阅读说明：本技术 产后康膏的指纹图谱检测方法 (Fingerprint spectrum detection method of postpartum rehabilitation ointment ) 是由 金俊杰 蔡宝昌 胡玉凯 李国维 于 2021-08-12 设计创作，主要内容包括：一种产后康膏的指纹图谱检测方法,包括以下步骤：步骤1、制备产后康膏供试品溶液：取适量产后康膏置于具塞锥形瓶中,加入水,振荡溶解,加入乙酸乙酯,振荡萃取,静置,上下分层清晰后,取上层萃取液,重复萃取两次,残渣用甲醇定容,离心后用有机微孔滤膜滤过,取续滤液,即得待测供试品溶液；步骤2、制备产后康膏对照品溶液；步骤3、对照品溶液,注入高效液相色谱仪,记录色谱图；步骤4,将指纹图谱导出,并导入中药色谱指纹图谱相似度评价系统2012A；通过本发明的方法可以控制产后康膏的质量稳定,确保其安全性与有效性,并完善相关制药工艺的优化。(A fingerprint detection method of postpartum rehabilitation ointment comprises the following steps: step 1, preparing a postpartum recovery paste test sample solution: placing a proper amount of puerperal rehabilitation ointment into a conical flask with a plug, adding water, oscillating for dissolution, adding ethyl acetate, oscillating for extraction, standing, layering the upper layer and the lower layer clearly, taking the upper layer of extract, repeatedly extracting for two times, diluting the residue with methanol to a constant volume, centrifuging, filtering with an organic microporous membrane, and taking the subsequent filtrate to obtain a sample solution to be tested; step 2, preparing a postpartum recovery paste reference solution; step 3, injecting the reference solution into a high performance liquid chromatograph, and recording a chromatogram; step 4, exporting the fingerprint, and importing the fingerprint into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system 2012A; the method can control the stable quality of the postpartum rehabilitation ointment, ensure the safety and the effectiveness of the postpartum rehabilitation ointment and perfect the optimization of related pharmaceutical technology.)

1. A fingerprint detection method of postpartum rehabilitation ointment is characterized by comprising the following steps:

step 1, preparation of a postpartum recovery paste test sample solution:

placing a proper amount of puerperal rehabilitation ointment into a conical flask with a plug, adding water, oscillating for dissolution, adding ethyl acetate, oscillating for extraction, standing, layering the upper layer and the lower layer clearly, taking the upper layer of extract, repeatedly extracting for two times, combining the extracts, evaporating in a water bath, diluting the residue with methanol to a constant volume, centrifuging, taking the supernatant, filtering with a 0.45 mu m organic microporous membrane, and taking the subsequent filtrate to obtain a sample solution to be tested;

step 2, preparation of postpartum recovery paste reference substance solution:

respectively taking appropriate amount of vanillic acid, syringic acid, leonurine hydrochloride, hesperidin, liquiritin, salvianolic acid B and quercetin reference substances, precisely weighing, respectively placing in volumetric flasks, and adding methanol to respectively prepare reference substance stock solutions; respectively and precisely sucking appropriate amount of control stock solutions of vanillic acid, syringic acid, leonurine hydrochloride, hesperidin, liquiritin, salvianolic acid B and quercetin, adding methanol to constant volume, and making into mixed control solution of vanillic acid, syringic acid, leonurine hydrochloride, hesperidin, liquiritin, salvianolic acid B and quercetin;

step 3, precisely absorbing the postpartum rehabilitation ointment test sample solution in the step 1 and the mixed reference substance solution in the step 2 respectively, injecting the solutions into a high performance liquid chromatograph, and recording a chromatogram;

step 4, exporting the fingerprint of the postpartum recovery paste test sample solution obtained in the step 3, and importing the fingerprint into a traditional Chinese medicine chromatography fingerprint similarity evaluation system 2012A; selecting chromatographic peaks existing in chromatograms of different batches of postpartum healing ointments as common peaks; generating a comparison fingerprint of the postpartum rehabilitation ointment by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak; and marking chemical components of peaks in the comparison fingerprint spectrum according to the retention time of the mixed comparison product solution chromatogram.

