CN112946134A - HPLC fingerprint spectrum of motherwort injection and establishing method thereof - Google Patents
HPLC fingerprint spectrum of motherwort injection and establishing method thereof Download PDFInfo
- Publication number
- CN112946134A CN112946134A CN202110156491.9A CN202110156491A CN112946134A CN 112946134 A CN112946134 A CN 112946134A CN 202110156491 A CN202110156491 A CN 202110156491A CN 112946134 A CN112946134 A CN 112946134A
- Authority
- CN
- China
- Prior art keywords
- peak
- hplc fingerprint
- injection
- phase
- retention time
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
Landscapes
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Library & Information Science (AREA)
- Engineering & Computer Science (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides an HPLC fingerprint spectrum of motherwort injection and an establishment method thereof. The HPLC fingerprint spectrum of the motherwort injection comprises 4 common characteristic peaks which are arranged according to ascending retention time, and the common characteristic peaks are respectively as follows: peak No. 1: the retention time is 3.4 min; peak No. 2: the retention time is 11.3 min; peak No. 3: the retention time is 12.9 min; peak No. 4: retention time 14.7 min. The invention establishes the motherwort injection HPLC fingerprint spectrum comprising the 4 common characteristic peaks for the first time, and the substances such as trigonelline hydrochloride, stachydrine hydrochloride and the like in the motherwort injection can be effectively controlled in quality by utilizing the HPLC fingerprint spectrum, so that the preparation is more stable and controllable. The method for establishing the HPLC fingerprint spectrum of the motherwort injection has good durability, precision and repeatability, and provides a new quality control method for the motherwort injection.
Description
Technical Field
The invention belongs to the field of quality control of injection preparations, and particularly relates to an HPLC fingerprint spectrum of a motherwort injection and an establishment method thereof.
Background
A motherwort injection is a clinical common traditional Chinese medicine for treating gynecological diseases. Research shows that the motherwort injection adopted after abortion of pregnant women can reduce postpartum hemorrhage, and because the motherwort injection is used as a medicine for contracting uterus, the motherwort injection has very strong effect of exciting uterine smooth muscle, is used for patients suffering from abortion, is beneficial to recovering and regenerating endometrium wounds of the patients, reduces vaginal hemorrhage amount of the patients and shortens bleeding time; in addition, the herba Leonuri injection also has effect in treating dysmenorrhea, and can be used for treating migraine in menstrual period.
The motherwort injection contains total alkaloids as main effective components, and the total alkaloids in the motherwort injection are mainly composed of stachydrine hydrochloride and choline chloride according to material base research results, wherein the stachydrine hydrochloride accounts for about 90% of the total alkaloids. According to the twentieth volume of Standard Chinese medicinal preparations of the Ministry of health, each injection (1mL) of herba Leonuri contains total alkaloid and stachydrine hydrochloride (C)7H13NO2HCl), should be 18-22 mg.
Because the components contained in the motherwort injection are complex, the stability and controllability of the motherwort injection are poor, different medicinal materials and finished product batches are different, and the quality of the motherwort injection also fluctuates greatly. In addition to total alkaloids, the motherwort injection contains many other known or unknown components. Therefore, the measurement of the content of a single component is not enough to indicate the quality of the motherwort injection and lacks specificity.
