CN113759037B - Characteristic spectrum of formula granules of semen lepidii and/or semen lepidii as well as construction method and identification method thereof - Google Patents

Characteristic spectrum of formula granules of semen lepidii and/or semen lepidii as well as construction method and identification method thereof Download PDF

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CN113759037B
CN113759037B CN202110988980.0A CN202110988980A CN113759037B CN 113759037 B CN113759037 B CN 113759037B CN 202110988980 A CN202110988980 A CN 202110988980A CN 113759037 B CN113759037 B CN 113759037B
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solution
peak
semen lepidii
mobile phase
characteristic
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CN113759037A (en
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张志强
吉艳慧
史国华
付静
安琪
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Beijing Tcmages Pharmaceutical Co Ltd
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    • 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
    • G01N30/50—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
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors

Abstract

The invention belongs to the technical field of traditional Chinese medicine identification, and particularly relates to a characteristic spectrum of a granule formula of semen lepidii and/or semen lepidii, a construction method and an identification method thereof. The construction method comprises the steps of preparing a test solution, and detecting under the condition of specific elution gradient to obtain a characteristic map of the semen lepidii and/or semen lepidii formula particles. The invention adopts high performance liquid chromatography to detect the effective components of the south pepperweed seed formula particles and/or the north pepperweed seed formula particles, acetonitrile is taken as a mobile phase A, acid-containing aqueous solution with the volume fraction of 0.1-0.3% is taken as a mobile phase B, and the characteristic spectrum of the obtained south pepperweed seed formula particles and/or the north pepperweed seed formula particles is constructed under a specific gradient elution program.

Description

Characteristic spectrum of formula granules of semen lepidii and/or semen lepidii as well as construction method and identification method thereof
Technical Field
The invention belongs to the technical field of traditional Chinese medicine identification, and particularly relates to a characteristic spectrum of a formula particle of semen lepidii and/or semen lepidii, a construction method and an identification method thereof.
Background
The semen Lepidii is dried mature seed of plant of Brassicaceae such as Descurainia sophia (L.) Webb, exPrantl or Lepidium apetalum Willd. The former is known as "Nanting Ting Li Zi", the latter as "Bei Ting Li Zi". The product has effects in purging lung, relieving asthma, promoting diuresis and relieving swelling, and can be used for treating phlegm accumulation in lung, cough, asthma, excessive phlegm, fullness and distention in chest and hypochondrium, insomnia, edema of chest and abdomen, and dysuria. According to the examination, ting Li Zi is listed as the inferior product from Shen nong Ben Cao Jing, and is a commonly used Chinese medicine in clinical practice. The original plant of semen Lepidii is semen Lepidii or semen Lepidii specified in the 'Chinese pharmacopoeia' 2015 edition.
At present, the legal standard only has relatively comprehensive control items on the medicinal materials of the lepidium linnei, and the standard only carries out qualitative and quantitative analysis on the component of quercetin-3-O-beta-D-gentiobioside, which is not beneficial to the quality control of the medicinal materials. In addition, the statutory standard defines the characters, microscopic and examination items of the semen lepidii. In the prior art, most researches on the medicinal material of the pepperweed seed are carried out, and a commonly used basic source identification method is adopted during traditional character identification and microscopic identification, for example, a paraffin section of the seed is observed by adopting a microscope, so that the result shows that the pepperweed seed is flat and oval, large, pointed and slightly concave at the other end, and has 2 longitudinal grooves, wherein 1 is obvious, the surface has granular protrusions, the thickness of a mucus layer is thick, and a cellulose column is short; the long round of semen Lepidii is slightly flat and smaller, the other end is more flat, the surface has fine reticulate pattern protrusions, the mucus layer is thin, and the cellulose column is longer. And the method also adopts chromatography to identify semen plantaginis, semen Lepidii and semen Lepidii which are confusing, and adopts molecular biology to identify semen Lepidii and pseudonymous substances.
However, the methods are all directed at the south and north pepperweed seed medicinal material, while the pepperweed seed formula particle loses the original appearance characteristics of the medicinal material, is a formula particle obtained by extracting, concentrating, drying, preparing and the like pepperweed seed decoction pieces, and the material basis of the pepperweed seed formula particle is greatly different from that of the medicinal material, while the pepperweed seed formula particle field is almost free of any method capable of carrying out base source identification.
Wang Aiqin et al, published in the literature "determination of HPLC fingerprint of south pepperweed seed" provide a method for determining the fingerprint of south and north pepperweed seed, but the determination method has long detection time and poor separation degree among characteristic peaks, and can not accurately identify the pepperweed seed formula particles.
Disclosure of Invention
Therefore, the invention aims to overcome the defects that the characteristic spectrum of the southern and northern lepidium medicinal material is long in determination time, poor in separation degree of characteristic peaks, incapable of effectively identifying the southern and northern lepidium formula particles and the like in the prior art, and provides the characteristic spectrum of the southern and northern lepidium formula particles and the construction method and the identification method thereof.
Therefore, the invention provides the following technical scheme.
The invention provides a construction method of a characteristic map of a formula particle of semen lepidii and/or semen lepidii, which comprises the following steps,
preparation of a test solution: preparing a test sample of the south pepperweed seed formula particle to obtain a test sample solution of the south pepperweed seed formula particle; and/or, preparing the test sample of the semen lepidii formula particle to obtain a test sample solution of the semen lepidii formula particle;
high performance liquid chromatography: injecting the test solution of the south pepperweed seed formula particle and/or the test solution of the north pepperweed seed formula particle into a liquid chromatograph, and detecting to obtain the product;
wherein, the chromatographic conditions of the high performance liquid chromatography comprise: performing gradient elution by taking acetonitrile as a mobile phase A and taking an acid-containing aqueous solution with the volume fraction of 0.1-0.3% as a mobile phase B, wherein the gradient elution conditions are as follows: 0-2min,6-8% of mobile phase A, and 94-92% of mobile phase B;2-5min,8% of mobile phase A and 92% of mobile phase B;5-17min,8-10% of mobile phase A, and 92-90% of mobile phase B;17-27min,10-18% of mobile phase A and 90-82% of mobile phase B;27-34min,18-30% of mobile phase A and 82-70% of mobile phase B.
The acid-containing aqueous solution is selected from acetic acid aqueous solution and/or phosphoric acid aqueous solution; preferably an aqueous acetic acid solution.
The chromatographic conditions further comprise: the column temperature is 30-40 ℃; the flow rate is 0.1-0.4ml/min; the detection wavelength is 255-270nm.
Further, the preparation process of the test solution of the south pepperweed seed formula particle or the north pepperweed seed formula particle comprises the steps of extracting and filtering the test sample by adopting a first solvent, and taking filtrate;
preferably, the first solvent is a 50-80% methanol aqueous solution in volume fraction; the extraction mode is heating reflux or ultrasonic extraction;
more preferably, the first solvent is 70% methanol aqueous solution by volume fraction, and the extraction mode is ultrasonic extraction;
the sample mass extracted from 1ml of solvent is 0.01-0.03g.
More specifically, taking a south pepperweed seed formula particle sample solution or a north pepperweed seed formula particle sample, precisely weighing, adding 70% methanol, carrying out ultrasonic extraction, complementing the weight loss amount, filtering, and taking a subsequent filtrate to obtain a sample solution.
The construction method also comprises a step of preparing a reference substance solution by adopting quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside and/or isoquercitrin, and a step of detecting the reference substance solution according to the high performance liquid chromatography to obtain a reference substance characteristic spectrum;
the preparation of the reference solution further comprises the following steps: taking a quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside reference substance, adding a second solvent to prepare a reference substance solution A containing 10-30 mu g of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in every 1ml of reference substance solution;
adding a second solvent into an isoquercitrin reference substance to prepare a reference substance solution B containing 10-30 mu g of isoquercitrin in each 1ml of reference substance solution;
the second solvent is 20-40% methanol aqueous solution by volume fraction, preferably 30% methanol aqueous solution by volume fraction.
The construction method also comprises the steps of preparing a reference solution and detecting the reference solution according to the high performance liquid chromatography to obtain a characteristic map of the reference medicinal material;
the preparation method of the reference solution comprises extracting semen Lepidii (descurainia sophia) as reference material, and filtering to obtain reference solution; specifically, the powder of the semen lepidii (descurainia sophia) reference medicinal material is precisely weighed, 70% methanol is added, ultrasonic extraction is carried out, the weight loss is complemented and reduced, filtration is carried out, and a subsequent filtrate is taken to obtain the semen lepidii (descurainia sophia) reference substance solution.
The preparation method of the reference solution of semen Lepidii comprises extracting semen Lepidii (herba Lepidii Wallichii) as reference material, and filtering to obtain reference solution of semen Lepidii. Specifically, the powder of the semen lepidii (striga asiatica) reference medicinal material is precisely weighed, 70% methanol is added, ultrasonic extraction is carried out, the weight loss is complemented and reduced, filtration is carried out, and a subsequent filtrate is taken to obtain the semen lepidii (striga asiatica) reference substance solution.
In addition, the invention also provides a characteristic spectrum of the semen lepidii formula particles and/or the semen lepidii formula particles obtained by the construction method.
The invention provides a characteristic map of a semen lepidii formula particle, wherein the characteristic map comprises 6 characteristic peaks, and the relative retention time of each characteristic peak and a peak 2 is within +/-10%, +/-5% or +/-3% of a specified value; the predetermined values were 0.66 (peak 1), 1.50 (peak 3), 1.70 (peak 4), 3.16 (peak 5), and 3.57 (peak 6).
The invention provides a characteristic map of a semen lepidii formula particle, wherein the characteristic map comprises 7 characteristic peaks, and the relative retention time of each characteristic peak and a peak 6 is within +/-10%, +/-5% or +/-3% of a specified value; the predetermined values were 0.74 (peak 1), 0.81 (peak 2), 0.85 (peak 3), 0.90 (peak 4), 0.92 (peak 5), and 1.05 (peak 7).
Further, the invention provides a method for identifying the south pepperweed seed formula particles and/or the north pepperweed seed formula particles, which comprises the step of comparing the characteristic spectrum of a product to be detected with the characteristic spectrum of the south pepperweed seed formula particles and/or the characteristic spectrum of the north pepperweed seed formula particles;
the characteristic map of the product to be detected is obtained by using the product to be detected according to the construction method; the characteristic spectrum of the semen lepidii formula particle is the characteristic spectrum of the semen lepidii formula particle; the characteristic spectrum of the semen lepidii formula particle is the characteristic spectrum of the semen lepidii formula particle.
And when the characteristic map of the product to be detected corresponds to the characteristic map of the semen lepidii formula particles, the product to be detected is the semen lepidii formula particles, and when the characteristic map of the product to be detected corresponds to the characteristic map of the semen lepidii formula particles, the product to be detected is the semen lepidii formula particles.
The technical scheme of the invention has the following advantages:
1. the construction method of the characteristic spectrum of the formula particles of the semen lepidii and/or the semen lepidii comprises the steps of preparing a test solution, and detecting under the condition of specific elution gradient to obtain the characteristic spectrum of the formula particles of the semen lepidii and/or the semen lepidii. The invention adopts high performance liquid chromatography to detect the effective components of the south pepperweed seed formula particles and/or the north pepperweed seed formula particles, acetonitrile is taken as a mobile phase A, acid-containing aqueous solution with the volume fraction of 0.1-0.3% is taken as a mobile phase B, and the characteristic spectrum of the obtained south pepperweed seed formula particles and/or the north pepperweed seed formula particles is constructed under a specific gradient elution program.
2. The characteristic spectrum of the south pepperweed seed formula particle and/or the north pepperweed seed formula particle provided by the invention has 6 characteristic peaks, and the characteristic peaks are completely separated and have good peak shapes. The characteristic spectrum of the northern lepidium seed formula particle has 7 characteristic peaks, the characteristic peaks are completely separated, the peak shape is good, compared with the characteristic spectrum of the southern lepidium seed formula particle, no interference peak exists between the peak 1 and the peak 4 in the characteristic spectrum of the northern lepidium seed formula particle, and the peak areas of the peak 1 and the peak 4 are relatively balanced, so that the characteristic spectrum can be used as the main characteristic for distinguishing the southern lepidium seed formula particle.
3. The invention provides an identification method of a semen lepidii formula particle and/or a semen lepidii formula particle, which is short in use time and can effectively achieve the effect of distinguishing the semen lepidii formula particle.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a characteristic spectrum of 17 batches of Nanchairhizi formula granules in example 1 of the present invention;
FIG. 2 is a characteristic spectrum of the granule of the formula of NanTing Li in example 1 of the present invention;
FIG. 3 is a characteristic spectrum of 8 batches of the granule formulation of semen Lepidii of example 1 according to the present invention;
FIG. 4 is a characteristic spectrum of the granule formulation of Pepperweed in example 1 of the present invention;
FIG. 5 is a characteristic map obtained from section 3.3 of different mobile phases B in Experimental example 3 of the present invention;
FIG. 6 is a feature map in section 3.4.1 of Experimental example 3 of the present invention;
FIG. 7 is a feature map of section 3.4.2 in Experimental example 3 of the present invention;
FIG. 8 is a feature map in section 3.4.3 of Experimental example 3 of the present invention;
FIG. 9 is a characteristic map obtained in example 2 of the present invention;
FIG. 10 is a feature map obtained in example 3 of the present invention.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
The experimental instruments, reagents and reference substances adopted in the embodiments of the invention are as follows:
the instrument comprises the following steps: waters ACQUITY
Figure BDA0003231806750000071
H-Class ultra-high performance liquid chromatograph; a PDA Detector; a TUV Detector Detector; empower 3 chromatography workstation; XP26 (Mettler-Tollido), BSA124S electronic balance (Saedoris scientific instruments (Beijing) Co., ltd.), KQ-500DB ultrasonic cleaner (Kunshan City ultrasound)Instrument limited);
and (3) chromatographic column: ACQUTIY UPLC HSS T3 (2.1X 100mm,1.8 μm)
Quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside reference substance (batch number: H-060-180517, purity > 98%, chengdui Feng Si Biotech Co., ltd.)
Semen Lepidii reference medicine (batch No. 121220-201403, china institute for testing food and drug)
Semen Lepidii reference drug (batch number: 121646-201101, china institute for testing food and drug)
Reagent: acetonitrile (Merck Co., ltd., JA 078530), acetic acid (Fisher Scientific, 171289) as chromatographically pure; the water is distilled water (drochen); other reagents were analytically pure.
The origin and batch number of the granule of the formula of semen lepidii: the lot numbers are S1-S17, the producing areas respectively correspond to Henan province Joke city warm county, shandong province Zibo city Gaoqing county, shandong province Ribos city Juxian county, shandong province Linyi city economic development area, shandong province Weifang city Changle county, shandong province Tex city Qingyun county, shandong province Ling city Tan county, shandong province Weifang city Anqiu city, shandong province chat city crown county, shandong province Lingyi city Hedong district, hebei province Canshan city Yanshan county, henan province Huiyang county, henan province Luyang city Ruyang county, jiangsu province Xuzhou county, henan province Luyang city county, jiangsu province Zhongyang city Zhongzhou county, jiangsu county, shandong province Ringyang county;
the origin and batch number of the granule of the formulation of semen lepidii: the production places are S18-S25, and respectively correspond to the Wen county of Joze city in Henan province, the Jun county of sunshine city in Shandong province, the Changle county in Weifang city in Shandong province, the Tan county in Linyi city in Shandong province, the Guanguan county of chat city in Shandong province, the Yanshan county in Canun county in Henan province, the Ruyang county in Luyang city in Henan province and the Ruyang county in Luyang city in Henan province.
Example 1
The embodiment provides a method for constructing a characteristic map of a granule prepared from semen lepidii and/or semen lepidii, which comprises the following steps,
preparation of a semen lepidii reference solution: accurately weighing 1.0g of semen Descurainiae reference medicinal material, placing in a conical flask with a plug, accurately adding 25ml of 70% methanol, sealing the plug, weighing, performing ultrasonic treatment for 30min (power 250W and frequency 40 kHz), taking out, cooling, weighing again, supplementing the reduced weight with 70% methanol, shaking up, filtering, and taking subsequent filtrate to obtain semen Descurainiae reference solution.
Preparation of a norpepperweed reference solution: precisely weighing 1.0g of semen Lepidii reference medicinal material, placing in a conical flask with a plug, precisely adding 25ml of 70% methanol, sealing the plug, weighing, ultrasonically treating for 30min (power 250W and frequency 40 kHz), taking out, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain semen Lepidii reference solution.
Preparation of control solutions: taking a proper amount of a quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside reference substance, precisely weighing, and adding 30% methanol to prepare a reference substance solution A containing 20 mu g of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside per 1 ml;
taking a proper amount of isoquercitrin reference substance, precisely weighing, and adding 30% methanol to prepare a reference substance solution B containing 20 μ g of isoquercitrin per 1 ml.
Preparation of a test solution: grinding the south pepperweed seed formula particles or the north pepperweed seed formula particles, taking about 0.5g, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 70% methanol, weighing the weight, carrying out ultrasonic treatment for 30min (the power is 250W, the frequency is 40 kHz), cooling, weighing again, complementing the weight loss by 70% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the south pepperweed seed formula particles or the north pepperweed seed formula particles to be tested.
Chromatographic conditions are as follows: chromatographic column octadecylsilane chemically bonded silica gel as filler (column length is 10cm, inner diameter is 2.1mm, particle diameter is 1.8 μm), acetonitrile as mobile phase A, and 0.2% acetic acid water solution as mobile phase B by volume fraction are subjected to gradient elution under the following conditions: 0-2min,6-8% of mobile phase A, and 94-92% of mobile phase B;2-5min,8% of mobile phase A and 92% of mobile phase B;5-17min,8-10% of mobile phase A, and 92-90% of mobile phase B;17-27min,10-18% of mobile phase A and 90-82% of mobile phase B;27-34min,18-30% of mobile phase A, and 82-70% of mobile phase B; the column temperature is 35 ℃, the flow rate is 0.3ml/min, and the wavelength is 265nm; precisely sucking 1 μ L of each of the reference solution, the reference solution and the sample solution, injecting into an ultra-high performance liquid chromatograph, and measuring to obtain characteristic chromatogram of the semen Lepidii formula granule or the semen Lepidii formula granule.
Constructing a characteristic spectrum of the formula granules of the semen lepidii:
taking S1-S17 different batches of south pepperweed seed formula particles, preparing a test solution according to the method, determining according to the chromatographic conditions to obtain characteristic maps of the south pepperweed seed formula particles of each batch, introducing the maps into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system for data matching, and establishing comparison characteristic maps of different basic south pepperweed seed formula particles, wherein as shown in figure 1, 17 batches of south pepperweed seed formula particles all present 6 characteristic peaks and correspond to retention time of the 6 characteristic peaks of a south pepperweed seed reference solution, wherein the 2 peak is quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside, the 5 peak is isoquercitrin, and the 2 peak is taken as a reference peak, the relative retention time is calculated, and the relative retention time of each characteristic peak is within +/-10% of a specified value: 0.66 (peak 1), 1.50 (peak 3), 1.70 (peak 4), 3.16 (peak 5), 3.57 (peak 6), see fig. 2.
Constructing a characteristic spectrum of the semen lepidii formula particles:
taking S18-S25 different batches of semen lepidii formula particles, preparing a test solution according to the method, determining according to the chromatographic conditions to obtain characteristic maps of the semen lepidii formula particles in each batch, introducing the maps into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system for data matching, and establishing control characteristic maps of different basic semen lepidii formula particles, wherein the chart is 3,8 that the semen lepidii formula particles in batches all present 7 characteristic peaks and correspond to the retention time of the 7 characteristic peaks of the semen lepidii reference solution, wherein the 6 peak is taken as a reference peak, the relative retention time is calculated, the relative retention time of each characteristic peak is within +/-10% of a specified value, and the specified value is: 0.74 (Peak 1), 0.81 (Peak 2), 0.85 (Peak 3), 0.90 (Peak 4), 0.92 (Peak 5), 1.05 (Peak 7), see FIG. 4.
Experimental example 1 methodological examination
1.1 precision test
1.1.1 repeatability
Taking 6 parts of the south pepperweed seed formula particle (S1), determining according to the method of the embodiment 1, obtaining the characteristic map, taking the No. 2 peak as a reference peak, and calculating the relative peak area and the relative retention time. And calculates the RSD. The results are shown in tables 1 and 2.
TABLE 1 retention time and relative retention time table for repeated survey of feature profiles
Figure BDA0003231806750000101
TABLE 2 feature map repeatability survey peak area and relative peak area
Figure BDA0003231806750000102
Figure BDA0003231806750000111
According to the repeatability investigation result, the relative retention time RSD of each characteristic peak is 0-0.3%, and the RSD of the relative peak area is in the range of 0-1.7%, which shows that the repeatability of the characteristic spectrum is good.
1.1.2 intermediate precision
A water UPLC H-Class TUV detector is adopted, 6 parts of the semen lepidii formula particles (S1) are taken, the characteristic spectrum is obtained by measuring according to the method of the example 1, and the relative peak area and the relative retention time are calculated by taking the No. 2 peak as a reference peak. And calculates the RSD. The results are shown in tables 3 and 4.
TABLE 3 retention time and relative retention time table for intermediate precision survey of feature profiles
Figure BDA0003231806750000112
Figure BDA0003231806750000121
TABLE 4 characteristic spectra middle precision survey peak area and relative peak area table
Figure BDA0003231806750000122
The above results show that the relative retention time RSD of each characteristic peak in the characteristic spectrum obtained by using the Waters UPLC H-Class, TUV detector is in the range of 0-0.6%, and the RSD of the relative peak area is in the range of 0-1.8%. The relative retention time RSD range between different instruments is 0-2.2%, and the relative peak area RSD% range is 0-7.4%, which shows that different instrument analysis has certain influence on the characteristic spectrum.
1.2 examination of stability of test solutions
The relative retention time of corresponding chromatographic peaks is less than 2.0 percent and the RSD of the relative peak area of the characteristic peak is less than 2.0 percent, the result shows that the test solution is stable within 24 hours. See tables 5 and 6.
TABLE 5 stability relative Retention time Table
Figure BDA0003231806750000123
Figure BDA0003231806750000131
TABLE 6 table of relative peak area of stability
Figure BDA0003231806750000132
According to the stability experiment results, the chemical components in the test solution are stable in 24 hours, the RSD of the relative retention time of each characteristic peak is within 0-2.0%, and the RSD of the relative peak area is within 0-1.8%, so that the stability and reliability of the analysis method are shown.
Experimental example 2 methodological examination
2.1 precision test
2.1.1 repeatability
Taking 6 parts of the semen lepidii formula particle (S18), determining according to the method of the example 1, obtaining the characteristic map of the semen lepidii formula particle, taking the No. 6 peak as a reference peak, calculating the relative peak area and the relative retention time, and calculating the RSD. The results are shown in tables 7 and 8.
TABLE 7 retention time and relative retention time table for repeated investigation of feature maps
Figure BDA0003231806750000141
TABLE 8 characteristic chromatogram repeatability survey peak area and relative peak area
Figure BDA0003231806750000142
Figure BDA0003231806750000151
Through the experiments, according to the repeatability investigation result, the relative retention time RSD of each characteristic peak is 0% -0.5%, and the RSD of the relative peak area is in the range of 0% -1.5%, which shows that the repeatability of the characteristic spectrum is good.
2.1.2 intermediate precision
By using Waters UPLC H-Class, TUV detector, 6 parts of the semen lepidii formula granules (S18) were taken, measured according to the method of example 1, to obtain the characteristic spectrum thereof, and the relative peak area and relative retention time thereof were calculated with peak No. 6 as the reference peak, and RSD was calculated. The results are shown in tables 9 and 10.
TABLE 9 intermediate precision survey of retention times and relative retention time tables for feature profiles
Figure BDA0003231806750000152
TABLE 10 characteristic spectra middle precision survey peak area and relative peak area table
Figure BDA0003231806750000153
Figure BDA0003231806750000161
According to the above experimental results, the relative retention time RSD of each characteristic peak in the characteristic spectrum obtained by using a Waters UPLC H-Class, TUV detector was in the range of 0-0.5%, and the RSD of the relative peak area was in the range of 0-2.0%. The relative retention time RSD range of different instruments is 0-1.1%, and the relative peak area RSD% range is 0-4.6%, which shows that different instruments have certain influence on the characteristic spectrum.
2.2 examination of the stability of the test solutions
The characteristic peaks in the test solution (prepared according to the method of example 1) of the semen lepidii formula particles are measured according to the method of example 1 after being placed for 0h,2h,4h,6h,8h,10h,12h and 24h respectively, the relative retention time of the corresponding chromatographic peaks is less than 2.0 percent, the RSD of the relative peak areas of the characteristic peaks is less than 2.0 percent, and the result shows that the test solution is stable within 24 h. See tables 11 and 12.
TABLE 11 stability relative Retention time Table
Figure BDA0003231806750000162
TABLE 12 table of stability versus peak area
Figure BDA0003231806750000171
According to the stability experiment result, the chemical components in the test solution are stable in 24 hours, the RSD of the relative retention time of each characteristic peak is within 0-0.7%, and the RSD of the relative peak area is within 0-1.9%, which shows that the analysis method is stable and reliable.
Experimental example 3 examination of chromatographic conditions
3.1 examination of column temperature
Examining the influence of the column temperature on the detection method, 3 parts of the test solution of the semen lepidii formula particle is prepared according to the method in the example 1, the column temperature is 33 ℃, 35 ℃ and 37 ℃ respectively except the column temperature, the characteristic spectrum is obtained by measuring, and the result is shown in table 13, which indicates that the method has good durability at the column temperature.
TABLE 13 relative retention time tables at different column temperatures
Figure BDA0003231806750000172
Figure BDA0003231806750000181
From the above results, it can be seen that the relative retention times of the 6 peaks of the profiles obtained at the other two temperatures differ by ± 10% with respect to the relative retention time of the profile obtained at 35 ℃, and that the method is well tolerated at different column temperatures.
3.2 examination of the concentration of mobile phase B
In addition to the difference in volume fraction of the acid in mobile phase B, in the same manner as in example 1 except that the concentration of mobile phase B was examined for the effect on the detection method, the mobile phase B was an aqueous solution of acetic acid having a volume fraction of 0.18%, 0.20% and 0.22%, a test solution of the south pepperweed seed formula granule was prepared and detected as in example 1, and the results were shown in table 14.
TABLE 14 relative retention time tables at different acid concentrations
Figure BDA0003231806750000182
From the above data, it can be seen that the relative retention time of the 6 peaks is within ± 10% of the specified value, the chromatographic peak separation is better at different acid concentrations, indicating that the aqueous acetic acid concentration has less influence on the process.
3.3 different mobile phases B
Examining the influence of different acid solutions on the detection method, the same as example 1 except that the mobile phase B is different, the mobile phase B is 0.1% acetic acid aqueous solution and 0.1% phosphoric acid aqueous solution by volume fraction, the Nanting Li formula particle sample solution is prepared according to the method of example 1, and the characteristic map is obtained by detection, as shown in figure 5.
Through comparison of the characteristic maps, it can be known that an aqueous acetic acid solution with a volume fraction of 0.1% is superior to an aqueous phosphoric acid solution with a volume fraction of 0.1%. Furthermore, there is no necessary connection between the choice of mobile phase and the sample to be tested.
3.4 examination of elution gradient
The influence of different elution gradients on the detection method is considered, except for the elution gradients, the method is the same as the example 1, the elution gradients are respectively as follows, the south pepperweed seed formula particle sample solution is prepared according to the method of the example 1, and the characteristic spectrum is obtained by detection.
3.4.1 the elution gradient is shown in Table 15, acetonitrile is taken as a mobile phase A, 1 percent of phosphoric acid aqueous solution in volume fraction is taken as a mobile phase B, and the obtained characteristic map is shown in figure 6 after detection;
TABLE 15 elution gradient procedure
Time (min) CH 3 CN(％) H 2 O-H 3 PO 4 (％)
0-2 5→10 95→90
2-3 10→13 90→87
3-4 13 87
4-6 13→20 87→80
6-9 20 80
9-11 20→30 80→70
11-12.5 30 70
12.5-14 30→40 70→60
14-14.5 40 60
14.5-15 40→5 60→95
15-17 5 95
As can be seen from the characteristic map, the separation degree between characteristic peaks is poor, and some characteristic peaks cannot be completely separated.
3.4.2 elution gradient is shown in Table 16, acetonitrile is used as a mobile phase A, and an acetic acid aqueous solution with volume fraction of 0.2% is used as a mobile phase B, and a characteristic spectrum obtained by detection is shown in FIG. 7;
TABLE 16 elution gradient procedure
Time (min) Mobile phase A (%) Mobile phase B (%)
0-5 10 90
5-20 10→20 90→80
20-30 20→30 80→70
As can be seen from the characteristic map, the elution gradient cannot obtain a characteristic peak.
3.4.3 the elution gradient is shown in Table 17, acetonitrile is taken as a mobile phase A, and an acetic acid aqueous solution with the volume fraction of 0.2% is taken as a mobile phase B, and the obtained characteristic map is shown in figure 8 after detection;
TABLE 17 elution gradient procedure
Time (min) Mobile phase A (%) Mobile phase B (%)
0-3 10→15 90→85
3-25 15→40 85→60
As can be seen from the characteristic map, the elution gradient cannot obtain a characteristic peak.
Example 2
The embodiment provides an identification method of a semen lepidii formula particle, which comprises the following steps,
preparation of a test solution: taking a sample, grinding, taking about 0.5g, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 70% methanol, weighing, carrying out ultrasonic treatment for 30min (power 250W and frequency 40 kHz), cooling, weighing again, supplementing the lost weight with 70% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain a sample solution.
Chromatographic conditions are as follows: chromatographic column octadecylsilane chemically bonded silica gel as filler (column length is 10cm, inner diameter is 2.1mm, particle diameter is 1.8 μm), acetonitrile as mobile phase A, and 0.2% acetic acid water solution as mobile phase B by volume fraction are subjected to gradient elution under the following conditions: 0-2min,6-8% of mobile phase A, and 94-92% of mobile phase B;2-5min,8% of mobile phase A and 92% of mobile phase B;5-17min,8-10% of mobile phase A, and 92-90% of mobile phase B;17-27min,10-18% of mobile phase A and 90-82% of mobile phase B;27-34min,18-30% of mobile phase A, and 82-70% of mobile phase B; the column temperature was 35 ℃, the flow rate 0.3ml/min, the wavelength 265nm.
Precisely sucking 1 mu L of the test solution, injecting into an ultra-high performance liquid chromatograph, and measuring to obtain a characteristic spectrum of the test solution, wherein the characteristic spectrum of the test solution presents 6 characteristic peaks, as shown in figure 9; and (3) processing and analyzing the data by using traditional Chinese medicine chromatographic fingerprint similarity evaluation software, comparing the data with the characteristic spectrum of the southern pepperweed seed formula particle constructed in the example 1, wherein the similarity result is 0.994, and the results are consistent, which indicates that the sample is the southern pepperweed seed formula particle.
Example 3
The embodiment provides an identification method of a semen lepidii formula particle, which comprises the following steps,
preparation of a test solution: taking a sample, grinding, taking about 0.5g, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 70% methanol, weighing, carrying out ultrasonic treatment for 30min (power 250W and frequency 40 kHz), cooling, weighing again, supplementing the lost weight with 70% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain a sample solution.
Chromatographic conditions are as follows: chromatographic column octadecylsilane chemically bonded silica gel as filler (column length is 10cm, inner diameter is 2.1mm, particle diameter is 1.8 μm), acetonitrile as mobile phase A, and 0.2% acetic acid water solution as mobile phase B by volume fraction are subjected to gradient elution under the following conditions: 0-2min,6-8% of mobile phase A, and 94-92% of mobile phase B;2-5min,8% of mobile phase A and 92% of mobile phase B;5-17min,8-10% of mobile phase A, and 92-90% of mobile phase B;17-27min,10-18% of mobile phase A and 90-82% of mobile phase B;27-34min,18-30% of mobile phase A, and 82-70% of mobile phase B; the column temperature was 35 ℃, the flow rate 0.3ml/min, the wavelength 265nm.
Precisely sucking 1 mu L of the test solution, injecting into an ultra-high performance liquid chromatograph, and measuring to obtain a characteristic spectrum of the test solution, wherein the characteristic spectrum of the test solution presents 7 characteristic peaks, as shown in figure 10; and (3) processing and analyzing the data by using traditional Chinese medicine chromatographic fingerprint similarity evaluation software, comparing the data with the characteristic spectrum of the semen lepidii formula particle obtained in the example 1, wherein the similarity result is 0.992, and the results are consistent, so that the sample is the semen lepidii formula particle.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims (12)

1. A construction method of a characteristic map of a granule prepared from semen lepidii and/or semen lepidii is characterized by comprising the following steps of,
preparation of a test solution: preparing the test sample of the semen lepidii formula particle to obtain a semen lepidii formula particle test sample solution; and/or preparing the test sample of the semen lepidii formula particle to obtain a test sample solution of the semen lepidii formula particle;
ultra-high performance liquid chromatography: injecting the test solution of the south pepperweed seed formula particle and/or the test solution of the north pepperweed seed formula particle into a liquid chromatograph, and detecting to obtain the product;
wherein, the chromatographic conditions of the ultra-high performance liquid chromatography comprise: and (3) chromatographic column: ACQUTIYUPLC HSS T3 with specification of 2.1 × 100mm,1.8 μm, and acetonitrile as mobile phase A, and 0.1-0.3% volume fraction of acid-containing aqueous solution as mobile phase B, and performing gradient elution under the following conditions: 0-2min,6-8% of mobile phase A, and 94-92% of mobile phase B;2-5min,8% of mobile phase A and 92% of mobile phase B;5-17min,8-10% of mobile phase A, and 92-90% of mobile phase B;17-27min,10-18% of mobile phase A and 90-82% of mobile phase B;27-34min,18-30% of mobile phase A, and 82-70% of mobile phase B;
wherein, the preparation process of the test solution of the south pepperweed seed formula particle or the test solution of the north pepperweed seed formula particle comprises the steps of extracting the test sample by adopting a first solvent; the first solvent is a methanol aqueous solution with the volume fraction of 50-80%.
2. The method for constructing according to claim 1, wherein the acid-containing aqueous solution is selected from an aqueous acetic acid solution and/or an aqueous phosphoric acid solution.
3. The method according to claim 2, wherein the acid-containing aqueous solution is an aqueous acetic acid solution.
4. The construction method according to claim 1 or 2, wherein the chromatographic conditions further comprise: the column temperature is 30-40 ℃; the flow rate is 0.1-0.4ml/min; the detection wavelength is 255-270nm.
5. The constructing method as claimed in claim 1 or 2, wherein the preparing process of the test solution of the south pepperweed seed formula particle or the north pepperweed seed formula particle comprises the steps of extracting and filtering the test sample by using a first solvent, and taking the filtrate;
the extraction mode is heating reflux or ultrasonic extraction.
6. The construction method according to claim 4, wherein the first solvent is 70% methanol aqueous solution by volume fraction, and the extraction mode is ultrasonic extraction;
the sample mass extracted from 1ml of solvent is 0.01-0.03g.
7. The method according to claim 1 or 2, further comprising a step of preparing a reference solution from quercetin-3-O- β -D-glucose-7-O- β -D-gentiobioside and/or isoquercitrin, and a step of detecting the reference solution by ultra high performance liquid chromatography according to claim 1 or 2 to obtain a reference characteristic spectrum;
the preparation of the reference solution further comprises the following steps: taking a reference substance of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside, adding a second solvent to prepare a reference substance solution A containing 10-30 mu g of quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside in every 1ml of reference substance solution;
adding a second solvent into an isoquercitrin reference substance to prepare a reference substance solution B containing 10-30 mu g of isoquercitrin in each 1ml of reference substance solution;
the second solvent is methanol water solution with the volume fraction of 20-40%.
8. The method of claim 7, wherein the second solvent is a 30% volume fraction aqueous methanol solution.
9. The method according to claim 1 or 2, wherein the method further comprises the steps of preparing a reference solution, and detecting the reference solution by the ultra-high performance liquid chromatography according to claim 1 or 2 to obtain a characteristic map of a reference drug;
the preparation method of the semen Descurainiae reference solution comprises the steps of taking semen Descurainiae reference medicinal material, extracting and filtering to obtain the semen Descurainiae reference solution;
the preparation method of the reference solution of semen Lepidii comprises extracting and filtering the reference medicinal material of semen Lepidii to obtain the reference solution of semen Lepidii.
10. The method for constructing according to claim 1 or 2, wherein the characteristic map of the granule of the formula of the semen lepidii has at least 6 characteristic peaks, and the relative retention time of each characteristic peak to peak 2 is within ± 10% of the specified value; the specified values for peak 1, peak 3, peak 4, peak 5 and peak 6 were 0.66, 1.50, 1.70, 3.16 and 3.57, respectively.
11. The method for constructing granule according to claim 1 or 2, wherein the characteristic map of the granule has at least 7 characteristic peaks, and the relative retention time of each characteristic peak to peak 6 is within ± 10% of the specified value; the specified values of peak 1, peak 2, peak 3, peak 4, peak 5 and peak 7 were 0.74, 0.81, 0.85, 0.90, 0.92 and 1.05, respectively.
12. A method for identifying a south pepperweed seed formula particle and/or a north pepperweed seed formula particle is characterized by comprising the step of comparing a characteristic spectrum of a product to be detected with a characteristic spectrum of the south pepperweed seed formula particle and/or a characteristic spectrum of the north pepperweed seed formula particle;
the characteristic map of the product to be detected is obtained by using the product to be detected according to the construction method of any one of claims 1 to 8; the characteristic spectrum of the south pepperweed seed formula particle is the characteristic spectrum of the south pepperweed seed formula particle as defined in claim 10; the characteristic map of the semen lepidii formula particle is the characteristic map of the semen lepidii formula particle as claimed in claim 11.
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