CN111007190A - Method for constructing UPLC (ultra performance liquid chromatography) characteristic spectrum of rhizoma bolbostemmae medicinal material and method for measuring component content of rhizoma bolbostemmae medicinal material - Google Patents

Method for constructing UPLC (ultra performance liquid chromatography) characteristic spectrum of rhizoma bolbostemmae medicinal material and method for measuring component content of rhizoma bolbostemmae medicinal material Download PDF

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CN111007190A
CN111007190A CN201911222672.6A CN201911222672A CN111007190A CN 111007190 A CN111007190 A CN 111007190A CN 201911222672 A CN201911222672 A CN 201911222672A CN 111007190 A CN111007190 A CN 111007190A
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volume fraction
tubeimoside
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CN111007190B (en
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黄敏烨
李振雨
童培珍
曹斯琼
潘礼业
孙冬梅
陈向东
魏梅
程学仁
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Guangdong Yifang Pharmaceutical Co Ltd
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Abstract

The invention relates to a method for constructing UPLC characteristic spectrum of rhizoma bolbostemmae medicinal material and a method for measuring component content of the rhizoma bolbostemmae medicinal material, wherein the method for measuring the component content of the rhizoma bolbostemmae medicinal material comprises the following steps: (1) respectively sucking a reference substance solution and a sample solution of the medicinal material of the bolbostemma paniculatum to be detected, injecting the reference substance solution and the sample solution into an ultra-high performance liquid chromatograph, and measuring corresponding peak areas; (2) calculating the content of tubeimoside A by an external standard method; (3) and calculating the contents of tubeimoside B and tubeimoside C by a one-test-multiple evaluation method. The method is rapid, stable, strong in specificity and stable in baseline, and provides a scientific new method for quality control of the medicinal material of the rhizoma bolbostemmae.

Description

Method for constructing UPLC (ultra performance liquid chromatography) characteristic spectrum of rhizoma bolbostemmae medicinal material and method for measuring component content of rhizoma bolbostemmae medicinal material
Technical Field
The invention belongs to the field of traditional Chinese medicine identification, and particularly relates to a method for constructing a UPLC (ultra performance liquid chromatography) characteristic spectrum of a rhizoma bolbostemmae medicinal material and a method for measuring component content of the rhizoma bolbostemmae medicinal material.
Background
The paniculate Bolbostemma rhizome is commonly called wild snakegourd fruit, also called Pseudobulbus fritillary, Bolbostemma paniculatum and Dikudan, and is originally carried in Bencao gang mu Shi Yi (supplement to compendium of materia Medica) edited by Zhao of the Qing Dynasty, and 2015 edition Chinese pharmacopoeia stipulates that the paniculate Bolbostemma rhizome is a dry tuber of the paniculate Bolbostemma paniculatum and is widely distributed in Hebei, Shanxi, Shandong, Henan, Hubei and Hunan. The literature reports that at present, a plurality of saponins, sterols, glycosides, esters, alkaloids, flavonoids and other components of the tubeimous fritillary bulb are obtained by separating the tubeimous bulb, wherein the saponins are the main active components of the tubeimous bulb. Modern pharmacological research shows that the rhizoma bolbostemmae has the functions of resisting inflammation, detumescence, virus, leukemia, immunosuppression, spermicidal action and the like.
In 2015, the 'Chinese pharmacopoeia' edition only takes tubeimoside A as a content determination index, and due to the diversity and complexity of effective ingredients of traditional Chinese medicines, the content determination of a single ingredient cannot completely reflect the internal quality of medicinal materials, and the specificity is also lacking. The traditional Chinese medicine fingerprint/characteristic spectrum can provide more comprehensive and abundant information, so that the traditional Chinese medicine fingerprint/characteristic spectrum is commonly used for the quality control research of traditional Chinese medicinal materials; the one-test-multiple-evaluation method is characterized in that the content of a plurality of components can be measured by measuring 1 component (internal reference substance) by means of relative correction factors, and the one-test-multiple-evaluation method can effectively solve the problems in view of the difficulty in preparation, high price, easiness in decomposition and other factors of most traditional Chinese medicine reference substances, and is gradually and widely applied to the analysis of traditional Chinese medicine components. The existing literature on the research on the fingerprint/characteristic spectrum and content determination of the rhizoma bolbostemmae adopts a high performance liquid chromatography (HPLC method), the method has long time, low sensitivity and limited separation capability, and the actual problem in the analysis process of the complex traditional Chinese medicine can not be well solved.
Disclosure of Invention
The invention aims to provide a method for constructing UPLC characteristic spectrum of rhizoma bolbostemmae medicinal material and a method for measuring component content of the rhizoma bolbostemmae medicinal material.
The technical problem to be solved by the invention is realized by the following technical scheme:
a method for constructing UPLC characteristic spectrum of rhizoma bolbostemmae comprises the following steps:
(1) accurately weighing rhizoma Bolbostematis powder to obtain rhizoma Bolbostematis test solution;
(2) analyzing the sample solution of the rhizoma bolbostemmae medicinal material by using an ultra-high performance liquid chromatograph to obtain a UPLC characteristic spectrum of the rhizoma bolbostemmae medicinal material.
Preferably, the chromatographic conditions for the ultra high performance liquid chromatograph analysis are as follows: gradient elution is carried out by adopting a Waters Cortecs T3 chromatographic column with the specification of 2.1 multiplied by 100mm and the diameter of 1.6 mu m, taking acetonitrile as a mobile phase A and taking a phosphoric acid aqueous solution with the concentration of 0.05-0.15% as a mobile phase B, wherein the column temperature is 20-40 ℃; the flow rate is 0.2-0.4 ml/min; the detection wavelength is 180-220 nm, and the sample injection amount is 0.5-1.5 mu l.
As a preferred embodiment, the gradient elution conditions are: the volume fraction of the mobile phase A is changed to be 5% -11% and the volume fraction of the mobile phase B is 95% -89% in 0-4 min; 4-7 min, the volume fraction of the mobile phase A is changed to 11-20%, and the volume fraction of the mobile phase B is changed to 89-80%; 7-21 min, the volume fraction of the mobile phase A is changed to 20-28%, and the volume fraction of the mobile phase B is changed to 80-72%; the volume fraction of the mobile phase A is changed to 28-32% and the volume fraction of the mobile phase B is changed to 72-68% in 21-30 min; 30-33 min, the volume fraction of the mobile phase A is changed to 32-35%, and the volume fraction of the mobile phase B is changed to 68-65%; and 33-38 min, wherein the volume fraction of the mobile phase A is 35%, and the volume fraction of the mobile phase B is 65%.
As a preferable scheme, the preparation method of the test solution comprises the following steps: taking 0.4-0.6 g of rhizoma bolbostemmae medicinal powder, precisely weighing, precisely adding 20-30 ml of 40-60% ethanol, weighing, carrying out ultrasonic treatment for 20-40 minutes, cooling, weighing again, complementing the loss weight with 40-60% ethanol, shaking up, filtering, and taking the subsequent filtrate.
The invention also provides a method for measuring the component content of the rhizoma bolbostemmae, which comprises the following steps:
(1) respectively sucking a reference substance solution and a sample solution of the medicinal material of the bolbostemma paniculatum to be detected, injecting the reference substance solution and the sample solution into an ultra-high performance liquid chromatograph, and measuring corresponding peak areas;
(2) calculating the content of tubeimoside A by an external standard method;
(3) and calculating the contents of tubeimoside B and tubeimoside C by a one-test-multiple evaluation method.
As a preferred embodiment, the preparation method of the reference solution comprises: taking the tubeimoside A, tubeimoside B and tubeimoside C reference substances, precisely weighing, and adding methanol to prepare a reference substance solution containing 80-120 mu g of tubeimoside A, 40-60 mu g of tubeimoside B and 60-100 mu g of tubeimoside C per 1 ml.
As a preferred scheme, the preparation method of the sample solution of the paniculate Bolbostemma rhizome to be detected comprises the following steps: taking 0.4-0.6 g of rhizoma bolbostemmae medicinal powder, precisely weighing, precisely adding 20-30 ml of 40-60% ethanol, weighing, carrying out ultrasonic treatment for 20-40 minutes, cooling, weighing again, complementing the loss weight with 40-60% ethanol, shaking up, filtering, and taking the subsequent filtrate.
Preferably, the chromatographic conditions of the ultra-high performance liquid chromatograph analysis are that a Waters Cortecs T3 chromatographic column with the specification of 2.1 multiplied by 100mm and the specification of 1.6 mu m is adopted, acetonitrile is taken as a mobile phase A, 0.05-0.15% phosphoric acid aqueous solution is taken as a mobile phase B, gradient elution is carried out, and the column temperature is 20-40 ℃; the flow rate is 0.2-0.4 ml/min; the detection wavelength is 180-220 nm, and the sample injection amount is 0.5-1.5 mu l.
As a preferred embodiment, the gradient elution conditions are: : the volume fraction of the mobile phase A is changed to be 5% -11% and the volume fraction of the mobile phase B is 95% -89% in 0-4 min; 4-7 min, the volume fraction of the mobile phase A is changed to 11-20%, and the volume fraction of the mobile phase B is changed to 89-80%; 7-21 min, the volume fraction of the mobile phase A is changed to 20-28%, and the volume fraction of the mobile phase B is changed to 80-72%; the volume fraction of the mobile phase A is changed to 28-32% and the volume fraction of the mobile phase B is changed to 72-68% in 21-30 min; 30-33 min, the volume fraction of the mobile phase A is changed to 32-35%, and the volume fraction of the mobile phase B is changed to 68-65%; and 33-38 min, wherein the volume fraction of the mobile phase A is 35%, and the volume fraction of the mobile phase B is 65%.
The application of the method for constructing UPLC characteristic spectrum of paniculate Bolbostemma rhizome or the method for measuring the content of components of paniculate Bolbostemma rhizome in detecting and identifying paniculate Bolbostemma rhizome is provided.
Has the advantages that: (1) the invention constructs UPLC characteristic map of the medicinal material of paniculate Bolbostemma rhizome, determines 4 common peaks, identifies the chemical components of the 4 common peaks, determines that the peak 2 is paniculate Bolbostemma rhizome glycoside B, the peak 3 is paniculate Bolbostemma rhizome glycoside C, and the peak 4 is paniculate Bolbostemma rhizome glycoside A, the constructed UPLC characteristic map fully reflects the characteristic peak information of the sample, and displays the chemical component characteristics of the medicinal material of paniculate Bolbostemma rhizome; (2) the UPLC characteristic spectrum of the panicled Bolbostemma rhizome constructed by the invention can be used for qualitatively detecting whether a sample is the panicled Bolbostemma rhizome medicinal material, has certain characteristics, and has the advantages of stable method, high precision and better reproducibility; (3) according to the method, the UPLC characteristic spectrum of the medicinal material of the paniculate Bolbostemma rhizome is constructed, the peak area is measured, the content of tubeimoside A is calculated by adopting an external standard method, the content of tubeimoside B and tubeimoside C is calculated by one-time multiple evaluation, and the quality standard of the paniculate Bolbostemma rhizome is established, namely the content of tubeimoside A is more than 1%, and the total content of tubeimoside A, tubeimoside B and tubeimoside C is 2.65-3.64%, so that the quantitative and qualitative analysis of the paniculate Bolbostemma rhizome is fundamentally carried out rapidly and accurately, certain characteristics are achieved, the content of all components is calculated by the external standard method, and the one-time multiple evaluation and the external standard method are verified to have no obvious difference in the; (4) the one-measurement-and-multiple-evaluation method only needs to measure the content of one component, namely the component is used as an internal reference, other relative correction factors are calculated, the simultaneous measurement of a plurality of components can be realized, and the difficulty caused by the shortage of reference substances in the quality analysis process can be effectively solved; (5) the method provided by the invention is rapid, stable, strong in specificity and stable in baseline, and provides a scientific and new method for quality control of the bolbostemma paniculatum medicinal material.
Drawings
FIG. 1 is a chromatogram of samples of tubeimoside A, tubeimoside B, tubeimoside C and tubeimoside C.
FIG. 2 is a UV chart of the test solution and the tubeimoside A reference solution.
FIG. 3 is a UV chart of the test solution and the tubeimoside B reference solution.
FIG. 4 shows UV images of the test solution and the tubeimoside C control solution.
FIG. 5 is a UPLC feature map overlay of 19 batches of rhizoma Bolbostematis herbs.
FIG. 6 is UPLC characteristic spectrum of paniculate Bolbostemma rhizome.
FIG. 7 is chromatogram for measuring contents of tubeimoside A, B and C.
FIG. 8 is UPLC characteristic spectrum of rhizoma Bolbostematis medicinal material to be identified.
The labels in the figure are: determining the peak 2 as tubeimoside B, the peak 3 as tubeimoside C, and the peak 4 as tubeimoside A.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The instrument, reagent and reference information used in the invention and the source of the bolbostemma paniculatum medicinal material are as follows:
TABLE 1 Instrument information summary sheet
Figure 548218DEST_PATH_IMAGE001
TABLE 2 summary of reagent information
Figure 523127DEST_PATH_IMAGE002
TABLE 3 control information
Figure 739345DEST_PATH_IMAGE003
TABLE 419 information Table of Tubeimu crude drug producing area
Figure 621850DEST_PATH_IMAGE004
Example 1
Method for constructing UPLC (unified Power level liquid chromatography) characteristic spectrum of paniculate Bolbostemma rhizome medicinal material
1. The chromatographic conditions of the ultra-high performance liquid chromatograph analysis are as follows: gradient elution is carried out by adopting a Waters Cortecs T3 chromatographic column with the specification of 2.1 multiplied by 100mm and the size of 1.6 mu m, taking acetonitrile as a mobile phase A and taking 0.1 percent phosphoric acid water solution as a mobile phase B, and the column temperature is 30 ℃; the flow rate is 0.3 ml/min; the detection wavelength is 203nm, and the sample injection amount is 1 mu l.
2. The gradient elution conditions were: the volume fraction of the mobile phase A is changed to be 5% -11% and the volume fraction of the mobile phase B is 95% -89% in 0-4 min; 4-7 min, the volume fraction of the mobile phase A is changed to 11-20%, and the volume fraction of the mobile phase B is changed to 89-80%; 7-21 min, the volume fraction of the mobile phase A is changed to 20-28%, and the volume fraction of the mobile phase B is changed to 80-72%; the volume fraction of the mobile phase A is changed to 28-32% and the volume fraction of the mobile phase B is changed to 72-68% in 21-30 min; 30-33 min, the volume fraction of the mobile phase A is changed to 32-35%, and the volume fraction of the mobile phase B is changed to 68-65%; and 33-38 min, wherein the volume fraction of the mobile phase A is 35%, and the volume fraction of the mobile phase B is 65%.
3. The preparation method of the test solution comprises the following steps: taking 0.5g of rhizoma bolbostemmae medicinal material powder, accurately weighing, accurately adding 25ml of 50% ethanol, weighing, ultrasonically treating for 30 minutes, cooling, weighing again, complementing the weight loss with 50% ethanol, shaking up, filtering, and taking the subsequent filtrate to obtain the final product.
4. Preparation of control solutions: taking tubeimoside A, tubeimoside B and tubeimoside C reference substances, precisely weighing, and adding methanol to prepare a mixed reference substance solution containing tubeimoside A100 μ g, tubeimoside B50 μ g and tubeimoside C80 μ g per 1 ml.
5. The methodology investigation of the rhizoma bolbostemmae medicinal material characteristic spectrum comprises the following steps:
5.1 precision test: taking a sample solution, continuously injecting samples for 6 times under the specified chromatographic condition, taking the tubeimoside A peak as a reference peak, calculating the relative retention time and the relative peak area RSD of each common peak to be less than 2.0 percent, and showing that the precision of the instrument is good.
5.2 repeatability test: taking 6 parts of medicinal material powder in the same batch, preparing a test solution, determining under a specified chromatographic condition, taking a tubeimoside A peak as a reference peak, and calculating that the relative retention time and the relative peak area RSD of each common peak are less than 2.0 percent, which indicates that the method has good repeatability.
5.3 stability test: taking the test solution, analyzing the test solution under the specified chromatographic condition for 0, 2, 4, 6, 8, 12 and 24 hours respectively, taking the tubeimoside A peak as a reference peak, and calculating the relative retention time and the relative peak area RSD of each common peak to be less than 2.0 percent, which indicates that the test solution has good stability within 24 hours.
6. Determination of rhizoma bolbostemmae medicinal material characteristic map
6.1 Peak assignment: respectively placing rhizoma Bolbostemmatis glucoside A, rhizoma Bolbostemmatis glucoside B and rhizoma Bolbostemmatis glucoside C reference substances in different volumetric flasks, precisely weighing, and adding methanol to obtain reference substance solution containing rhizoma Bolbostemmatis glucoside A100 μ g, rhizoma Bolbostemmatis glucoside B100 μ g and rhizoma Bolbostemmatis C100 μ g per 1 ml. Taking a test solution and the 3 reference solutions, performing determination under a specified chromatographic condition, starting ultraviolet 3D scanning, comparing the retention time and UV absorption spectrum of the reference solution and the test solution, and identifying each spectral peak in the characteristic spectrum of the rhizoma bolbostemmae. The results are shown in fig. 1-4, and characteristic peaks consistent with retention time of tubeimoside A, tubeimoside B and tubeimoside C can be found from the tubeimoside characteristic spectrum, and UV absorption spectra are consistent, wherein the peak 2 is tubeimoside B, the peak 3 is tubeimoside C, and the peak 4 is tubeimoside A.
6.2 sample determination: taking 19 bolbostemma paniculatum medicinal materials in the table 4, preparing a test solution, obtaining and analyzing UPLC characteristic spectrums of the 19 bolbostemma paniculatum medicinal materials under the specified chromatographic condition, taking a No. 4 peak bolbostemma paniculatum glucoside A peak as a reference peak, and calculating the relative retention time and the relative peak area of each characteristic peak and an S peak, wherein the results are shown in the table 5 and the table 6.
Relative retention time of each characteristic peak of characteristic spectrum of medicinal materials of Bolbostemma paniculatum in Table 519 batches
Figure 443176DEST_PATH_IMAGE005
Relative peak area of each characteristic peak of the characteristic spectrum of the medicinal materials of paniculate Bolbostemma rhizome in Table 619 batches
Figure 385724DEST_PATH_IMAGE006
Analysis and discussion of results: the common peak with better separation degree and purer chromatographic peak is the characteristic peak of the characteristic spectrum of the medicinal material rhizoma bolbostemmae, and the result marks 4 characteristic peaks in total. Taking the peak 3 as a reference peak S peak, the relative retention time RSD values of the other 3 characteristic peaks of 19 batches of the rhizoma bolbostemmae medicinal materials relative to the S peak are all less than 5.0% and are within the range of 0.135% -3.056%, the relative peak area RSD values of the other 3 characteristic peaks of 19 batches of the rhizoma bolbostemmae medicinal materials relative to the S peak are within the range of 9.231% -46.474%, and the results show that the difference of chemical components represented by each characteristic peak of the rhizoma bolbostemmae medicinal materials in different producing areas is large; wherein the relative peak area of peak 1 is 0.106-2.395, the relative peak area of peak 2 is 0.198-0.269, and the relative peak area of peak 3 is 0.329-0.506.
Therefore, according to the fluctuation range of the relative peak areas of 4 characteristic peaks and peak 4 of 19 batches of different local rhizoma bolbostemmae medicinal materials, the relative peak area range of 3 characteristic peaks is specified by taking the minimum and maximum relative peak areas of the characteristic peaks in consideration of the representativeness of the 19 batches of different local samples. Namely: the relative peak area of each characteristic peak and the S peak is calculated by taking the peak 3 corresponding to the tubeimoside A reference substance peak as the S peak, and the relative peak areas are within the specified range, namely 0.106-2.395 (peak 1), 0.198-0.269 (peak 2) and 0.329-0.506 (peak 3).
6.3 similarity evaluation of Tubeimu herb medicine feature map
Preparing a test solution from 19 batches of rhizoma bolbostemmae medicinal material powder; the measurement was carried out under a predetermined chromatographic condition, and a chromatogram was recorded. Introducing the cdf format of 19 batches of medicinal materials into software of a traditional Chinese medicine chromatogram characteristic spectrum similarity evaluation system (2012 edition), taking the characteristic spectrum of a sample with the batch number of G1902024 as a reference spectrum, taking the peak No. 2 as a Mark peak to carry out retention time correction and carry out full peak matching, and generating a control characteristic spectrum of the medicinal materials of the rhizoma bolbostemmae by using an average method, wherein the superposition graph of the characteristic spectrums of the 19 batches of the medicinal materials of the rhizoma bolbostemmae is shown in figure 5, and the control characteristic spectrum of the medicinal materials of. The similarity evaluation was performed on 19 batches of paniculate Bolbostemma rhizome, and the results are shown in Table 7.
Similarity evaluation of bulk Fritillaria paniculata in Table 719
Figure 823659DEST_PATH_IMAGE007
Analysis and discussion of results: in 19 batches of samples, the similarity of two batches (G1902024 and G1903048) is lower than 0.95, and the similarity of the feature spectrum of the rest 17 batches of samples is more than 0.95, so that the similarity is high and the quality difference is small. According to the analysis of spectrogram, the difference between two batches of samples with the similarity lower than 0.95 and the rest 17 batches is mainly shown in the peak height of peak 1 or the difference between the peak area and other batches is obvious, wherein the peak height of the paniculate Bolbostemma rhizome medicinal material G1902024 is obviously lower than that of the rest batches, the peak height of the paniculate Bolbostemma rhizome medicinal material G1903048 is obviously higher than that of the other batches, according to the determination results of 19 batches of samples, the similarity between the characteristic spectrum of the sample of the paniculate Bolbostemma rhizome medicinal material and the reference characteristic spectrum is not lower than 0.90 by the specified calculation of a traditional Chinese medicine chromatogram fingerprint.
Example 2
Method for measuring content of medicinal components of bolbostemma paniculatum
1. The chromatographic conditions of the ultra-high performance liquid chromatograph analysis are as follows: gradient elution is carried out by adopting a Waters Cortecs T3 chromatographic column with the specification of 2.1 multiplied by 100mm and the size of 1.6 mu m, taking acetonitrile as a mobile phase A and taking 0.1 percent phosphoric acid water solution as a mobile phase B, and the column temperature is 30 ℃; the flow rate is 0.3 ml/min; the detection wavelength is 203nm, and the sample injection amount is 1 mu l.
2. The gradient elution conditions were: the volume fraction of the mobile phase A is changed to be 5% -11% and the volume fraction of the mobile phase B is 95% -89% in 0-4 min; 4-7 min, the volume fraction of the mobile phase A is changed to 11-20%, and the volume fraction of the mobile phase B is changed to 89-80%; 7-21 min, the volume fraction of the mobile phase A is changed to 20-28%, and the volume fraction of the mobile phase B is changed to 80-72%; the volume fraction of the mobile phase A is changed to 28-32% and the volume fraction of the mobile phase B is changed to 72-68% in 21-30 min; 30-33 min, the volume fraction of the mobile phase A is changed to 32-35%, and the volume fraction of the mobile phase B is changed to 68-65%; and 33-38 min, wherein the volume fraction of the mobile phase A is 35%, and the volume fraction of the mobile phase B is 65%.
3. The preparation method of the sample solution of the paniculate Bolbostemma rhizome to be detected comprises the following steps: taking 0.5g of rhizoma bolbostemmae medicinal material powder, accurately weighing, accurately adding 25ml of 50% ethanol, weighing, ultrasonically treating for 30 minutes, cooling, weighing again, complementing the weight loss with 50% ethanol, shaking up, filtering, and taking the subsequent filtrate to obtain the final product.
4. Preparation of control solutions: taking tubeimoside A, tubeimoside B and tubeimoside C reference substances, precisely weighing, and adding methanol to prepare a mixed reference substance solution containing tubeimoside A100 μ g, tubeimoside B50 μ g and tubeimoside C80 μ g per 1 ml.
5 methodological investigation
5.1 Linear relationship examination: taking the tubeimoside A, tubeimoside B and tubeimoside C reference substances, precisely weighing, and adding methanol to prepare reference substance stock solution containing 3.758mg of tubeimoside A, 0.971mg of tubeimoside B and 1.574mg of tubeimoside C per 1 ml. Taking the reference stock solution, sequentially diluting to different concentrations, performing sample injection measurement under specified chromatographic conditions, performing linear regression with peak area as ordinate and reference concentration as abscissa, and obtaining standard curves and linear ranges shown in Table 8.
Linear regression equation and range for 83 ingredients in Table
Figure 713117DEST_PATH_IMAGE008
5.2 precision test: taking a sample solution to be detected, carrying out continuous sample injection for 6 times under the specified chromatographic condition, and calculating the RSD values of the peak areas of the tubeimoside A, the tubeimoside B and the tubeimoside C to be 0.37%, 0.59% and 0.29% respectively, which indicates that the precision of the instrument is good.
5.3 stability test: taking a sample solution to be detected, respectively measuring for 0, 2, 4, 6, 8, 12 and 24 hours under the specified chromatographic condition, and respectively calculating the RSD values of the areas of the tubeimoside A, the tubeimoside B and the tubeimoside C to be 0.91%, 1.15% and 1.18%, which shows that the stability of the sample solution to be detected is good within 24 hours.
5.4 repeatability test: taking 6 parts of the same batch of medicinal material powder, preparing 6 parts of sample solution to be tested, carrying out sample injection measurement under the specified chromatographic condition, and calculating the RSD values of the contents of the tubeimoside A, the tubeimoside B and the tubeimoside C, wherein the results are shown in a table 9.
Repetitive results of the 93 components in Table
Figure 185687DEST_PATH_IMAGE009
The experimental result shows that the average values of the contents of the tubeimoside A, the tubeimoside B and the tubeimoside C are 18.820mg/g, 4.754mg/g and 7.718mg/g, the RSD values are 0.66 percent, 1.03 percent and 1.03 percent respectively, and the method has good repeatability.
5.5 sample recovery rate determination: taking 0.25g of rhizoma bolbostemmae medicinal material powder, precisely weighing, adding the rhizoma bolbostemmae glycoside A, B and C reference substances according to the content ratio of the sample to the reference substances bolbostemmae glycoside A, B and C of 1:0.5, 1:1 and 1:1.5, wherein each ratio is 3 parts in parallel, preparing 9 parts of sample solution to be detected, carrying out sample injection measurement under the specified chromatographic condition, and calculating the recovery rate range and RSD value of 3 components, wherein the results are shown in Table 10.
TABLE 103 sample recovery results for the components
Figure 33557DEST_PATH_IMAGE010
The experimental result shows that the sample adding recovery rates of the tubeimoside A, the tubeimoside B and the tubeimoside C are 94.04-104.70%, 95.74-104.87% and 97.19-107.73% respectively, and the RSD values are all less than 4.0%, which indicates that the analysis method has good accuracy.
6. Calculation of the relative correction factor (f): taking tubeimoside A as an internal standard substance according to a formula
Figure RE-GDA0002393005410000152
Wherein A issPeak area of control as internal standard s, CsConcentration of control as internal standard s, AiThe peak area of the control sample of the other component i, CiThe concentrations of the other components i are used as reference. According to the linear investigation result, the relative correction factors of the internal standard substance tubeimoside A, tubeimoside B and tubeimoside C are calculated according to the concentration of the reference substance and the corresponding chromatographic peak area. The results are shown in Table 11.
TABLE 112 relative correction factors for the components
Figure 643268DEST_PATH_IMAGE013
The experimental result shows that the average relative correction factors of the tubeimoside B and the tubeimoside C are respectively 1.140 and 1.259, and the RSD values are respectively 1.22 percent and 2.05 percent. The chromatogram for measuring the contents of tubeimoside A, B and C is shown in figure 7.
7. Durability investigation: precisely sucking 1 μ L of the mixed control solution, injecting sample under specified chromatographic conditions, and respectively examining the influence of 3 different chromatographic columns of Waters H-Class, Thermos Vanqish, Agilent 1290 finety ultra performance liquid chromatographs and Waters Cortecs T3 (2.1 mm x 100mm, 1.6 μm), Waters HSS T3 (2.1 mm x 100mm, 1.8 μm), Waters BEH C18 (2.1 mm x 100mm, 1.7 μm), and different column temperatures and flow rates on relative correction factors of tubeimoside B and tubeimoside C. The results are shown in Table 12.
TABLE 12 relative correction factors for different chromatographic conditions
Figure 405688DEST_PATH_IMAGE014
The experimental result shows that the RSD of the relative correction factor under different chromatographic conditions is less than 2.0%, and different chromatographs, chromatographic columns and column temperatures and flow rates have no significant influence on the relative correction factor.
8. Chromatographic peak location: based on the above results of durability examination, the relative retention times of tubeimoside C and tubeimoside B with respect to the reference peak were calculated using tubeimoside A as the reference peak, and the results are shown in Table 13.
TABLE 13 relative Retention time for different chromatographic conditions
Figure 56112DEST_PATH_IMAGE015
The experimental result shows that the relative retention time mean values of the tubeimoside B and the tubeimoside C are 0.735 and 0.771 respectively, and the RSD is less than 3.0 percent, which indicates that different chromatographs and chromatographic columns and different column temperatures and flow rates have no significant influence on the relative retention time. Therefore, the relative retention time of the tubeimoside B, the tubeimoside C and the S peak is calculated by taking the tubeimoside A reference substance as the reference peak S, the relative retention time of the tubeimoside B should be within the range of 0.735 +/-10%, and the relative retention time of the tubeimoside C should be within the range of 0.771 +/-10%.
9. Comparison of results of one-test-multiple-evaluation method and external standard method
Precisely weighing 19 batches of rhizoma bolbostemmae medicinal material powder, preparing into a sample solution to be tested, carrying out sample injection measurement under the specified chromatographic conditions, respectively adopting an External Standard Method (ESM) and a one-test-multiple evaluation method (QAMS) to calculate the contents of the 19 batches of rhizoma bolbostemmae medicinal material rhizoma bolbostemmae glycoside B and the rhizoma bolbostemmae glycoside C, and calculating the relative deviation of the two measurement methods, wherein the result is shown in a table 14.
TABLE 1419 determination of the crude Bolbostemma paniculatum
Figure 511364DEST_PATH_IMAGE016
Analysis and discussion of results: the relative deviation of the contents of tubeimoside B and tubeimoside C determined by the two methods is within the range of 0.88-3.70%, the relative deviation of the contents of tubeimoside C and tubeimoside C is within the range of 0.59-4.35%, both the contents are less than 5.0%, so that the results of the one-test and multi-evaluation method are not obviously different from the results of the external standard method, the results of the one-test and multi-evaluation method are reliable, the contents of 3 components can be accurately determined, and the external standard method can be replaced for controlling the contents of tubeimo fritillary medicinal materials. The content of the 19 batches of the medicinal material rhizoma bolbostemmae, namely the rhizoma bolbostemmae glucoside A is more than 1.0 percent and meets the requirement of the content limit of 'Chinese pharmacopoeia' of 2015 edition; the total amount of 3 effective components of 19 batches of the paniculate Bolbostemma rhizome medicinal material is 2.65-3.64 percent, and the paniculate Bolbostemma rhizome medicinal material can be used as the quality evaluation standard of the paniculate Bolbostemma rhizome medicinal material.
Example 3
Identifying rhizoma Bolbostematis by UPLC characteristic spectrum.
The rhizoma bolbostemmae is randomly purchased in the market.
1. The chromatographic conditions of the ultra-high performance liquid chromatograph analysis are as follows: gradient elution is carried out by adopting a Waters Cortecs T3 chromatographic column with the specification of 2.1 multiplied by 100mm and the size of 1.6 mu m, taking acetonitrile as a mobile phase A and taking 0.1 percent phosphoric acid water solution as a mobile phase B, and the column temperature is 30 ℃; the flow rate is 0.3 ml/min; the detection wavelength is 203nm, and the sample injection amount is 1 mu l.
2. The gradient elution conditions were: the volume fraction of the mobile phase A is changed to be 5% -11% and the volume fraction of the mobile phase B is 95% -89% in 0-4 min; 4-7 min, the volume fraction of the mobile phase A is changed to 11-20%, and the volume fraction of the mobile phase B is changed to 89-80%; 7-21 min, the volume fraction of the mobile phase A is changed to 20-28%, and the volume fraction of the mobile phase B is changed to 80-72%; the volume fraction of the mobile phase A is changed to 28-32% and the volume fraction of the mobile phase B is changed to 72-68% in 21-30 min; 30-33 min, the volume fraction of the mobile phase A is changed to 32-35%, and the volume fraction of the mobile phase B is changed to 68-65%; and 33-38 min, wherein the volume fraction of the mobile phase A is 35%, and the volume fraction of the mobile phase B is 65%.
3. The preparation method of the sample solution of the rhizoma bolbostemmae medicinal material to be identified comprises the following steps: taking 0.5g of rhizoma bolbostemmae medicinal material powder, accurately weighing, accurately adding 25ml of 50% ethanol, weighing, ultrasonically treating for 30 minutes, cooling, weighing again, complementing the weight loss with 50% ethanol, shaking up, filtering, and taking the subsequent filtrate to obtain the final product.
4. Preparation of control solutions: taking a tubeimoside A reference substance, adding methanol to prepare a reference substance solution containing 100 mu g of tubeimoside A per 1 ml.
5. Precisely sucking 1 μ l of each of the reference solution and the sample solution of rhizoma Bolbostematis to be identified, injecting into a liquid chromatograph, and measuring.
6. And (3) measuring results: comparing the characteristic spectrum (figure 8) of the rhizoma bolbostemmae medicinal material sample to be identified with the comparison characteristic spectrum (figure 6) of the rhizoma bolbostemmae medicinal material: 4 characteristic peaks can be detected when the paniculate Bolbostemma rhizome medicinal material is identified, and correspond to the 4 characteristic peaks in the paniculate Bolbostemma rhizome medicinal material in the comparison characteristic spectrum; the data result shows (table 15), the relative retention time and the relative peak area range of the 4 characteristic peaks of the sample are both in the range specified by the standard, and the sample to be identified is the bolbostemma paniculatum medicinal material.
TABLE 15 relative retention time of each characteristic peak and relative peak area specified range of native herba Verbenae
Figure 411187DEST_PATH_IMAGE017
Example 4
The contents of tubeimoside A, tubeimoside B and tubeimoside C in the tubeimoside are determined by using the tubeimoside component content determination method.
The rhizoma bolbostemmae is randomly purchased in the market.
1. The chromatographic conditions of the ultra-high performance liquid chromatograph analysis are as follows: gradient elution is carried out by adopting a Waters Cortecs T3 chromatographic column with the specification of 2.1 multiplied by 100mm and the size of 1.6 mu m, taking acetonitrile as a mobile phase A and taking 0.1 percent phosphoric acid water solution as a mobile phase B, and the column temperature is 30 ℃; the flow rate is 0.3 ml/min; the detection wavelength is 203nm, and the sample injection amount is 1 mu l.
2. The gradient elution conditions were: the volume fraction of the mobile phase A is changed to be 5% -11% and the volume fraction of the mobile phase B is 95% -89% in 0-4 min; 4-7 min, the volume fraction of the mobile phase A is changed to 11-20%, and the volume fraction of the mobile phase B is changed to 89-80%; 7-21 min, the volume fraction of the mobile phase A is changed to 20-28%, and the volume fraction of the mobile phase B is changed to 80-72%; the volume fraction of the mobile phase A is changed to 28-32% and the volume fraction of the mobile phase B is changed to 72-68% in 21-30 min; 30-33 min, the volume fraction of the mobile phase A is changed to 32-35%, and the volume fraction of the mobile phase B is changed to 68-65%; and 33-38 min, wherein the volume fraction of the mobile phase A is 35%, and the volume fraction of the mobile phase B is 65%.
3. The preparation method of the sample solution of the paniculate Bolbostemma rhizome to be detected comprises the following steps: taking 0.5g of rhizoma bolbostemmae medicinal material powder, accurately weighing, accurately adding 25ml of 50% ethanol, weighing, ultrasonically treating for 30 minutes, cooling, weighing again, complementing the weight loss with 50% ethanol, shaking up, filtering, and taking the subsequent filtrate to obtain the final product.
4. Preparation of control solutions: taking tubeimoside A, tubeimoside B and tubeimoside C reference substances, precisely weighing, and adding methanol to prepare a mixed reference substance solution containing tubeimoside A100 μ g, tubeimoside B50 μ g and tubeimoside C80 μ g per 1 ml.
5. And (3) determination: precisely sucking 1 μ l of each of the reference solution and the sample solution of rhizoma Bolbostematis to be measured, injecting into a liquid chromatograph, measuring corresponding peak area, calculating the content of tubeimoside A by external standard method, and calculating the content of tubeimoside B and tubeimoside C by one-measurement-multiple-evaluation method; in addition, the content verification reliability of tubeimoside B and tubeimoside C is calculated by an external standard method.
6. The results are shown in tables 16 and 17.
TABLE 16 relative Retention time of tubeimosides B and C
Figure 446139DEST_PATH_IMAGE019
TABLE 17 content of tubeimoside A, B, C determined by one-test-multiple-evaluation method and external standard method
Figure 482228DEST_PATH_IMAGE020
The results show that the relative retention time of the tubeimoside B and the tubeimoside C is in a specified range, and the measurement result of one measurement and multiple evaluations has no obvious difference from the measurement of an external standard method, which indicates that the method is more reliable;
the content of tubeimoside A is 1.57 percent and is more than 1.0 percent, which meets the requirement of the content limit of 'Chinese pharmacopoeia' of 2015 edition; the total amount of 3 effective components in the paniculate Bolbostemma rhizome is 2.87 percent and is between 2.65 percent and 3.64 percent, and the paniculate Bolbostemma rhizome is a qualified paniculate Bolbostemma rhizome medicinal material.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (10)

1. A method for constructing UPLC characteristic spectrum of rhizoma bolbostemmae medicinal material is characterized by comprising the following steps:
(1) accurately weighing rhizoma Bolbostematis powder to obtain rhizoma Bolbostematis test solution;
(2) analyzing the sample solution of the rhizoma bolbostemmae medicinal material by using an ultra-high performance liquid chromatograph to obtain a UPLC characteristic spectrum of the rhizoma bolbostemmae medicinal material.
2. The method for constructing UPLC characteristic spectrum of paniculate Bolbostemma rhizome as claimed in claim 1, wherein the chromatographic conditions of the hplc analysis are as follows: gradient elution is carried out by adopting a Waters Cortecs T3 chromatographic column with the specification of 2.1 multiplied by 100mm and the diameter of 1.6 mu m, taking acetonitrile as a mobile phase A and taking a phosphoric acid aqueous solution with the concentration of 0.05-0.15% as a mobile phase B, wherein the column temperature is 20-40 ℃; the flow rate is 0.2-0.4 ml/min; the detection wavelength is 180-220 nm, and the sample injection amount is 0.5-1.5 mu l.
3. The method for constructing UPLC feature map of paniculate Bolbostemma rhizome as claimed in claim 2, wherein the gradient elution conditions are as follows: the volume fraction of the mobile phase A is changed to be 5% -11% and the volume fraction of the mobile phase B is 95% -89% in 0-4 min; 4-7 min, the volume fraction of the mobile phase A is changed to 11-20%, and the volume fraction of the mobile phase B is changed to 89-80%; 7-21 min, the volume fraction of the mobile phase A is changed to 20-28%, and the volume fraction of the mobile phase B is changed to 80-72%; the volume fraction of the mobile phase A is changed to 28-32% and the volume fraction of the mobile phase B is changed to 72-68% in 21-30 min; 30-33 min, the volume fraction of the mobile phase A is changed to 32-35%, and the volume fraction of the mobile phase B is changed to 68-65%; and 33-38 min, wherein the volume fraction of the mobile phase A is 35%, and the volume fraction of the mobile phase B is 65%.
4. The method for constructing UPLC characteristic spectrum of paniculate Bolbostemma rhizome as claimed in claim 1, wherein the preparation method of the test solution comprises: taking 0.4-0.6 g of rhizoma bolbostemmae medicinal powder, precisely weighing, precisely adding 20-30 ml of 40-60% ethanol, weighing, carrying out ultrasonic treatment for 20-40 minutes, cooling, weighing again, complementing the loss weight with 40-60% ethanol, shaking up, filtering, and taking the subsequent filtrate.
5. A method for measuring the component content of rhizoma bolbostemmae is characterized by comprising the following steps:
(1) respectively sucking a reference substance solution and a sample solution of the medicinal material of the bolbostemma paniculatum to be detected, injecting the reference substance solution and the sample solution into an ultra-high performance liquid chromatograph, and measuring corresponding peak areas;
(2) calculating the content of tubeimoside A by an external standard method;
(3) and calculating the contents of tubeimoside B and tubeimoside C by a one-test-multiple evaluation method.
6. The method for measuring the component content of the rhizoma bolbostemmae as the medicinal material of claim 5, wherein the preparation method of the reference solution comprises the following steps: taking the tubeimoside A, tubeimoside B and tubeimoside C reference substances, precisely weighing, and adding methanol to prepare a reference substance solution containing 80-120 mu g of tubeimoside A, 40-60 mu g of tubeimoside B and 60-100 mu g of tubeimoside C per 1 ml.
7. The method for determining the component content of the rhizoma bolbostemmae medicinal material according to claim 5, wherein the preparation method of the sample solution of the rhizoma bolbostemmae medicinal material to be determined comprises the following steps: taking 0.4-0.6 g of rhizoma bolbostemmae medicinal powder, precisely weighing, precisely adding 20-30 ml of 40-60% ethanol, weighing, carrying out ultrasonic treatment for 20-40 minutes, cooling, weighing again, complementing the loss weight with 40-60% ethanol, shaking up, filtering, and taking the subsequent filtrate.
8. The method for measuring the component content of the rhizoma bolbostemmae as the medicinal material of the tubeimo fritillary bulb as the claim 5, wherein the chromatographic conditions of the ultra-high performance liquid chromatograph analysis are that a Waters Cortecs T3 chromatographic column with the specification of 2.1 x 100mm and the diameter of 1.6 mu m is adopted, acetonitrile is taken as a mobile phase A, a 0.05-0.15% phosphoric acid water solution is taken as a mobile phase B, gradient elution is carried out, and the column temperature is 20-40 ℃; the flow rate is 0.2-0.4 ml/min; the detection wavelength is 180-220 nm, and the sample injection amount is 0.5-1.5 mu l.
9. The method for measuring the component content of the rhizoma bolbostemmae as the medicinal material of claim 8, wherein the gradient elution condition is as follows: : the volume fraction of the mobile phase A is changed to be 5% -11% and the volume fraction of the mobile phase B is 95% -89% in 0-4 min; 4-7 min, the volume fraction of the mobile phase A is changed to 11-20%, and the volume fraction of the mobile phase B is changed to 89-80%; 7-21 min, the volume fraction of the mobile phase A is changed to 20-28%, and the volume fraction of the mobile phase B is changed to 80-72%; the volume fraction of the mobile phase A is changed to 28-32% and the volume fraction of the mobile phase B is changed to 72-68% in 21-30 min; 30-33 min, the volume fraction of the mobile phase A is changed to 32-35%, and the volume fraction of the mobile phase B is changed to 68-65%; and 33-38 min, wherein the volume fraction of the mobile phase A is 35%, and the volume fraction of the mobile phase B is 65%.
10. The method for constructing UPLC characteristic spectrum of paniculate Bolbostemma rhizome or the method for determining the content of constituents of paniculate Bolbostemma rhizome as claimed in any one of claims 1 to 9, is used for detecting and identifying paniculate Bolbostemma rhizome.
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