The traditional Chinese medicine fingerprint spectrum refers to a spectrum of a common peak which can mark the characteristics of a certain traditional Chinese medicine or a traditional Chinese medicine by adopting a certain analysis means after the traditional Chinese medicine or the traditional Chinese medicine is properly processed. According to the quality control purpose, the method can be divided into a fingerprint spectrum and a characteristic spectrum. The fingerprint spectrum is the overall information based on the spectrum, is used for the overall evaluation of the quality of the traditional Chinese medicine, and ensures the uniformity and stability of the internal quality. The fingerprint spectrum can establish a comprehensive evaluation system with objective, integral and multi-index from the viewpoint that the holistic concept of the traditional Chinese medicine and the drug effect of the traditional Chinese medicine come from the comprehensive action of various chemical substances, can more comprehensively reflect the types and the quantity of chemical components contained in the traditional Chinese medicine through the holistic characteristics of the spectrum, and is a comprehensive and quantifiable quality control means. Compared with the determination of single component, it has the advantages of providing more abundant and effective quality and quantity information, so that the fingerprint spectrum technology can more objectively, scientifically and reasonably control the quality of the traditional Chinese medicine. The traditional Chinese medicine fingerprint spectrum can reflect the internal quality of the traditional Chinese medicine macroscopically and comprehensively, and is approved by researchers at home and abroad.
Therefore, establishing the fingerprint spectrum which can accurately and comprehensively reflect the chemical components of the motherwort injection has important significance.
Yang Chao et al (research on fingerprint of herba Leonuri injection) disclose a method for establishing fingerprint of herba Leonuri injection, which utilizes high performance liquid chromatography-evaporative light scattering detection method to establish fingerprint of herba Leonuri injection, and determines that the chromatographic peak in the fingerprint has Cl-5-hydroxymethyl-2-furoic acid, stachydrine and Na+、K+And choline. Fingerprint detection is carried out on 31 batches of motherwort injection, and similarity calculation is carried out on the fingerprint of each batch of samples by adopting a traditional Chinese medicine fingerprint similarity evaluation system, wherein the similarity is more than 0.90.
However, with the intensive research on the chemical components of the motherwort injection, the alkaloid component trigonelline is found to exist in the motherwort injection, and the existing motherwort injection cannot effectively control the trigonelline in the motherwort injection by using an HPLC (high performance liquid chromatography) fingerprint spectrum.
Disclosure of Invention
The invention aims to provide a novel HPLC fingerprint spectrum of motherwort injection and an establishment method thereof. The common characteristic peaks in the HPLC fingerprint spectrum comprise 2 unknown peaks, a stachydrine hydrochloride peak and a trigonelline hydrochloride peak, and the HPLC fingerprint spectrum can be used for effectively controlling the quality of substances such as trigonelline hydrochloride, stachydrine hydrochloride and the like in the motherwort injection, so that the preparation is more stable and controllable.
The invention provides a method for establishing an HPLC fingerprint of motherwort injection, which utilizes a high performance liquid chromatography and specifically comprises the following steps:
(1) preparing a test solution: collecting herba Leonuri injection;
(2) absorbing the test solution, injecting into a high performance liquid chromatograph for detection, wherein the detection conditions are as follows:
a chromatographic column: the filler is a cation exchanger of sulfonic group bonded silica gel;
mobile phase: the composite material consists of a phase A and a phase B, wherein the phase A is acetonitrile, and the phase B is a potassium dihydrogen phosphate solution.
Further, in the step (2), the concentration of the potassium dihydrogen phosphate solution is 10 mmol/L-40 mmol/L; the volume ratio of the phase A to the phase B is (5-11): (89-95); the detected flow rate is 0.6-1.2 mL/min; the detected column temperature is 25-35 ℃; the detection wavelength is 185-200 nm.
Further, in the step (2), the concentration of the potassium dihydrogen phosphate solution is 20 mmol/L; the volume ratio of the phase A to the phase B is 8: 92; the detected flow rate is 0.8 mL/min; the detected column temperature is 30 ℃; the detection wavelength is 192 nm.
Further, the method comprises the step of injecting a reference substance solution into a high performance liquid chromatograph for detection, wherein the reference substance is one or two of stachydrine hydrochloride and trigonelline.
Further, the preparation method of the reference solution comprises the following steps: dissolving stachydrine hydrochloride reference substance in water to obtain a stachydrine hydrochloride reference substance solution; dissolving trigonelline reference substance in water to obtain trigonelline reference substance solution.
Further, the concentration of the stachydrine hydrochloride reference substance solution is 0.5-2.0 mg/mL; the concentration of the trigonelline reference substance solution is 5-20 mug/mL.
Further, the concentration of the stachydrine hydrochloride reference substance solution is 0.7 mg/mL; the concentration of the trigonelline reference solution is 8 mug/mL.
The invention also provides an HPLC fingerprint of the motherwort injection, which is constructed according to the method and comprises the following 4 common characteristic peaks: with peak 4 as a reference peak, peak 1 relative retention time 0.23 + -0.05, peak 2 relative retention time 0.77 + -0.05, peak 3 relative retention time 0.88 + -0.05, and peak 4 relative retention time 1.00 + -0.05.
Further, it contains the following 4 common characteristic peaks: according to the ascending order of retention time, the common characteristic peaks are respectively as follows: peak No. 1: the retention time is 3.4 plus or minus 0.05 min; peak No. 2: the retention time is 11.3 plus or minus 0.05 min; peak No. 3: the retention time is 12.9 plus or minus 0.05 min; peak No. 4: retention time 14.7 ± 0.05 min.
Further, the ratio of peak areas of the common characteristic peaks is peak No. 1: peak No. 2: peak No. 3: peak No. 4 ═ 23:2:17: 9.
Further, the ratio of the peak area of the common characteristic peak to the total peak area is respectively as follows: 23% of peak 1, 2% of peak 2, 17% of peak 3, and 9% of peak 4.
Further, the peak 3 is an absorption peak of stachydrine hydrochloride, and the peak 4 is an absorption peak of trigonelline.
Further, the HPLC fingerprint is shown in figure 1.
The invention also provides a motherwort injection, and the HPLC fingerprint spectrum of the motherwort injection is as above.
The invention establishes the HPLC fingerprint of the motherwort injection comprising 4 common characteristic peaks of 2 unknown peaks, a stachydrine hydrochloride peak and a trigonelline hydrochloride peak for the first time, and the HPLC fingerprint can be used for effectively controlling the quality of the trigonelline hydrochloride, the stachydrine hydrochloride and other substances in the motherwort injection, so that the preparation is more stable and controllable.
The HPLC fingerprint of the motherwort injection established by the invention has 4 common characteristic peaks, similarity analysis is carried out by taking the HPLC fingerprint as a comparison fingerprint, the similarity of 13 batches of motherwort injection samples is respectively 0.995, 0.994, 0.999, 0.995, 0.972, 0.982, 0.998, 0.995, 0.998, 0.962, 0.960, 0.954 and 0.962, the average is 0.982, and the similarity of the HPLC fingerprint and the comparison fingerprint of each motherwort injection sample is more than 0.9. The established HPLC fingerprint spectrum of the motherwort injection has strong representativeness, and the invention provides a new quality control method for the motherwort injection.
The HPLC fingerprint spectrum method for establishing the motherwort injection has good durability, precision and repeatability.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
Fig. 1 is an HPLC fingerprint of the motherwort injection established in example 2, wherein peak 1 is an unknown peak, peak 2 is an unknown peak, peak 3 is stachydrine hydrochloride, and peak 4 is trigonelline.
FIG. 2 is a HPLC fingerprint obtained at a column temperature of 25 ℃ in the evaluation of durability of the method.
FIG. 3 is a HPLC fingerprint obtained at a column temperature of 30 ℃ in the evaluation of durability of the method.
FIG. 4 is a HPLC fingerprint obtained at a column temperature of 35 ℃ in the evaluation of durability of the method.
FIG. 5 shows the HPLC fingerprint obtained from the 1 st injection in the precision measurement.
FIG. 6 shows the HPLC fingerprint obtained from the 2 nd injection in the precision measurement.
FIG. 7 shows the HPLC fingerprint obtained from the 3 rd injection in the precision measurement.
FIG. 8 shows the HPLC fingerprint obtained from the 4 th injection in the precision measurement.
