CN114965739B - Method for detecting kadsura pepper stem and preparation and quality control method thereof - Google Patents
Method for detecting kadsura pepper stem and preparation and quality control method thereof Download PDFInfo
- Publication number
- CN114965739B CN114965739B CN202210400951.2A CN202210400951A CN114965739B CN 114965739 B CN114965739 B CN 114965739B CN 202210400951 A CN202210400951 A CN 202210400951A CN 114965739 B CN114965739 B CN 114965739B
- Authority
- CN
- China
- Prior art keywords
- kadsura pepper
- pepper stem
- peak
- internal standard
- solution
- 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.)
- Active
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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
-
- 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
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
-
- 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
- G01N30/60—Construction of the column
- G01N30/6052—Construction of the column body
-
- 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
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
-
- 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
- G01N2030/042—Standards
- G01N2030/045—Standards internal
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- General Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to the technical field of traditional Chinese medicine detection, in particular to a detection method and a quality control method of kadsura pepper stem and a preparation thereof. The detection method adopts ultra-high performance liquid chromatography to detect, and notopterygium alcohol is added into the sample solution adopted during detection. According to the detection method for the kadsura pepper stem and the preparation thereof, provided by the invention, the notopterygol is selected as the internal standard substance, the notopterygol peak is arranged in the detected fingerprint, and the notopterygol peak is used as the internal standard reference peak, so that the accurate positioning of the positions of all characteristic peaks in the fingerprint can be realized, and the accuracy of the detection result is obviously improved.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine detection, in particular to a detection method and a quality control method of kadsura pepper stem and a preparation thereof.
Background
The caulis Piperis Kadsura (Choisy) Ohwi dry rattan of Piperaceae is Kadsura (Piper Kadsura) has pungent smell and bitter taste, has effects of dispelling pathogenic wind and dampness, dredging channels and collaterals, and relieving arthralgia, and can be used for treating arthralgia due to wind-cold-dampness, limb joint pain, spasm of tendons and collaterals, and difficulty in flexion and extension.
The Chinese pharmacopoeia (2020 edition) receives the quality standard of the kadsura pepper stem medicinal material, but the effective components and the content of the kadsura pepper stem medicinal material are not regulated, and only the characteristics and the extract are used as quality detection items, so that the characteristic identification depends on experience, has certain subjectivity on one hand, and the characteristic of the appearance characteristics of the prescription granule is lost through the processes of decoction, concentration, granulation and the like on the other hand, and the extract cannot reflect the speciality of the medicinal material.
The volatile oil component in the kadsura pepper stem is determined by a gas chromatography in literature, but the clinical application of the kadsura pepper stem is mostly in a decoction form, and the volatile oil is almost not reserved after decoction, so the volatile oil determination is not suitable for quality detection of kadsura pepper stem formula particles. The kadsura pepper medicinal material quality detection method is characterized in that a scholars use kadsura pepper ketone as a detection index, however, the kadsura pepper ketone reference substance is used as an external standard reference, the kadsura pepper ketone reference substance is difficult to obtain, and the quality control of single components has certain unilateral performance. Some scholars use a high-efficiency liquid phase method to establish the futokadsura stem medicinal material fingerprint, but the time is longer and is more than 60 minutes, so that the detection efficiency is lower, and the obtained futokadsura stem medicinal material fingerprint has no known peak as a reference, so that the position of a characteristic peak cannot be accurately positioned, and the accuracy of a detection result is poor. In addition, no report on quality detection of the kadsura pepper stem preparation, such as kadsura pepper stem formula particles, is known.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to overcome the defect that the characteristic peak position cannot be accurately positioned by taking the known peak as a reference in the futokadsura stem medicinal material fingerprint obtained by the existing futokadsura stem medicinal material quality detection method, thereby providing a futokadsura stem and preparation detection method and quality control method thereof.
Therefore, the invention provides a method for detecting the kadsura pepper stem and the preparation thereof, which adopts an ultra-high performance liquid chromatography to detect, and the notopterygium alcohol is added into the sample solution adopted during the detection.
Alternatively, the preparation process of the sample solution may be: adding a first internal standard solution into a sample to be tested, extracting, filtering and taking liquid; wherein the first internal standard solution is a solution containing notopterol.
Optionally, the first internal standard solution is a mixed solution of notopterygol and a solvent, and the solvent is methanol or a methanol water solution with the volume percent of methanol not less than 50%; the concentration of notopterol in the first internal standard solution is 10-50 mug/mL; the extraction is ultrasonic extraction, the ultrasonic power is 300W-500W, and the ultrasonic time is 20-40 min; for 1g of the sample, the addition amount of the first internal standard solution is 5-50 mL.
Optionally, the preparation method of the sample solution may include: taking a proper amount of the product, grinding, taking 0.5-5 g, precisely weighing, adding 5-50 mL of the first internal standard solution, sealing, performing ultrasonic treatment (with the power of 300W and the frequency of 40 kHz) for 20-40 minutes, taking out, cooling, shaking uniformly, filtering, and taking the subsequent filtrate as a sample solution.
Optionally, the preparation process of the sample solution may further be: adding solvent into the sample, extracting, filtering, collecting liquid, and adding a second internal standard solution; wherein the second internal standard solution is a solution containing notopterol.
Optionally, the second internal standard solution is a mixed solution of notopterygol and a solvent, and the solvent is methanol or a methanol water solution with the volume percent of methanol not less than 50%; the concentration of notopterol in the second internal standard solution is 10-500 mug/mL; the extraction is ultrasonic extraction, the ultrasonic power is 300W-500W, and the ultrasonic time is 20-40 min; for 1g of the sample, the addition amount of the solvent is 5-50 mL, and the addition amount of the second internal standard solution is 0.5-10 mL.
Optionally, the preparation method of the sample solution may further include: taking a proper amount of the product, grinding, taking 0.5-5 g, precisely weighing, adding 5-50 mL of solvent, sealing, performing ultrasonic treatment (with the power of 300W and the frequency of 40 kHz) for 20-40 minutes, taking out, cooling, shaking uniformly, filtering, taking a subsequent filtrate, adding 0.5-10 mL of a second internal standard solution, shaking uniformly, and taking the subsequent filtrate as a sample solution.
Optionally, the detection conditions of the ultra performance liquid chromatography include at least one of the following conditions:
1) Gradient elution is carried out by taking acetonitrile-water as a mobile phase, and the gradient elution program comprises the following steps: 0 to 0.04min, wherein the volume percentage of acetonitrile in the mobile phase is 35 percent; 0.04-2 min, wherein the volume percentage of acetonitrile in the mobile phase is 35% -40%; 2-3 min, wherein the volume percentage of acetonitrile in the mobile phase is 40%; 3-9 min, wherein the volume percentage of acetonitrile in the mobile phase is 40% -43%; 9-15 min, wherein the volume percentage of acetonitrile in the mobile phase is 43% -95%;
2) Chromatographic column with octadecylsilane chemically bonded silica as filler, column length of 100mm, inner diameter of 2.1mm, and particle diameter of 1.8 μm;
3) The column temperature is 38-42 ℃;
4) The flow rate is 0.38-0.42 mL/min;
5) The detection wavelength is 210-310 nm;
6) The sample injection amount is 1-3 mu l;
7) The number of theoretical plates should be not less than 3000 calculated as notopterygium alcohol.
Optionally, the sample comprises any one of caulis Piperis Futokadsurae medicinal material, caulis Piperis Futokadsurae decoction piece or caulis Piperis Futokadsurae preparation; optionally, the kadsura pepper stem preparation comprises kadsura pepper stem formulation particles. Wherein the kadsura pepper stem decoction pieces can be obtained by removing impurities from kadsura pepper stem, soaking, moistening thoroughly, slicing into thick slices, and sun drying; the kadsura pepper stem preparation can be prepared from the kadsura pepper stem medicinal material or the extractive solution (such as decoction obtained by decocting) obtained by extracting kadsura pepper stem decoction pieces according to the conventional pharmaceutical process, with or without adding conventional pharmaceutical adjuvants. Preferably, the kadsura pepper stem preparation can be selected from, but not limited to, dry powder, tablets, granules, capsules, ointments, solutions and the like. More preferably, the kadsura pepper stem formulation is a kadsura pepper stem formulation granule.
Optionally, the method further comprises a step of preparing a control medicinal material solution by adopting the kadsura pepper stem control medicinal material, and a step of detecting the control medicinal material solution by using the ultra performance liquid chromatography in the detection method according to any one of the invention to obtain a control medicinal material reference map.
Optionally, the step of preparing a control medicinal material solution from the kadsura pepper stem control medicinal material may include: taking 0.5-2 g of kadsura pepper stem reference medicinal material, precisely weighing, adding 10-50 mL of water, carrying out reflux extraction for 20-60 minutes, filtering, recovering the solvent from the filtrate to dryness, adding 5-50 mL of a first internal standard solution, carrying out ultrasonic treatment (with the power of 300W and the frequency of 40 kHz) for 20-40 minutes, taking out, cooling, shaking uniformly, filtering, and taking the subsequent filtrate as the reference medicinal material solution.
Alternatively, the step of preparing the control medicinal material solution by using the kadsura pepper stem control medicinal material may further include: taking 0.5-2 g of kadsura pepper stem reference medicinal material, precisely weighing, adding 10-50 mL of water, carrying out reflux extraction for 20-60 minutes, filtering, recovering the solvent from the filtrate until the filtrate is dried, adding 5-50 mL of the solvent, carrying out ultrasonic treatment (with the power of 300W and the frequency of 40 kHz) for 20-40 minutes, taking out, cooling, shaking uniformly, filtering, taking the subsequent filtrate, adding 0.5-10 mL of a second internal standard solution, and shaking uniformly to obtain a reference medicinal material solution.
The invention also provides a quality control method of the kadsura pepper stem and the preparation thereof, which comprises the steps of obtaining a characteristic map of a kadsura pepper stem product to be detected according to the detection method of the kadsura pepper stem and the preparation thereof; comparing the characteristic spectrum with a contrast characteristic spectrum;
the contrast characteristic spectrum is obtained by fitting the characteristic spectrum obtained by the method according to any one of the invention by using at least one batch of standard substances of the kadsura pepper stem and the preparation thereof through an average value or a median method.
Optionally, the reference characteristic spectrum at least comprises 1 internal standard reference peak and 4 characteristic peaks, the internal standard reference peak is a notopterygol peak, the relative retention time of each characteristic peak and the internal standard reference peak is within a range of +/-10% of a specified value, and the specified value is: 0.57, 1.07, 1.14, 1.16.
Optionally, the kadsura pepper stem product to be detected is selected from any one of kadsura pepper stem medicinal materials, kadsura pepper stem decoction pieces or kadsura pepper stem preparations. Wherein the kadsura pepper stem decoction pieces can be obtained by removing impurities from kadsura pepper stem, soaking, moistening thoroughly, slicing into thick slices, and sun drying; the kadsura pepper stem preparation can be prepared from the kadsura pepper stem medicinal material or the extractive solution (such as decoction obtained by decocting) obtained by extracting kadsura pepper stem decoction pieces according to the conventional pharmaceutical process, with or without adding conventional pharmaceutical adjuvants. The kadsura pepper stem preparation can be selected from, but not limited to, dry powder, tablets, granules, capsules, ointments, solutions and the like.
Preferably, the kadsura pepper stem product to be detected is selected from any one of kadsura pepper stem medicinal materials, kadsura pepper stem decoction pieces or kadsura pepper stem formula particles.
Optionally, the contrast characteristic spectrum of the kadsura pepper stem and the preparation thereof is generated by utilizing the traditional Chinese medicine chromatographic fingerprint similarity evaluation software.
Optionally, the step of marking common characteristic peaks is further included after the contrast characteristic spectrum of the kadsura pepper stem and the preparation thereof is generated by utilizing the traditional Chinese medicine chromatographic fingerprint similarity evaluation software.
The technical scheme of the invention has the following advantages:
1. according to the detection method for the kadsura pepper stem and the preparation thereof, provided by the invention, the notopterygol is selected as the internal standard substance, the notopterygol peak is arranged in the detected fingerprint, and the notopterygol peak is used as the internal standard reference peak, so that the accurate positioning of the positions of all characteristic peaks in the fingerprint can be realized, and the accuracy of the detection result is obviously improved.
2. The detection method of the kadsura pepper stem and the preparation thereof provided by the invention selects the notopterygium alcohol as an internal standard substance: (1) The maximum absorption wavelength of the notopterol is 250nm and 310nm, wherein 250nm is similar to the optimal detection wavelength of the ultra-high performance liquid chromatography, so that the notopterol is selected as an internal standard substance of the invention, and the internal standard substance can be ensured to have larger absorption under the detection condition; (2) The notopterygium alcohol has proper polarity conditions, so that the notopterygium alcohol can be reserved and detected in the gradient elution of the invention, and the chromatographic peak of the notopterygium alcohol is well separated from each characteristic peak detected by the invention, and the chromatographic peak of the notopterygium alcohol is positioned between each characteristic peak, is more suitable as a reference peak, and ensures the stability of the relative retention time of each characteristic peak; (3) The notopterygium alcohol reference substance is stable in supply, easy to obtain and wide in application, and can reduce the detection cost and promote the universality of the detection method.
3. According to the detection method for the kadsura pepper stem and the preparation thereof, provided by the invention, octadecylsilane chemically bonded silica gel is used as a filling agent, acetonitrile-water is used as a mobile phase for gradient elution, a specific elution program is selected to obtain 4 common characteristic peaks, good separation of the common characteristic peaks and internal standard reference peaks is realized, the elution program is simple, the obtained characteristic spectrum has stable base line, the peak shapes of the characteristic peaks and the internal standard reference peaks are good, the separation degree between the characteristic peaks and the internal standard reference peaks is high, the peak positions of the 4 characteristic peaks can be accurately positioned, and a basis is provided for quality detection and control of the kadsura pepper stem and the preparation thereof.
4. The detection method of the kadsura pepper stem and the preparation thereof provided by the invention has the advantages of short elution time and stronger operability; the mobile phase is acetonitrile-water, so that the toxicity is low, the corrosiveness to a detection system is low, and the damage to a chromatographic column is low; meanwhile, the method is stable, high in precision, strong in durability and good in reproducibility, and provides a rapid and comprehensive detection means for the quality of the kadsura pepper stems and the preparation thereof.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph comparing the internal standard solution, the control medicinal solution and the test sample solution in example 1 of the present invention;
FIG. 2 is a graph comparing the internal standard solution, the control medicinal solution and the test sample solution in example 2 of the present invention;
FIGS. 3-5 are liquid phase diagrams of three batches of the kadsura pepper stem formulation particles of example 3 of the present invention;
FIG. 6 is a graph of the comparative characteristics of the kadsura pepper stem formulation particles obtained in example 3 of the present invention;
FIG. 7 is a graph showing the comparison of characteristic patterns obtained by detecting different flow rates in example 4 of the present invention;
FIG. 8 is a comparison of characteristic patterns obtained by different column temperature measurements in example 4 of the present invention;
FIG. 9 shows an embodiment of the invention in which Waters ACQUITY UPLC is used in example 4
C18 (2.1 x 100mm,1.7 μm) detecting the obtained characteristic map;
FIG. 10 shows an embodiment of the invention in example 4 using Waters ACQUITY UPLC
Detecting a characteristic map obtained by T3 (2.1 x 100mm,1.8 mu m);
FIG. 11 is utilized in example 4 of the present invention; waters CORTECS
UPLC/>
Detecting a characteristic map obtained by T3 (2.1 x 100mm,1.6 μm);
FIG. 12 is a liquid phase diagram of the kadsura pepper stem formulation particles measured in comparative example 1 of the present invention.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
The instruments, reagents and reagents involved in the examples are as follows:
instrument: waters ACQUITY
An H-Class ultra-high performance liquid chromatograph, a TUV Detector ultraviolet Detector, an Empower 3 chromatographic workstation; ME104E electronic balance (Metrehler Tolyduo), JY2002 electronic balance (Metrehler Tolyduo), KQ-500DB ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.), electronic constant temperature water bath DZKW-4 (Beijing Zhongxing Weijie instruments Co., ltd.); waters ACQUITY UPLC->
C18 column (2.1X100 mm,1.7 μm), waters ACQUITY UPLC->
T3 column (2.1X100 mm,1.8 μm), waters CORTECS->
UPLC
T3 column (2.1X100 mm,1.6 μm).
Reagent: notopterygium alcohol (lot number: 111820-201705, china food and drug inspection institute); kadsura pepper stem formulation granules (lot numbers: 19031461, 19046531, 20032261), available from Beijing Kang Rentang pharmaceutical company, inc.; kadsura pepper stem control medicinal material (lot number: 121635-201603, china food and drug inspection institute).
Reagent: acetonitrile is chromatographic purity, methanol is analytical purity, and water is purified water of the chen type.
The preparation method of the kadsura pepper stem formula granule comprises the following steps: decocting caulis Piperis Futokadsurae decoction pieces with water, filtering, concentrating the filtrate into fluid extract, adding dextrin, drying, adding dextrin, mixing, and granulating.
Example 1
The detection method of the kadsura pepper stem formula granule (batch number: 20032261) comprises the following steps:
(1) Preparation of reference solution: comprises the preparation of an internal standard solution and a control medicinal material solution. Taking a proper amount of notopterol reference substance, adding methanol to prepare an internal standard solution containing 25 mug per 1mL for later use. Taking about 1g of a kadsura pepper stem reference medicinal material, precisely weighing, adding 50mL of water, carrying out reflux extraction for 30 minutes, filtering, recovering the solvent from the filtrate until the filtrate is dry, adding 10mL of an internal standard solution, carrying out ultrasonic treatment (with the power of 300W and the frequency of 40 kHz) for 30 minutes, taking out, cooling, shaking uniformly, filtering, and taking the subsequent filtrate as the reference medicinal material solution.
(2) Preparation of test solution: taking a proper amount of the product, grinding, taking about 1g, precisely weighing, adding 10mL of an internal standard solution, sealing, performing ultrasonic treatment (with the power of 300W and the frequency of 40 kHz) for 30 minutes, taking out, cooling, shaking uniformly, filtering, and taking a subsequent filtrate as a sample solution.
(3) Ultra-high performance liquid chromatography detection: octadecylsilane chemically bonded silica is used as a filler (column length is 100mm, inner diameter is 2.1mm, and particle diameter is 1.8 μm); acetonitrile as mobile phase A and water as mobile phase B, and performing gradient elution according to Table 1; the flow rate was 0.4mL per minute; column temperature is 40 ℃; the detection wavelength was 254nm. The number of theoretical plates is not less than 3000 calculated by notopterygium alcohol.
Respectively precisely sucking 2 μl of each of the internal standard solution, the control medicinal material solution and the sample solution, and measuring with ultra-high performance liquid chromatograph.
TABLE 1 gradient elution table
The detection results are shown in fig. 1, the fig. 1 is a graph comparison of an internal standard solution, a control medicinal material solution and a test sample solution, and as shown in fig. 1, 4 characteristic peaks are shown in the test sample graph and correspond to 4 characteristic peaks in the control medicinal material graph, and the test sample graph and the control medicinal material graph both have notopterygium alcohol internal standard peaks, and the relative retention time of each characteristic peak in the test sample graph and the notopterygium alcohol internal standard peak is calculated as follows: 0.57 (Peak 1), 1.07 (Peak 2), 1.14 (Peak 3), 1.16 (Peak 4).
Example 2
The detection method of the kadsura pepper stem formula granule (batch number: 20032261) comprises the following steps:
(1) Preparation of reference solution: comprises the preparation of an internal standard solution and a control medicinal material solution. Taking a proper amount of notopterol reference substance, adding methanol to prepare an internal standard solution containing 250 mug per 1mL for later use. Taking about 1g of a kadsura pepper stem reference medicinal material, precisely weighing, adding 50mL of water, carrying out reflux extraction for 30 minutes, filtering, recovering the solvent from the filtrate until the filtrate is dry, adding 10mL of methanol, carrying out ultrasonic treatment (with the power of 300W and the frequency of 40 kHz) for 30 minutes, taking out, cooling, shaking uniformly, filtering, adding 1mL of an internal standard solution into the subsequent filtrate, and shaking uniformly to obtain the reference medicinal material solution.
(2) Preparation of test solution: taking a proper amount of the product, grinding, taking about 1g, precisely weighing, adding 10mL of methanol, sealing, performing ultrasonic treatment (with the power of 300W and the frequency of 40 kHz) for 30 minutes, taking out, cooling, shaking uniformly, filtering, adding 1mL of internal standard solution into the continuous filtrate, and shaking uniformly to obtain a sample solution.
Performing ultra-high performance liquid chromatography detection according to the method of step (3) in example 1, wherein the detection result is shown in fig. 2, fig. 2 is a graph comparison of an internal standard solution, a reference medicinal material solution and a test sample solution, and as shown in fig. 2, 4 characteristic peaks are shown in the test sample graph and correspond to the 4 characteristic peaks in the reference medicinal material graph, and notopterygol internal standard peaks exist in both the test sample graph and the reference medicinal material graph, and the relative retention time of each characteristic peak in the test sample graph and the notopterol internal standard peak is calculated as follows: 0.560 (Peak 1), 1.084 (Peak 2), 1.052 (Peak 3), 1.175 (Peak 4).
Example 3
The embodiment is used for explaining the establishment of the characteristic spectrum of the kadsura pepper stem formula granule contrast:
three batches of kadsura pepper stem formula particles (batch numbers: 19031461, 19046531 and 20032261) are taken, test solution is prepared according to the method of step (2) in example 1, ultra-high performance liquid chromatography detection is carried out according to the method of step (3) in example 1, and liquid phase patterns are obtained respectively, as shown in fig. 3-5.
The liquid phase spectrum shown in fig. 3-5 is imported into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012.1 version) for data matching, the S1 liquid phase spectrum is used as a reference spectrum, a comparison characteristic spectrum is obtained according to the median calculation, the identification of common characteristic peaks is carried out, and 4 common characteristic peaks are identified in total, as shown in fig. 6.
Example 4
This example was used to methodologically verify the detection method in example 1:
(1) Precision verification
6 parts of kadsura pepper stem formula particles (batch number: 20032261) are taken, test solution is prepared according to the method of the step (2) in the example 1, ultra-high performance liquid chromatography detection is carried out according to the method of the step (3) in the example 1, a characteristic spectrum is obtained, a notopterygium alcohol peak is taken as a reference peak, the relative retention time of each characteristic peak (peak 1-peak 4) is calculated, and RSD is calculated, and the calculation results are shown in the table 2.
TABLE 2 feature profile precision validation retention time and relative retention time table
The result shows that the relative retention time RSD of each characteristic peak is in the range of 0.06% -0.09%, which indicates that the precision of the characteristic spectrum is good.
(2) Repeatability verification
Taking 1 part of kadsura pepper stem formula particles (batch number: 20032261), preparing a test solution according to the method of the step (2) in the example 1, performing ultra-high performance liquid chromatography detection according to the method of the step (3) in the example 1, continuously injecting sample for 6 times to obtain a characteristic map, calculating the relative retention time of each characteristic peak (peak 1-peak 4) by taking a notopterygium alcohol peak as a reference peak, and calculating RSD, wherein the calculation result is shown in a table 3.
TABLE 3 feature profile repeatability validation retention time and relative retention time table
The results show that the relative retention time RSD of each characteristic peak is in the range of 0.02% -0.05%, which shows that the repeatability of the characteristic map is good.
(3) Stability verification
1 part of kadsura pepper stem formula particles (batch number: 20032261) is taken, a test solution is prepared according to the method of the step (2) in the example 1, and the ultra-high performance liquid chromatography detection is carried out according to the method of the step (3) in the example 1 at 0, 3, 6, 9, 12, 15, 18, 21 and 24 hours respectively, the relative retention time of each characteristic peak (peak 1-peak 4) is calculated by taking notopterygol as a reference peak, and the RSD is calculated, and the calculation results are shown in the table 4.
TABLE 4 feature profile stability validation retention times and relative retention schedules
The result shows that the relative retention time RSD of each characteristic peak is between 0.14% and 0.30%, which shows that the characteristic components in the test sample solution have better stability within 24 hours.
(4) Durability verification
1) Flow rate investigation
Taking 1 part of kadsura pepper stem formula particles (batch number: 20032261), preparing a test solution according to the method of step (2) in example 1, respectively carrying out ultra-high performance liquid chromatography detection according to the method of step (3) in example 1 at flow rates of 0.38mL/min, 0.40mL/min and 0.42mL/min, examining the durability of the experimental method on different flow rates, obtaining a characteristic map, calculating the relative retention time of each characteristic peak (peak 1-peak 4) by taking notopterygol as a reference peak, calculating RSD (reactive power distribution), and calculating the calculation result as shown in table 5.
TABLE 5 characterization profile durability flow rate investigation retention time and relative retention time table
The results show that the relative retention time RSD of each characteristic peak is between 1.17% and 1.70%, indicating that the method of the present invention has good durability for different flow rates.
2) Column temperature investigation
Taking 1 part of kadsura pepper stem formula particles (batch number: 20032261), preparing a test solution according to the method of step (2) in example 1, respectively carrying out ultra-high performance liquid chromatography detection at a column temperature of 38 ℃ and a temperature of 40 ℃ and a temperature of 42 ℃ according to the method of step (3) in example 1, examining the durability of the experimental method on different column temperatures, obtaining a characteristic map, calculating the relative retention time of each characteristic peak (peak 1-peak 4) by taking notopterygol as a reference peak as shown in fig. 8, calculating RSD, and calculating the calculation result as shown in table 6.
TABLE 6 durability column temperature investigation retention time and relative retention time table for feature patterns
The results show that the relative retention time RSD of each characteristic peak is between 0.41% and 0.98%, indicating that the method of the present invention has good durability against different column temperatures.
3) Chromatographic column inspection
1 part of kadsura pepper stem formulation particles (batch number: 20032261) is taken, a test solution is prepared according to the method of step (2) in example 1, and chromatographic columns Waters ACQUITY UPLC are used respectively
C18(2.1*100mm，1.7μm)、Waters ACQUITY UPLC/>
T3(2.1*100mm，1.8μm)、Waters CORTECS/>
UPLC/>
Ultra performance liquid chromatography detection under T3 (2.1X100 mm,1.6μm) according to the method of step (3) in example 1, examining the durability of the experimental method on different chromatographic columns, and obtaining characteristic patterns, as shown in FIGS. 9-11, wherein FIG. 9 is a graph using Waters ACQUITY UPLC/>
C18 (2.1.100 mm,1.7 μm) and FIG. 10 shows a characteristic pattern obtained by detection using Waters ACQUITY UPLC +.>
T3 (2.1X100 mm,1.8μm) and FIG. 11 shows the characteristic pattern obtained by Waters CORTECS +.>
UPLC/>
T3 (2.1 x 100mm,1.6 μm) detection.
The relative retention time of each characteristic peak (peak 1-peak 4) was calculated using notopterol as a reference peak, and RSD was calculated, and the calculation results are shown in table 7.
TABLE 7 characterization durability chromatography column investigation retention time and relative retention time table
The results showed that the difference between the relative retention time of each characteristic peak and the predetermined value was large in the characteristic pattern obtained by the detection using the ACQUITY BEH C18 column and the CORTECS T3 column, and therefore Waters ACQUITY UPLC was proposed
(2.1 x 100mm,1.8 μm).
Example 5
This example was used to verify the detection effect of the method of example 1 on batches of kadsura pepper stem formulation particles:
three batches of kadsura pepper stem formula particles (batch numbers: 19031461, 19046531 and 20032261) were taken, test solutions were prepared according to the method of step (2) in example 1, ultra-high performance liquid chromatography detection was performed according to the method of step (3) in example 1, characteristic patterns of the three batches of kadsura pepper stem formula particles were obtained, relative retention time of each characteristic peak (peak 1-peak 4) was calculated by taking notopterygol as a reference peak, and RSD value of relative retention time of each characteristic peak (peak 1-peak 4) between the three batches was calculated, and the calculation results are shown in table 8.
TABLE 8 three batches of kadsura pepper formulation particles retention time and relative retention time schedule
As shown in table 8, each batch of sample chromatograph has 4 common characteristic peaks, the difference of the relative retention time of each characteristic peak between different batches is smaller and is within ±10%, the sample chromatograph meets the quality control requirement, and the average value of the relative retention time is selected as the measured value: 0.57 (Peak 1), 1.07 (Peak 2), 1.14 (Peak 3), 1.16 (Peak S).
The detection result of fig. 1 in example 1 shows that the characteristic peaks in the kadsura pepper stem decoction pieces and the kadsura pepper stem formula granule map correspond well, so that the method can rapidly and comprehensively detect the kadsura pepper stem and kadsura pepper stem formula granule, and provides a more scientific basis for quality control of the kadsura pepper stem and kadsura pepper stem formula granule.
Comparative example 1
The test solution prepared in example 1 was tested by high performance liquid chromatography according to the following chromatographic conditions:
a Kromasil C18 (250 mm x 46mm,5 μm) column; mobile phase: acetonitrile-aqueous solution containing 3% tetrahydrofuran; gradient elution procedure: 0-10min, isocratic elution of acetonitrile-3% tetrahydrofuran-containing aqueous solution (35:65), 10-60min, increase of acetonitrile volume percent from 35% to 50%,60-70min, isocratic elution of acetonitrile-3% tetrahydrofuran-containing aqueous solution (50:50); the column temperature was 25℃and the sample injection amount was 15. Mu.l, the flow rate was 1.0mL per minute, and the detection wavelength was 280nm.
As shown in fig. 12, it can be seen from fig. 12 that the method of the present comparative example cannot effectively separate the characteristic peak 1 from the surrounding material peaks.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (9)
1. A detection method of kadsura pepper stem and a preparation thereof is characterized in that the detection method is used for constructing a characteristic spectrum of a kadsura pepper stem product, and adopts an ultra-high performance liquid chromatography for detection, and notopterygium alcohol is added into a sample solution adopted during detection;
the chromatographic conditions of the ultra performance liquid chromatography include: the chromatographic column takes octadecylsilane chemically bonded silica gel as a filler, the column length is 100mm, the inner diameter is 2.1mm, and the particle size is 1.8 mu m; gradient elution is carried out by taking acetonitrile-water as a mobile phase, and the gradient elution program comprises the following steps: 0 to 0.04min, wherein the volume percentage of acetonitrile in the mobile phase is 35 percent; 0.04-2 min, wherein the volume percentage of acetonitrile in the mobile phase is 35% -40%; 2-3 min, wherein the volume percentage of acetonitrile in the mobile phase is 40%; 3-9 min, wherein the volume percentage of acetonitrile in the mobile phase is 40% -43%; 9-15 min, wherein the volume percentage of acetonitrile in the mobile phase is 43% -95%;
the preparation process of the sample solution comprises the following steps: adding a first internal standard solution into a sample to be tested, and taking liquid after first extraction and filtration; wherein the first internal standard solution is a mixed solution of notopterygol and a first solvent; the first solvent is methanol or methanol aqueous solution with the volume percentage of methanol not less than 50%; alternatively, the preparation process of the sample solution comprises the following steps: taking a sample, adding a second solvent, performing second extraction, filtering, taking liquid, and adding a second internal standard solution; the second internal standard solution is a solution containing notopterygium alcohol, and the second solvent is methanol or methanol aqueous solution with the volume percent of methanol not less than 50%.
2. The detection method according to claim 1, wherein the concentration of notopterygol in the first internal standard solution is 10-50 μg/mL.
3. The method according to claim 1, wherein the first extraction is ultrasonic extraction, the ultrasonic power is 300W to 500W, and the ultrasonic time is 20 to 40min;
the addition amount of the first internal standard solution is 5-50 mL for 1g of the sample.
4. The detection method according to claim 1, wherein the second internal standard solution is a mixed solution of notopterygol and a solvent, and the solvent is methanol or a methanol aqueous solution with a methanol volume percentage content of not less than 50%;
the concentration of notopterygol in the second internal standard solution is 10-500 mug/mL.
5. The method according to claim 1, wherein the second extraction is an ultrasonic extraction, the ultrasonic power is 300W to 500W, and the ultrasonic time is 20 to 40min;
for 1g of the sample, the addition amount of the second solvent is 5-50 mL, and the addition amount of the second internal standard solution is 0.5-10 mL.
6. The method according to claim 1, wherein the detection conditions of the ultra-high performance liquid chromatography further comprise at least one of the following conditions:
1) The column temperature is 38-42 ℃;
2) The flow rate is 0.38-0.42 mL/min;
3) The detection wavelength is 210-310 nm;
4) The sample injection amount is 1-3 mu l;
5) The number of theoretical plates should be not less than 3000 calculated as notopterygium alcohol.
7. The method according to any one of claims 1 to 6, wherein the sample comprises any one of a kadsura pepper stem medicinal material, a kadsura pepper stem decoction piece or a kadsura pepper stem preparation.
8. A quality control method of a kadsura pepper stem and a preparation thereof, which is characterized by comprising the steps of obtaining a characteristic map of a kadsura pepper stem product to be detected according to the detection method of the kadsura pepper stem and the preparation thereof as claimed in any one of claims 1 to 7; comparing the characteristic spectrum with a contrast characteristic spectrum;
wherein the reference characteristic spectrum is obtained by fitting the characteristic spectrum obtained by the detection method according to any one of claims 1 to 7 by using at least one batch of standard substances of kadsura pepper stem and preparations thereof through an average value or a median method.
9. The quality control method according to claim 8, wherein the control characteristic map includes at least 1 internal reference peak and 4 characteristic peaks, the internal reference peak is a notopterygol peak, the relative retention time of each characteristic peak and the internal reference peak is within ±10% of a prescribed value, and the prescribed value is: 0.57, 1.07, 1.14, 1.16.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210400951.2A CN114965739B (en) | 2022-04-14 | 2022-04-14 | Method for detecting kadsura pepper stem and preparation and quality control method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210400951.2A CN114965739B (en) | 2022-04-14 | 2022-04-14 | Method for detecting kadsura pepper stem and preparation and quality control method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114965739A CN114965739A (en) | 2022-08-30 |
| CN114965739B true CN114965739B (en) | 2023-05-16 |
Family
ID=82977521
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210400951.2A Active CN114965739B (en) | 2022-04-14 | 2022-04-14 | Method for detecting kadsura pepper stem and preparation and quality control method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114965739B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110988238A (en) * | 2019-12-09 | 2020-04-10 | 广东一方制药有限公司 | Method for constructing HPLC (high performance liquid chromatography) characteristic map of standard decoction of lilac daphne flower bud and method for measuring component content of lilac daphne flower bud |
| CN113466355A (en) * | 2021-06-03 | 2021-10-01 | 山东宏济堂制药集团股份有限公司 | Construction method of high performance liquid phase characteristic spectrum of caulis sinomenii |
-
2022
- 2022-04-14 CN CN202210400951.2A patent/CN114965739B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110988238A (en) * | 2019-12-09 | 2020-04-10 | 广东一方制药有限公司 | Method for constructing HPLC (high performance liquid chromatography) characteristic map of standard decoction of lilac daphne flower bud and method for measuring component content of lilac daphne flower bud |
| CN113466355A (en) * | 2021-06-03 | 2021-10-01 | 山东宏济堂制药集团股份有限公司 | Construction method of high performance liquid phase characteristic spectrum of caulis sinomenii |
Non-Patent Citations (4)
| Title |
|---|
| 广东海风藤挥发油GC-MS指纹图谱研究;焦豪妍 等;中药材;第30卷(第09期);1431-1434 * |
| 广西海风藤HPLC指纹图谱研究;范卫锋 等;湖北中医药大学学报;第21卷(第4期);46-50 * |
| 海风藤HPLC指纹图谱研究;刘艳菊 等;中国药师;第12卷(第11期);1525-1527 * |
| 海风藤的化学成分研究(Ⅰ);任风芝 等;中草药;第36卷(第02期);184-185 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114965739A (en) | 2022-08-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114778731B (en) | Construction method and application of UPLC characteristic spectrum of endothelium corneum Gigeriae Galli, parched endothelium corneum Gigeriae Galli, vinegar endothelium corneum Gigeriae Galli decoction pieces, and standard soup and granule thereof | |
| CN109655558B (en) | Method for detecting effective part group of periploca forrestii schltr | |
| CN108663440B (en) | Method for constructing UPLC fingerprint spectrum of callicarpa nudiflora medicinal material and standard fingerprint spectrum | |
| CN114942291B (en) | Quality detection method of yang-suppressing yin-nourishing particles | |
| CN114965739B (en) | Method for detecting kadsura pepper stem and preparation and quality control method thereof | |
| CN115524424A (en) | Capsella bursa-pastoris sample quality control method | |
| CN115372534A (en) | Folium artemisiae argyi and construction method of characteristic map of preparation of folium artemisiae argyi, characteristic map and application | |
| CN113759011B (en) | Method for establishing characteristic spectrum of starwort root and preparation thereof | |
| CN115508463A (en) | Detection method and quality control method of Tanggute radix Et rhizoma Rhei product | |
| CN114965758B (en) | Construction method and application of characteristic spectrum of alisma orientale or pharmaceutical preparation thereof | |
| CN114646695B (en) | Ultra-high performance liquid chromatography detection method of starwort root standard decoction and application thereof | |
| CN112881541B (en) | Detection method of north bupleurum and south bupleurum prescription granule | |
| CN113759036B (en) | Method for measuring content of protodioscin in rhizoma Dioscoreae Septemlobae | |
| CN115389652B (en) | Method for establishing fingerprinting of rhizoma phragmitis medicinal material | |
| CN112180022B (en) | Method for measuring content of tribuloside K in stir-fried caltrops or caltrops | |
| CN115097040B (en) | UPLC characteristic spectrum construction method and application of semen momordicae | |
| CN115015405B (en) | Construction method and application of characteristic spectrum of red bean or pharmaceutical preparation thereof | |
| CN118169257A (en) | Construction method and detection method of characteristic spectrum of holly leaf sample | |
| CN116858969A (en) | Construction method and application of feature maps of Beijing pinellia decoction pieces, standard decoction and formula granules | |
| CN118010902A (en) | Construction method and detection method of long pepper sample characteristic spectrum | |
| CN118090985A (en) | Construction method and detection method of UPLC characteristic spectrum of calyx kaki sample | |
| CN116879457A (en) | Construction method and application of high-efficiency liquid-phase characteristic spectrum of lycopodium clavatum | |
| CN117825563A (en) | Method for constructing UPLC characteristic spectrum of fried feather cockscomb seed decoction pieces and standard decoction and formula granules thereof | |
| CN117849242A (en) | HPLC (high Performance liquid chromatography) characteristic spectrum construction method for Chuanminshen violaceum medicinal materials, decoction pieces, standard decoction and prescription granule thereof | |
| CN116539761A (en) | Method for detecting rhizoma smilacis glabrae product |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |