CN117269352B - Detection method of medicine - Google Patents

Detection method of medicine Download PDF

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CN117269352B
CN117269352B CN202311201528.0A CN202311201528A CN117269352B CN 117269352 B CN117269352 B CN 117269352B CN 202311201528 A CN202311201528 A CN 202311201528A CN 117269352 B CN117269352 B CN 117269352B
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equation
gradient
mobile phase
current
elution
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CN117269352A (en
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唐海涛
李兴尚
王殿广
葛海涛
王正俊
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Jiangsu Suzhong Pharmaceutical Research Institute Co ltd
Suzhong Pharmaceutical Group Co ltd
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Jiangsu Suzhong Pharmaceutical Research Institute Co ltd
Suzhong Pharmaceutical Group Co ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
    • G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography

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Abstract

The invention relates to the technical field of medicine quality control, in particular to a medicine detection method which can be applied to quality control of Danlu capsules. The method has the advantages of rich characteristic peaks, short analysis time and good separation effect, and is an important means for comprehensively controlling the quality of the Dan deer capsule.

Description

Detection method of medicine
Technical Field
The invention relates to the field of medical analytical chemistry, in particular to a detection method of a medicine, and particularly relates to a quality control method of Danlu capsules.
Background
The Danlu capsule is a unique product of Su Chinese medicine group Co., ltd, and is composed of 8 kinds of Chinese medicines of deer antler, prepared fleece-flower root, cnidium fruit, tree peony bark, red peony root, curcuma root, oyster shell and kelp, and has the main functions of regulating thoroughfare and conception vessels, resolving hard mass and relieving pain. Can be used for treating hyperplasia of mammary glands, and symptoms of disorder of thoroughfare and conception vessel, stagnation and phlegm coagulation, such as breast pain, breast lump, soreness of waist and knees, listlessness, chest and hypochondrium distending pain, menoxenia, pale tongue with thin white or greasy coating, and wiry and thin pulse.
The traditional Chinese medicine has the characteristics of multiple components and multiple targets, and relates to multiple medicinal flavors, complex chemical components and higher quality control difficulty. The identification and content measurement of individual components are difficult to meet the requirements of quality control of traditional Chinese medicines, and the characteristic spectrum (or fingerprint) method is an important means for controlling the quality of traditional Chinese medicines on the whole in recent years, and can comprehensively reflect the types and the amounts of chemical components contained in the traditional Chinese medicines and preparations thereof, thereby integrally describing and evaluating the quality of medicines. The method establishes standard characteristic patterns through a large number of samples, determines common peaks, and has integrity and ambiguity through a similarity evaluation method. In order to better control the quality of the medicine of the Danlu capsule and ensure the clinical effectiveness and safety of the Danlu capsule, a method for comprehensively evaluating the quality of the preparation needs to be established.
Disclosure of Invention
The invention aims to: the invention aims to provide a method for detecting a medicine.
The invention also solves the technical problem of providing a method for establishing a drug characteristic spectrum.
The invention also solves the technical problem of providing a contrast characteristic map of the medicine.
In order to solve the first technical problem, the invention discloses a drug detection method, which adopts high performance liquid chromatography, and the chromatographic conditions are as follows:
Chromatographic column: reversed phase silica gel bonded phase chromatographic column
Mobile phase a: aqueous acid solution
Mobile phase B: organic solvents
Eluting by adopting a gradient elution program;
The medicine is any one or more of cornu Cervi extract, radix Polygoni Multiflori Preparata extract, fructus Cnidii extract, cortex moutan extract, radix Paeoniae Rubra extract, radix Curcumae extract, concha Ostreae extract, and thallus laminariae extract;
in each gradient elution process of the gradient elution program, a variable equation of the volume percent of the mobile phase B and the current gradient elution time is equation a or equation B;
Wherein,
Ve is the volume percent of mobile phase B in the current gradient elution;
vf is the volume percentage of mobile phase B at the beginning of the current gradient elution;
vt is the volume percent of mobile phase B at the end of the current gradient elution;
Te is the time of elution in the current gradient elution;
Tf is the start time of the current gradient elution;
tt is the end time of the current gradient elution;
K in equation a is selected from 0 to 1, k in equation b is selected from 0 to 0.75, preferably 0.2 to 0.5, preferably 0.2 to 0.3, preferably 0.22 to 0.28, preferably 0.25.
In order to solve the second technical problem, the invention discloses a method for establishing a drug characteristic spectrum, which adopts high performance liquid chromatography, and the chromatographic conditions are as follows:
Chromatographic column: reversed phase silica gel bonded phase chromatographic column
Mobile phase a: aqueous acid solution
Mobile phase B: organic solvents
Eluting by adopting a gradient elution program;
The medicine is any one or more of cornu Cervi extract, radix Polygoni Multiflori Preparata extract, fructus Cnidii extract, cortex moutan extract, radix Paeoniae Rubra extract, radix Curcumae extract, concha Ostreae extract, and thallus laminariae extract.
In each gradient elution process of the gradient elution program, a variable equation of the volume percent of the mobile phase B and the current gradient elution time is equation a or equation B;
Wherein,
Ve is the volume percent of mobile phase B in the current gradient elution;
vf is the volume percentage of mobile phase B at the beginning of the current gradient elution;
vt is the volume percent of mobile phase B at the end of the current gradient elution;
Te is the time of elution in the current gradient elution;
Tf is the start time of the current gradient elution;
tt is the end time of the current gradient elution;
K in equation a is selected from 0 to 1, k in equation b is selected from 0 to 0.75, preferably 0.2 to 0.5, preferably 0.2 to 0.3, preferably 0.22 to 0.28, preferably 0.25.
In the first to second technical problems described above,
Wherein the medicine is one or more of radix Polygoni Multiflori Preparata extract, fructus Cnidii extract, cortex moutan extract, radix Paeoniae Rubra extract, radix Curcumae extract, thallus laminariae extract; or the medicine is Danlu capsule.
Wherein, in the gradient elution, the initial mobile phase is: 97-99% of mobile phase A and 1-3% of mobile phase B; the final mobile phase was: 3-7% of mobile phase A and 93-97% of mobile phase B; in each gradient elution process of the gradient elution, the variable equation of the volume percent of the mobile phase B and the current gradient elution time is equation a or equation B, and preferably equation B.
Specifically, the gradient elution procedure is:
Preferably, in the 0.fwdarw.9.about.11 min, the 9.fwdarw.11.about.19.about.21 min and the 19.about.21.5.about.23 min, the volume percentage of the mobile phase B and the current elution time are respectively changed as equation a/k=0.5, equation B/k=0.25 and equation a/k=1;
Or alternatively
Preferably, the volume percentage of the mobile phase B and the current elution time change in 0.8-1.2 min, 0.8-1.2-4-6 min, 4-6-19-21 min, 19-21-23.5-24.5 min, 23.5-24.5-25-27 min, 25-27-28-30 min, 28-30-32-34 min and 32-34-35-37 min are all equation a/k=1;
Or alternatively
Preferably, in 0.fwdarw.1.6-2.4 min, 1.6-2.4-8-12 min, 8-12-38-42 min, 38-42-47-49 min, 47-49-50-54 min, 50-54-56-60 min, 56-60-65-67 min, 65-67-67.5-68.5 min, 67.5-68.5-70-74 min, the volume percentage of the mobile phase B and the current elution time change are equation a/k=1;
Or:
preferably, the volume percent of the mobile phase B and the current elution time change in 0-18-19 min and 18-19-21-23 min are respectively expressed as the equations B/k=0.25 and a/k=1;
Or:
Preferably, the volume percent of the mobile phase B and the change of the current elution time are respectively equation B/k=0.25 in 0-0.5-1.5 min and 0.5-1.5-5-8 min; in the 5 th to 8 th to 15 th to 18 th minutes, the change of the volume percentage of the mobile phase B and the current elution time is equation a/k=1 or equation B/k=0.25; at 15-18 to 26-27.5 min, the change of the volume percent of the mobile phase B and the current elution time is equation B/k=0.5; at 26-27.5-28-30 min, the change of the volume percent of the mobile phase B and the current elution time is equation a/k=1;
Further specifically, the gradient elution procedure is:
Preferably, in the 0.fwdarw.9.about.11 min, the 9.fwdarw.11.about.19.about.21 min and the 19.about.21.21.5.about.23 min, the volume percentage of the mobile phase B and the current elution time are respectively changed as equation a/k=0.5, equation B/k=0.25 and a/k=1;
Or alternatively
Preferably, the volume percentage of the mobile phase B and the current elution time change in 0.8-1.2 min, 0.8-1.2-4-6 min, 4-6-19-21 min, 19-21-23.5-24.5 min, 23.5-24.5-25-27 min, 25-27-28-30 min, 28-30-32-34 min and 32-34-35-37 min are all equation a/k=1;
Or alternatively
Preferably, in 0.fwdarw.1.6-2.4 min, 1.6-2.4-8-12 min, 8-12-38-42 min, 38-42-47-49 min, 47-49-50-54 min, 50-54-56-60 min, 56-60-65-67 min, 65-67-67.5-68.5 min, 67.5-68.5-70-74 min, the volume percentage of the mobile phase B and the current elution time change are equation a/k=1;
Or:
preferably, the volume percent of mobile phase B and the change of the current elution time are respectively expressed as B/k=0.25 and a/k=1 in the ranges from 0 to 18 to 19min and from 18 to 19 to 21 to 23 min;
Or:
Preferably, the volume percent of the mobile phase B and the change of the current elution time are respectively equation B/k=0.25 in 0-0.5-1.5 min and 0.5-1.5-5-8 min; in the 5 th to 8 th to 15 th to 18 th minutes, the change of the volume percentage of the mobile phase B and the current elution time is equation a/k=1 or equation B/k=0.25; at 15-18 to 26-27.5 min, the change of the volume percent of the mobile phase B and the current elution time is equation B/k=0.5; at 26-27.5-28-30 min, the change of the volume percent of the mobile phase B and the current elution time is equation a/k=1;
Still more particularly, the gradient elution procedure is:
Preferably, in the 0 to 10min, the 10 to 20min and the 20 to 22min, the volume percentage of the mobile phase B and the current elution time are respectively changed as equation a/k=0.5, equation B/k=0.25 and equation a/k=1;
Or:
Preferably, in the 0.fwdarw.1min, 1.fwdarw.5min, 5.fwdarw.20min, 20.fwdarw.24min, 24.fwdarw.26min, 26.fwdarw.29 min, 29.fwdarw.33 min, 33.fwdarw.36 min, the volume percentage of the mobile phase B and the current elution time are both changed as equation a/k=1;
Or:
Preferably, in 0 to 2min, 2 to 10min, 10 to 40min, 40 to 48min, 48 to 52min, 52 to 58min, 58 to 66min, 66 to 68min, 68 to 72min, the volume percentage of the mobile phase B and the current elution time are all changed as equation a/k=1;
Or:
preferably, at 0 to 20min and 20 to 22min, the volume percent of the mobile phase B and the current elution time change are respectively expressed as equations B/k=0.25 and a/k=1;
Or:
Preferably, at 0 to 1min and 1 to 6min, the change of the volume percentage of the mobile phase B and the current elution time is equation B/k=0.25; at 6-16 min, the change in volume percent of mobile phase B from the current elution time is equation a/k=1; at 16-27 min, the change in volume percent of mobile phase B from the current elution time is equation B/k=0.5; at 27-29 min, the change in volume percent of mobile phase B from the current elution time is equation a/k=1;
Or:
Preferably, at 0 to 1min, 1 to 7min, 7 to 17min, the volume percent of mobile phase B and the current elution time all vary by equation B/k=0.25; at 17 to 27min, the change in volume percent of mobile phase B from the current elution time is equation B/k=0.5; at 27-29 min, the change in volume percent of mobile phase B from the current elution time is equation a/k=1.
The high performance liquid chromatograph adopted by the high performance liquid chromatograph is an ultra-high performance liquid chromatograph with a pressure resistance of 500-700 bar or an ultra-high performance liquid chromatograph with a pressure resistance of 1000-1300 bar, and is preferably a bloom S6000 plus ultra-high performance liquid chromatograph or a Sieimer Feishier Vanquish ultra-high performance liquid chromatograph.
Wherein the reverse phase silica gel bonded phase chromatographic column is a C18 or C8 chromatographic column, preferably a C18 chromatographic column; preferably, the diameter and the column length of the reverse phase silica gel bonded phase chromatographic column are respectively 1.7-2.5 mm, 50-200 mm, preferably 1.9-2.3 mm, 100-150 mm, more preferably 2.1mm, 100-150 mm, still more preferably 2.1mm, 100mm, or 150mm; preferably, the particle size of the reverse phase packing in the reverse phase silica gel bound phase chromatography column is 1.6 to 5. Mu.m, preferably 1.7 to 2.5. Mu.m, more preferably 1.7 μm or 2.5. Mu.m.
Wherein the flow rate of the mobile phase is 0.1-0.7 ml/min, preferably 0.2-0.6 ml/min, and more preferably 0.3-0.5 ml/min; preferably, the column temperature is 20 ℃ to 50 ℃, preferably 25 ℃ to 42.5 ℃, further preferably 30 ℃ to 35 ℃; preferably, the detection is performed using an ultraviolet detector; preferably, the detection wavelength of the ultraviolet detector is 245 to 270nm, preferably 249.5 to 262nm, and more preferably 254nm.
Wherein the acid is any one or more of phosphoric acid, formic acid, acetic acid and trifluoroacetic acid, preferably phosphoric acid; preferably, the volume percentage of the acid in the acid-containing aqueous solution is 0.05-0.5%, preferably 0.075-0.2%, and more preferably 0.1%; the organic solvent is any one or a combination of several of acetonitrile, methanol, ethanol and isopropanol, and is preferably acetonitrile.
The method for detecting the medicine and the method for establishing the characteristic spectrum of the medicine further comprise the step of detecting a sample solution by adopting a high performance liquid chromatography; preferably, the preparation method of the test solution comprises the steps of taking a test of the medicine, adding a solvent for extraction, and carrying out solid-liquid separation, wherein the obtained liquid is the test solution; preferably, the method for preparing a test solution further satisfies any one or more of the following conditions:
a. The solvent is an alcohol aqueous solution or an alcohol solvent; preferably, the alcohol is C1-C6 alcohol, preferably any one or a combination of methanol, ethanol, propanol and butanol, and more preferably methanol; preferably, the volume ratio of the alcohol solvent in the alcohol aqueous solution is 30% -90%, preferably 40% -80%, and more preferably 50% -70%;
b. The ratio of the test sample to the solvent is 1:30-70, preferably 1:40-66.25, more preferably 1:50-62.5 in g/ml;
c. The extraction method comprises ultrasonic extraction or heating reflux extraction; preferably, when ultrasonic extraction is adopted, the temperature of the extraction is 20-40 ℃;
d. The extraction time is more than 15min, preferably 15 min-2 h, more preferably 30-60 min;
e. the solid-liquid separation mode is centrifugation and/or filtration.
The method for detecting the medicine and the method for establishing the characteristic spectrum of the medicine also comprise the step of detecting a reference substance solution by adopting a high performance liquid chromatography; preferably, the reference substance solution is a mixed solution of solvent and one or more of paeoniflorin oxide, paeoniflorin, stilbene glucoside, paeonol, emodin-8-O-beta-D-glucoside, emodin, osthole and physcion; preferably, the solvent is the alcohol, preferably C1-C6 alcohol, more preferably any one or a combination of several of methanol, ethanol, propanol and butanol, still more preferably methanol; preferably, the concentration of each component in the reference solution is as follows: 0.01-0.03 mg/ml of paeoniflorin oxide, preferably 0.015-0.025 mg/ml, and preferably 0.02mg/ml; paeoniflorin 0.2-0.6 g/ml, preferably 0.3-0.5 g/ml, preferably 0.4g/ml; 0.25 to 0.45mg/ml, preferably 0.3 to 0.4mg/ml, preferably 0.35mg/ml of stilbene glucoside; paeonol 0.05-0.25 mg/ml, preferably 0.1-0.2 mg/ml, preferably 0.15mg/ml; huang Su 8 to O to beta to D to glucoside 0.003 to 0.007mg/ml, preferably 0.004 to 0.006mg/ml, preferably 0.005mg/ml; emodin 0.005-0.025 mg/ml, preferably 0.01-0.02 mg/ml, preferably 0.015mg/ml; osthole 0.003-0.011 mg/ml, preferably 0.005-0.009 mg/ml, preferably 0.007mg/ml, physcion 0.002-0.01 mg/ml, preferably 0.004-0.008 mg/ml, preferably 0.006mg/ml.
In order to solve the third technical problem, the invention discloses a contrast characteristic map of a drug, 1. The contrast characteristic map comprises more than 8 common characteristic peaks, a peak corresponding to paeoniflorin is taken as an S peak, the relative retention time of each characteristic peak and the S peak is calculated, the relative retention time is within +/-5% of a specified value, and the specified value is:
i,0.72, 1.00 (S), 1.27, 1.80, 1.88, 2.25, 2.27, 2.40; preferably, it further comprises any one or more of the following specified values: 0.16, 0.55, 0.92, 1.41;
ii,0.71, 1.00 (S), 1.22, 1.80, 1.84, 2.28, 2.30, 2.42; preferably, it further comprises any one or more of the following specified values: 0.12, 0.45, 0.92, 1.16, 1.41;
iii,0.67, 1.00 (S), 1.30, 2.24, 2.30, 2.81, 2.82, 2.90; preferably, it further comprises any one or more of the following specified values: 0.17, 0.52, 0.90, 1.20, 1.68;
iv, constructing the obtained characteristic map by using the medicine according to the method of the first technical problem or the second technical problem.
Wherein the medicine is any one or more of cornu Cervi extract, radix Polygoni Multiflori Preparata extract, fructus Cnidii extract, cortex moutan extract, radix Paeoniae Rubra extract, radix Curcumae extract, concha Ostreae extract, and thallus laminariae extract; preferably, the medicine is any one or more of radix Polygoni Multiflori Preparata extract, fructus Cnidii extract, cortex moutan extract, radix Paeoniae Rubra extract, radix Curcumae extract, thallus laminariae extract; or the medicine is Danlu capsule.
The gradient elution program comprises a plurality of gradients, wherein the change trend of a mobile phase in each gradient can be changed according to the same trend or different trends, the different change trends are shown in a graph in fig. 1, curves 1-5 are shown as an equation a, k are respectively shown as 0, 1/4, 1/2, 3/4 and 1, curves 6-9 are shown as an equation b, and k are respectively shown as 3/4, 1/2, 1/4 and 0; taking the following table as an example, the mobile phase B in each elution gradient changes according to different trends, specifically, in a first elution gradient from 0 to 10min, the change of the volume percentage of the mobile phase B and the current elution time is a curve 3 (equation a/k=0.5), in a second elution gradient from 10 to 20min, the change of the volume percentage of the mobile phase B and the current elution time is a curve 8 (equation B/k=0.25), and in a third elution gradient from 20 to 22min, the change of the volume percentage of the mobile phase B and the current elution time is a curve 5 (equation a/k=1); more specifically, taking the second elution gradient of 10 to 20min as an example in the following table, in equation b, vf=16, vt=95, tf=10, tt=20, k=0.25, ve=16+ (1-0.25) × (95-16) × (2 (-10×(20-20)/(20-10)))/(1-2-10) +0.25× (95-16) × (20-10)/(20-10) =95 when eluting at 20min (i.e., te=20).
The variable equation of the volume percentage of the mobile phase B and the current gradient elution time in the present invention is equation a or equation B, that is, the variable of the volume percentage of the mobile phase B and the current gradient elution time is curve 1, curve 9 or any curve between curve 1 and curve 9, which includes various situations, such as curve 4, curve 5, curve 6, curve 7, other curves between curve 3 and curve 4, other curves between curve 6 and curve 7, and the like.
The Danlu capsule is composed of 8 traditional Chinese medicines, the chemical components are complex and various, the method provided by the invention realizes the good separation of the main components, and the better characteristic patterns (such as example 1, example 2, example 3, etc.) are obtained.
The analysis methods in the embodiment of the invention are verified by methodology, and the method is a detection method of a drug, and can be used as a method for establishing a characteristic spectrum and a method for establishing a fingerprint spectrum. Wherein the medicine is any one or more of cornu Cervi extract, radix Polygoni Multiflori Preparata extract, fructus Cnidii extract, cortex moutan extract, radix Paeoniae Rubra extract, radix Curcumae extract, concha Ostreae extract, and thallus laminariae extract; preferably, the medicine is any one or more of radix Polygoni Multiflori Preparata extract, fructus Cnidii extract, cortex moutan extract, radix Paeoniae Rubra extract, radix Curcumae extract, thallus laminariae extract; or the medicine is Danlu capsule.
In the invention, the cortex moutan can be extracted to obtain paeoniflorin, oxidized paeoniflorin and paeonol; radix Paeoniae Rubra can be extracted to obtain paeoniflorin and oxidized paeoniflorin; fructus Cnidii can be extracted to obtain osthole; the radix Polygoni Multiflori Preparata can be extracted to obtain stilbene glucoside, emodin, physcion, and emodin-8-O-beta-D-glucoside. In the present invention, the standard names of stilbene glucoside are as follows: 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D-glucoside.
The beneficial effects are that:
The invention provides a detection method of a medicine, which can be used for detecting 8 chemical components of a Dan deer capsule. The method has the advantages of rich characteristic peaks, short analysis time and good separation effect, and is an important means for comprehensively controlling the quality of the Dan deer capsule. Specifically, according to the method disclosed by the invention, gradient turning points are obviously reduced, analysis time is obviously shortened, the separation effect is not reduced, and meanwhile, each main peak is more uniformly distributed on a chromatogram, and the overall characteristic chromatogram is more attractive; time and labor are saved, and unexpected effects are obtained.
Drawings
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
FIG. 1 is a flow elution profile of the present invention.
FIG. 2 is a chromatogram of the sample of example 1.
FIG. 3 is a chromatogram of the sample of example 2.
FIG. 4 is a chromatogram of the sample of example 3.
FIG. 5 is a chromatogram of the sample of example 4.
FIG. 6 is a chromatogram of the test sample of example 5.
FIG. 7 is a chromatogram of the test sample of example 6.
FIG. 8 is a chromatogram of the test sample of comparative example 1.
FIG. 9 is a chromatogram of the test sample of comparative example 2.
FIG. 10 is a chromatogram of the test sample of comparative example 3.
FIG. 11 is a chromatogram of the test sample of comparative example 4.
FIG. 12 is a chromatogram of the test sample of comparative example 5.
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.
The Dan deer capsule (Z20150004) is an exclusive product of Su traditional Chinese medicine group Co., ltd.
In the invention, 50% of methanol and 70% of methanol refer to volume concentration.
In the following examples, analysis and detection were performed by an ultra-high performance liquid chromatograph.
The concentrations of the components in the control solution in the following examples are 0.02mg/ml of paeoniflorin oxide, 0.4g/ml of paeoniflorin, 0.35mg/ml of stilbene glucoside, 0.15mg/ml of paeonol, 0.005mg/ml of emodin-8-O-beta-D-glucoside, 0.015mg/ml of emodin, 0.007mg/ml of osthole and 0.006mg/ml of physcion.
Example 1
Taking 0.4g of the content of the Dan deer capsule, precisely weighing, adding into a conical flask, precisely adding 25ml of 50% methanol, shaking for a moment, extracting under reflux for 60 minutes, supplementing the weight loss with 50% methanol, centrifuging, and filtering to obtain a sample solution.
Taking 8 reference substances of paeoniflorin oxide, paeoniflorin, stilbene glucoside, emodin-8-O-beta-D-glucoside, paeonol, osthole and physcion, precisely weighing, adding methanol, respectively dissolving, and quantitatively diluting to prepare reference substance solution.
Sample injection analysis was performed under the following conditions
Instrument: ultra-high performance liquid chromatograph of Hua-pectrum S6000 plus
Chromatographic column: hua Spectrum ALPHASIL VC-C18 2.1X105 mm,2.5 μm
Mobile phase: gradient elution with 0.1% phosphoric acid-acetonitrile, flow rate 0.3ml/min
The column temperature is 30 ℃, the detection wavelength is 254nm, and the sample injection amount is 10ul
The chromatogram of the obtained test sample is shown in figure 2. In FIG. 1, the peak 1 is oxidized paeoniflorin, the peak 2 is paeoniflorin, the peak 3 is stilbene glucoside, the peak 4 is emodin-8-O-beta-D-glucoside, the peak 5 is paeonol, the peak 6 is emodin, the peak 7 is osthole, and the peak 8 is physcion.
Example 2
Taking 0.5g of capsule content of the deer antler, precisely weighing, adding into a conical flask, precisely adding 25ml of 70% methanol, shaking for a moment, performing ultrasonic extraction at 20-40 ℃ for 30 minutes, supplementing weight loss with 70% methanol, centrifuging, and filtering to obtain a sample solution.
Taking 8 reference substances of paeoniflorin oxide, paeoniflorin, stilbene glucoside, emodin-8-O-beta-D-glucoside, paeonol, osthole and physcion, precisely weighing, adding methanol, respectively dissolving, and quantitatively diluting to prepare reference substance solution.
Sample injection analysis was performed under the following conditions
Instrument: super high performance liquid chromatograph of Simerfeishier Vanquish
Chromatographic column: acquity UPLC BEH C18.2.1X100 mm,1.7 μm
Mobile phase: gradient elution with 0.1% phosphoric acid-acetonitrile, flow rate 0.4ml/min
The column temperature is 30 ℃, the detection wavelength is 254nm, and the sample injection quantity is 1ul
The chromatogram of the obtained sample is shown in figure 3. In FIG. 2, peak 1 is oxidized paeoniflorin, peak 2 is paeoniflorin, peak 3 is stilbene glucoside, peak 4 is paeonol, peak 5 is emodin-8-O-beta-D-glucoside, peak 6 is emodin, peak 7 is osthole, and peak 8 is physcion.
Example 3
Taking 0.5g of capsule content of the deer antler, precisely weighing, adding into a conical flask, precisely adding 25ml of 70% methanol, shaking for a moment, performing ultrasonic extraction at 20-40 ℃ for 30 minutes, supplementing weight loss with 70% methanol, centrifuging, and filtering to obtain a sample solution.
Taking 8 reference substances of paeoniflorin oxide, paeoniflorin, stilbene glucoside, emodin-8-O-beta-D-glucoside, paeonol, osthole and physcion, precisely weighing, adding methanol, respectively dissolving, and quantitatively diluting to prepare reference substance solution.
Sample injection analysis was performed under the following conditions
Instrument: super high performance liquid chromatograph of Simerfeishier Vanquish
Chromatographic column: acquity UPLC BEH C18.2.1X100 mm,1.7 μm
Mobile phase: gradient elution with 0.1% phosphoric acid-acetonitrile, flow rate 0.5ml/min
The column temperature is 35 ℃, the detection wavelength is 254nm, and the sample injection quantity is 1ul
The chromatogram of the obtained sample is shown in figure 4. In FIG. 3, peak 1 is oxidized paeoniflorin, peak 2 is paeoniflorin, peak 3 is stilbene glucoside, peak 4 is paeonol, peak 5 is emodin-8-O-beta-D-glucoside, peak 6 is emodin, peak 7 is osthole, and peak 8 is physcion.
Example 4
The sample solution and the control solution were prepared in the same manner as in example 3, and the chromatographic conditions were the same as in example 3 except for the elution gradient as follows:
The chromatogram of the obtained sample is shown in figure 5.
Example 5
The sample solution and the control solution were prepared in the same manner as in example 3, and the chromatographic conditions were the same as in example 3 except for the elution gradient as follows:
The chromatogram of the obtained sample is shown in figure 6.
Example 6
The sample solution and the control solution were prepared in the same manner as in example 3, and the chromatographic conditions were the same as in example 3 except for the elution gradient as follows:
The chromatogram of the obtained sample is shown in figure 7.
Comparative example 1
The sample solution and the control solution were prepared in the same manner as in example 3, and the chromatographic conditions were the same as in example 3 except for the elution gradient as follows:
The chromatogram of the obtained sample is shown in figure 8.
Comparative example 2
The sample solution and the control solution were prepared in the same manner as in example 3, and the chromatographic conditions were the same as in example 3 except for the elution gradient as follows:
The chromatogram of the obtained sample is shown in figure 9.
Comparative example 3
The sample solution and the control solution were prepared in the same manner as in example 3, and the chromatographic conditions were the same as in example 3 except for the elution gradient as follows:
the chromatogram of the obtained sample is shown in figure 10.
Comparative example 4
The sample solution and the control solution were prepared in the same manner as in example 3, and the chromatographic conditions were the same as in example 3 except for the elution gradient as follows:
The chromatogram of the obtained sample is shown in figure 11.
Comparative example 5
The sample solution and the control solution were prepared in the same manner as in example 3, and the chromatographic conditions were the same as in example 3 except for the elution gradient as follows:
the chromatogram of the obtained sample is shown in figure 12.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (25)

1. A detection method of paeoniflorin, stilbene glucoside, paeonol, emodin-8-O-beta-D-glucoside, emodin, osthole and physcion in a medicine is characterized in that ultra-high performance liquid chromatography is adopted, and the chromatographic conditions are as follows:
chromatographic column: c18 reverse phase silica gel bonded phase chromatographic column;
mobile phase a: an acid-containing aqueous solution; the volume percentage of acid in the acid-containing aqueous solution is 0.05-0.5%; the acid is any one or a combination of more of phosphoric acid, formic acid, acetic acid and trifluoroacetic acid;
Mobile phase B: acetonitrile;
the column temperature is 30-35 ℃;
Eluting by adopting a gradient elution program; the gradient elution procedure was:
Gradient 1:
Or gradient 2:
Or gradient 3:
The flow rate of the mobile phase is 0.2-0.6 ml/min;
detecting by an ultraviolet detector; the detection wavelength of the ultraviolet detector is 249.5-262 nm;
the medicine is cornu Cervi extract, radix Polygoni Multiflori Preparata extract, fructus Cnidii extract, cortex moutan extract, radix Paeoniae Rubra extract, radix Curcumae extract, concha Ostreae extract, thallus laminariae extract mixture or DANLU capsule;
The method also comprises the steps of detecting the sample solution by adopting a high performance liquid chromatography; the preparation method of the sample solution comprises extracting the sample of the medicine with methanol water solution, and separating solid from liquid to obtain the sample solution; the method also comprises detecting the reference substance solution by high performance liquid chromatography; the reference substance solution is a mixed solution of paeoniflorin oxide, paeoniflorin, stilbene glucoside, paeonol, emodin-8-O-beta-D-glucoside, emodin, osthole, emodin dimethyl ether reference substance and solvent.
2. The method according to claim 1, wherein the variable equation of the volume percent of mobile phase B and the current gradient elution time during the current gradient elution is equation a or equation B;
Wherein,
Ve is the volume percent of mobile phase B in the current gradient elution;
vf is the volume percentage of mobile phase B at the beginning of the current gradient elution;
vt is the volume percent of mobile phase B at the end of the current gradient elution;
Te is the time of elution in the current gradient elution;
Tf is the start time of the current gradient elution;
tt is the end time of the current gradient elution;
In the gradient 1, in 0 to 9 to 11min, 9 to 11 to 19 to 21min and 19 to 21 to 21.5 to 23min, the volume percentage of the mobile phase B and the change of the current elution time are respectively shown as an equation a, wherein k=0.5 and an equation B, wherein k=0.25 and an equation a, and wherein k=1;
In the gradient 2, in 0 to 0.8 to 1.2min, 0.8 to 1.2 to 4 to 6min, 4 to 6 to 19 to 21min, 19 to 21 to 23.5 to 24.5min, 23.5 to 24.5 to 25 to 27min, 25 to 27 to 28 to 30min, 28 to 30 to 32 to 34min and 32 to 34 to 35 to 37min, the volume percentage of the mobile phase B and the change of the current elution time are all equation a, wherein k=1;
In the gradient 3, in 0 to 1.6 to 2.4min, 1.6 to 2.4 to 8 to 12min, 8 to 12 to 38 to 42min, 38 to 42 to 47 to 49min, 47 to 49 to 50 to 54min, 50 to 54 to 56 to 60min, 56 to 60 to 65 to 67min, 65 to 67 to 67.5 to 68.5min and 67.5 to 68.5 to 70 to 74min, the volume percentage of the mobile phase B and the current elution time change are all equation a, wherein k=1.
3. The method of claim 2, wherein the gradient elution procedure is:
Gradient 4:
Or gradient 5:
Or gradient 6:
4. A method according to claim 3, wherein in the gradient 4, the volume percent of mobile phase B and the current elution time in 0→9 to 11min, 9 to 11→19 to 21min, 19 to 21→21.5 to 23min are respectively changed as equation a, wherein k=0.5, equation B, wherein k=0.25, equation a, wherein k=1;
In the gradient 5, in 0.8-1.2 min, 0.8-1.2-4-6 min, 4-6-19-21 min, 19-21-23.5-24.5 min, 23.5-24.5-25-27 min, 25-27-28-30 min, 28-30-32-34 min and 32-34-35-37 min, the volume percentage of the mobile phase B and the change of the current elution time are all equation a, wherein k=1;
In the gradient 6, in 0 to 1.6 to 2.4min, 1.6 to 2.4 to 8 to 12min, 8 to 12 to 38 to 42min, 38 to 42 to 47 to 49min, 47 to 49 to 50 to 54min, 50 to 54 to 56 to 60min, 56 to 60 to 65 to 67min, 65 to 67 to 67.5 to 68.5min and 67.5 to 68.5 to 70 to 74min, the volume percentage of the mobile phase B and the current elution time change are all equation a, wherein k=1.
5. The method of claim 1, wherein the gradient elution procedure is:
gradient 7:
Or gradient 8:
Or gradient 9:
6. The method according to claim 5, wherein the variable equation of the volume percent of mobile phase B and the current gradient elution time during the current gradient elution is equation a or equation B;
Wherein,
Ve is the volume percent of mobile phase B in the current gradient elution;
vf is the volume percentage of mobile phase B at the beginning of the current gradient elution;
vt is the volume percent of mobile phase B at the end of the current gradient elution;
Te is the time of elution in the current gradient elution;
Tf is the start time of the current gradient elution;
tt is the end time of the current gradient elution;
In the gradient 7, in the 0 to 10min, the 10 to 20min and the 20 to 22min, the change of the volume percentage of the mobile phase B and the current elution time is respectively shown as an equation a, wherein k=0.5 and an equation B, wherein k=0.25 and an equation a, and wherein k=1;
in the gradient 8, in 0 to 1min, 1 to 5min, 5 to 20min, 20 to 24min, 24 to 26min, 26 to 29min, 29 to 33min and 33 to 36min, the volume percentage of the mobile phase B and the change of the current elution time are all equation a, wherein k=1;
In the gradient 9, in 0 to 2min, 2 to 10min, 10 to 40min, 40 to 48min, 48 to 52min, 52 to 58min, 58 to 66min, 66 to 68min, 68 to 72min, the volume percentage of the mobile phase B and the current elution time change are all equation a, wherein k=1.
7. The method according to claim 1, wherein the high performance liquid chromatograph used in the ultra-high performance liquid chromatography is an ultra-high performance liquid chromatograph having a pressure resistance of 500 to 700bar or an ultra-high performance liquid chromatograph having a pressure resistance of 1000 to 1300 bar.
8. The method according to claim 1, wherein the diameter and column length of the C18 reverse phase silica gel bonded phase chromatographic column are 1.7-2.5 mm, 50-200 mm, respectively; the particle size of the reverse phase filler in the reverse phase silica gel bonding phase chromatographic column is 1.6-2.5 mu m.
9. The method according to claim 1, wherein the flow rate of the mobile phase is 0.3 to 0.5ml/min; the detection wavelength of the ultraviolet detector is 254nm; the volume percentage of the acid in the acid-containing aqueous solution is 0.075-0.2%.
10. The method of claim 1, wherein the method of preparing the test solution further satisfies any one or more of the following conditions:
a. The ratio of the test sample to the methanol aqueous solution is 1:30-70 in terms of g/ml;
b. the extraction method comprises ultrasonic extraction or heating reflux extraction;
c. The extraction time is more than 15 min;
d. The solid-liquid separation mode is centrifugation and/or filtration.
11. The method according to claim 10, wherein the volume proportion of methanol in the aqueous methanol solution is 40% to 80%; the ratio of the test sample to the methanol aqueous solution is 1:50-62.5; when ultrasonic extraction is adopted, the temperature of the extraction is 20-40 ℃; the extraction time is 15 min-2 h.
12. The method of claim 1, wherein the reference solution is a C1-C6 alcohol; the concentration of each component in the reference substance solution is as follows: 0.01-0.03 mg/ml of paeoniflorin oxide; paeoniflorin 0.2-0.6 g/ml; 0.25-0.45 mg/ml stilbene glucoside; 0.05 to 0.25mg/ml of paeonol; emodin-8-O-beta-D-glucoside 0.003-0.007 mg/ml; emodin 0.005-0.025 mg/ml; osthole 0.003-0.011 mg/ml; 0.002-0.01 mg/ml of physcion.
13. A method for establishing a drug characteristic spectrum is characterized in that ultra-high performance liquid chromatography is adopted, and chromatographic conditions are as follows: chromatographic column: c18 reverse phase silica gel bonded phase chromatographic column;
mobile phase a: an acid-containing aqueous solution; the volume percentage of acid in the acid-containing aqueous solution is 0.05-0.5%; the acid is any one or a combination of more of phosphoric acid, formic acid, acetic acid and trifluoroacetic acid;
Mobile phase B: acetonitrile;
the column temperature is 30-35 ℃;
Eluting by adopting a gradient elution program; the gradient elution procedure was:
Gradient 1:
Or gradient 2:
Or gradient 3:
The flow rate of the mobile phase is 0.2-0.6 ml/min;
detecting by an ultraviolet detector; the detection wavelength of the ultraviolet detector is 249.5-262 nm;
the medicine is cornu Cervi extract, radix Polygoni Multiflori Preparata extract, fructus Cnidii extract, cortex moutan extract, radix Paeoniae Rubra extract, radix Curcumae extract, concha Ostreae extract, thallus laminariae extract mixture or DANLU capsule;
The method also comprises the steps of detecting the sample solution by adopting a high performance liquid chromatography; the preparation method of the sample solution comprises extracting the sample of the medicine with methanol water solution, and separating solid from liquid to obtain the sample solution; the method also comprises detecting the reference substance solution by high performance liquid chromatography; the reference substance solution is a mixed solution of any one of paeoniflorin oxide, paeoniflorin, stilbene glucoside, paeonol, emodin-8-O-beta-D-glucoside, emodin, osthole and physcion reference substance and a solvent;
The drug characteristic spectrum comprises more than 8 common characteristic peaks, the peak corresponding to paeoniflorin is taken as an S peak, the relative retention time of each characteristic peak and the S peak is calculated, the relative retention time is within +/-5% of a specified value, and the specified value of the relative retention time is as follows:
i，0.72、1.00(S)、1.27、1.80、1.88、2.25、2.27、2.40；
or ii,0.71, 1.00 (S), 1.22, 1.80, 1.84, 2.28, 2.30, 2.42;
or iii,0.67, 1.00 (S), 1.30, 2.24, 2.30, 2.81, 2.82, 2.90;
The correspondence between the predetermined value of the relative retention time and the characteristic peak is as follows:
14. The method of claim 13, wherein the variable equation of the volume percent of mobile phase B to the current gradient elution time during the current gradient elution is equation a or equation B;
Wherein,
Ve is the volume percent of mobile phase B in the current gradient elution;
vf is the volume percentage of mobile phase B at the beginning of the current gradient elution;
vt is the volume percent of mobile phase B at the end of the current gradient elution;
Te is the time of elution in the current gradient elution;
Tf is the start time of the current gradient elution;
tt is the end time of the current gradient elution;
In the gradient 1, in 0 to 9 to 11min, 9 to 11 to 19 to 21min and 19 to 21 to 21.5 to 23min, the volume percentage of the mobile phase B and the change of the current elution time are respectively shown as an equation a, wherein k=0.5 and an equation B, wherein k=0.25 and an equation a, and wherein k=1;
In the gradient 2, in 0 to 0.8 to 1.2min, 0.8 to 1.2 to 4 to 6min, 4 to 6 to 19 to 21min, 19 to 21 to 23.5 to 24.5min, 23.5 to 24.5 to 25 to 27min, 25 to 27 to 28 to 30min, 28 to 30 to 32 to 34min and 32 to 34 to 35 to 37min, the volume percentage of the mobile phase B and the change of the current elution time are all equation a, wherein k=1;
In the gradient 3, in 0 to 1.6 to 2.4min, 1.6 to 2.4 to 8 to 12min, 8 to 12 to 38 to 42min, 38 to 42 to 47 to 49min, 47 to 49 to 50 to 54min, 50 to 54 to 56 to 60min, 56 to 60 to 65 to 67min, 65 to 67 to 67.5 to 68.5min and 67.5 to 68.5 to 70 to 74min, the volume percentage of the mobile phase B and the current elution time change are all equation a, wherein k=1.
15. The method of claim 14, wherein the gradient elution procedure is:
Gradient 4:
Or gradient 5:
Or gradient 6:
16. The method according to claim 15, wherein in the gradient 4, the volume percent of the mobile phase B and the current elution time in 0→9 to 11min, 9 to 11→19 to 21min, 19 to 21→21.5 to 23min are respectively changed as equation a, wherein k=0.5, equation B, wherein k=0.25, equation a, wherein k=1;
In the gradient 5, in 0.8-1.2 min, 0.8-1.2-4-6 min, 4-6-19-21 min, 19-21-23.5-24.5 min, 23.5-24.5-25-27 min, 25-27-28-30 min, 28-30-32-34 min and 32-34-35-37 min, the volume percentage of the mobile phase B and the change of the current elution time are all equation a, wherein k=1;
In the gradient 6, in 0 to 1.6 to 2.4min, 1.6 to 2.4 to 8 to 12min, 8 to 12 to 38 to 42min, 38 to 42 to 47 to 49min, 47 to 49 to 50 to 54min, 50 to 54 to 56 to 60min, 56 to 60 to 65 to 67min, 65 to 67 to 67.5 to 68.5min and 67.5 to 68.5 to 70 to 74min, the volume percentage of the mobile phase B and the current elution time change are all equation a, wherein k=1.
17. The method of claim 13, wherein the gradient elution procedure is:
gradient 7:
Or gradient 8:
Or gradient 9:
18. The method of claim 17, wherein the variable equation of the volume percent of mobile phase B to the current gradient elution time during the current gradient elution is equation a or equation B;
Wherein,
Ve is the volume percent of mobile phase B in the current gradient elution;
vf is the volume percentage of mobile phase B at the beginning of the current gradient elution;
vt is the volume percent of mobile phase B at the end of the current gradient elution;
Te is the time of elution in the current gradient elution;
Tf is the start time of the current gradient elution;
tt is the end time of the current gradient elution;
In the gradient 7, in the 0 to 10min, the 10 to 20min and the 20 to 22min, the change of the volume percentage of the mobile phase B and the current elution time is respectively shown as an equation a, wherein k=0.5 and an equation B, wherein k=0.25 and an equation a, and wherein k=1;
in the gradient 8, in 0 to 1min, 1 to 5min, 5 to 20min, 20 to 24min, 24 to 26min, 26 to 29min, 29 to 33min and 33 to 36min, the volume percentage of the mobile phase B and the change of the current elution time are all equation a, wherein k=1;
In the gradient 9, in 0 to 2min, 2 to 10min, 10 to 40min, 40 to 48min, 48 to 52min, 52 to 58min, 58 to 66min, 66 to 68min, 68 to 72min, the volume percentage of the mobile phase B and the current elution time change are all equation a, wherein k=1.
19. The method according to claim 13, wherein the high performance liquid chromatograph used in the ultra-high performance liquid chromatography is an ultra-high performance liquid chromatograph having a pressure resistance of 500 to 700bar or an ultra-high performance liquid chromatograph having a pressure resistance of 1000 to 1300 bar.
20. The method of claim 13, wherein the C18 reverse phase silica gel bonded phase chromatographic column has a diameter and column length of 1.7-2.5 mm, 50-200 mm, respectively; the particle size of the reverse phase filler in the reverse phase silica gel bonding phase chromatographic column is 1.6-2.5 mu m.
21. The method according to claim 13, wherein the flow rate of the mobile phase is 0.3 to 0.5ml/min; the detection wavelength of the ultraviolet detector is 254nm; the volume percentage of the acid in the acid-containing aqueous solution is 0.075-0.2%.
22. The method of claim 13, wherein the method of preparing the test solution further satisfies any one or more of the following conditions:
a. The ratio of the test sample to the methanol aqueous solution is 1:30-70 in terms of g/ml;
b. the extraction method comprises ultrasonic extraction or heating reflux extraction;
c. The extraction time is more than 15 min;
d. The solid-liquid separation mode is centrifugation and/or filtration.
23. The method of claim 22, wherein the volume fraction of methanol in the aqueous methanol solution is 40% to 80%; the ratio of the test sample to the methanol aqueous solution is 1:50-62.5; when ultrasonic extraction is adopted, the temperature of the extraction is 20-40 ℃; the extraction time is 15 min-2 h.
24. The method of claim 13, wherein the reference solution is a C1-C6 alcohol; the concentration of each component in the reference substance solution is as follows: 0.01-0.03 mg/ml of paeoniflorin oxide; paeoniflorin 0.2-0.6 g/ml; 0.25-0.45 mg/ml stilbene glucoside; 0.05 to 0.25mg/ml of paeonol; emodin-8-O-beta-D-glucoside 0.003-0.007 mg/ml; emodin 0.005-0.025 mg/ml; osthole 0.003-0.011 mg/ml; 0.002-0.01 mg/ml of physcion.
25. The method according to claim 13, wherein in i, it further comprises a specified value of any one or more of the following relative retention times: 0.16, 0.55, 0.92, 1.41; the above ii further comprises a predetermined value of any one or more of the following relative retention times: 0.12, 0.45, 0.92, 1.16, 1.41; the above iii further includes a predetermined value of any one or more of the following relative retention times: 0.17, 0.52, 0.90, 1.20, 1.68.
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