2. The method for detecting fingerprint of postpartum rehabilitation ointment according to claim 1, characterized in that,

step 1, the preparation method of the postpartum recovery paste test sample solution comprises the following steps:

placing 3ml of the postpartum rehabilitation ointment into a conical flask with a plug, adding 10ml of purified water, oscillating for dissolving, adding 20ml of ethyl acetate, oscillating for extraction, standing, taking the upper layer of extract liquor after the upper layer and the lower layer are clearly separated, repeatedly extracting for two times, combining the extract liquor, evaporating by drying in a water bath, diluting the residue with 2ml of methanol to a constant volume, centrifuging, taking the supernatant, filtering with a 0.45 mu m organic microporous filter membrane, and taking the subsequent filtrate to obtain the solution of the sample to be tested.

3. The method for detecting fingerprint of postpartum rehabilitation ointment according to claim 1, characterized in that,

step 2, preparation of a mixed reference solution:

respectively taking appropriate amount of vanillic acid, syringic acid, leonurine hydrochloride, hesperidin, liquiritin, salvianolic acid B and quercetin reference substances, precisely weighing, placing in a10 ml volumetric flask, and adding methanol to prepare reference substance stock solutions with the concentrations of 457 mu g/ml, 591 mu g/ml, 519 mu g/ml, 456.5 mu g/ml, 750 mu g/ml, 423 mu g/ml and 624 mu g/ml respectively; respectively precisely measuring appropriate amounts of vanillic acid, syringic acid, leonurine hydrochloride, hesperidin, liquiritin, salvianolic acid B and quercetin reference substance stock solution, adding methanol to constant volume to 20ml, and preparing into mixed reference substance solution containing 45.7 μ g of vanillic acid, 59.1 μ g of syringic acid, 51.9 μ g of leonurine hydrochloride, 45.65 μ g of hesperidin, 75 μ g of liquiritin, 42.3 μ g of salvianolic acid B and 62.4 μ g of quercetin per 1 ml.

4. The method for detecting fingerprint of postpartum rehabilitation ointment according to claim 1, characterized in that,

and 3, detecting chromatographic conditions by using a high performance liquid chromatograph, wherein the chromatographic conditions are as follows:

using octadecylsilane chemically bonded silica as a filler, formic acid water as a mobile phase A, acetonitrile as a mobile phase B, and carrying out gradient elution at a flow rate of 0.5-1.5 ml/min < -1 >, a column temperature of 25-40 ℃ and a detection wavelength of 200-360 nm.

5. The method for detecting fingerprint of postpartum rehabilitation ointment according to claim 4, characterized in that,

step 3, the chromatographic conditions detected by the high performance liquid chromatograph are as follows: octadecylsilane chemically bonded silica is used as a filling agent, 0.1% formic acid water is used as a mobile phase A, methanol is used as a mobile phase B, gradient elution is carried out, the flow rate is 1 ml.min < -1 >, the column temperature is 30 ℃, and the detection wavelength is 254 nm.

6. The fingerprint detection method of postpartum rehabilitation ointment according to claim 4 or 5, characterized in that the gradient elution is preferably as follows:

time/min Volume percent of mobile phase A Volume percent of mobile phase B 0 95 5 15 95 5 55 90 10 80 85 15 160 70 30 170 65 35 175 95 5 180 95 5

。

7. The method for detecting fingerprint of postpartum rehabilitation ointment as claimed in claim 4, characterized in that the chromatographic column is YMC-Pack ODS-A chromatographic column with specification of 50mm x 4.6mm and 5 μm.

8. The method for detecting the fingerprint of postpartum rehabilitation ointment as claimed in claim 1, wherein the acquired fingerprint of postpartum rehabilitation ointment has 13 common peaks, and the retention time of each common peak is as follows: has a total peak 1 and a retention time of 23.162 min; peak 2 was shared, retention time 26.381 min; peak 3 was shared, retention time 37.807 min; peak 4 was shared, retention time 47.379 min; peak 5 was shared, retention time 76.069 min; peak 6 was shared, retention time 88.859 min; peak 7 was shared, retention time 100.243 min; peak 8 was shared, retention time 109.256 min; peak 9 was shared, retention time 117.862 min; peak 10 was shared, retention time 125.050 min; peak 11 was shared, retention time 131.556 min; peak 12 was shared, retention time 135.199 min; there was a peak 13 with a retention time of 168.875 min.

9. The method for detecting fingerprint of postpartum recovery ointment as claimed in claim 8, wherein the peak 3 is vanillic acid, the peak 4 is syringic acid, the peak 5 is leonurine hydrochloride, the peak 6 is hesperidin, the peak 8 is liquiritin, the peak 10 is salvianolic acid B, and the peak 11 is quercetin.

Technical Field

The invention belongs to the technical field of Chinese patent medicine detection, and particularly relates to a fingerprint detection method and a fingerprint of postpartum rehabilitation ointment.

Background

The postpartum recovery ointment is a compound preparation consisting of 20 medicinal materials such as astragalus, codonopsis pilosula (fried), angelica (fried) and the like, has the effects of tonifying qi and nourishing blood, nourishing kidney and liver, soothing nerves and astringing Han, strengthening spleen and stomach and the like, and is used for treating postpartum anemia, poor dew, dizziness, palpitation and hyperhidrosis, insomnia and fatigue and inappetence.

According to statistics, about 2500 million women in the childbearing age in China are pregnant and delivered every year, and women who receive induced abortion operations have tens of millions of times, so that the health-care rehabilitation work of postpartum (including induced labor and abortion) is well done, and the health-care rehabilitation method has important significance for guaranteeing the health of women and children and improving the quality of human bodies.

The postpartum rehabilitation ointment is a pure traditional Chinese medicine preparation with the functions of tonifying qi and nourishing blood, soothing nerves and strengthening spleen, and nourishing yin and liver, and can effectively treat gynecological postpartum symptoms such as postpartum anemia, poor lochia, light headedness and the like.

Postpartum recovery contains various flavonoids, phenolic acids and alkaloids, and the content of each effective component cannot be comprehensively and accurately evaluated by the existing content measurement method.

Therefore, it is necessary to establish a fingerprint detection method of the postpartum rehabilitation ointment by adopting high performance liquid chromatography on the basis of the prior art, and the method has important significance for ingredient identification, quality evaluation and quality standard formulation of the postpartum rehabilitation ointment.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to overcome the defects of the prior art and develop a fingerprint detection method for postpartum rehabilitation ointment, and the method can control the stable quality of the postpartum rehabilitation ointment, ensure the safety and the effectiveness of the postpartum rehabilitation ointment and perfect the optimization of related pharmaceutical technology.

The technical scheme is as follows: in order to realize the purpose, the invention adopts the technical scheme that:

a fingerprint detection method of postpartum rehabilitation ointment comprises the following steps:

step 1, preparation of a postpartum recovery paste test sample solution:

placing a proper amount of puerperal rehabilitation ointment into a conical flask with a plug, adding purified water, oscillating for dissolution, adding ethyl acetate, oscillating for extraction, standing, layering the upper layer and the lower layer clearly, taking the upper layer of extract, repeatedly extracting for two times, combining the extracts, evaporating in a water bath, diluting the residue with methanol to a constant volume, centrifuging, taking the supernatant, filtering with a 0.45-micrometer organic microporous membrane, and taking the subsequent filtrate to obtain a sample solution to be tested;

step 2, preparation of postpartum recovery paste reference substance solution:

respectively taking appropriate amount of vanillic acid, syringic acid, leonurine hydrochloride, hesperidin, liquiritin, salvianolic acid B and quercetin reference substances, precisely weighing, respectively placing in volumetric flasks, and adding methanol to respectively prepare reference substance stock solutions; respectively and precisely sucking appropriate amount of control stock solutions of vanillic acid, syringic acid, leonurine hydrochloride, hesperidin, liquiritin, salvianolic acid B and quercetin, adding methanol to constant volume, and making into mixed control solution of vanillic acid, syringic acid, leonurine hydrochloride, hesperidin, liquiritin, salvianolic acid B and quercetin;

step 3, precisely absorbing the postpartum rehabilitation ointment test sample solution in the step 1 and the mixed reference substance solution in the step 2 respectively, injecting the solutions into a high performance liquid chromatograph, and recording a chromatogram;

step 4, exporting the fingerprint of the postpartum recovery paste test sample solution obtained in the step 3, and importing the fingerprint into a traditional Chinese medicine chromatography fingerprint similarity evaluation system 2012A; selecting chromatographic peaks existing in chromatograms of different batches of postpartum healing ointments as common peaks; generating a comparison fingerprint of the postpartum rehabilitation ointment by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak; and marking chemical components of peaks in the comparison fingerprint spectrum according to the retention time of the mixed comparison product solution chromatogram.

