces the detection cost, has high sensitivity, strong specificity and good accuracy and repeatability, and effectively ensures the quality of the product.

Description

Method for simultaneously detecting content of multiple active ingredients in Rupishu capsule

Technical Field

The invention belongs to the technical field of medicine quality analysis, particularly relates to a detection method of a pharmaceutical composition for treating hyperplasia of mammary glands and mastitis, and particularly relates to a content determination method for simultaneously detecting various active ingredients in a Rupishu capsule.

Background

The hyperplasia of mammary glands and mastitis are the most common mammary gland diseases of women, and the incidence rate of the diseases is on the rise year by year in recent years, wherein the incidence rate of hyperplasia of mammary glands accounts for the first place of the mammary gland diseases; according to survey data, chinese women suffering from hyperplasia of mammary glands gradually increase year by year, the incidence rate of hyperplasia of mammary glands of Chinese women gradually increases from 70% to 80%, the growth rate of hyperplasia of mammary glands in all breast diseases also increases to 75%, and the hyperplasia of mammary glands accounts for 40% in women of childbearing age.

At present, a plurality of methods for treating hyperplasia of mammary glands and mastitis exist, common western medicines such as oral hormone medicines or operation treatment are adopted, the oral hormone medicines can relieve some symptoms, but the aim of thorough cure is difficult to achieve, the side effect of the medicines is great, and canceration of the medicines is possibly increased. The traditional Chinese medicine treatment mainly comprises soothing liver, relieving depression, eliminating stagnation and removing blood stasis, and clinically common medicines comprise Rupishu capsules, rupixiao tablets, xiaojin capsules and the like, wherein the Rupishu capsules are produced by Xian Qianhao pharmaceutical industry GmbH, and the prescription of the Rupishu capsules comprises 7 medicines of salvia miltiorrhiza, snakegourd fruit, red paeony root, rhizoma bolbostemmae, dandelion, corydalis tuber and radix bupleuri, and is mainly used for treating hyperplasia of mammary glands and mastitis caused by liver qi stagnation and toxic stasis mutual resistance. The variety is well accepted by patients and medical workers in the clinical test process due to the definite curative effect, but in the current national drug standard YBZ05022017, only single-component thin-layer identification is carried out on the salvia miltiorrhiza, the rhizoma bolbostemmae and the red paeony root in the prescription, the content of paeoniflorin in the red paeony root is measured, and the quality control is not carried out on other key medicinal flavors in the prescription. Patent CN201911250147.5 discloses a method for detecting a pharmaceutical composition Rupishu capsule for treating hyperplasia of mammary glands and mastitis, but the patent only carries out content measurement on the common active ingredient quercetin in pericarpium trichosanthis and dandelion in the prescription and cannot comprehensively reflect the quality condition of the medicine, so that it is necessary to continuously perfect the detection method of the product to ensure the quality and clinical efficacy of the product.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a method for simultaneously detecting the content of various effective components in Rupishu capsules, which simultaneously detects 7 effective components in total, namely tubeimoside A in the tubeimo fritillary bulb, saikoside a and saikoside d in the bupleurum, tetrahydropalmatine in the corydalis tuber, tanshinone IIA and salvianolic acid B in the salvia miltiorrhiza and paeoniflorin in the red paeony root in a prescription. The detection method can make up the defects of the existing method, provides a more comprehensive and effective drug control quality standard, and further effectively ensures the product quality.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for detecting the content of multiple active ingredients in a Rupishu capsule simultaneously is characterized in that the pharmaceutical composition is prepared from the following seven medicinal materials: saviae Miltiorrhizae radix, pericarpium Trichosanthis, radix Paeoniae Rubra, paniculate Bolbostemma rhizome, herba Taraxaci, rhizoma corydalis, and bupleuri radix; a liquid chromatography method is adopted to simultaneously determine the effective components of tubeimoside A in tubeimoside A, the effective components of saikoside A and saikoside D in bupleurum, the effective components of tetrahydropalmatine in corydalis tuber, the effective components of tanshinone IIA and salvianolic acid B in salvia miltiorrhiza and the effective components of paeoniflorin in red paeony root, which comprises the following steps:

(1) Chromatographic conditions and System suitability test

Octadecylsilane chemically bonded silica is used as a filling agent; gradient elution is carried out by taking an organic solvent as phase A and a diluted acid aqueous solution with a certain concentration as phase B, wherein the organic solvent is selected from methanol, acetonitrile or acetonitrile-methanol mixed solution, and the diluted acid aqueous solution is selected from phosphoric acid, formic acid and glacial acetic acid with weight or volume percentage concentration of 0.01-1.0% or the mixture of the three; the column temperature is 10-40 ℃; diode array detector (PDA) for measuring the detection wavelength of tubeimoside A, saikoside a and saikoside d at 210-216nm; detecting tetrahydropalmatine with wavelength of 275-285nm; measuring tanshinone IIA with detection wavelength of 265-275nm; measuring the detection wavelength of the salvianolic acid B at 280-290nm; the detection wavelength of paeoniflorin is 225-235nm; the flow rate is 0.8ml/min-1.2ml/min; the sample injection volume is 10-20 mul; the number of theoretical plates is not less than 3000;

preferably, the phase A is acetonitrile, and the phase B is a phosphoric acid aqueous solution with the volume percentage concentration of 0.2%; the flow rate is 0.85ml/min; the column temperature is 20 ℃; measuring the detection wavelength of tubeimoside A, saikoside a and saikoside d to be 212nm; measuring detection wavelength of tetrahydropalmatine and tanshinone IIA at 275nm; measuring the detection wavelength of the salvianolic acid B to be 286nm; measuring the paeoniflorin detection wavelength to be 230nm; the number of theoretical plates is not less than 3000;

preferably, when gradient elution is performed, the gradient elution procedure is as follows: 0 to 10min,20% by weight of A; 10-25min, 20-60 percent of A; 25-35min, 60% A;35 to 50min,60 to 20% of A; 50-60min, 20 percent A; the specific gradient elution procedure was:

time (minutes)	Mobile phase A (%)	Mobile phase B (%)
			0～10	20	80
10～25	20→60	80→40
			25～35	60	40
35～50	60→20	40→80
			50～60	20	80

(2) Preparation of control solutions

Accurately weighing appropriate amount of each reference substance, and adding 80% methanol respectively to obtain stock solutions containing tubeimoside A0.3 mg/ml, saikoside a0.65mg/ml, saikoside d0.8mg/ml, tetrahydropalmatine 0.1mg/ml, tanshinone IIA 0.045mg/ml, salvianolic acid B0.2 mg/ml, and paeoniflorin 0.7 mg/ml; precisely sucking 1ml of each of the 7 stock solutions in turn, placing in a same 10ml brown volumetric flask, adding 80% methanol to dissolve and fix the volume, shaking up to obtain mixed reference substance solutions with mass concentrations of 30, 65, 80, 10, 4.5, 20 and 70 mug/ml respectively.

(3) Preparation of test solution

Grinding the content of the capsule, weighing 0.3-1.0g, precisely weighing, placing in a conical flask with a plug, precisely adding 20-50ml of 80% methanol, weighing, treating with ultrasound (250W, 50kHz) for 30-50min, cooling, supplementing the weight loss with 80% methanol, shaking, filtering, and collecting the filtrate.

Preferably, the content of the capsule for treating hyperplasia of mammary glands is taken, ground, weighed by 0.4g, precisely weighed, placed in a conical flask with a plug, precisely added with 20ml of 80% methanol, weighed, ultrasonically treated (250W, 50kHz) for 40min, cooled, supplemented with 80% methanol to reduce the weight loss, shaken uniformly, filtered, and a subsequent filtrate is taken, thus obtaining the capsule.

(4) Measurement of

Respectively sucking 10-20 μ l of each of the reference solution and the test solution, injecting into high performance liquid chromatograph, and measuring to obtain measurement result.

The prescription of the capsule for treating mammary nodules in the invention is as follows: 450g of snakegourd peel, 150g of paniculate Bolbostemma rhizome, 450g of dandelion, 225g of Salvia miltiorrhiza Bunge, 225g of red paeony root, 135g of corydalis tuber and 135g of radix bupleuri.