FIG. 9 shows the HPLC fingerprint obtained from the 5 th injection in the precision measurement.
FIG. 10 shows the HPLC fingerprint obtained from the 6 th injection in the precision measurement.
FIG. 11 is the HPLC fingerprint measured for the 1 st sample solution in the repeatability test.
FIG. 12 is a HPLC fingerprint measured for the 2 nd sample solution in the repeatability test.
FIG. 13 is an HPLC fingerprint measured for the 3 rd sample solution in the repeatability test.
FIG. 14 shows HPLC finger prints obtained from the 4 th sample solution in a repeat study.
FIG. 15 shows HPLC finger prints obtained from the 5 th sample solution in the reproducibility test.
FIG. 16 is a HPLC fingerprint measured for the 6 th sample solution in the repeatability test.
Detailed Description
The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.
The stachydrine hydrochloride reference substance is purchased from China food and drug testing research institute, and the trigonelline hydrochloride reference substance is purchased from China food and drug testing research institute.
Cation exchangers for sulfonic acid-bonded silica gel (SCX) were purchased from agilent.
Example 1 preparation of motherwort injection
Decocting motherwort herb with 3 times of water for three times, wherein the first time is 2 hours, the second time is 1.7 hours, and the third time is 1.5 hours, filtering, concentrating the filtrate under reduced pressure to obtain clear paste with the relative density of 1.36-1.38 (80 ℃), cooling, and adding water to dilute until the water content is 35%; precipitating with ethanol for three times, adding ethanol for the first time to make ethanol content reach 78%, adding ethanol for the second time to make ethanol content reach 88%, and adding ethanol for the third time to make ethanol content reach 92%; standing, collecting supernatant, and recovering ethanol to obtain soft extract (named as ethanol precipitation extract).
Taking alcohol precipitation paste, adding injection water with the weight being 3-5 times of that of the alcohol precipitation paste, stirring, standing, taking supernate, decoloring with 15 wt.% of activated carbon until the solution is light yellow to yellow, adjusting the pH to 4.8-5.2 with 40% sodium hydroxide solution or dilute hydrochloric acid, adding 5 wt.% of activated carbon, boiling for 10-15 minutes, decarburizing and filtering to obtain concentrated solution of the leonurus (each 1ml of the concentrated solution contains total alkaloids calculated by stachydrine hydrochloride, and the total alkaloids content is not less than 35 mg).
Accurately weighing herba Leonuri concentrated solution [ containing total alkaloids (calculated as stachydrine hydrochloride) 20g ], placing in a preparation tank, adding appropriate amount of water for injection, adding 10ml of benzyl alcohol, stirring, adding activated carbon (1-5 g), circularly decarbonizing for 10 min, adding water for injection to 1000ml, stirring, fine filtering, bottling, sterilizing, and packaging to obtain herba Leonuri injection.
Example 2 method for establishing HPLC fingerprint of motherwort injection
(1) Preparing a test solution: taking 1mL of herba Leonuri injection (prepared by the method of example 1), heating in water bath for 0.5 hr, taking out, cooling, adding water to dissolve, transferring to a 25mL volumetric flask, adding water to dilute to the scale, shaking, filtering, and collecting the filtrate.
(2) Preparation of control solutions:
weighing 7mg of stachydrine hydrochloride reference substance, precisely weighing, placing in a 10mL volumetric flask, adding water for dissolving, and fixing the volume to obtain a stachydrine hydrochloride reference substance solution; the concentration of the stachydrine hydrochloride in the stachydrine hydrochloride reference substance solution is 0.7 mg/mL;
weighing 80 μ g of trigonelline reference substance, precisely weighing, placing in a 10mL volumetric flask, adding water to dissolve, and fixing volume to obtain trigonelline reference substance solution; the concentration of trigonelline in the trigonelline reference solution is 8 μ g/mL.
(3) Respectively sucking 10 μ L of reference solution and test solution, injecting into high performance liquid chromatograph, and measuring under the following chromatographic conditions:
a chromatographic column: cation exchanger bonded with silica gel (SCX) in sulfonic group is used as filler (Agilent Zorbax 300-SCX, 4.6 × 250mm, 5 μm);
mobile phase: the composition comprises a phase A and a phase B, wherein the phase A is acetonitrile, the phase B is 20mmol/L potassium dihydrogen phosphate aqueous solution, and the volume ratio of the phase A to the phase B is 8: 92;
flow rate: 0.8 mL/min;
column temperature: 30 ℃;
detection wavelength: 192 nm.
The separation degree of the stachydrine hydrochloride peak and the trigonelline hydrochloride peak is not less than 2.0.
According to the above method, HPLC fingerprint (shown in figure 1) of herba Leonuri injection is obtained, wherein the No. 1 peak is unknown peak, the No. 2 peak is unknown peak, the No. 3 peak is stachydrine hydrochloride, and the No. 4 peak is trigonelline. Arranged according to ascending retention time sequence, the retention time of each chromatographic peak is as follows: peak No. 1: the retention time is 3.4 min; peak No. 2: the retention time is 11.3 min; peak No. 3: the retention time is 12.9 min; peak No. 4: retention time 14.7 min. The peak areas of the chromatographic peaks are respectively as follows: 23% of peak 1, 2% of peak 2, 17% of peak 3, and 9% of peak 4.
The beneficial effects of the detection method of the invention are demonstrated by experimental examples below.
Experimental example 1 evaluation of similarity of HPLC fingerprint of motherwort injection of the present invention
1. Experimental methods
(1)24 batches of motherwort injection (all commercially available in Chengdu first pharmaceutical market) were examined according to the method of example 2 to generate each HPLC fingerprint.
(2) 13 batches of motherwort injection are prepared according to the method of the embodiment 1, and HPLC fingerprint spectrums of the 13 batches of motherwort injection are detected according to the method of the embodiment 2. Then, similarity analysis was performed using the HPLC fingerprint obtained in example 2 as a control fingerprint.
2. Results of the experiment
(1) The HPLC fingerprint of 24 batches of motherwort injection has 4 common characteristic peaks which are arranged according to ascending retention time, and the retention time of each spectrum peak is as follows: peak No. 1: the retention time is 3.4 min; peak No. 2: the retention time is 11.3 min; peak No. 3: the retention time is 12.9 min; peak No. 4: retention time 14.7 min; the peak areas of the chromatographic peaks are respectively as follows: 23% of peak 1, 2% of peak 2, 17% of peak 3, and 9% of peak 4.
(2) And (3) performing similarity analysis by using the HPLC fingerprint obtained in example 2 as a control fingerprint, wherein the HPLC fingerprint similarities of 13 motherwort injection samples are respectively 0.995, 0.994, 0.999, 0.995, 0.972, 0.982, 0.998, 0.995, 0.998, 0.962, 0.960, 0.954 and 0.962, the average is 0.982, and the similarity between the HPLC fingerprint and the control fingerprint of each sample is more than 0.9. The HPLC fingerprint spectrum of the motherwort injection established by the invention has strong representativeness.
Experimental example 2 methodological investigation of the motherwort injection HPLC fingerprint spectrum establishment method
(1) Method of evaluation of durability
a. Flow rate of flow
A motherwort injection (lot No. 190302) was prepared according to the method of example 1. With reference to the method of example 2, the motherwort injection (lot No. 190302) was used as a sample, and HPLC analysis was performed at different flow rates (shown in Table 1) (the remaining detection conditions were the same as those of example 2), and an HPLC chromatogram was recorded. The results are as follows:
TABLE 1 table of the similarity results of motherwort injection (batch No. 190302) at different flow rates
From the above results, the similarity of HPLC fingerprints obtained at a flow rate of 0.6-1.2 ml/min is as high as 0.999, and the separation of the No. 3 peak (stachydrine hydrochloride) and the No. 4 peak (trigonelline) is as high as 2.02.