As a preferred scheme, the fingerprint spectrum detection method of the postpartum rehabilitation ointment is characterized in that,

step 1, the preparation method of the postpartum recovery paste test sample solution comprises the following steps:

placing 3ml of postpartum rehabilitation ointment into a conical flask with a plug, adding 10ml of purified water, oscillating for dissolution, adding 20ml of ethyl acetate, oscillating for extraction, standing, taking the upper layer of extract liquor after the upper layer and the lower layer are clearly layered, repeatedly extracting for two times, combining the extract liquor, evaporating by drying in a water bath, diluting residues to a constant volume with 2ml of methanol, centrifuging, taking the supernatant, filtering with a 0.45-micron organic microporous membrane, and taking the subsequent filtrate to obtain a sample solution to be tested;

as a preferred scheme, the fingerprint spectrum detection method of the postpartum rehabilitation ointment is characterized in that,

step 2, preparation of a mixed reference solution:

respectively taking appropriate amount of vanillic acid, syringic acid, leonurine hydrochloride, hesperidin, liquiritin, salvianolic acid B and quercetin reference substances, precisely weighing, placing in a10 ml volumetric flask, and adding methanol to prepare reference substance stock solutions with the concentrations of 457 mu g/ml, 591 mu g/ml, 519 mu g/ml, 456.5 mu g/ml, 750 mu g/ml, 423 mu g/ml and 624 mu g/ml respectively; respectively precisely measuring appropriate amounts of vanillic acid, syringic acid, leonurine hydrochloride, hesperidin, liquiritin, salvianolic acid B and quercetin reference substance stock solution, adding methanol to constant volume to 20ml, and preparing into mixed reference substance solution containing 45.7 μ g of vanillic acid, 59.1 μ g of syringic acid, 51.9 μ g of leonurine hydrochloride, 45.65 μ g of hesperidin, 75 μ g of liquiritin, 42.3 μ g of salvianolic acid B and 62.4 μ g of quercetin per 1 ml.

As a preferred scheme, in the method for detecting the fingerprint of the postpartum rehabilitation ointment, step 3, the chromatographic conditions detected by the high performance liquid chromatograph are as follows: using octadecylsilane chemically bonded silica as a filler, formic acid water as a mobile phase A, acetonitrile as a mobile phase B, and carrying out gradient elution at a flow rate of 0.5-1.5 ml/min < -1 >, a column temperature of 25-40 ℃ and a detection wavelength of 200-360 nm;

as a preferred scheme, the fingerprint detection method of postpartum rehabilitation ointment is characterized in that the chromatographic conditions detected by the high performance liquid chromatograph in the step 3 are as follows: octadecylsilane chemically bonded silica is used as a filling agent, 0.1% formic acid water is used as a mobile phase A, acetonitrile is used as a mobile phase B, gradient elution is carried out, the flow rate is 1.0 ml/min < -1 >, the column temperature is 30 ℃, and the detection wavelength is 254 nm.

Preferably, the fingerprint detection method of postpartum rehabilitation ointment is characterized in that gradient elution is preferably as follows:

time/min	Volume percent of mobile phase A	Volume percent of mobile phase B
			0	95	5
15	95	5
			55	90	10
80	85	15
			160	70	30
170	65	35
			175	95	5
180	95	5

。

Preferably, in the method for detecting fingerprint of postpartum rehabilitation ointment, the chromatographic column is YMC-Pack ODS-A chromatographic column (250mm × 4.6mm, 5 μm).