Compared with the prior art, the invention has the following beneficial technical effects:

according to the method for measuring the content of the Rupishu capsule, only single-component thin-layer identification is carried out on the paniculate Bolbostemma rhizome, the red sage root and the red peony root in the original quality standard YBZ05022017, and the content of paeoniflorin is measured on the red peony root; the detection method of the product is further improved on the basis that the existing quality control standard is poor in specificity, the quality and the curative effect of the product cannot be effectively guaranteed, a method for simultaneously measuring the contents of 7 effective components, namely tubeimoside A of the effective component of tubeimordia fritillary bulb, saikosaponin a and saikosaponin d of the effective component of bupleurum, tetrahydropalmatine of corydalis tuber, tanshinone IIA of salvia miltiorrhiza, salvianolic acid B and paeoniflorin of red paeony root in a prescription is added, the detection is carried out by adopting a wavelength switching technology, and the wavelength switching detection is carried out by comprehensively considering the wavelengths with strongest absorption of different effective components and smaller impurity interference by using a diode array detector. The determination components can be well separated in a chromatogram; the detection method has the advantages of high sensitivity, strong specificity, high accuracy and good repeatability, can make up for the defects of the existing method, simultaneously realizes the detection of a plurality of effective components at one time, saves the detection time, improves the detection efficiency and effectively ensures the quality of the product.

Drawings

The invention is further described below with reference to the accompanying drawings:

FIGS. 1-2 are specific test chromatograms of the present invention under chromatographic conditions, wherein A is a chromatogram of a mixed reference substance, B is a chromatogram of a test substance, C is a chromatogram of a negative sample lacking radix Paeoniae Rubra, D is a chromatogram of a negative sample lacking rhizoma corydalis, E is a chromatogram of a negative sample lacking radix Salviae Miltiorrhizae, F is a chromatogram of a negative sample lacking Fritillaria paniculata, and G is a chromatogram of a negative sample lacking radix bupleuri; wherein 1 is paeoniflorin peak, 2 is tetrahydropalmatine peak, 3 is salvianolic acid B peak, 4 is tubeimoside A peak, 5 is saikosaponin a peak, 6 is saikosaponin d peak, and 7 is tanshinone IIA peak.

FIGS. 3-4 are linear graphs of 7 effective components.

Detailed Description

The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are merely illustrative of the present invention and do not limit the scope of the present invention in any way. The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials, reagents and materials used in the following examples are all commercially available products unless otherwise specified.

Example 1: prescription of Rupishu capsule and its preparation method

Prescription: 450g of snakegourd peel, 150g of rhizoma bolbostemmae, 450g of dandelion, 225g of salvia miltiorrhiza, 225g of red paeony root, 135g of rhizoma corydalis and 135g of radix bupleuri

The preparation method comprises the following steps: pulverizing rhizoma corydalis into fine powder; extracting the red sage root and the red peony root twice by adding ethanol, adding 5 times of ethanol each time, extracting for 2 hours respectively, merging ethanol extracting solutions, filtering, recovering ethanol from filtrate, and concentrating to obtain thick paste with the relative density of 1.33-1.36 (60 ℃); decocting the rest four ingredients such as the pericarpium trichosanthis and the like in water for three times, adding 10 times of water for the first time, decocting for 2 hours, adding 8 times of water for the second time, decocting for 1.5 hours, adding 6 times of water for the third time, decocting for 1 hour, merging decoction, filtering, concentrating filtrate under reduced pressure to obtain thick paste with the relative density of 1.33-1.36 (60 ℃), merging the thick paste, adding the fine powder of rhizoma corydalis, 12g of silicon dioxide and 8g of pregelatinized starch, stirring uniformly, drying in vacuum, crushing into fine powder, and filling into capsules to prepare 1000 capsules.

Example 2: selection of detection wavelength

By scanning ultraviolet absorption spectra of 7 reference solutions of tubeimoside A, saikoside a, saikoside d, tetrahydropalmatine, tanshinone IIA, salvianolic acid B and paeoniflorin, and referring to the part of China pharmacopoeia 2020 edition, tubeimoside A has maximum absorption at 214nm, saikoside a and saikoside d have maximum absorption at 210nm, tetrahydropalmatine has maximum absorption at 280nm, tanshinone IIA has maximum absorption at 270nm, salvianolic acid B has maximum absorption at 286nm, and paeoniflorin has maximum absorption at 230nm; because the maximum absorption wavelengths of the tubeimoside A, the saikoside a and the saikoside d are close, the detection wavelengths of the tubeimoside A, the saikoside a and the saikoside d are finally determined to be 212nm by combining relevant documents and comprehensively considering the simplicity of the method; determining the detection wavelength of tetrahydropalmatine and tanshinone IIA to 275nm; the maximum absorption wavelength of the salvianolic acid B and the paeoniflorin is greatly different from that of other components, and the original maximum absorption wavelength is adopted as the detection wavelength in consideration of the content sensitivity and accuracy of the method comprehensively. Tests show that the wavelength switching technology of a diode array detector (PDA) is adopted for detection, and the diode array detector is used for carrying out wavelength switching detection by comprehensively considering the wavelengths with strongest absorption of different active ingredients and less impurity interference. The determination components can be well separated in a chromatogram; and interference impurities are removed, so that accurate calculation of the component content is facilitated.

Example 3: selection of mobile phase

In order to effectively separate 7 active ingredients in a sample and simultaneously meet the requirements of peak shape, theoretical plate number and the like, the flow properties of acetonitrile-0.5% glacial acetic acid solution, methanol-0.5% phosphoric acid solution, methanol-0.1% formic acid solution, methanol-acetonitrile-0.2% phosphoric acid solution, acetonitrile-0.2% phosphoric acid solution and the like in different proportions are respectively adopted for the preliminary test. The results show that the separation effect is best by using the linear gradient of acetonitrile-0.2% phosphoric acid aqueous solution, the baseline is the most stable, so that the mobile phase A is finally selected to be acetonitrile, and the mobile phase B is selected to be phosphoric acid aqueous solution with the volume percentage concentration of 0.2%. The detailed results are shown in Table 1.

Table 1 mobile phase selection test results

The results of the mobile phase selection test as in table 1 show that: the mobile phase system consisting of acetonitrile-0.2 percent phosphoric acid aqueous solution is adopted, the separation effect is best under the condition of gradient elution, the baseline is stable, chromatographic peaks of 7 effective components can be completely separated, and the peak time, the peak type and the separation degree of each component meet the requirements, so the condition can meet the related requirements of analysis and determination.

Example 4: study of specialization

4.1 preparation of each negative sample:

according to the prescription process and formula proportion of the sample (example 1), negative samples without tubeimo fritillary bulb, salvia miltiorrhiza, corydalis tuber, bupleurum and red paeony root are respectively prepared; for example, preparation of a salvia miltiorrhiza negative sample: pulverizing rhizoma corydalis into fine powder; extracting red peony root twice with ethanol under reflux, adding 5 times of ethanol each time, extracting for 2 hours respectively, combining ethanol extracting solutions, filtering, recovering ethanol from filtrate, and concentrating to obtain thick paste with the relative density of 1.33-1.36 (60 ℃); the preparation method comprises the following steps of adding water into rhizoma bolbostemmae, pericarpium trichosanthis, dandelion and radix bupleuri, decocting for three times, adding 10 times of water for 2 hours for the first time, adding 8 times of water for the second time, decocting for 1.5 hours, adding 6 times of water for the third time, decocting for 1 hour, merging decoction, filtering, concentrating filtrate under reduced pressure to obtain thick paste with the relative density of 1.33-1.36 (60 ℃), merging the thick paste, adding rhizoma corydalis fine powder, 12g of silicon dioxide and 8g of pregelatinized starch, stirring uniformly, drying in vacuum, and crushing into fine powder to obtain the traditional Chinese medicine.

4.2 preparation of negative sample solution:

weighing 0.4g of each negative sample respectively, precisely weighing, placing in a conical flask with a plug, precisely adding 20ml of 80% methanol, weighing, ultrasonically treating (250W, 50kHz) for 40min, cooling, supplementing the lost weight with 80% methanol, shaking up, filtering, and taking the subsequent filtrate.

4.3 preparation of test solution:

the test method comprises the following steps: grinding the content of the same batch of capsules, weighing 0.4g, precisely weighing, placing in a conical flask with a plug, precisely adding 20ml of extraction solvent, weighing, heating under reflux or ultrasonic extraction, cooling, supplementing weight, filtering, and collecting the filtrate; precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into high performance liquid chromatograph, and measuring to obtain measurement result. The results are detailed in table 2.

The method comprises the following steps: precisely adding 20ml of methanol, precisely weighing, ultrasonically extracting for 30min, cooling to complement weight, and filtering;

the method 2 comprises the following steps: precisely adding 80% methanol 20ml, precisely weighing, ultrasonically extracting for 30min, cooling to make up the weight, and filtering;

the method 3 comprises the following steps: precisely adding 20ml of 60% methanol, precisely weighing, ultrasonically extracting for 40min, cooling to complement weight, and filtering;

the method 4 comprises the following steps: precisely adding 80% methanol 20ml, precisely weighing, heating under reflux for 60min, cooling to make up weight, and filtering;

the method 5 comprises the following steps: precisely adding 80% methanol 20ml, precisely weighing, heating under reflux for 90min, cooling to make up the weight, and filtering;

the method 6 comprises the following steps: precisely adding 80% methanol 20ml, precisely weighing, ultrasonically extracting for 40min, cooling to make up the weight, and filtering;

the method 7 comprises the following steps: precisely adding 80% methanol 20ml, precisely weighing, ultrasonically extracting for 50min, cooling to make up weight, and filtering;

the method 8 comprises the following steps: adding 80% methanol 30ml precisely, weighing precisely, ultrasonic extracting for 60min, cooling to make up weight, and filtering;

TABLE 2 selection test results of test article preparation methods

The results show that: when the method 1 is adopted, the salvianolic acid B is not fully extracted, and the peak area is obviously lower than that of other methods; when the method 2 is adopted, all effective components can be effectively extracted, but the peak area is slightly lower than that of the method 6, which indicates that 100% of the effective components cannot be transferred after ultrasonic extraction for 30 min; when the method 3 is adopted, the peak area of each effective component is obviously lower due to lower methanol concentration, which indicates that the transfer rate of the effective component is lower; when the method 4 and the method 5 are adopted, the peak area of the tanshinone IIA is small, and the tanshinone IIA is likely to be lost in the heating process; the peak area in the method 7 and the method 8 is not obviously different from that in the method 6, but the ultrasonic extraction is longer; therefore, the preparation method for determining the optimal test solution comprises the following steps: collecting the content of RUPISHU Capsule, grinding, weighing 0.4g, precisely weighing, placing into a conical flask with a plug, precisely adding 80% methanol 20ml, weighing, ultrasonic processing (250W, 50kHz) for 40min, cooling, supplementing the weight loss with 80% methanol, shaking, filtering, and collecting the filtrate.

4.4 chromatographic conditions and System suitability test

Taking ZORBAX Eclipse XDB C18 column (250 mm multiplied by 4.6mm,5 μm) as a chromatographic column; the mobile phase was as identified in Table 1 at a flow rate of 0.9ml/min; the column temperature is 20 ℃; the detection wavelength of tubeimoside A, saikosaponin a and saikosaponin d is 212nm; the detection wavelength of tetrahydropalmatine and tanshinone IIA is 275nm; the detection wavelength of the salvianolic acid B is 286nm; the detection wavelength of the paeoniflorin measurement is 230nm; the flow rate is 0.9ml/min; the sample injection volume is 10 mul; theoretical plates should be no less than 3000;

preparation of control solutions:

precisely weighing appropriate amount of each reference substance, and adding 80% methanol to obtain extract containing tubeimoside A0.3142 mg/ml, saikosaponin a0.6644mg/ml, saikosaponin d0.8104mg/ml, tetrahydropalmatine 0.1016mg/ml, tanshinone II

A0.0446mg/ml, salvianolic acid B0.2224mg/ml and paeoniflorin 0.7086 mg/ml; precisely sucking 1ml of each of the 7 stock solutions in turn, placing the stock solutions in a same 10ml brown volumetric flask, adding 80% methanol to dissolve the stock solutions, fixing the volume, and shaking the stock solutions uniformly to obtain mixed reference substance solutions with mass concentrations of 31.42, 66.44, 81.04, 10.16,4.46, 22.24 and 70.86 mu g/ml respectively.

And (3) determination:

each negative sample solution, the mixed control solution, and the sample solution were each taken 10. Mu.l precisely, and injected into a Perkinelmer Altus A-10 high performance liquid chromatograph (PDA detector, empower workstation, shanghai Co., ltd., perkin Elmer corporation) for measurement.

The results show that: in the chromatogram of the test solution, the same chromatographic peaks are arranged at the corresponding positions of the chromatographic peaks of the 7 reference substances; the negative is not interfered, and the separation degree and the number of theoretical plates both meet the requirements; indicating that the chromatographic conditions are well specified. See in particular figures 1-2 below.

Example 5: repeatability test

5.1 chromatographic conditions and System suitability test

Same as example 4

5.2 precisely absorbing 10 mul of the same sample solution, injecting into a liquid chromatograph, repeatedly injecting for 6 times, and calculating the peak area RSD.4.3, the results show that the RSD values of the peak areas of the 7 effective components are respectively 0.18%, 0.28%, 0.18%, 0.31%, 0.20% and 0.34%, and are all less than 2.0%; indicating good precision. The specific results are shown in Table 3 below.

Table 3: repeatability experiment 7 effective component peak area data table

Example 6: linear range

6.1 chromatographic conditions and System suitability test

Same as example 4

6.2 precisely sucking 1.3ml of each reference stock solution respectively, placing the solution in a 10ml brown measuring flask, fixing the volume with 80% methanol to obtain a mixed reference solution, precisely sucking 2 mul, 5 mul, 8 mul, 10 mul, 15 mul and 20 mul of the mixed reference solution respectively, detecting according to the chromatographic conditions described in the embodiment 3, recording a chromatogram, performing linear regression analysis on the concentration (X) by using a peak area (Y), drawing a linear relation graph and reporting a linear equation.

The results are shown in Table 4 (standard curve equation and correlation coefficient and linear range for 7 components) and FIGS. 3-4 are graphs of the linear relationship for 7 components.

Table 4: standard curve equation, correlation coefficient and linear range of 7 components

Name of ingredient	Regression equation	R	Linear Range (μ g/ml)
				Paeoniflorin	Y＝2.19E+05X-8.05E+04	0.99	18.42-184.24
Tetrahydropalmatine B	Y＝7.20E+04X-2.91E+04	0.99	2.64-264.16
				Salvianolic acid B	Y＝2.16E+04X-6.78E+03	1	5.78-57.82
Tubeimoside A	Y＝3.77E+04X-1.17E+04	0.99	8.17-81.69
				Saikosaponin a	Y＝1.40E+05X-6.15E+04	0.99	17.27-172.74
Saikosaponin d	Y＝2.17E+05X-1.23E+05	1	21.07-316.06
				Tanshinone IIA	Y＝2.72E+03X-8.32E+02	1	1.16-11.60

Example 7: accuracy (recovery)

7.1 chromatographic conditions and System suitability test

Same as example 4

7.2 preparation of test solution:

taking the content of the capsule for treating hyperplasia of mammary glands, grinding, respectively weighing 6 parts of 0.2g of sample, precisely weighing, placing in a 20ml brown measuring flask, respectively precisely sucking 2ml of a reservoir solution of paeoniflorin 0.7086mg/ml, 0.5ml of a reservoir solution of tetrahydropalmatine 0.1016mg/ml, 5ml of a reservoir solution of salvianolic acid B0.2224mg/ml, 3ml of a reservoir solution of tubeimosiderin 0.3142mg/ml, 0.2ml of a reservoir solution of saikosaponin A0.6644mg/ml, 0.2ml of a reservoir solution of saikosaponin d0.8104mg/ml and 5ml of a reservoir solution of tanshinone IIA 0.0446mg/ml, placing in the brown measuring flask, adding a proper amount of 80% methanol, performing ultrasonic treatment (250W, 50kHz) for 40min, cooling, diluting to scale with 80% methanol, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the capsule.

And (3) determination: precisely sucking the control solutions 10 μ l each, injecting into high performance liquid chromatograph, and measuring the recovery rates and RSD of tubeimoside A, saikosaponin a, saikosaponin d, tetrahydropalmatine, tanshinone IIA, salvianolic acid B and paeoniflorin, the results are shown in Table 4, and the standard requirements are as follows: the recovery rate is 85-110%, and the RSD is less than 4.0%;

the experimental result shows that the method has good accuracy. The results are shown in Table 5.

Percent recovery = (C-A)/B x 100%,

wherein A is the measured component content of the test sample; b is the amount of the added reference substance; c is an observed value.

TABLE 5 recovery test data sheet

Example 8: intermediate precision

8.1 chromatographic conditions and System suitability test

Same as example 4

8.2 in the same laboratory, different experimenters A and B carry out experiments on different dates by using different chromatographs, and the content of each effective component is calculated. The results are shown in Table 6, and show that the patented method has good precision.

Table 6: intermediate precision test data sheet

Example 9: durability

9.1 chromatographic conditions and System suitability test

Same as example 4

9.2 taking the same sample solution, standing at room temperature for 0, 4, 8, 12 and 24 hours respectively, and precisely sucking 10 mul to inject into a high performance liquid chromatograph; the result is shown in Table 6, and the result shows that the RSD maximum value of the peak retention area of the test solution within 24 hours is only 0.19 percent and is far less than the standard 2.0 percent; the results are shown in Table 7, which indicates that the test solutions are stable at room temperature for 36 h.

Table 7: stability test data sheet

Sample introduction time point	0h	2h	4h	8h	12h	24h	Average	RSD％
									Paeoniflorin	602713	601903	602163	602658	602006	603273	602452.67	0.09
Tetrahydropalmatine B	181494	181153	181303	181887	181099	181503	181406.50	0.16
									Salvianolic acid B	56887	56695	56746	56853	56692	56896	56794.83	0.17
Tubeimoside A	107523	107351	107561	107504	107489	107947	107562.50	0.19
									Saikosaponin a	375436	374951	376063	375817	375480	376092	375639.83	0.12
Saikosaponin d	542167	541998	542081	542542	542101	542813	542283.67	0.06
									Tanshinone IIA	7135	7119	7128	7131	7117	7143	7128.83	0.14

In summary, the following steps: the method for measuring the contents of various active ingredients in the Rupishu capsule verifies the scientificity of the method in the aspects of specificity, repeatability, accuracy (recovery rate), intermediate precision, durability and the like. By measuring the content of the effective components in 7 of the 5 medicinal materials in the prescription, the product quality assurance is further improved, the safety and the effectiveness of the Rupishu capsule are ensured, and the defects of the prior art are overcome.

Example 10: stability test

10.1 chromatographic conditions and System suitability test As in example 4

10.2 commercially available samples of Rupishu capsules (batch nos. 20191001, 20191002, 20191003, manufactured by Xian Qian He pharmaceutical industry Co., ltd.) were taken for accelerated and room temperature long term stability experiments. Accelerated test conditions: placing the sample in a stability test box with the relative humidity of 75% +/-5% and the temperature of 40 +/-2 ℃ for 6-month accelerated test investigation; room temperature experimental conditions: the samples were placed in a stability box at 60% + -5% relative humidity and 25 deg. + -2 deg.C for a long period of 24 months. The test results are shown in Table 8.

Table 8: data of accelerated and long-term stability test of Rupishu capsule

The experimental results show that the contents of the detected effective components of paeoniflorin, tetrahydropalmatine, tubeimoside A, saikoside a, saikoside d, salvianolic acid B and tanshinone IIA do not have significant changes after the accelerated test for 6 months and the long-term test for 24 months, which meet the requirements, and the detection method can effectively control the quality of the medicine and can be considered to be included in the quality standard. In addition, from the detection results of 24 months end of continuous three batches of products (batch numbers: 20191001, 20191002 and 20191003), the average content of paeoniflorin is 3.21 mg/grain, the average content of tetrahydropalmatine is 0.11 mg/grain, the average content of tubeimoside A is 1.91 mg/grain, the average content of saikosaponin a is 0.25 mg/grain, the average content of saikosaponin d is 0.34 mg/grain mg/ml, the average content of salvianolic acid B is 2.04 mg/grain and the average content of tanshinone IIA is 0.54 mg/grain; considering the differences between different raw materials and the errors of production and inspection process, it is suggested that the content standard can be defined as: contains paeoniflorin more than or equal to 2.75 mg/granule, tetrahydropalmatine more than or equal to 0.10 mg/granule, tubeimoside A more than or equal to 1.65 mg/granule, saikoside a more than or equal to 0.24 mg/granule, saikoside d more than or equal to 0.30 mg/granule mg/ml, salvianolic acid B more than or equal to 1.76/granule, and tanshinone IIA more than or equal to 0.48 mg/granule.

The embodiments given above are preferred examples for implementing the present invention, and the present invention is not limited to the above-described embodiments. Any non-essential addition and replacement made by the technical characteristics of the technical scheme of the invention by a person skilled in the art belong to the protection scope of the invention.

Claims (4)

1. A method for simultaneously detecting the contents of various effective components in Rupishu capsules is characterized in that a liquid chromatography method is adopted to simultaneously detect the contents of an effective component tubeimoside A in Rupishu capsules, effective components saikoside a and saikoside d in bupleurum, an effective component tetrahydropalmatine in corydalis tuber, effective components tanshinone IIA and salvianolic acid B in salvia miltiorrhiza and an effective component paeoniflorin in red paeony root, and specifically comprises the following steps:

s1: chromatographic condition and system applicability test

Octadecylsilane chemically bonded silica is used as a filling agent; taking an organic solvent as a phase A, taking a dilute acid water solution with a certain concentration as a phase B, wherein the phase A is acetonitrile, and the phase B is a phosphoric acid water solution with a volume percentage concentration of 0.2%; gradient elution, the gradient elution procedure is: 0 to 10min,20% A; 10-25min, 20-60% of A; 25-35min, 60% A; 35-50min, 60-20% A; 50-60min, 20 percent A; switching wavelength by diode array detector, and detecting at 210-216nm for tubeimoside A, saikosaponin a and saikosaponin d; 275-285nm is adopted for measuring tetrahydropalmatine; measuring tanshinone IIA at 265-275nm; the salvianolic acid B is measured at 280-290nm; measuring paeoniflorin with a wavelength of 225-235nm; the flow rate is 0.8ml/min-1.2ml/min; the number of theoretical plates is not less than 3000;

s2: preparation of control solutions

Accurately weighing appropriate amount of each reference substance, and adding 80% methanol respectively to obtain stock solutions containing tubeimoside A0.3 mg/ml, saikoside a0.65mg/ml, saikoside d0.8mg/ml, tetrahydropalmatine 0.1mg/ml, tanshinone IIA 0.045mg/ml, salvianolic acid B0.2 mg/ml, and paeoniflorin 0.7 mg/ml; precisely sucking 1ml of each of the 7 stock solutions in sequence, placing into a same 10ml brown volumetric flask, adding 80% methanol for dissolving, fixing the volume, shaking up, and preparing into mixed reference substance solutions with mass concentrations of 30, 65, 80, 10, 4.5, 20 and 70 μ g/ml respectively to obtain;

s3: preparation of test solution

Taking the content of the capsule for treating hyperplasia of mammary glands, grinding, weighing 0.3-1.0g, precisely weighing, placing in a conical flask with a plug, precisely adding 20-50ml of 80% methanol, weighing, ultrasonically treating for 30-50min, cooling, supplementing the loss weight with 80% methanol, shaking up, filtering, and taking the subsequent filtrate;

s4: measurement of

Precisely sucking 10-20 μ l of each of the reference solution and the sample solution, respectively, injecting into high performance liquid chromatograph, and measuring to obtain measurement result.

2. The method for simultaneously detecting the contents of a plurality of active ingredients in the Rupishu capsule according to claim 1, wherein the method comprises the following steps: the Rupishu capsule is prepared from the following seven medicinal materials: pericarpium Trichosanthis, paniculate Bolbostemma rhizome, herba Taraxaci, saviae Miltiorrhizae radix, radix Paeoniae Rubra, rhizoma corydalis, and bupleuri radix.

3. The method for simultaneously detecting the contents of a plurality of active ingredients in the rupishu capsule according to claim 1, wherein: measuring the detection wavelength of tubeimoside A, saikoside a and saikoside d to be 212nm; measuring tetrahydropalmatine and tanshinone IIA, and measuring wavelength at 275nm; measuring the detection wavelength of the salvianolic acid B to 286nm; measuring the detection wavelength of paeoniflorin to be 230nm; the flow rate is 0.85ml/min; the column temperature was 20 ℃.

4. The method for simultaneously detecting the contents of a plurality of active ingredients in the rupishu capsule according to claim 1, wherein: grinding the content of the capsule, weighing 0.4g, precisely weighing, placing in a conical flask with a plug, precisely adding 20ml of 80% methanol, weighing, ultrasonically treating for 40min, cooling, supplementing the weight loss with 80% methanol, shaking, filtering, and collecting the filtrate.

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