CN111624271B - Liquid chromatography method for detecting corresponding substance of peony and licorice decoction, standard fingerprint spectrum and application - Google Patents

Liquid chromatography method for detecting corresponding substance of peony and licorice decoction, standard fingerprint spectrum and application Download PDF

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CN111624271B
CN111624271B CN202010522414.6A CN202010522414A CN111624271B CN 111624271 B CN111624271 B CN 111624271B CN 202010522414 A CN202010522414 A CN 202010522414A CN 111624271 B CN111624271 B CN 111624271B
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肖惠琳
阳丽华
许舒瑜
陈晓琳
南淑华
郑珊珊
许玉珍
肖顺彪
赖志成
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Xiamen Traditional Chinese Medicine Co ltd
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Abstract

The invention relates to a liquid chromatography method for detecting a corresponding substance of a peony-licorice decoction, a standard fingerprint and application thereof. The method adopts a high performance liquid chromatograph equipped with an ultraviolet detector to carry out detection, and the chromatographic conditions are as follows: mobile phase: acetonitrile (A) -0.05% phosphoric acid water solution (B), gradient elution is carried out for 0-1min, 14-17.5% A,1-5min, 17.5-19% A,5-6min, 19-20% A,6-7min, 20% A,7-8min, 20-20.5% A,8-12min, 20.5-23% A,12-30min, 23-36% A,30-32min, 36% A and 32-45min, 36-43% A; a chromatographic column: venusil XBP C184.6 × 150mm,3 μm; detection wavelength 230-240 nm; flow rate: 1.0 ml/min; the column temperature is 20-30 ℃. The standard fingerprint of the corresponding substance of the peony and licorice decoction can be used for quality control of the corresponding substance of the peony and licorice decoction.

Description

Liquid chromatography method for detecting corresponding substance of peony and licorice decoction, standard fingerprint spectrum and application
Technical Field
The invention belongs to the field of traditional Chinese medicine analysis, and relates to a liquid chromatography method for detecting a substance corresponding to a peony and licorice decoction, a method for establishing a standard fingerprint spectrum containing the method, a standard fingerprint spectrum of the substance corresponding to the peony and licorice decoction and application.
Background
The radix Paeoniae and Glycyrrhrizae radix decoction is prepared by decocting Chinese medicinal decoction pieces in water according to prescription, and lyophilizing, wherein the Chinese medicinal decoction pieces are radix Paeoniae alba and radix Glycyrrhizae Preparata.
The Chinese pharmacopoeia is a code of the quality standard of Chinese medicines and a legal basis for inspecting and judging whether the medicines are qualified or not. The existing version is 2015, 2 traditional Chinese medicine decoction pieces in the peony and licorice decoction are collected, and the main chemical component control of the decoction pieces in the prescription in the pharmacopoeia is of great significance for researching the key quality attributes of corresponding real objects of the peony and licorice decoction. The main content measurement indexes and limit requirements of radix paeoniae alba and radix glycyrrhizae preparata are shown in table 1.
TABLE 1 pharmacopoeia content determination of single-ingredient decoction pieces
Figure BDA0002532560460000011
In recent years, as higher requirements on drug quality control are provided by regulatory departments, manufacturers are required to strictly control the stability among product batches, so that the adoption of an effective quality control method is particularly important.
The fingerprint spectrum technology of traditional Chinese medicine is increasingly used for quality control of traditional Chinese medicine due to the characteristics of comprehensively marking the characteristics and proportion of the main chemical components of the traditional Chinese medicine. The chromatography is one of analysis methods which are developed rapidly and widely applied in the field of analytical chemistry, and is also the most basic technology of the traditional Chinese medicine fingerprint. Common Chromatography methods include thin layer Chromatography, Liquid Chromatography, gas Chromatography and capillary electrophoresis, wherein High Performance Liquid Chromatography (HPLC) has the advantages of wide application range, High analysis speed, High sensitivity and the like, and is the most widely applied method for researching traditional Chinese medicine fingerprints at present. An Evaporative Light Scattering Detector (ELSD) is a universal mass Detector that can compensate for the requirement that the detection material must have a chromophore to absorb uv Light for the most widely used uv detectors in HPLC. ELSD as a concentration type detector shows great superiority in the purity detection process of unknown compounds without ultraviolet or ultraviolet terminal absorption.
The main components of the peony and licorice decoction formula comprise paeoniflorin, liquiritin and glycyrrhizic acid. At present, a liquid chromatography method for detecting a corresponding substance of the peony and licorice decoction, a method for establishing a peony and licorice decoction standard fingerprint spectrum and a research for performing quality control by adopting the method do not exist.
Disclosure of Invention
In order to more comprehensively and effectively control the quality of the substance corresponding to the peony and licorice decoction, the inventor carefully studies the conditions such as a mobile phase, a detection wavelength, a chromatographic column, a gradient condition, a flow rate and a sample volume by using an HPLC method to obtain a liquid chromatography method for detecting the substance corresponding to the peony and licorice decoction, and simultaneously studies an extraction solution, an extraction mode, ultrasonic time, a solvent amount and the like to obtain a method for preparing a test solution, thereby obtaining a method for establishing a standard fingerprint of the substance corresponding to the peony and licorice decoction and a standard fingerprint of the substance corresponding to the peony and licorice decoction, which can be used for controlling the quality of the substance corresponding to the peony and licorice decoction, and thus completing the invention.
The invention provides a liquid chromatography method for detecting a substance corresponding to the peony and licorice decoction, which adopts a high performance liquid chromatograph equipped with an ultraviolet detector to carry out detection, and the chromatographic conditions are as follows:
a chromatographic column: venusil XBP C184.6 × 150mm,3 μm;
mobile phase: acetonitrile (A) -0.05% phosphoric acid aqueous solution (B),
gradient elution: according to the specifications in the table below
Figure BDA0002532560460000021
Flow rate: 1.0 ml/min;
column temperature: 20-30 ℃, preferably 25 ℃;
detection wavelength: 230-240nm, preferably 235 nm.
In the present invention, the percentages in the mobile phase and gradient elution are volume percentages, for example, 0.05% means 0.05 volume%.
Particularly, the number of theoretical plates of the chromatographic column is not less than 2000 calculated according to the paeoniflorin peak.
In the liquid chromatography method for detecting the peony and licorice root decoction, the test solution of the peony and licorice root decoction for detection can be prepared as follows: extracting the corresponding substance of the peony and licorice decoction with 70% methanol at a liquid-solid ratio (volume/mass, ml/g) of 100-500 times. The extraction method can be ultrasonic treatment, shaking extraction, reflux extraction, etc., preferably ultrasonic extraction.
Specifically, the test solution of the peony and licorice decoction can be prepared as follows: precisely weighing about 0.1g of the peony and licorice decoction, placing the weighed material in a 50ml conical flask with a plug, precisely adding 25ml of 70% methanol, weighing, carrying out ultrasonic treatment (250W and 40kHz) for 20 minutes, cooling, weighing again, supplementing the weight loss by 70% methanol, shaking up, and filtering to obtain the final product. In this case, the amount of the sample is preferably 5. mu.l.
In the invention, the corresponding real object of the peony and licorice decoction refers to a water extract prepared by extracting the Chinese medicinal decoction pieces of the white peony root and the honey-fried licorice root with water according to the classical name formula of the peony and licorice decoction. In an embodiment, the peony licorice decoction counterpart may be prepared as follows: decocting radix Paeoniae alba and radix Glycyrrhizae Preparata in water (for example, decocting radix Paeoniae alba and radix Glycyrrhizae Preparata at a weight ratio of 1:1, and decocting in water at a solid-to-liquid ratio of 1:5-1:25 (unit g/ml) for 60 min), filtering with 200 mesh sieve to obtain medicinal liquid, and drying (for example, freeze drying) to obtain radix Paeoniae and radix Glycyrrhizae decoction dry powder, i.e. radix Paeoniae and radix Glycyrrhizae decoction corresponding substance. The prescription refers to a peony and licorice decoction prescription in a classical name prescription, wherein the weight ratio of the white peony root to the honey-fried licorice root is about 1: 1.
The aqueous white peony extract may be prepared as follows: decocting radix Paeoniae alba with water (such as water decoction for 60 min at a solid-to-liquid ratio of 1:10-1:50, unit g/ml), filtering with 200 mesh sieve to obtain medicinal liquid, and drying to obtain radix Paeoniae alba dry powder, i.e. radix Paeoniae alba water extract.
The honey-fried licorice root aqueous extract can be prepared as follows: decocting radix Glycyrrhizae Preparata with water (for example, decocting with water at a solid-to-liquid ratio of 1:10-1:50 in unit g/ml for 60 min), filtering with 200 mesh sieve to obtain medicinal liquid, and drying to obtain radix Glycyrrhizae Preparata dry powder, i.e. radix Glycyrrhizae Preparata water extract. The invention also provides a method for establishing a standard fingerprint of a corresponding real object of the peony and licorice decoction, which comprises the following steps:
(1) preparing a test solution:
precisely weighing about 0.1g of radix Paeoniae and Glycyrrhrizae radix decoction, placing in 50ml conical flask with plug, precisely adding 25ml of 70% methanol, weighing, performing ultrasonic treatment at 250W and 40kHz for 20 min, cooling, weighing again, supplementing lost weight with 70% methanol, shaking, and filtering to obtain the final product;
(2) preparing a reference substance solution:
accurately weighing appropriate amount of penoniflorin, liquiritin and ammonium glycyrrhizinate as reference substances, and adding 70% methanol to obtain mixed reference substance solution; precisely weighing appropriate amount of isoliquiritin, isoliquiritigenin, isoliquiritin apioside, neoliquiritin, albiflorin, benzoylpaeoniflorin, oxypaeoniflorin, gallic acid, and catechin reference, and adding 70% methanol to obtain single reference solution; for example, solutions each having a concentration of 0.1 mg/ml;
(3) preparing a single medicinal solution:
respectively taking about 0.1g of white paeony root aqueous extract and about 0.1g of honey-fried licorice root aqueous extract, precisely weighing, placing in a 50ml conical flask with a plug, and preparing each negative sample solution with the sample solution preparation;
(4) HPLC detection
Precisely sucking 1-5 μ l of each of the sample solution, the reference solution and the negative sample solution of (1), (2) and (3), and detecting by the liquid chromatography method for detecting the corresponding substance of the peony and licorice decoction to obtain a sample fingerprint chromatogram and a reference fingerprint chromatogram;
(5) generating a standard fingerprint
Generating a standard fingerprint spectrum based on common peaks in fingerprint chromatograms of N batches of test samples, wherein a chromatographic peak with a main component and a better separation degree in the peony and licorice decoction is selected as a characteristic peak and is determined as a common peak, and N is more than 10, preferably 10-20;
(6) identification and attribution of common peaks
And attributing and identifying common peaks in the fingerprint of the peony and licorice decoction corresponding to the real object.
In the above method, the method for generating the standard fingerprint based on the sample fingerprint chromatogram is not particularly limited, and a conventional method in the art may be employed. For example, the chromatogram of the test sample of the peony licorice decoction can be introduced into a similarity evaluation system (2012 edition) of traditional Chinese medicine chromatogram issued by the national pharmacopoeia committee, one of the chromatograms of the test sample is set as a reference spectrum, a standard spectrum generation method is a median method, for example, and a multi-point correction method is adopted to establish the standard fingerprint of the peony licorice decoction.
In the method, identification and attribution of the common peaks can be completed by comparing the negative sample control, the single medicinal material and the chromatogram of the control solution.
In the method for establishing the standard fingerprint spectrum of the peony licorice decoction corresponding to the real object, the Chinese herbal pieces meeting the quality requirements of Chinese pharmacopoeia are adopted to prepare the peony licorice decoction corresponding to the real object. In order to be representative, the peony and licorice decoction should be prepared by feeding Chinese herbal pieces prepared from different producing areas respectively. Generally, the number of samples N may be 10 or more, for example, 15, and the upper limit is, for example, 30 or less, 20 or less, and preferably 10 to 20.
The invention also provides a standard fingerprint spectrum of a corresponding real object of the peony and licorice decoction, which comprises 13 characteristic peaks, wherein the 6 th peak is taken as a reference, and the relative retention time of each peak is as follows:
Figure BDA0002532560460000041
by comparison, among the 13 characteristic peaks, peaks 1, 2, 5, 6, 8, 9, 10, 12 and 13 are from liquorice, peaks 2, 3, 4, 7 and 11 are from white peony root, peak 3 is albiflorin, peak 4 is paeoniflorin, peak 5 is neoliquiritin, peak 6 is liquiritin, peak 8 is isoliquiritin apioside, peak 9 is isoliquiritin, and peak 11 is benzoylpaeoniflorin; peak 13 is glycyrrhizic acid.
In an embodiment, the physical standard fingerprint of the peony and licorice decoction is obtained by the method for establishing the physical standard fingerprint of the peony and licorice decoction.
In an embodiment, the standard fingerprint of the peony/licorice decoction is substantially as shown in fig. 23.
The invention further provides a method for determining the fingerprint of the substance corresponding to the peony and licorice decoction, which comprises the following steps:
(1) preparing a test solution:
precisely weighing about 0.1g of radix Paeoniae and Glycyrrhrizae radix decoction, placing in 50ml conical flask with plug, precisely adding 25ml of 70% methanol, weighing, performing ultrasonic treatment at 250W and 40kHz for 20 min, cooling, weighing again, supplementing lost weight with 70% methanol, shaking, and filtering to obtain the final product;
(2) chromatographic conditions are as follows:
adopting the chromatographic condition in the liquid chromatographic method for detecting the corresponding substance of the peony and licorice decoction according to the invention;
(3) and (3) determination:
precisely sucking 5 μ l of the sample solution of (1), injecting into a liquid chromatograph, and measuring to obtain fingerprint of corresponding substance of the peony and licorice decoction.
On the other hand, the invention provides a quality control method of a peony and licorice root soup corresponding substance, which comprises the following steps:
(1) preparing a test solution:
precisely weighing 0.1g of the peony and licorice decoction to be detected, placing the weighed material in a 50ml conical flask with a plug, precisely adding 25ml of 70% methanol, weighing, carrying out ultrasonic treatment at 250W and 40kHz for 20 minutes, cooling, weighing again, supplementing the weight loss by 70% methanol, shaking up, and filtering to obtain the final product;
(2) chromatographic conditions are as follows:
using chromatographic conditions as described in claim 1;
(3) and (3) determination:
precisely absorbing 5 mu l of the solution (1), injecting into a liquid chromatograph, and measuring to obtain a fingerprint of a substance corresponding to the peony and licorice soup to be detected;
(4) and (4) qualification judgment:
and calculating the similarity between the fingerprint of the corresponding real object of the peony and licorice decoction to be detected and the standard map, wherein the similarity is more than or equal to 0.8, thus obtaining the qualified product.
The similarity can be obtained by introducing the fingerprint of the substance corresponding to the peony and licorice decoction to be detected and the standard fingerprint of the substance corresponding to the peony and licorice decoction into a traditional Chinese medicine chromatography fingerprint similarity evaluation system issued by the State pharmacopoeia Committee for comparison, so as to calculate the similarity between the sample spectrum and the standard spectrum, wherein the similarity is more than or equal to 0.8, preferably more than 0.9, namely the product is qualified, otherwise, the product is unqualified.
The invention also provides a method for detecting the content of effective components in a corresponding substance of the peony and licorice decoction, which comprises the following steps:
(1) preparing a test solution:
precisely weighing about 0.1g of radix Paeoniae and Glycyrrhrizae radix decoction, placing in 50ml conical flask with plug, precisely adding 25ml of 70% methanol, weighing, performing ultrasonic treatment at 250W and 40kHz for 20 min, cooling, weighing again, supplementing lost weight with 70% methanol, shaking, and filtering to obtain the final product;
(2) preparing a reference substance solution:
taking appropriate amount of penoniflorin reference substance, liquiritin reference substance and ammonium glycyrrhizinate reference substance, precisely weighing, adding 70% methanol to obtain mixed solution containing penoniflorin 0.18mg, liquiritin 0.03mg and glycyrrhizic acid 0.03mg in l ml;
(3) HPLC detection
Precisely sucking the sample solution and the reference solution of (1) and (2) respectively by 5 μ l, and detecting by liquid chromatography for detecting corresponding substance of the peony and licorice decoction to obtain sample fingerprint;
(4) content calculation
The contents of paeoniflorin, liquiritin and glycyrrhizic acid were calculated according to the following formula:
content (wt.)
Figure BDA0002532560460000061
CTo pair-control concentration; vTo pair-control sample volume; a. theTo pair-control peak area; a. theFor supplying to-sample peak area; m isFor supplying to-sample size of the test sample.
As proved by experimental results, in the chromatogram obtained by the method for detecting the fingerprint of the peony and licorice decoction, the number of peaks is large, the sensitivity is high, the base line is stable, the noise is low, the response is sharp, the response value is large, and the separation degree is good. In addition, the blank solvent has no interference on the test of the test solution, and has good specificity. The method has good precision, stability and reproducibility.
The present invention has been described in detail hereinabove, but the above embodiments are merely illustrative in nature and are not intended to limit the present invention. Furthermore, there is no intention to be bound by any theory presented in the preceding prior art or the summary or the following examples.
Unless expressly stated otherwise, a numerical range throughout this specification includes any sub-range therein and any numerical value incremented by the smallest sub-unit within a given value. Unless expressly stated otherwise, numerical values throughout this specification represent approximate measures or limitations to the extent that such deviations from the given values, as well as embodiments having approximately the stated values and having the exact values stated, are included. Other than in the operating examples provided at the end of the detailed description, all numbers expressing quantities or conditions of parameters (e.g., quantities or conditions) used in the specification (including the appended claims) are to be understood as being modified in all instances by the term "about" whether or not "about" actually appears before the number. "about" means that the numerical value so stated is allowed to be somewhat imprecise (with some approach to exactness in that value; about or reasonably close to that value; approximately). As used herein, "about" refers to at least variations that can be produced by ordinary methods of measuring and using such parameters, provided that the imprecision provided by "about" is not otherwise understood in the art with this ordinary meaning. For example, "about" can include less than or equal to 10%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, or less than or equal to 0.1% variation, and in some aspects, less than or equal to 0.01% variation.
The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein. Various modifications, changes, or substitutions may be made to the technical solution of the present invention without departing from the scope of the technical idea and technical spirit of the present invention, and the modified, changed, or substituted technical solution is still included in the scope of the present invention.
It will be understood that the words or terms used in the specification and claims should not be construed as meaning defined in commonly used dictionaries. It will be further understood that the terms or terms should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the technical spirit of the present invention, based on the principle that the inventor can appropriately define the meaning of the terms or terms to best explain the present invention.
Drawings
FIG. 1 shows a liquid chromatogram for detection using methanol-0.05% phosphoric acid in water as the mobile phase, control A, sample B; 1 paeoniflorin, 2 liquiritin and 3 glycyrrhizic acid.
FIG. 2 shows a liquid chromatogram for detection using acetonitrile-0.05% phosphoric acid in water as the mobile phase, control A, sample B; 1 paeoniflorin, 2 liquiritin and 3 glycyrrhizic acid.
FIG. 3 shows a liquid chromatogram for detection using acetonitrile-water as the mobile phase, control A, sample B; 1 paeoniflorin, 2 liquiritin and 3 glycyrrhizic acid.
FIG. 4 shows a liquid chromatogram for detection using methanol-water as the mobile phase, control A, sample B; 1 paeoniflorin, 2 liquiritin and 3 glycyrrhizic acid.
FIG. 5 shows a liquid chromatogram for detection using methanol-0.1% formic acid as the mobile phase, control A, sample B; 1 paeoniflorin, 2 liquiritin and 3 glycyrrhizic acid.
FIG. 6 shows a liquid chromatogram for detection using acetonitrile-0.1% formic acid as the mobile phase, control A, sample B; 1 paeoniflorin, 2 liquiritin and 3 glycyrrhizic acid.
FIG. 7 shows a control liquid chromatogram detected at 199 nm.
FIG. 8 shows a liquid chromatogram of a control detected at 220-250nm, A220 nm, B230 nm, C240 nm, D250 nm; 1 paeoniflorin, 2 liquiritin and 3 glycyrrhizic acid.
FIG. 9 shows a liquid chromatogram of a control detected at 230-240nm, A230 nm, B231 nm, C232 nm, D233 nm, E234 nm, F235 nm, G236 nm, H237 nm; 1 paeoniflorin, 2 liquiritin and 3 glycyrrhizic acid.
FIG. 10 shows liquid chromatograms of control samples tested at different temperatures, A20 deg.C, B25 deg.C, C30 deg.C, D35 deg.C; 1 paeoniflorin, 2 liquiritin and 3 glycyrrhizic acid.
FIG. 11 shows liquid chromatograms of samples examined at different temperatures, A20 deg.C, B25 deg.C, C30 deg.C, D35 deg.C; 1 paeoniflorin, 2 liquiritin and 3 glycyrrhizic acid.
FIG. 12 shows a sample liquid chromatogram for gradient study a-8.
Figure 13 shows a liquid chromatogram of samples tested using different chromatography columns, a.ultimare AQ-C184.6 x 150mm,5 μm; thermo BDS Hypersil C184.6 x 150mm,5 μm; venusil XBP C184.6 × 150mm,3 μm; 1. paeoniflorin; 2. and (3) liquiritin.
FIG. 14 shows the sample liquid chromatogram of gradient survey b-3, 1 paeoniflorin, 2 glycyrrhizin, 3 glycyrrhizic acid.
FIG. 15 shows the sample liquid chromatogram of gradient survey b-5, 1 paeoniflorin, 2 glycyrrhizin, 3 glycyrrhizic acid.
FIG. 16 shows a sample liquid chromatogram for flow rate investigation, A.0.8ml/min; b.1.0 ml/min; c.1.2ml/min; 1. paeoniflorin; 2. liquiritin; 3. glycyrrhizic acid.
FIG. 17 shows a liquid chromatogram of a sample for sample volume investigation, A.5. mu.l; b.10 μ l; c.15 μ l; d.20 mu l; 1. paeoniflorin; 2. liquiritin; 3. glycyrrhizic acid.
FIG. 18 shows a sample liquid chromatogram for a gradient survey, A c-1; b c-2; c c-3; 1. paeoniflorin; 2. liquiritin; 3. glycyrrhizic acid.
Fig. 19 shows a specific experimental chromatogram, a. control solution; B. a test solution; C. honey-fried licorice root negative solution; D. white peony root negative solution; E. a blank solution; 1. paeoniflorin; 2. liquiritin; 3. glycyrrhizic acid.
FIG. 20 shows a control chromatogram of radix Paeoniae alba, A-Paeoniae radix and Glycyrrhrizae radix decoction sample, B-Paeoniae radix negative, C-mixed control, D-albiflorin, E-benzoylpaeoniflorin, F-oxypaeoniflorin, G-gallic acid, and H-catechin; 1. paeoniflorin; 2. liquiritin; 3. glycyrrhizic acid.
FIG. 21 shows control chromatogram of radix Glycyrrhizae Preparata, A-Paeoniae radix Glycyrrhizae decoction sample, B-Glycyrrhizae radix Glycyrrhizae Preparata negative, C-mixed control, D-isoliquiritin, E-neoliquiritin, F-isoliquiritin apioside, and G-isoliquiritigenin.
FIG. 22 shows HPLC finger prints of 15 corresponding batches obtained by the method according to the invention.
FIG. 23 shows a standard fingerprint obtained by the method according to the invention.
Detailed Description
The present invention will be described in detail below with reference to the drawings and technical solutions, but the present invention is not limited thereto.
Preparation of 1 peony and licorice decoction
The prescription composition is as follows: white peony root 55.20g, prepared licorice root 55.20g
Putting the decoction pieces in the prescription amount into a 2L decoction kettle, adding 600ml of water, decocting for 60 min, filtering while hot (200 meshes) to obtain a liquid medicine, drying to obtain a peony and licorice decoction dry powder, namely a peony and licorice decoction corresponding substance, which is brown to tan powder, sealing in a self-sealing bag, and storing in a drying cabinet.
Paeoniflorin, liquiritin, ammonium glycyrrhizinate, gallic acid and catechin are purchased from China food and drug testing research institute; isoliquiritin, isoliquiritigenin, isoliquiritin apioside, neoliquiritin, albiflorin, benzoylpaeoniflorin, and oxypaeoniflorin are available from Shanghai HongyanYongsheng Biotech Co., Ltd.
The following equipment was used.
Figure BDA0002532560460000081
2 investigation of liquid phase conditions
Solution preparation
Preparation of a test solution: taking about 0.5g of peony and licorice decoction freeze-dried powder, precisely preparing into powder, placing into a conical flask with a stopper, precisely adding 50ml of 70% methanol, sealing the stopper, weighing, carrying out ultrasonic treatment for 30 minutes (250W, 40KHz), cooling, weighing again, supplementing the lost weight with 70% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the final product.
Control solution: taking appropriate amount of penoniflorin, liquiritin, and ammonium glycyrrhizinate (weight of glycyrrhizic acid is equal to weight of ammonium glycyrrhizinate/1.0207) as reference, and preparing into mixed reference solution containing penoniflorin 0.18mg, liquiritin 0.03mg, and glycyrrhizic acid 0.03mg per ml with 70% methanol solution.
2.1 mobile phase systems inspection
6 flows of acetonitrile (A) -0.05% aqueous phosphoric acid solution (B), acetonitrile (A) -water (B), acetonitrile (A) -0.1% aqueous formic acid solution (B), methanol (A) -0.05% aqueous phosphoric acid solution (B), methanol (A) -water (B), and methanol (A) -0.1% aqueous formic acid solution (B) were examined for analysis of the samples.
Liquid chromatography conditions: gradient elution: 19% of A at 0-8min, 19-50% of A at 8-35min, and 50-100% of A at 35-36 min; a chromatographic column: elite Hypersil ODS 250X 4.6mm, 5 μm, detection wavelength 237nm, flow rate: 1.0ml/min, column temperature 30 ℃.
Respectively adopting the above mobile phases, injecting 10 μ l of reference solution and sample solution, respectively, into a liquid chromatograph, and measuring, the results are shown in FIGS. 1-6.
As can be seen from fig. 3 to 4, when the aqueous phase is pure water, the glycyrrhizic acid chromatographic peak has a severe or even no peak despite the organic phase being methanol or acetonitrile, and thus these two mobile phases are not selected. As can be seen from fig. 5, the baseline was not flat with the mobile phase methanol-0.1% formic acid, which fell over time and was therefore not selected for use. FIG. 1 shows that the paeoniflorin chromatogram has a poor peak shape compared with FIG. 6 and FIG. 2. FIG. 6 shows that the baseline is less stable than FIG. 2, so the best mobile phase for this sample is acetonitrile-0.05% phosphoric acid in water.
2.2 wavelength investigation
Ultraviolet spectrograms of glycyrrhizic acid, liquiritin and glycyrrhizic acid are respectively measured, and from the ultraviolet spectrograms, paeoniflorin is at 199nm and 231nm, liquiritin is at 199nm, 216nm and 276nm, and glycyrrhizic acid has maximum absorption at 199nm and 250 nm.
However, as shown in fig. 7, the chromatogram detected at 199nm is extracted, and it can be seen that the baseline noise of the chromatogram at 199nm is large, and the absorption intensity of paeoniflorin is far higher than that of glycyrrhizin and glycyrrhizic acid, so that the peak height ratio of glycyrrhizin is too small, and thus the chromatogram is not selected.
From the other maximum absorption wavelengths, the absorption wavelengths of the more compatible 3 compounds are centered between 220-250 nm. FIG. 8 shows the chromatogram detected at 250nm of 220-.
FIG. 9 shows the chromatogram detected at 230-240nm, and it can be seen from the graph that 3 target peaks in each chromatogram have good response, and do not affect the response of the chromatographic peak in front of paeoniflorin, and the detection result is optimal at 235 nm.
2.3 column temperature investigation
Liquid chromatography conditions: mobile phase: acetonitrile (a) -0.05% aqueous phosphoric acid (B), gradient elution: 19% of A at 0-8min, 19-50% of A at 8-35min, and 50-100% of A at 35-36 min; a chromatographic column: elite Hypersil ODS 250X 4.6mm, 5 μm, detection wavelength 235nm, flow rate: 1.0 ml/min.
The control solution and the test solution were each drawn up 10. mu.l at 20 ℃, 25 ℃,30 ℃ and 35 ℃ with precision, and injected into a liquid chromatograph, and the results were measured as shown in FIG. 10 (control) and FIG. 11 (test).
As can be seen from FIGS. 10-11, the higher the temperature, the more serious the paeoniflorin tailing, and the lower the temperature, the more unfavorable the liquiritin separation. Table 2 lists the tailing factors and peak heights of paeoniflorin, liquiritin, glycyrrhizic acid at different column temperatures from FIGS. 10-11. As can be seen from the data in the table, both the control and the sample showed no effect on the peak height and the tailing factor at different temperatures with respect to the glycyrrhizic acid chromatographic peak, indicating that the temperature had no effect on the glycyrrhizic acid chromatographic peak; as for the liquiritin chromatographic peak, the higher the temperature, the better the response height; and as for the paeoniflorin chromatographic peak, the higher the temperature, the larger the tailing factor and the worse the peak shape. The analysis is more suitable at 20-30 ℃ and the best at 25 ℃ by combining the chromatographic peak of the chromatographic peak shapes of the 3 components and the response condition.
TABLE 2 tailing factor and peak height results at different column temperatures
Figure BDA0002532560460000101
2.4 column and gradient Condition Observation a
Liquid chromatography conditions: mobile phase: acetonitrile (a) -0.05% aqueous phosphoric acid (B), gradient elution: 19% of A at 0-8min, 19-50% of A at 8-35min, and 50-100% of A at 35-36 min; detection wavelength 235nm, flow rate: 1.0ml/min, column temperature: at 25 ℃.
Respectively adopting A elette Hypersil ODS 250 x 4.6mm, 5 μm; b Saimeifen C18 thermo Hypersil BDS C18250X 4.6mm, 5 μm; c Saimer FeiAQ thermo Hypersil GOLD aQ 250X 4.6mm, 5 μm; d Diima Diamond C18250X 4.6mm, 5 μm, Asahi Ultimate AQ-C18150X 4.6mm, 5 μm column chromatography. Precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, measuring the chromatographic curve, and calculating the peak area and peak height of penoniflorin, liquiritin and glycyrrhizic acid from the chromatographic curve, as shown in Table 3.
TABLE 3 comparison of peak areas and peak heights for different chromatographic columns
Figure BDA0002532560460000102
As can be seen from the analysis in table 3, the dima column has a lower response height for three components compared to other columns and is therefore not selected, and nauzu as an analytical column has a higher response area and height for three components and is therefore selected as an analytical column.
The chromatographic conditions were as above, except that Asahi Ultimate AQ-C18150X 4.6mm, 5 μm column was used and the gradient in Table 4 was followed. Respectively and precisely sucking 10 μ l of the sample solution, injecting into a liquid chromatograph, and analyzing to find suitable gradient to make chromatographic peak of three components including paeoniflorin, liquiritin and glycyrrhizic acid reach content determination requirement.
The results obtained are as follows.
Under the gradient condition of a-1, the chromatogram obtained has a small peak after the chromatographic peak of liquiritin, so the gradient is not selected. Under the gradient condition of a-2 and a-3, the obtained chromatogram has a small peak after the chromatographic peak of paeoniflorin, and the liquiritin does not reach separation, so the gradient is not selected. Under the gradient condition of a-4, the chromatographic peak of paeoniflorin in the obtained chromatogram is trailing, and liquiritin does not reach separation, so the gradient is not selected. Under the gradient condition of a-5, the paeoniflorin chromatographic peak purity is 6, the peak shape is good, the requirement is met, and the retention time can be properly shortened. Under the gradient condition of a-6, the paeoniflorin chromatographic peak has a beautiful shape, meets the requirement, and can determine the gradient program of the paeoniflorin peak. Under the gradient condition of a-7, the liquiritin chromatographic peak does not reach the separation degree, so the liquiritin chromatographic peak is not applicable. Under the gradient condition of a-8, the chromatogram is shown in FIG. 12, and the peak shape of paeoniflorin chromatographic peak is good, and the gradient proportion can be determined to be 0-1min, 14-15% A, and 15-15.8% A within 1-5 min.
Under the gradient conditions of a-9, a-10, a-11 and a-12, the liquiritin chromatographic peak does not reach the resolution, so the liquiritin chromatographic peak is not applicable.
TABLE 4 gradient survey a
Figure BDA0002532560460000111
In conclusion, the chromatographic column was replaced in consideration of the failure of effective separation of glycyrrhizin by replacing various gradient ratios.
2.5 column and gradient Condition Observation b
Liquid chromatography conditions: mobile phase: acetonitrile (a) -0.05% aqueous phosphoric acid (B), gradient elution: 14% -15% of A in 0-1min, 1-5min, 15% -18% of A, 18% -20% of A in 5-6min, 20% -22% of A in 6-7min, and 22% of A in 7-13 min; the detection wavelength is 235 nm; flow rate: 1.0 ml/min; the column temperature was 25 ℃.
Respectively adopting A.Ultimare AQ-C184.6 × 150mm,5 μm; thermo BDS Hypersil C184.6 x 150mm,5 μm; venusil XBP C184.6 × 150mm,3 μm column. Respectively sucking 10 μ l of test solution, injecting into liquid chromatograph, and measuring to obtain the result shown in FIG. 13.
As can be seen from FIG. 13, under the chromatographic conditions, the separation of paeoniflorin by the three chromatographic columns is better. For liquiritin separation, liquiritin in the chromatographic column B and the previous peak are mixed into a large peak, liquiritin peaks can be separated by the chromatographic column A and the chromatographic column C, and the chromatographic column C is better separated, so that a chromatographic column Venusil XBP C184.6 x 150mm,3 mu m is selected as a peony licorice soup liquid phase analytical column.
The chromatographic conditions were as above except that the column was packed with Venusil XBP C184.6 × 150mm,3 μm and the gradients in Table 5 were followed. Respectively and precisely sucking 10 μ l of the sample solution, injecting into a liquid chromatograph, and analyzing to find suitable gradient to make chromatographic peak of three components including paeoniflorin, liquiritin and glycyrrhizic acid reach content determination requirement.
TABLE 5 gradient survey b
Figure BDA0002532560460000121
The results obtained are as follows.
Under the gradient condition of b-1, the chromatograms obtained by the five conditions have no influence on the peak separation of the paeoniflorin chromatographic peak, but a small peak is arranged in front of the liquiritin chromatographic peak, so the gradient is not selected. Under the gradient condition of b-2, chromatograms obtained under six conditions have no influence on chromatographic peak-to-peak separation of paeoniflorin, but liquiritin has a small peak in front, the separation degree is about 1.3, and the requirement of 1.5 is not met, so that the method is not applicable.
Under the gradient condition of b-3, the obtained chromatogram is shown in fig. 14, and the liquiritin separation degree is found to be 1.570, the peak purity is found to be 41, the method separation degree is good, and the peak purity meets the requirements, so that the gradient elution condition of paeoniflorin and liquiritin is determined. Adjusting the subsequent elution procedure to make the separation degree of glycyrrhizic acid meet the requirement.
Under the gradient condition of b-4, small peaks are found in front of glycyrrhizic acid chromatographic peaks, and the separation degree meets the requirements, so that the method is not applicable. Under the gradient condition of b-5, the obtained chromatogram is shown in FIG. 15, and the calculation results of the separation degree and purity of the obtained product are shown in Table 6. Under the condition of the gradient, the separation degree is good, and the chromatographic peak purity is high, so that the gradient elution is determined to be 14-15% A in 0-1min, 15-15.8% in 1-5min, 15.8-20% A in 5-6min, 20% A in 6-7min, 20-21% A in 7-8min, 21% A in 8-12min, 21-36% A in 12-30min, 36% A in 30-32min and 36-43% A in 32-45 min.
TABLE 6 gradient survey b-5 results
Compound (I) Degree of separation Purity of
Paeoniflorin 1.256 2
Liquiritin 1.563 1
Glycyrrhizic acid 1.679 40
2.6 flow Rate investigation
Liquid chromatography conditions: mobile phase: acetonitrile (a) -0.05% aqueous phosphoric acid (B), gradient elution: 14-15% of A in 0-1min, 15-15.8% in 1-5min, 15.8-20% of A in 5-6min, 20% of A in 6-7min, 20-21% of A in 7-8min, 21% of A in 8-12min, 21-36% of A in 12-30min, 36% of A in 30-32min and 36-43% of A in 32-45 min; a chromatographic column: venusil XBP C184.6 × 150mm,3 μm; the detection wavelength is 235 nm; the column temperature was 25 ℃.
The liquid phase flow rates are respectively 0.8ml/min, 1.0ml/min and 1.2ml/min, respectively, and 10 μ l of the sample solution is precisely absorbed and injected into a liquid chromatograph, and the measurement result is shown in FIG. 16.
As shown in fig. 16, the faster the flow rate, the faster the peak-out speed. When the flow rate is 0.8ml/min, a small peak behind the liquiritin peak can be wrapped in the peak, so that the peak is impure; when the flow rate is 1.2ml/min, the separation degree of the liquiritin peak and the former peak is 1.175 which is less than the separation degree requirement; at a flow rate of 1.0ml/min, the degree of separation of the three components is good, so the optimum flow rate is selected to be 1.0 ml/min.
2.7 sample size investigation
Liquid chromatography conditions: mobile phase: acetonitrile (a) -0.05% aqueous phosphoric acid (B), gradient elution: 14-15% of A in 0-1min, 15-15.8% in 1-5min, 15.8-20% of A in 5-6min, 20% of A in 6-7min, 20-21% of A in 7-8min, 21% of A in 8-12min, 21-36% of A in 12-30min, 36% of A in 30-32min and 36-43% of A in 32-45 min; a chromatographic column: venusil XBP C184.6 × 150mm,3 μm; the detection wavelength is 235 nm; flow rate: 1.0 ml/min; the column temperature was 25 ℃.
Precisely sucking 5, 10, 15 and 20 μ l of test solution, respectively, injecting into liquid chromatograph, and measuring to obtain the result shown in FIG. 17.
As shown in fig. 17, the amount of the sample was small, but the effect on paeoniflorin and glycyrrhizic acid was large. The smaller the sample amount, the lower the baseline noise of the liquiritin peak position, and the better the liquiritin separation, so the optimal sample amount of 5 μ l is selected.
2.8 gradient Condition Observation c
The mobile phase gradient is further optimized in order to make the chromatographic peak base line more stable and make the peak shape of the peak better.
Liquid phase conditions: mobile phase: acetonitrile (a) -0.05% aqueous phosphoric acid (B), column: venusil XBP C184.6 × 150mm,3 μm; the detection wavelength is 235 nm; flow rate: 1.0 ml/min; the column temperature was 25 ℃.
According to the gradient shown in Table 7, 5. mu.l of each sample solution was precisely aspirated and injected into a liquid chromatograph to perform measurement.
TABLE 7 gradient survey c
Figure BDA0002532560460000141
As seen in FIG. 18 and Table 8, the results show that under the chromatographic conditions of c-1, the degree of separation is good and the peak shape is good, and therefore, the gradient elution is determined to be 0-1min 14-17.5% A,1-5min 17.5-19% A,5-6min 19-20% A,6-7min 20% A,7-8min 20-20.5% A,8-12min 20.5-23% A,12-30min 23-36% A,30-32min 36% A,32-45min 36-43% A.
TABLE 8 results of gradient survey c
Figure BDA0002532560460000142
Final defined chromatographic conditions:
mobile phase: acetonitrile (A) -0.05% phosphoric acid water solution (B), gradient elution is carried out for 0-1min, 14-17.5% A,1-5min, 17.5-19% A,5-6min, 19-20% A,6-7min, 20% A,7-8min, 20-20.5% A,8-12min, 20.5-23% A,12-30min, 23-36% A,30-32min, 36% A and 32-45min, 36-43% A; a chromatographic column: venusil XBP C184.6 × 150mm,3 μm; the detection wavelength is 235 nm; flow rate: 1.0 ml/min; the column temperature was 25 ℃.
3. Investigation of preparation of test solution
Liquid chromatography conditions: mobile phase: acetonitrile (a) -0.05% aqueous phosphoric acid (B), gradient elution: 14-15% of A at 0-1min, 15-15.8% at 1-5min, 15.8-20% of A at 5-6min, 20% of A at 6-7min, 20-20.5% of A at 7-8min, 20.5-23% of A at 8-12min, 23-36% of A at 12-30min, 36% of A at 30-32min, and 36-43% of A at 32-45 min; a chromatographic column: venusil XBP C184.6 × 150mm,3 μm; the detection wavelength is 235 nm; flow rate: 1.0 ml/min; the column temperature was 25 ℃.
3.1 examination of extraction solvent
Preparation of a test solution: taking about 0.25g of peony and licorice decoction freeze-dried powder, precisely weighing, placing in a 50ml conical flask with a plug, precisely adding 25ml of solvent (methanol, 30% methanol, 70% methanol, absolute ethanol, 30% ethanol, 70% ethanol, acetonitrile, 30% acetonitrile, 70% acetonitrile and water), weighing, carrying out ultrasonic treatment (250W, 40KHz) for 30 minutes, taking out, cooling, weighing again, complementing weight loss reduction with corresponding solvent, shaking up, filtering, and taking the subsequent filtrate to obtain the final product.
Preparation of control solutions: precisely preparing 126.632 mu g/ml paeoniflorin with 70% methanol; 29.196 μ g/ml of liquiritin; 84.918 μ g/ml glycyrrhizic acid control mixed solution.
Precisely sucking 5 μ l of each of the test solution and the reference solution, injecting into a liquid chromatograph, and measuring to obtain the results shown in Table 9.
TABLE 9 examination of extraction solvent
Figure BDA0002532560460000151
As can be seen from the data in the above table, both anhydrous ethanol and acetonitrile have a particularly low extraction rate of glycyrrhizic acid, and are therefore not used. In the remaining 8 solvents, the glycyrrhizic acid content extracted by methanol is the lowest, and is not selected, and in the remaining 7 solvents, the extraction efficiency of 70% ethanol and 70% methanol is equivalent and higher than that of other solvents, but the comparison of the sample chromatograms of the two solvents shows that the chromatographic peak of the sample prepared by 70% ethanol is lost compared with the sample prepared by 70% methanol, so that the best extraction solvent is selected to be 70% methanol.
3.2 examination of extraction methods
Preparation of a test solution: taking about 0.25g of peony and licorice decoction freeze-dried powder, precisely weighing, placing in a 50ml conical flask with a plug, precisely adding 25ml of 70% methanol, weighing, respectively carrying out ultrasonic treatment (250W, 40KHz) for 20 minutes, shaking for extraction for 2 hours, refluxing for extraction for 1 hour, cooling, weighing again, complementing weight loss reduction amount with 70% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the final product.
Preparation of control solutions: preparing control mixed solution of paeoniflorin 155.84 μ g/ml, liquiritin 119.54 μ g/ml and glycyrrhizic acid 141.66 μ g/ml with 70% methanol.
Precisely sucking 5 μ l of each of the test solution and the reference solution, injecting into a liquid chromatograph, and measuring to obtain the results shown in Table 10.
TABLE 10 examination results of extraction modes
Figure BDA0002532560460000152
The data in the table show that under the three extraction modes, the extraction amounts of paeoniflorin, liquiritin and glycyrrhizic acid are not greatly different, and the ultrasonic operation is simple and convenient, and the time is short, so that the optimal treatment mode is selected.
3.3 ultrasonic time survey
Preparation of a test solution: taking about 0.25g of peony and licorice decoction freeze-dried powder, precisely weighing, placing in a 50ml conical flask with a plug, precisely adding 25ml of 70% methanol, weighing, respectively carrying out ultrasonic treatment (250W, 40KHz) for 10 min, 20 min, 30min and 40 min, cooling, weighing again, complementing weight loss reduction amount with 70% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the final product.
Preparation of control solutions: preparing control mixed solution of paeoniflorin 155.84 μ g/ml, liquiritin 119.54 μ g/ml and glycyrrhizic acid 141.66 μ g/ml with 70% methanol.
Precisely sucking 5 μ l of each of the test solution and the reference solution, injecting into a liquid chromatograph, and measuring to obtain the results shown in Table 11.
TABLE 11 ultrasonic time examination results
Figure BDA0002532560460000161
The data in the above table show that the longer the sonication time, the more complete the extraction, and that the sonication time of 20 minutes, the complete extraction has been achieved, so the optimal sonication time is selected to be 20 minutes.
3.4 solvent dosage examination
Preparation of a test solution: precisely weighing about 0.05g, 0.10g, 0.25g and 0.5g of peony and liquorice decoction freeze-dried powder, placing the powder into a 50ml conical flask with a plug, precisely adding 25ml of 70% methanol, adding 500, 250, 100 and 50 times of solvent respectively, weighing the weight, respectively carrying out ultrasonic treatment (250W, 40KHz) for 20 minutes, cooling, weighing the weight again, complementing weight loss reduction with 70% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the Chinese herbal medicine composition.
Preparation of control solutions: preparing control mixed solution containing paeoniflorin 155.84 μ g/ml, liquiritin 119.54 μ g/ml and glycyrrhizic acid 141.66 μ g/ml with 70% methanol.
Precisely sucking 5 μ l of each of the test solution and the reference solution, injecting into a liquid chromatograph, and measuring to obtain the results shown in Table 12.
TABLE 12 investigation results of solvent amount
Figure BDA0002532560460000162
The data in the table show that the extraction is more complete when the solvent is added, 50 times of solvent amount (volume/mass, ml/g) is less complete from the aspect of the content of 3 components, the extraction of 100 times, 250 times and 500 times of solvent amount tends to be stable, and the liquid-solid ratio is 250 times (volume/mass, ml/g) based on the intermediate principle.
In conclusion, the preparation conditions of the test solution are determined as follows: extracting solvent: 70% methanol, ultrasonic treatment (250W, 40KHz) for 20 minutes, and liquid-solid ratio (volume/mass, ml/g)250 times.
4 methodology study
Chromatographic conditions are as follows: mobile phase: acetonitrile (A) -0.05% phosphoric acid water solution (B), gradient elution is carried out for 0-1min, 14-17.5% A,1-5min, 17.5-19% A,5-6min, 19-20% A,6-7min, 20% A,7-8min, 20-20.5% A,8-12min, 20.5-23% A,12-30min, 23-36% A,30-32min, 36% A and 32-45min, 36-43% A; a chromatographic column: venusil XBP C184.6 × 150mm,3 μm; sample introduction amount: 5 mul, detection wavelength 235 nm; flow rate: 1.0 ml/min; the column temperature was 25 ℃.
Preparation of a test solution: weighing about 0.1g of peony and licorice decoction freeze-dried powder, accurately weighing, placing in a 50ml conical flask with a plug, accurately adding 25ml of 70% methanol, weighing, carrying out ultrasonic treatment (250W, 40kHz) for 20 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking up, and filtering to obtain the final product.
Preparation of control solutions: preparing with 70% methanol to obtain control solution containing paeoniflorin 188.41 μ g/ml, liquiritin 34.11 μ g/ml, and glycyrrhizic acid 34.00 μ g/ml.
The determination method comprises the following steps: precisely sucking 5 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Preparation of white peony root negative sample solution: precisely weighing about 0.1g of white peony root negative freeze-dried powder, placing the white peony root negative freeze-dried powder into a 50ml conical flask with a plug, precisely adding 25ml of 70% methanol, weighing, carrying out ultrasonic treatment (250W and 40kHz) for 20 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking up, and filtering to obtain the white peony root negative freeze-dried powder.
Preparation of honey-fried licorice root negative sample solution: taking about 0.1g of honey-fried licorice root negative freeze-dried powder, precisely weighing, placing in a 50ml conical flask with a plug, precisely adding 25ml of 70% methanol, weighing, carrying out ultrasonic treatment (250W and 40kHz) for 20 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking up, and filtering to obtain the honey-fried licorice root negative freeze-dried powder.
4.1 specificity test
Precisely sucking 5 μ l of each of the control solution, the test solution, the radix Paeoniae alba negative sample solution, the radix Glycyrrhizae Preparata negative sample solution, and 70% methanol blank solvent, injecting into a liquid chromatograph, and measuring to obtain the result shown in figure 19.
As can be seen from FIG. 19, in the chromatogram of the test sample, there are chromatographic peaks at the positions corresponding to the chromatograms of the paeoniflorin control, the liquiritin control and the glycyrrhizic acid control, and there is no corresponding peak on the chromatogram of the negative sample, indicating that the negative sample is not interfered, and the method has strong specificity.
4.2 Linear test
Precisely sucking 1, 2, 5, 10, 15, 20 and 25 μ l of the control solution, respectively, and injecting into liquid chromatograph for measurement. The results of linear regression were shown in Table 13, using the amount of the control (μ g) as the abscissa (X) and the measured peak area as the ordinate (Y).
TABLE 13 results of Linear regression equation
Figure BDA0002532560460000171
The results show that the sampling amount and the peak area of the paeoniflorin are in a good linear relation in the range of 188.41-4710.25 mu g; the sampling amount of liquiritin is in a good linear relation with the peak area within the range of 34.11-852.75 mu g; the glycyrrhizic acid has a good linear relationship between the sample amount and the peak area within the range of 34.00-850.00 mu g.
4.3 precision test
4.3.1 intermediate precision test
Taking about 0.1g of peony and licorice soup corresponding to S6, S7 and S8 batches, precisely weighing, placing in a 50ml conical flask with a plug, and preparing 6 parts of test solution by the same method as the method for preparing the test solution.
Precisely sucking 5 μ l of the sample solution, respectively injecting into Shimadzu LC-20A college liquid chromatograph and Shimadzu LC-20A high performance liquid chromatograph, measuring, and calculating content, the results are shown in Table 14.
TABLE 14 intermediate precision test
Figure BDA0002532560460000181
The data show that the RSD values of paeoniflorin content of three batches of paeony and licorice soup corresponding to the content measurement values of the real object on the two instruments are respectively 1.2%, 1.9% and 1.0%, the RSD values of liquiritin content are respectively 2.7%, 5.9% and 2.0%, the RSD values of glycyrrhizic acid content are respectively 0.7%, 2.0% and 0.7%, and the RSD values are respectively less than 6%, which shows that the analysis method has good reproducibility on different instruments.
4.3.2 repeatability test
6 parts of the test solution was prepared according to the method of "preparation of test solution". Precisely sucking 5 mul of sample solution, injecting into a liquid chromatograph, measuring peak area and calculating content, and the result is shown in Table 15.
TABLE 15 repeatability tests
Figure BDA0002532560460000182
The results of the repeatability tests show that the RSD% values of paeoniflorin, liquiritin and glycyrrhizic acid in 6 test samples are respectively 1.5%, 1.2% and 1.5%, and are all less than 3.0%, which indicates that the method has good repeatability.
4.3.3 sample recovery test
Preparation of a test solution: taking 0.05g of peony and liquorice soup freeze-dried powder (the result of repeatability tests shows that paeoniflorin is 40.37mg/g, liquiritin is 7.99mg/g and glycyrrhizic acid is 13.93mg/g), taking 6 parts in parallel, precisely weighing, placing in a 50ml conical flask with a stopper, precisely adding control solution containing paeoniflorin 0.13038mg/ml, liquiritin is 0.02784mg/ml and glycyrrhizic acid is 0.04683mg/ml, weighing, carrying out ultrasonic treatment (250W, 40kHz) for 20 minutes, cooling, weighing again, supplementing the weight loss by 70% ethanol, shaking up, and filtering to obtain the final product.
The determination method comprises the following steps: precisely sucking 5 μ l of each of the control solution and the sample solution, injecting into a liquid chromatograph, measuring peak area, and calculating recovery rate, the results are shown in Table 16.
TABLE 16 sample recovery test
Figure BDA0002532560460000191
The result shows that the recovery rate of paeoniflorin is within the range of 94.4-97.2%, the RSD value is 1.1%, the recovery rate of the content of liquiritin is within the range of 93.4-95.4%, the RSD value is 0.8%, the recovery rate of the content of glycyrrhizic acid is within the range of 102.1-105.0%, and the RSD value is 1.0%, which all meet the requirements of the 2015-edition pharmacopoeia of 92-105%, and the method has good recovery rate and high accuracy.
5 study of feature maps
Chromatographic conditions are as follows: mobile phase: acetonitrile (A) -0.05% phosphoric acid water solution (B), gradient elution is carried out for 0-1min, 14-17.5% A,1-5min, 17.5-19% A,5-6min, 19-20% A,6-7min, 20% A,7-8min, 20-20.5% A,8-12min, 20.5-23% A,12-30min, 23-36% A,30-32min, 36% A and 32-45min, 36-43% A; a chromatographic column: venusil XBP C184.6 × 150mm,3 μm; the detection wavelength is 235 nm; flow rate: 1.0 ml/min; the column temperature was 25 ℃.
Preparation of a test solution: weighing about 0.1g of peony and licorice decoction freeze-dried powder, accurately weighing, placing in a 50ml conical flask with a plug, accurately adding 25ml of 70% methanol, weighing, carrying out ultrasonic treatment (250W, 40kHz) for 20 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking up, and filtering to obtain the final product.
Preparing a single medicinal solution: 0.1g of white paeony root aqueous extract and 0.1g of honey-fried licorice root aqueous extract are respectively taken, precisely weighed and placed in a 50ml conical flask with a plug, and each negative sample solution is prepared in the same way as the preparation of the test sample solution.
Control solution: taking appropriate amount of penoniflorin, liquiritin, and ammonium glycyrrhizinate as reference substances, adding 70% methanol to obtain mixed reference substance solution (penoniflorin 188.41 μ g/ml, liquiritin 34.11 μ g/ml, glycyrrhizic acid 34.00 μ g/ml); additionally, appropriate amount of control substances such as isoliquiritin, isoliquiritigenin, isoliquiritin apioside, neoliquiritin, albiflorin, benzoylpaeoniflorin, oxypaeoniflorin, gallic acid, and catechin are added with 70% methanol to obtain single control solution (1 ml containing isoliquiritin 0.16mg, isoliquiritigenin 0.27mg, isoliquiritin apioside 0.23mg, neoliquiritin 0.16mg, albiflorin 0.27mg, benzoylpaeoniflorin 0.33mg, oxypaeoniflorin 0.23mg, gallic acid 0.13mg, and catechin 0.14 mg.
Precisely sucking 1-5 μ l of each of the sample solution, the reference solution and the negative sample solution, injecting into a liquid chromatograph, and measuring.
5.1 identification of chromatographic peaks
The position and the attribution of the compound are determined by comparison, and the white paeony root is compared with the white paeony root shown in figure 20; radix Glycyrrhizae Preparata control is shown in FIG. 21.
As can be seen from FIG. 20, the chromatogram is shown at the position corresponding to the control of albiflorin and benzoylpaeoniflorin in the test sample, and the result is negative without interference. The gallic acid has interference of other components in the test sample picture, and the oxidized paeoniflorin and catechin have small peaks in the test sample picture. In addition, the spectrogram of the sample of albiflorin, paeoniflorin and benzoylpaeoniflorin is consistent with that of the reference substance.
As can be seen from FIG. 21, the chromatogram is shown at the corresponding position of the control of neoliquiritin, isoliquiritin and isoliquiritin apioside in the test sample picture, and the negative is not interfered. The isoliquiritigenin shows extremely small peaks in the test sample images. In addition, the spectrogram of the sample was consistent with that of the control.
The positions of the compounds of the peony and licorice decoction and the attribution of the medicinal materials are confirmed by analyzing and comparing chromatographic peaks which are not present in the negative sample chromatogram but present in the single medicine chromatogram in the retention time of chromatographic peaks in the reference substance and the test substance, and according to the ultraviolet spectrum information and the reference substance spectrum information, see table 17.
TABLE 17 tables of Compounds
Compound (I) Retention time Source
Paeoniflorin 6.9 White peony root
Paeoniflorin 7.9 White peony root
New liquiritin 10.3 Prepared licorice root
Liquiritin 10.8 Prepared licorice root
Isoliquiritin apioside 18.2 Prepared licorice root
Isoliquiritin 19.7 Prepared licorice root
Benzoylpaeoniflorin 28.4 White peony root
Glycyrrhizic acid 38.0 Prepared licorice root
5.2 feature map
Preparing the test solution for 15 batches of corresponding substances of the peony and licorice decoction according to the description in the preparation of the test solution, precisely sucking 5 mu l of the test solution, injecting the test solution into a liquid chromatograph, and measuring.
Chromatogram of corresponding substance of 15 batches of Paeoniae radix and Glycyrrhizae radix decoction is shown in FIG. 22.
Introducing the chromatogram data corresponding to 15 batches of the peony and licorice decoction into software of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), taking S1 as a reference chromatogram, setting the time window width to be 0.1min, carrying out multipoint correction, selecting 13 common peaks with larger content, good separation degree and characteristics in the chromatogram for Mark peak matching, and generating a standard fingerprint by adopting an average value method, wherein the standard fingerprint is shown in figure 23. In the standard fingerprint, the neoliquiritin is relatively stable in 13 characteristic peaks, relatively high in content and good in separation degree, so that the relative retention time of the 13 characteristic peaks is calculated by determining the liquiritin (peak 6) as a reference peak, and the retention time of each peak is 0.375 (peak 1), 0.516 (peak 2), 0.647 (peak 3), 0.731 (peak 4), 0.948 (peak 5), 1.000 (peak 6), 1.497 (peak 7), 1.715 (peak 9), 1.847 (peak 10), 2.150 (peak 10), 2.663 (peak 11), 2.860 (peak 12) and 3.566 (peak 13) as shown in table 18.
TABLE 1815 results of relative retention time of corresponding real object of Paeonia lactiflora and Glycyrrhiza uralensis decoction
Figure BDA0002532560460000211
The relative peak areas of the 13 characteristic peaks are calculated and shown in Table 19, and the RSD values of the relative peak areas of the 13 characteristic peaks of 15 batches are 9.9% -73.0%, which shows that the content of the compounds in each batch is different.
Table 1915 relative peak area results of corresponding real object of Paeonia lactiflora and Glycyrrhiza uralensis decoction
Figure BDA0002532560460000212
5.3 assay
The content is calculated as follows: content (wt.)
Figure BDA0002532560460000221
Wherein, CTo pair-control concentration; vTo pair-control sample volume; a. theTo pair-control peak area; a. theFor supplying to-sample peak area; m isFor supplying to-sample size of the test sample.
Table 20 shows the results of measurement of the contents of the major active ingredients in the corresponding real objects of the 15 batches of the peony/licorice root decoction.
5.4 similarity
The similarity of the fingerprint is calculated as follows: and (3) importing the chromatogram data (AIA data) of the peony and licorice decoction to be detected into software of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), and comparing the chromatogram data with the standard fingerprint to obtain the similarity between the fingerprint of the corresponding substance to be detected and the standard fingerprint on the software.
The similarity results of the fingerprints of the 15 batches of peony and licorice decoction corresponding to the real objects and the obtained standard fingerprints are shown in table 20, and it can be seen that the similarity of the 15 batches of peony and licorice decoction corresponding to the real objects is more than 0.9, which indicates that the chemical components are stable.
Table 2015 shows the detection results and similarity of the main active ingredient content of the corresponding material
Figure BDA0002532560460000222

Claims (6)

1. A method for establishing standard fingerprint of corresponding real object of peony licorice decoction, which comprises:
(1) preparing a test solution:
precisely weighing 0.1g of the peony and licorice decoction corresponding substance, placing in a 50ml conical flask with a plug, precisely adding 25ml of 70% methanol, weighing, performing ultrasonic treatment at 250W and 40kHz for 20 minutes, cooling, weighing again, supplementing the weight loss by 70% methanol, shaking up, and filtering to obtain the final product;
(2) preparing a reference substance solution:
accurately weighing appropriate amount of penoniflorin, liquiritin and ammonium glycyrrhizinate as reference substances, and adding 70% methanol to obtain mixed reference substance solution; precisely weighing appropriate amount of isoliquiritin, isoliquiritigenin, isoliquiritin apioside, neoliquiritin, albiflorin, benzoylpaeoniflorin, oxypaeoniflorin, gallic acid, and catechin reference, and adding 70% methanol to obtain single reference solution;
(3) preparing a single medicinal solution:
respectively taking 0.1g of white paeony root aqueous extract and 0.1g of honey-fried licorice root aqueous extract, precisely weighing, placing in a 50ml conical flask with a plug, and preparing each negative sample solution with the sample solution preparation;
(4) HPLC detection
Precisely sucking 1-5 μ l of each of the sample solution, the reference solution and the negative sample solution of (1), (2) and (3), and detecting by the following liquid chromatography to obtain a sample fingerprint chromatogram and a reference fingerprint chromatogram; the liquid chromatography method adopts a high performance liquid chromatograph equipped with an ultraviolet detector to detect, and the chromatographic conditions are as follows:
a chromatographic column: venusil XBP C184.6 × 150mm,3 μm;
mobile phase: the mobile phase A is acetonitrile, the mobile phase B is 0.05 percent of phosphoric acid water solution,
gradient elution: according to the specifications in the table below
Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～1 14→17.5 86→82.5 1～5 17.5→19 82.5→81 5～6 19→20 81→80 6～7 20 80 7～8 20→20.5 80→79.5 8～12 20.5→23 79.5→77 12～30 23→36 77→64 30～32 36 64 32～45 36→43 64→57
Flow rate: 1.0 ml/min;
column temperature: 20-30 ℃;
detection wavelength: 230-240 nm;
(5) generating a standard fingerprint
Generating a standard fingerprint spectrum based on common peaks in fingerprint chromatograms of N batches of test samples, wherein a chromatographic peak with a main component and a better separation degree in the peony and licorice decoction is selected as a characteristic peak and is determined as a common peak, and N is more than 10;
(6) identification and attribution of common peaks
And attributing and identifying common peaks in the fingerprint of the peony and licorice decoction corresponding to the real object.
2. The method of claim 1, wherein in the HPLC assay of step (4), the column temperature: 25 ℃; detection wavelength: 235 nm.
3. The method as claimed in claim 1, wherein the number of theoretical plates of the chromatographic column in the HPLC assay of step (4) is not less than 2000 as paeoniflorin peak.
4. The method of claim 1, wherein the peony licorice root decoction is prepared by: taking radix paeoniae alba and radix glycyrrhizae preparata according to the mass ratio of 1:1, adding water, decocting, filtering with a 200-mesh sieve while the decoction is hot to obtain liquid medicine, and drying to obtain radix paeoniae alba and radix glycyrrhizae decoction dry powder, namely the corresponding substance of the radix paeoniae and radix glycyrrhizae decoction.
5. The method of claim 1, wherein step (5) generates a standard fingerprint, wherein N is 10-20.
6. The method of claim 1, wherein the standard fingerprint of peony licorice decoction against real object comprises 13 characteristic peaks, wherein with reference to peak 6, the relative retention time of each peak is as follows:
peak number Relative retention time 1 0.375 2 0.516 3 0.647 4 0.731 5 0.948 6 1.000 7 1.497 8 1.715 9 1.847 10 2.150 11 2.663 12 2.860 13 3.566
。
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