b. Column temperature
Motherwort injection is prepared according to the method of example 1. Referring to example 2, the herba Leonuri injection is used as test sample, HPLC detection is performed at flow rate of 0.8ml/min and column temperatures of 25 deg.C, 30 deg.C and 35 deg.C respectively (the rest detection conditions are the same as example 2), and chromatogram is recorded, and is shown in figure 2-4. The results are as follows:
table 2 different column temperature investigation results table
From the above results, the similarity of the HPLC fingerprints obtained at the column temperature of 25-35 ℃ is as high as above 0.990; the separation degree and the analysis time are comprehensively considered, and when the column temperature is 30 ℃, each performance parameter is optimal.
c. Flow phase ratio
Motherwort injection is prepared according to the method of example 1. Referring to the method of example 2, the motherwort injection is used as a sample, the volume ratio of a mobile phase a to a mobile phase B is 8:92, 5 different mobile phase ratios are designed according to the specification of 0512 high performance liquid chromatography of the four parts of the chinese pharmacopoeia 2015 edition (see table 3), HPLC detection is performed respectively (the rest detection conditions are the same as those of example 2), and a chromatogram is recorded. The results are given in the following table:
TABLE 3 table of the similarity measurement results of the finger prints obtained under different mobile phase ratios
From the above results, the similarity of the HPLC fingerprints obtained at the above mobile phase ratio was as high as 0.99 or more, and the separation of the No. 3 peak (stachydrine hydrochloride) and the No. 4 peak (trigonelline) was as high as 3.68 or more.
(2) Measurement of precision
Using herba Leonuri injection (prepared according to the method of example 1, lot number: 190302) as sample, performing HPLC determination according to the method of example 2, repeating sample injection for 6 times, and recording chromatogram, as detailed in figures 5-10. The results are as follows:
TABLE 4 results of precision measurement
From the results, the similarity of the HPLC fingerprint spectra obtained by 6 times of sample injection is 0.999, and RSD is 0, which shows that the precision of the motherwort injection HPLC fingerprint spectrum establishment method is good.
(3) Repeatability survey
Motherwort injection (prepared according to the method of example 1, lot # 190302) was used as a test sample, 6 test sample solutions were prepared in parallel according to the method of example 2, HPLC measurements were performed according to the method of example 2, and chromatograms were recorded, as shown in FIGS. 11-16. The similarity was calculated from the measurement results as follows:
TABLE 5 repeatability test results
From the above results, the similarity of HPLC fingerprints of 6 sample solutions in the repeatability test is 0.999, and RSD is 0. The repeatability of the motherwort injection HPLC fingerprint spectrum establishment method is good.
In conclusion, the invention provides an HPLC fingerprint spectrum of motherwort injection and an establishment method thereof. And (3) performing similarity analysis by taking the HPLC fingerprint as a control fingerprint, wherein the similarity of 13 motherwort injection samples is respectively 0.995, 0.994, 0.999, 0.995, 0.972, 0.982, 0.998, 0.995, 0.998, 0.962, 0.960, 0.954 and 0.962, the average similarity is 0.982, and the similarity of the HPLC fingerprint and the control fingerprint of each motherwort injection sample is more than 0.9. The invention establishes the HPLC fingerprint of the motherwort injection comprising 4 common characteristic peaks of 2 unknown peaks, a stachydrine hydrochloride peak and a trigonelline hydrochloride peak for the first time, and the HPLC fingerprint can be used for effectively controlling the quality of the trigonelline hydrochloride, the stachydrine hydrochloride and other substances in the motherwort injection, so that the preparation is more stable and controllable. The invention provides a new quality control method for motherwort injection.
