CN109030702B

CN109030702B - Quality detection method of enema for removing blood stasis and dissipating stagnation

Info

Publication number: CN109030702B
Application number: CN201811304036.3A
Authority: CN
Inventors: 张云清; 陈海生; 吴彤
Original assignee: Qinghai Reach Pharmaceutical Group Co ltd
Current assignee: Qinghai Reach Pharmaceutical Group Co ltd
Priority date: 2018-11-03
Filing date: 2018-11-03
Publication date: 2020-07-24
Anticipated expiration: 2038-11-03
Also published as: CN109030702A

Abstract

The invention discloses a quality detection method of a stasis-removing and stasis-dissipating enema, which comprises the following steps: 1) checking aristolochic acid; 2) measuring the content of paeoniflorin and chlorogenic acid; the content determination comprises the following steps: (1) chromatographic conditions and system applicability experiments; (2) preparing a reference substance solution; (3) preparing a test solution; (4) and (4) measuring. The quality detection method of the stasis-removing stasis-dissipating enema liquid increases the inspection of aristolochic acid, the content determination of chlorogenic acid which is one of the effective components of honeysuckle in the traditional Chinese medicine formula and the determination method thereof. The method has simple operation, strong specificity and good reproducibility, and makes quality control and detection of the preparation safer and more effective.

Description

Quality detection method of enema for removing blood stasis and dissipating stagnation

Technical Field

The invention relates to the technical field of quality detection of medicines, in particular to a quality detection method of a Chinese patent medicine 'stasis-dissolving stasis-dissipating enema'.

Background

The stasis-removing and stasis-resolving enema is a Chinese patent medicine produced by the limited Richesan pharmacy company, an approved literature number [ national standard Z20025840 ] is prepared from 15 traditional Chinese medicinal materials of Chinese angelica, red paeony root, rehmannia root, ligusticum wallichii, peach kernel, safflower, salvia miltiorrhiza, medicinal cyathula root, rhizoma sparganii, curcuma zedoary, turtle shell, tortoise shell, caulis aristolochiae manshuriensis, fructus forsythiae and honeysuckle, has the effects of activating blood and dissolving stasis, resolving hard lumps, promoting qi circulation and removing stagnancy and clearing heat and detoxifying, can be used for auxiliary treatment of chronic pelvic inflammation and acute pelvic inflammation, has obvious curative effects on diseases such as adnexitis masses, pelvic tuberculosis masses, pelvic blood stasis masses, uterine fibroids, endometriosis, uterine fibroids, ovarian cysts and dysmenorrheal, the current quality detection mainly comprises three parts, namely (1) identification of ferulic acid and catechualdehyde by adopting a thin-layer chromatography, a (2) inspection item, pH and relative density are measured, a content measurement item, namely, a high-performance liquid phase chromatography (addicture), a chromatographic condition is determined by a pharmacopoeia year appendix), a chromatographic test medium, a chromatographic method is adopted, a chromatographic method for identifying ferulic acid and catechuality, a chromatographic method is prepared by adding a theoretical chromatography, a theoretical chromatography method of a theoretical chromatography (No. 35: 24: 35: 10: 24: 500: 24.70), a silica gel column, a standard Z20020: 24: 500: a standard silica gel column, a standard silica gel column chromatography, a sample is prepared, a contrast medium is added.

Disclosure of Invention

The invention provides a quality detection method of a stasis-removing and stasis-dissipating enema liquid, which can better control the quality of the stasis-removing and stasis-dissipating enema liquid preparation, ensure the stability and effectiveness of the preparation, and revise and improve the existing quality standard of products. The content determination item comprises the content determination of paeoniflorin, so the detection of the paeoniflorin is removed in the identification item, and the detection of the aristolochic acid I is added in the detection item in the essential quantity standard on the basis of the fact that the formula of the product contains a akebia stem medicinal material which contains the aristolochic acid I and the aristolochic acid I is reported in research documents of renal toxicity of the aristolochic acid I; the content determination item is added with the content determination and determination method of chlorogenic acid which is an important Chinese medicine honeysuckle flower effective component in a formula. The method adopts high performance liquid chromatography to simultaneously determine the effective components paeoniflorin and chlorogenic acid in the two traditional Chinese medicinal materials of red paeony root and honeysuckle in the prescription.

[ IDENTIFICATION ] collecting 1 bottle of the product, performing ultrasonic treatment for 30min, centrifuging, collecting supernatant, extracting with diethyl ether for 2 times (50 ml each time), mixing diethyl ether solution, volatilizing, dissolving the residue with 1ml ethanol to obtain sample solution, collecting protocatechuic aldehyde control solution, adding ethanol to obtain 1mg/ml control solution, performing thin layer chromatography (2015 pharmacopoeia, general rules of the four parts of the pharmacopoeia 0502), collecting 10 μ L of the two solutions, respectively placing on the same silica gel G thin layer plate, developing with chloroform-acetone-formic acid (8:1:1) as developing agent, taking out, air drying, spraying 10% ferric trichloride ethanol solution, heating at 105 deg.C to obtain clear spots, and displaying spots with the same color in the sample chromatogram at the position corresponding to the control chromatogram.

[ EXAMINATION ] aristolochic acid I: the measurement was carried out by high performance liquid chromatography (2015 edition pharmacopeia four-part general regulation 0512). Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; taking 0.1% phosphoric acid as a mobile phase (A) and acetonitrile as a mobile phase (B), and adopting gradient elution, wherein the gradient elution is 0min to 40min, the volume ratio of the (A) to the (B) is 59:41, 40min to 50min, the volume ratio of the (A) to the (B) is 59:41 to 20:80, the volume ratio of the (A) to the (B) is 20:80 to 59:41, and the volume ratio of the (A) to the (B) is 55min to 60min, the volume ratio of the (A) to the (B) is 59: 41; the detection wavelength was 260nm and the column temperature was 30 ℃. The theoretical plate number is not less than 3000 calculated according to aristolochic acid I peak. Preparation of control solutions: precisely weighing aristolochic acid I reference substance, placing in brown volumetric flask, and adding methanol to obtain solution containing aristolochic acid 12 μ g per 1 ml. Preparing a test solution, taking a bottle of the product, carrying out ultrasonic treatment for 30min, precisely transferring 20ml of the product into a triangular bottle with a plug, precisely adding 80ml of methanol, sealing the plug, carrying out ultrasonic treatment for 30min, filtering, precisely measuring 50ml of subsequent filtrate, adding a proper amount of methanol to dissolve the subsequent filtrate, transferring the subsequent filtrate into a 10ml volumetric flask, adding methanol to a constant volume to scale, and shaking up to obtain the test solution.

[ CONTENT DETERMINATION ] paeoniflorin and chlorogenic acid: injecting 20 μ l of the reference solution into a liquid chromatograph, adjusting the detection sensitivity to make the peak height of the chromatographic peak reach 20% of the full scale, and measuring with 20 μ l of the sample solution to prevent the chromatographic peak with the same retention time as the reference chromatogram from appearing in the sample chromatogram. If chromatographic peaks with the same retention time appear, comparing the ultraviolet-visible absorption spectra of the corresponding chromatographic peaks in the wavelength range of 190-300nm by using a diode array detector, wherein the absorption spectra are different. Chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; taking 0.1% phosphoric acid as a mobile phase (A) and acetonitrile as a mobile phase (B), and adopting gradient elution, wherein the gradient elution is 0min to 26min, the volume ratio of the (A) to the (B) is 87:13, the volume ratio of the (A) to the (B) is 26min to 27min, the volume ratio of the (A) to the (B) is 87:13 to 20:80, and the volume ratio of the (A) to the (B) is 20: 80; 32 min-32.1 min. the volume ratio of (A) to (B) is 20: 80-87: 13, 32.1 min-36 min(B) Is 87: 13. The detection wavelength was 230nm and the column temperature was 30 ℃. The theoretical plate number is not less than 3000 calculated according to chlorogenic acid peak. Preparation of control solutions: precisely weighing chlorogenic acid and penoniflorin as reference substances, placing in a brown volumetric flask, and adding 50% methanol to obtain a mixed solution containing chlorogenic acid 15 μ g and penoniflorin 30 μ g per 1 ml. Preparation of a test solution: taking a bottle of the product, carrying out ultrasonic treatment for 30 minutes, shaking up, filtering, taking a subsequent filtrate, precisely transferring 1ml of the subsequent filtrate into a 50ml volumetric flask, adding water to dilute to a scale, and shaking up to obtain the product. 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. The product contains paeoniflorin (C) per 1ml₂₃H₂₈O₁₁) Not less than 1.2 mg; contains chlorogenic acid (C)₁₆H₁₈O₉) Not less than 0.5 mg.

The invention has the advantages that:

1. the HP L C chromatographic condition and method adopted for the detection of aristolochic acid I in the method have accurate and feasible detection results, and the HP L C chromatographic condition and method adopted for the two effective components of paeoniflorin and chlorogenic acid in the preparation have accurate and feasible detection results.

2. The safety of the medicine can be better ensured by increasing the identification of aristolochic acid I, the quality of the enema liquid for removing blood stasis and resolving masses can be comprehensively and effectively controlled by selecting paeoniflorin and chlorogenic acid in the prescription for content measurement, the quality of the product can be stabilized, the safety and effectiveness of clinical medication can be ensured, and the requirements of medical treatment and market can be better met.

Drawings

FIG. 1 is a graph of aristolochic acid I control HP L C of example 1.

FIG. 2 is a spectrum of HP L C of the sample (batch No. 20170326) from example 1.

FIG. 3 is a graph of aristolochic acid I control HP L C in example 2.

FIG. 4 is a spectrum of the sample (lot 20170326) in example 2 with HP L C added as a label.

FIG. 5 is a graph of aristolochic acid I control HP L C in example 3.

FIG. 6 is a spectrum of the sample (lot 20170326) in example 3 with HP L C added as a label.

FIG. 7 is a graph of aristolochic acid I control HP L C in example 4.

FIG. 8 is a spectrum of the sample (batch No. 20170326) in example 4 with HP L C added as a label.

FIG. 9 is a graph of aristolochic acid I control HP L C in example 5.

FIG. 10 is a spectrum of HP L C of the sample (batch No. 20170709) from example 5.

FIG. 11 is a graph of aristolochic acid I control HP L C in example 6.

FIG. 12 is a spectrum of HP L C of a sample (batch No. 20171018) from example 6.

FIG. 13 is the HP L C spectrum of the chlorogenic acid and paeoniflorin control mixed solution in example 7.

FIG. 14 is a spectrum of HP L C of a sample (batch No. 20170326) from example 7.

FIG. 15 is the HP L C spectrum of the chlorogenic acid and paeoniflorin control mixed solution in example 8.

FIG. 16 is a spectrum of HP L C of a sample (batch No. 20170326) from example 8.

FIG. 17 is a spectrum of HP L C as a control mixed solution of chlorogenic acid and paeoniflorin in example 9.

FIG. 18 is a spectrum of HP L C of a sample (batch No. 20170326) from example 9.

FIG. 19 is the HP L C spectrum of the chlorogenic acid and paeoniflorin control mixed solution in example 10.

FIG. 20 is a spectrum of HP L C of a sample (batch No. 20170326) from example 10.

FIG. 21 is a spectrum of HP L C as a control mixed solution of chlorogenic acid and paeoniflorin in example 11.

FIG. 22 is a spectrum of HP L C of a sample (batch No. 20170709) from example 11.

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it is understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the present disclosure, and such equivalents may fall within the scope of the present invention as defined by the appended claims.

EXAMPLE 1 detection of aristolochic acid I

Detection example of aristolochic acid I:

chromatographic conditions of a chromatographic column including Dikma Platisil C18, 4.6 × 250mm and 5 μm, a column temperature of 30 ℃, a flow rate of 1.0ml/min, a detection wavelength of 260nm, mobile phases including 0.1% phosphoric acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B, and an elution program of the chromatographic column are as shown in Table 1:

TABLE 1 mobile phase elution gradient

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0～40	59	41
40～50	59→20	41→80
			50～55	20→59	80→41
55～60	59	41

Preparation of control solutions: precisely weighing aristolochic acid I reference substance, placing in brown volumetric flask, and adding methanol to obtain solution containing aristolochic acid I2 μ g per 1 ml;

preparation of a test solution: taking a bottle of the product (batch No. 20170326), performing ultrasonic treatment for 30min, precisely transferring 20ml into a triangular flask with a plug, precisely adding 80ml of methanol, sealing the plug, performing ultrasonic treatment for 30min, filtering, precisely measuring 5ml of subsequent filtrate, adding an appropriate amount of methanol to dissolve, transferring into a 10ml volumetric flask, adding methanol to a constant volume to scale, and shaking uniformly to obtain the product;

the determination method comprises the following steps: injecting 20 μ l of the reference solution into a liquid chromatograph, adjusting detection sensitivity to make the peak height of chromatographic peak reach 20% of full scale, and measuring with 20 μ l of the sample solution to obtain aristolochic acid I.

As a result, aristolochic acid I was not detected in the sample, as shown in fig. 1 and 2.

EXAMPLE 2 examination of aristolochic acid I (II)

Detection example of aristolochic acid I:

the chromatographic conditions of a chromatographic column are Jiazhou, Kyoho C18, 4.6 × 250mm and 5 mu m, the column temperature is 30 ℃, the flow rate is 1.0ml/min, the detection wavelength is 260nm, the mobile phase comprises 0.1% phosphoric acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B, and the elution procedure is shown in Table 2.

TABLE 2 mobile phase elution gradient

Preparation of control solutions: precisely weighing aristolochic acid I reference substance, placing in brown volumetric flask, and adding methanol to obtain solution containing aristolochic acid I2 μ g per 1 ml.

Preparation of a test solution: taking a bottle of the product (batch No. 20170326), performing ultrasonic treatment for 30min, precisely transferring 20ml into a triangular flask with a plug, precisely adding 80ml of methanol, sealing the plug, performing ultrasonic treatment for 30min, filtering, precisely measuring 5ml of subsequent filtrate, adding an appropriate amount of methanol to dissolve, transferring into a 10ml volumetric flask, adding methanol to a constant volume to scale, and shaking up to obtain the product.

The determination method comprises injecting 20 μ l of control solution into liquid chromatograph, adjusting detection sensitivity to make peak height of chromatographic peak reach 20% of full scale, and determining with 20 μ l of test solution to obtain aristolochic acid I.

The results are shown in FIGS. 3 and 4.

EXAMPLE 3 examination of aristolochic acid I (III)

Detection example of aristolochic acid I:

chromatographic conditions of a chromatographic column including Dikma Platisil C18, 4.6 × 250mm and 5 μm, a column temperature of 25 ℃, a flow rate of 1.0ml/min, a detection wavelength of 260nm, mobile phases including 0.1% phosphoric acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B, and elution procedures as shown in Table 3:

TABLE 3 mobile phase elution gradient

The results are shown in FIGS. 5 and 6.

EXAMPLE 4 examination of aristolochic acid I (IV)

Detection example of aristolochic acid I:

the chromatographic conditions of the chromatographic column are Dikma Platisil C18, 4.6 × 250mm and 5 mu m, the column temperature is 30 ℃, the flow rate is 0.8ml/min, the detection wavelength is 260nm, the mobile phase is 0.1% phosphoric acid aqueous solution as mobile phase A, the acetonitrile as mobile phase B, and the elution procedure is shown in Table 4.

TABLE 4 mobile phase elution gradient

The results are shown in FIGS. 7 and 8.

EXAMPLE 5 examination of aristolochic acid I (five)

Detection example of aristolochic acid I:

chromatographic conditions of a chromatographic column including Dikma Platisil C18, 4.6 × 250mm and 5 μm, a column temperature of 30 ℃, a flow rate of 1.0ml/min, a detection wavelength of 260nm, mobile phases including 0.1% phosphoric acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B, and elution procedures as shown in Table 5:

TABLE 5 mobile phase elution gradient

Preparation of a test solution: taking a bottle of the product (batch No. 20170709), performing ultrasonic treatment for 30min, precisely transferring 20ml into a triangular flask with a plug, precisely adding 80ml of methanol, sealing the plug, performing ultrasonic treatment for 30min, filtering, precisely measuring 5ml of subsequent filtrate, adding an appropriate amount of methanol to dissolve, transferring into a 10ml volumetric flask, adding methanol to a constant volume to scale, and shaking up to obtain the product.

The results are shown in FIGS. 9 and 10.

EXAMPLE 6 examination of aristolochic acid I (six)

Detection example of aristolochic acid I:

the chromatographic conditions of the chromatographic column were Dikma Platisil C18, 4.6 × 250mm and 5 μm, the column temperature was 30 ℃, the flow rate was 1.0ml/min, the detection wavelength was 260nm, the mobile phase was 0.1% phosphoric acid aqueous solution as mobile phase A, and acetonitrile as mobile phase B, and the elution procedure was as shown in Table 6.

TABLE 6 mobile phase elution gradient

preparation of a test solution: taking a bottle of the product (batch No. 20171018), performing ultrasonic treatment for 30min, precisely transferring 20ml into a triangular flask with a plug, precisely adding 80ml of methanol, sealing the plug, performing ultrasonic treatment for 30min, filtering, precisely measuring 5ml of subsequent filtrate, adding an appropriate amount of methanol to dissolve, transferring into a 10ml volumetric flask, adding methanol to a constant volume to scale, and shaking uniformly to obtain the product;

The results are shown in FIGS. 11 and 12.

Example 7 content determination of chlorogenic acid and paeoniflorin (I)

The chromatographic conditions of the chromatographic column are Dikma Platisil C18, 4.6 × 250mm and 5 mu m, the column temperature is 30 ℃, the flow rate is 1.0ml/min, the detection wavelength is 230nm, the mobile phase comprises 0.1 percent of phosphoric acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B, and the elution program is as follows in the following table 7:

TABLE 7 mobile phase elution gradient

Time (min)	0.1% phosphoric acid aqueous solution (%)	Acetonitrile (%)
			0	87	13
26	87	13
			27	20	80
32	20	80
			32.1	87	13
36	87	13

Preparation of control solutions: weighing appropriate amount of chlorogenic acid and paeoniflorin reference, adding methanol to obtain standard solutions with concentrations of 0.0150375 and 0.03366mg/ml respectively;

preparation of a test solution: precisely absorbing the enema (batch number: 20170326) for removing blood stasis and resolving hard mass, ultrasonically mixing, filtering, taking 1.0ml to 50ml of subsequent filtrate in a volumetric flask, adding a proper amount of water for dilution, ultrasonically dissolving completely, adding water to a scale mark, and filtering to obtain the subsequent filtrate;

the determination method comprises the following steps: precisely sucking 10 μ l of sample and reference substance, and determining with liquid chromatograph.

As shown in FIGS. 13 and 14, the contents of chlorogenic acid and paeoniflorin were 0.76mg/ml and 2.08mg/ml, respectively.

Example 8 content determination of chlorogenic acid and paeoniflorin (II)

The chromatographic conditions of chromatographic columns comprise Jiazhou province C18, 4.6 × 250mm and 5 mu m, the column temperature is 30 ℃, the flow rate is 1.0ml/min, the detection wavelength is 230nm, the mobile phase comprises 0.1% phosphoric acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B, and the elution procedure is as follows in the following table 8:

TABLE 8 mobile phase elution gradient

As shown in FIGS. 15 and 16, the contents of chlorogenic acid and paeoniflorin were 0.78mg/ml and 2.09mg/ml, respectively.

Example 9 content determination of chlorogenic acid and paeoniflorin (III)

The chromatographic conditions of the chromatographic column were Dikma Platisil C18, 4.6 × 250mm, 5 μm, the column temperature 25 ℃, the flow rate 1.0ml/min, the detection wavelength 230nm, the mobile phase 0.1% phosphoric acid aqueous solution as mobile phase A, and acetonitrile as mobile phase B, and the elution procedure is as shown in Table 9 below.

TABLE 9 mobile phase elution gradient

Preparation of control solutions: weighing appropriate amount of chlorogenic acid and paeoniflorin reference, and adding methanol to obtain standard solutions with concentrations of 0.0150375 and 0.03366mg/ml respectively.

Preparation of a test solution: precisely absorbing the enema (batch number: 20170326) for removing blood stasis and resolving hard mass, mixing, filtering, taking the subsequent filtrate 1.0ml to 50ml volumetric flask, adding appropriate amount of water for dilution, performing ultrasonic to fully dissolve, adding water to the scale mark, filtering, and taking the subsequent filtrate to obtain the final product.

As shown in FIGS. 17 and 18, the contents of chlorogenic acid and paeoniflorin were 0.81mg/ml and 2.14mg/ml, respectively.

Example 10 content determination of chlorogenic acid and paeoniflorin (IV)

The chromatographic conditions of the chromatographic column were Dikma Platisil C18, 4.6 × 250mm, 5 μm, the column temperature was 30 ℃, the flow rate was 1.2ml/min, the detection wavelength was 230nm, the mobile phase was 0.1% phosphoric acid aqueous solution as mobile phase A, and acetonitrile as mobile phase B, and the elution procedure was as shown in Table 10 below.

TABLE 10 mobile phase elution gradient

As shown in FIGS. 19 and 20, the contents of chlorogenic acid and paeoniflorin were 0.80mg/ml and 2.19mg/ml, respectively.

Example 11 content determination of chlorogenic acid and paeoniflorin (V)

The chromatographic conditions of the chromatographic column were Dikma Platisil C18, 4.6 × 250mm, 5 μm, the column temperature 30 ℃, the flow rate 1.0ml/min, the detection wavelength 230nm, the mobile phase 0.1% phosphoric acid aqueous solution as mobile phase A, and acetonitrile as mobile phase B, and the elution procedure is as shown in Table 11 below.

TABLE 11 mobile phase elution gradient

preparation of a test solution: precisely absorbing the enema (batch number: 20170709) for removing blood stasis and resolving hard mass, ultrasonically mixing, filtering, taking 1.0ml to 50ml of subsequent filtrate in a volumetric flask, adding a proper amount of water for dilution, ultrasonically dissolving completely, adding water to a scale mark, and filtering to obtain the subsequent filtrate;

As shown in FIGS. 21 and 22, the contents of chlorogenic acid and paeoniflorin were 0.80mg/ml and 1.97mg/ml, respectively.

The implementation example shows that the HP L C chromatographic condition and the method adopted for detecting aristolochic acid in the method are feasible, the obtained identification result is accurate, and the HP L C chromatographic condition and the method adopted for two effective components of paeoniflorin and chlorogenic acid in the preparation are feasible, and the obtained identification result is accurate.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims

1. A quality detection method of a stasis-removing and stasis-resolving enema liquid is characterized by comprising the following steps:

(1) detecting aristolochic acid by using HP L C, wherein the conditions of the HP L C are as follows:

a) preparation of a test solution: taking a bottle of a tested sample, carrying out ultrasonic treatment for 30min, precisely transferring 20ml into a triangular bottle with a plug, precisely adding 80ml of methanol, sealing the plug, carrying out ultrasonic treatment for 30min, filtering, precisely measuring the amount of a subsequent filtrate to obtain 5ml, adding a proper amount of methanol to dissolve the subsequent filtrate, transferring the subsequent filtrate into a 10ml volumetric flask, adding methanol to a constant volume to reach a scale, and shaking up to obtain the product;

b) preparation of control solutions: precisely weighing aristolochic acid I reference substance, placing in brown volumetric flask, and adding methanol to obtain solution containing aristolochic acid I2 μ g per 1 ml;

c) octadecylsilane chemically bonded silica is used as a filling agent; taking 0.1% phosphoric acid as a mobile phase A and acetonitrile as a mobile phase B, and adopting gradient elution, wherein the volume ratio of A to B is 59:41 when the gradient elution is carried out for 0-40 min, the volume ratio of A to B is 59: 41-20: 80 when the gradient elution is carried out for 40-50 min, the volume ratio of A to B is 20: 80-59: 41 when the gradient elution is carried out for 50-55 min, and the volume ratio of A to B is 59:41 when the gradient elution is carried out for 55-60 min; the detection wavelength is 240-280nm, and the column temperature is 25-35 ℃; the number of tower plates is not less than 3000 calculated according to aristolochic acid I peak;

(2) the contents of paeoniflorin and chlorogenic acid are simultaneously measured by using HP L C, and the conditions of the HP L C are as follows:

a) preparation of a test solution: taking a bottle of a tested sample, carrying out ultrasonic treatment for 30 minutes, shaking up, filtering, taking a subsequent filtrate, precisely transferring 1ml of the subsequent filtrate into a 50ml volumetric flask, adding water to dilute to a scale, and shaking up to obtain the product;

b) preparation of control solutions: precisely weighing chlorogenic acid and penoniflorin reference substances, placing in a brown volumetric flask, and adding 50% methanol to obtain a mixed solution containing chlorogenic acid 15 μ g and penoniflorin 30 μ g per 1 ml;

c) chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; taking 0.1% phosphoric acid water solution as a mobile phase A and acetonitrile as a mobile phase B, and adopting gradient elution, wherein the volume ratio of A to B is 87:13 when the gradient elution is carried out for 0-26 min, the volume ratio of A to B is 87: 13-20: 80 when the gradient elution is carried out for 26-27 min, and the volume ratio of A to B is 20:80 when the gradient elution is carried out for 27-32 min; the volume ratio of A to B is 20: 80-87: 13 when the time is 32 min-32.1 min, and the volume ratio of A to B is 87:13 when the time is 32.1 min-36 min; the detection wavelength is 210 nm-250 nm, and the column temperature is 20-35 ℃; the number of the tower plates is not less than 3000 calculated according to the peak of chlorogenic acid;

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

wherein, in the process of detecting aristolochic acid by using HP L C and in the process of simultaneously determining the contents of paeoniflorin and chlorogenic acid by using HP L C, the used chromatographic columns are Dikma Platisil C18, 4.6 × 250mm and 5 mu m, or Jiazhou Jiazhong C18, 4.6 × 250mm and 5 mu m.

2. The quality detection method of the stasis-resolving and stasis-resolving enema liquid as claimed in claim 1, wherein in the simultaneous determination of the contents of paeoniflorin and chlorogenic acid by HP L C, the detection wavelength is 230nm, and the column temperature is 30 ℃.

3. The method for detecting the quality of the enema liquid for removing blood stasis and dissipating stagnation according to claim 1, wherein the detection wavelength is 260nm, the column temperature is 30 ℃ and the flow rate of the mobile phase is 0.8-1.2ml/min in the detection of aristolochic acid by HP L C.

4. The method for detecting the quality of the enema liquid for removing blood stasis and dissipating stagnation according to claim 1, wherein the flow rate of a mobile phase of a chromatographic column is 0.8-1.2ml/min in the simultaneous determination of the contents of paeoniflorin and chlorogenic acid by HP L C.