As a preferred scheme, the fingerprint spectrum of the postpartum rehabilitation ointment obtained by the method for detecting the fingerprint spectrum of the postpartum rehabilitation ointment has 13 common peaks, and the retention time of each common peak is as follows: has a total peak 1 and a retention time of 23.162 min; peak 2 was shared, retention time 26.381 min; peak 3 was shared, retention time 37.807 min; peak 4 was shared, retention time 47.379 min; peak 5 was shared, retention time 76.069 min; peak 6 was shared, retention time 88.859 min; peak 7 was shared, retention time 100.243 min; peak 8 was shared, retention time 109.256 min; peak 9 was shared, retention time 117.862 min; peak 10 was shared, retention time 125.050 min; peak 11 was shared, retention time 131.556 min; peak 12 was shared, retention time 135.199 min; there was a peak 13 with a retention time of 168.875 min.

Wherein, the peak 3 is vanillic acid, the peak 4 is syringic acid, the peak 5 is leonurine hydrochloride, the peak 6 is hesperidin, the peak 8 is liquiritin, the peak 10 is salvianolic acid B, and the peak 11 is quercetin.

The experimental method and the optimization experiment of the fingerprint spectrum detection condition are as follows:

(1) selection of different mobile phases:

the invention screens mobile phase systems such as pure water-acetonitrile, pure water-methanol, 0.1% formic acid water-acetonitrile, 0.1% formic acid water-methanol, 0.1% phosphoric acid water-acetonitrile, 0.1% phosphoric acid water-methanol and the like for detection through a large number of experiments, and the detection result shows that the mobile phase system containing 0.1% volume of formic acid water and acetonitrile is selected, and the system has stable baseline, peak shape and best separation degree of each peak. Therefore, the mobile phase A containing 0.1% by volume of formic acid and the mobile phase B containing acetonitrile are selected as the best mobile phase.

The effective components of the postpartum rehabilitation ointment not only contain flavone and phenolic acid acidic components, but also contain alkaloid components, so that the separation difficulty on the same chromatographic column is higher. On the basis of determining a mobile phase, different gradient elution modes are screened in order to achieve better separation degree, and experimental results show that the different gradient elution modes have large influence on the separation degree of each compound, and the optimal gradient elution mode is preferably obtained by adjusting the separation degree and the chromatographic peak number in different time periods: the ratio of mobile phase B was varied as: 0-15 min, 5% -5% acetonitrile; 15-55 min, 5% -10% acetonitrile; 55-80 min, 10-15% acetonitrile, 80-160 min, 15-30% acetonitrile; 160-170 min, 30-35% acetonitrile; 170-175 min, 35-5% acetonitrile; 175-180 min, 5% -5% acetonitrile;

(2) selection of different detection wavelengths:

according to the invention, chromatographic detection results with different wavelengths of 230nm, 254nm, 280nm and 350nm are screened through a large number of experiments, and the detection results show that the intensity of each characteristic peak under 254nm is better, so that 254nm is selected as the detection wavelength, as shown in figure 1.

The invention has the beneficial effects that:

(1) according to the structural property characteristics of active ingredients contained in the postpartum rehabilitation ointment, the optimal mobile phase composition is screened out through a large number of experiments, analysis conditions such as gradient elution procedures, flow rate, detection wavelength, chromatographic column, column temperature and the like are verified through a plurality of experiments, and the ginseng poria cocos wine fingerprint spectrum detection method provided by the invention can comprehensively, objectively and accurately detect and evaluate the quality of the postpartum rehabilitation ointment and has important significance for ensuring the curative effect of the clinical postpartum rehabilitation ointment.

(2) The fingerprint spectrum established by the invention can pay attention to the front-back sequence and the mutual relation of all the formed fingerprint characteristic peaks and the overall facial features, thereby not only avoiding the one-sidedness of judging the quality of the produced health paste due to the measurement of individual chemical components, but also reducing the possibility of manual treatment for reaching the quality standard.

(3) The method has the advantages of simplicity, convenience, stability, high precision and good reproducibility.

Drawings

FIG. 1 is a chromatogram of a postpartum recovery ointment sample at 254 nm.

FIG. 2 is a chromatogram of a control solution at 254 nm.

FIG. 3 is a fingerprint of 10 batches of postpartum rehabilitation ointment sample solutions.

Detailed Description

Embodiments of the present invention will be described in detail with reference to examples, in which specific conditions are not specified, according to conventional conditions or conditions recommended by manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

Apparatus used in the following examples

LC-20AT high performance liquid chromatograph (SPD-M20A diode array detector) (Shimadzu, Japan), BP211D model ten thousandth precision analytical balance (Beijing Saedoolis instruments systems Co., Ltd.), FA1004N model one hundred thousandth precision electronic analytical balance (Beijing Saudis instruments systems Co., Ltd.), HH-6 model digital display constant temperature water bath (Jiangnan laboratory instruments, Changz-D (III)) model circulating water vacuum pump (Chengxi Yuhua instruments Co., Ltd.), KQ5200DE model digital control ultrasonic cleaner (frequency: 40KHz, Kunshan ultrasonic instruments Co., Ltd.).

The following examples used the following reagents: vanillic acid analysis standard (batch No. 110776-. Acetonitrile is chromatographically pure, water is ultrapure water, and formic acid is analytically pure. Postpartum recovery paste (Jiangsu Haishen pharmaceutical Co., Ltd., batch Nos. 190301, 190401, 190402, 190501, 190502, 200101, 200102, 200301, 200401, 200601) and other reagents (analytically pure).

Example 1

A fingerprint detection method of postpartum recovery ointment comprises the following steps:

(1) preparation of a test solution:

putting 3ml of postpartum rehabilitation ointment (Jiangsu Haichang pharmaceutical industry Co., Ltd., batch No. 190301, 190401, 190402, 190501, 190502, 200101, 200102, 200301, 200401 and 200601) into a conical flask with a stopper, adding 10ml of purified water, oscillating for dissolution, adding 20ml of ethyl acetate, oscillating for extraction, standing, separating the upper layer and the lower layer clearly, taking the upper layer of extract, extracting twice, combining the extracts, evaporating in water bath, diluting the residue with 2ml of methanol to a constant volume, centrifuging, taking the supernatant, filtering with a 0.45 mu m organic microporous filter membrane, and taking the subsequent filtrate to obtain the postpartum rehabilitation ointment sample solution.

(2) Preparation of control solutions:

respectively taking appropriate amount of vanillic acid, syringic acid, leonurine hydrochloride, hesperidin, liquiritin, salvianolic acid B and quercetin reference substances, precisely weighing, placing in a10 ml volumetric flask, and adding methanol to prepare reference substance stock solutions with the concentrations of 457 mu g/ml, 591 mu g/ml, 519 mu g/ml, 456.5 mu g/ml, 750 mu g/ml, 423 mu g/ml and 624 mu g/ml respectively; respectively precisely measuring appropriate amounts of vanillic acid, syringic acid, leonurine hydrochloride, hesperidin, liquiritin, salvianolic acid B and quercetin reference substance stock solution, adding methanol to constant volume to 20ml, and preparing into mixed reference substance solution containing 45.7 μ g of vanillic acid, 59.1 μ g of syringic acid, 51.9 μ g of leonurine hydrochloride, 45.65 μ g of hesperidin, 75 μ g of liquiritin, 42.3 μ g of salvianolic acid B and 62.4 μ g of quercetin per 1 ml.

(3) Respectively and precisely sucking 10 batches of postpartum rehabilitation ointment test sample solution in the step 1 and the mixed reference substance solution in the step 2, injecting the solutions into a high performance liquid chromatograph, and recording a chromatogram;

(4) exporting the fingerprints of the 10 batches of postpartum rehabilitation ointment test sample solutions obtained in the step 3, and importing the fingerprints into a traditional Chinese medicine chromatography fingerprint similarity evaluation system 2012A; selecting chromatographic peaks existing in chromatograms of 10 batches of postpartum rehabilitation ointments as common peaks; the comparison fingerprint of the postpartum rehabilitation ointment is generated by using an average calculation method, as shown in figure 3, and the similarity evaluation result is shown in table 2. Calculating the relative retention time and the relative peak area of each common peak; and labeling the chemical components of the peak in the control fingerprint according to the retention time of a mixed control solution chromatogram (shown in figure 2, the peak 3 is vanillic acid, the retention time is 37.807min, the peak 4 is syringic acid, the retention time is 47.379min, the peak 5 is leonurine hydrochloride, the retention time is 76.069min, the peak 6 is hesperidin, the retention time is 88.859min, the peak 8 is liquiritin, the retention time is 117.862min, the peak 10 is salvianolic acid B, the retention time is 131.556min, the peak 11 is quercetin, and the retention time is 135.199 min).

Chromatographic conditions and System applicability

A chromatographic column: YMC-Pack ODS-A column (250 mm. times.4.6 mm, 5 μm).

Mobile phase: phase A: 0.1 vol% formic acid water; phase B: acetonitrile; detection wavelength: 254nm, flow rate: 1.0 ml/min^-1Column temperature: 30 ℃; the gradient elution procedure is as follows in table 1:

TABLE 1 gradient elution procedure

Time/min	Volume percent of mobile phase A	Volume percent of mobile phase B
			0	95	5
15	95	5
			55	90	10
80	85	15
			160	70	30
170	65	35
			175	95	5
180	95	5

。

TABLE 210 evaluation results of similarity of postpartum rehabilitation ointment test sample solutions

The acquired postpartum rehabilitation plaster fingerprint contains 13 common peaks, and the retention time of each common peak is as follows: has a total peak 1 and a retention time of 23.162 min; peak 2 was shared, retention time 26.381 min; peak 3 was shared, retention time 37.807 min; peak 4 was shared, retention time 47.379 min; peak 5 was shared, retention time 76.069 min; peak 6 was shared, retention time 88.859 min; peak 7 was shared, retention time 100.243 min; peak 8 was shared, retention time 109.256 min; peak 9 was shared, retention time 117.862 min; peak 10 was shared, retention time 125.050 min; peak 11 was shared, retention time 131.556 min; peak 12 was shared, retention time 135.199 min; there was a peak 13 with a retention time of 168.875 min.

Wherein, the peak 3 is vanillic acid, the peak 4 is syringic acid, the peak 5 is leonurine hydrochloride, the peak 6 is hesperidin, the peak 8 is liquiritin, the peak 10 is salvianolic acid B, and the peak 11 is quercetin.

Example 2 methodological studies of fingerprint detection:

1. methodology investigation

1.1 precision investigation

Taking a postpartum recovery paste sample with the batch number of 200601, preparing a sample solution according to the sample preparation method of the example 1, carrying out continuous sample introduction for 6 times, wherein the sample introduction amount is 10 mu L each time, detecting according to the chromatographic conditions of the example 1, measuring an HPLC chromatogram, and inspecting 10 common fingerprint peaks in the chromatogram, wherein the result shows that the retention time RSD of the common fingerprint peaks is less than 3.42%, the common peak area RSD is less than 1.89%, and the instrument precision is better.

1.2 stability Studies

Taking a postpartum recovery paste sample with the batch number of 200601, preparing a sample solution according to the sample preparation method of the example 1, injecting samples at 0, 4, 8, 12, 16 and 24 hours according to the chromatographic conditions in the example 1, recording a chromatogram, and inspecting 10 common fingerprint peaks in the chromatogram, wherein the results show that the retention time RSD of the common fingerprint peaks is less than 1.22%, the area RSD of the common peak is less than 2.76%, and the sample solution has better stability within 24 hours.

1.3 repeatability test

6 parts of the postpartum recovery paste sample with the batch number of 200601 are taken, the sample solution is prepared according to the sample preparation method of the example 1, the sample solution is respectively measured, the chromatogram is recorded, and 6 common fingerprint peaks in the chromatogram are examined, and the result shows that the retention time RSD of the common fingerprint peaks is less than 0.59 percent, and the area RSD of the common fingerprint peaks is less than 2.56 percent, which indicates that the method has good repeatability.

The experimental results show that the fingerprint detection method for postpartum rehabilitation ointment, which is established by the invention, has good precision, stability and repeatability, can effectively characterize the quality of postpartum rehabilitation ointment, and is beneficial to comprehensively monitoring the quality of postpartum rehabilitation ointment.