Claims (10)
1. A method for establishing HPLC fingerprint of motherwort injection is characterized by comprising the following steps: the method utilizes high performance liquid chromatography, and specifically comprises the following steps:
(1) preparing a test solution: collecting herba Leonuri injection;
(2) absorbing the test solution, injecting into a high performance liquid chromatograph for detection, wherein the detection conditions are as follows:
a chromatographic column: the filler is a cation exchanger of sulfonic group bonded silica gel;
mobile phase: the composite material consists of a phase A and a phase B, wherein the phase A is acetonitrile, and the phase B is a potassium dihydrogen phosphate solution.
2. The method of claim 1, wherein: in the step (2), the concentration of the potassium dihydrogen phosphate solution is 10 mmol/L-40 mmol/L; the volume ratio of the phase A to the phase B is (5-11): (89-95); the detected flow rate is 0.6-1.2 mL/min; the detected column temperature is 25-35 ℃; the detection wavelength is 185-200 nm.
3. The method of claim 2, wherein: in the step (2), the concentration of the potassium dihydrogen phosphate solution is 20 mmol/L; the volume ratio of the phase A to the phase B is 8: 92; the detected flow rate is 0.8 mL/min; the detected column temperature is 30 ℃; the detection wavelength is 192 nm.
4. The method according to any one of claims 1 to 3, wherein: the method also comprises the step of injecting a reference substance solution into a high performance liquid chromatograph for detection, wherein the reference substance is one or two of stachydrine hydrochloride and trigonelline.
5. An HPLC fingerprint of a motherwort injection is characterized in that: it is constructed according to the method of any one of claims 1 to 4 and comprises the following 4 common characteristic peaks: with peak 4 as a reference peak, peak 1 relative retention time 0.23 + -0.05, peak 2 relative retention time 0.77 + -0.05, peak 3 relative retention time 0.88 + -0.05, and peak 4 relative retention time 1.00 + -0.05.
6. An HPLC fingerprint according to claim 5, characterized in that: the peak area ratio of the common characteristic peak is No. 1 peak: peak No. 2: peak No. 3: peak No. 4 ═ 23:2:17: 9.
7. An HPLC fingerprint according to claim 6, characterized in that: the proportions of the peak areas of the common characteristic peaks in the total peak area are respectively as follows: 23% of peak 1, 2% of peak 2, 17% of peak 3, and 9% of peak 4.
8. An HPLC fingerprint according to claim 5, characterized in that: the No. 3 peak is the absorption peak of stachydrine hydrochloride, and the No. 4 peak is the absorption peak of trigonelline.
9. An HPLC fingerprint according to any one of claims 5 to 8, wherein: the HPLC fingerprint is shown in figure 1.
10. A motherwort injection is characterized in that: its HPLC fingerprint spectrum is as defined in any one of claims 5 to 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110156491.9A CN112946134A (en) | 2021-02-04 | 2021-02-04 | HPLC fingerprint spectrum of motherwort injection and establishing method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110156491.9A CN112946134A (en) | 2021-02-04 | 2021-02-04 | HPLC fingerprint spectrum of motherwort injection and establishing method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112946134A true CN112946134A (en) | 2021-06-11 |
Family
ID=76244058
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110156491.9A Pending CN112946134A (en) | 2021-02-04 | 2021-02-04 | HPLC fingerprint spectrum of motherwort injection and establishing method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112946134A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113759043A (en) * | 2021-09-06 | 2021-12-07 | 北京康仁堂药业有限公司 | Characteristic spectrum of motherwort fruit and construction method thereof, method for measuring content of stachydrine hydrochloride, motherwort fruit formula granules and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110618188A (en) * | 2019-09-20 | 2019-12-27 | 成都第一制药有限公司 | Method for detecting content of cations in motherwort injection |
| CN111487344A (en) * | 2020-04-27 | 2020-08-04 | 江苏弘典中药产业研究院有限公司 | Method for detecting fingerprint spectrum of motherwort particles |
-
2021
- 2021-02-04 CN CN202110156491.9A patent/CN112946134A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110618188A (en) * | 2019-09-20 | 2019-12-27 | 成都第一制药有限公司 | Method for detecting content of cations in motherwort injection |
| CN111487344A (en) * | 2020-04-27 | 2020-08-04 | 江苏弘典中药产业研究院有限公司 | Method for detecting fingerprint spectrum of motherwort particles |
Non-Patent Citations (3)
| Title |
|---|
| 何卓琳 等: "基于指纹图谱的益母草胶囊质量控制方法研究", 《中国当代医药》 * |
| 刘琼 等: "探讨液相色谱中部分中药生物碱在SCX柱上的色谱分离行为", 《中国现代应用药学》 * |
| 杨超 等: "益母草注射液指纹图谱的研究", 《药物分析杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113759043A (en) * | 2021-09-06 | 2021-12-07 | 北京康仁堂药业有限公司 | Characteristic spectrum of motherwort fruit and construction method thereof, method for measuring content of stachydrine hydrochloride, motherwort fruit formula granules and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114689780A (en) | Multi-component quality detection method for Baoyin decoction extract | |
| CN110568099B (en) | Fingerprint spectrum construction method of radix acanthopanacis senticosi, radix angelicae sinensis and radix astragali refining agent and multi-index component synchronous content determination method | |
| CN112946134A (en) | HPLC fingerprint spectrum of motherwort injection and establishing method thereof | |
| CN114280209B (en) | Method for establishing fingerprint of oral liquid for clearing heat and relieving cough of children and fingerprint thereof | |
| CN102359942B (en) | Qingpeng ointment and quality control method of Qingpeng ointment preparation | |
| CN102507834B (en) | Quality control method for eight-flavor agilawood preparations | |
| CN110579545A (en) | Quality detection method of traditional Chinese medicine composition for clearing heat and ventilating lung | |
| CN110031564A (en) | The quality determining method of natural plants anticoccidial feed addictive based on HPLC finger-print | |
| CN110196299B (en) | Fingerprint spectrum of capsule for improving vision and its application in quality control and component analysis | |
| CN109270181B (en) | Method for establishing HPLC fingerprint of aloe laxative capsule and standard chromatogram thereof | |
| CN103230453A (en) | Yinchengao Decoction formula particles, and preparation method and detection method thereof | |
| CN102645505A (en) | Method for determining content and content homogeneity degree of chlorpheniramine maleate in antitussive and expectorant particles | |
| CN114062553B (en) | HPLC fingerprint determination method of children six-ingredient food-enhancing ointment | |
| CN114636760B (en) | Fingerprint spectrum establishment method of traditional Chinese medicine composition for treating kidney diseases and application of fingerprint spectrum establishment method | |
| CN114689776B (en) | Construction method of high-efficiency liquid-phase characteristic spectrum of lindera root decoction | |
| CN115266975A (en) | Method for measuring content of genistin in endothelium corneum gigeriae galli and processed decoction pieces, standard decoction and formula granules thereof | |
| CN110412162B (en) | Construction method of HPLC (high performance liquid chromatography) characteristic spectrum of Baixiangdan capsule and quality detection method of Baixiangdan capsule | |
| CN113447596A (en) | Method for determining 3 active ingredients in pharmaceutical preparation | |
| CN112730694A (en) | A herba Leonuri injection with controlled D-arabitol content and its quality control method | |
| CN113030291A (en) | HPLC-ELSD characteristic spectrum of motherwort injection and establishing method thereof | |
| CN112858517A (en) | A herba Leonuri injection with controlled glycerol content and its quality control method | |
| CN114689708B (en) | Long Guican Qijiu fingerprint detection method | |
| CN105004810A (en) | Rheumatism bone pain tablet quality standard and test method thereof | |
| CN112903862A (en) | A herba Leonuri injection with controlled lactic acid content and quality control method thereof | |
| CN118362674B (en) | Feature spectrum of Kemin capsule and content detection method of various quality markers |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |