CN110261505A - A kind of method of quality control of Radix Isatidis - Google Patents
A kind of method of quality control of Radix Isatidis Download PDFInfo
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- CN110261505A CN110261505A CN201910546826.0A CN201910546826A CN110261505A CN 110261505 A CN110261505 A CN 110261505A CN 201910546826 A CN201910546826 A CN 201910546826A CN 110261505 A CN110261505 A CN 110261505A
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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
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/04—Preparation or injection of sample to be analysed
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Abstract
The present invention relates to pharmaceutical chemistry composition detection technical fields, especially a kind of method of quality control of Radix Isatidis, including step 2: test solution preparation: taking Isatis Root 1g, and 30% methanol 20mL is added and sets in round-bottomed flask, weighed weight, it ultrasonic extraction 45 minutes, lets cool, weighed weight, and weight is mended with 70% methanol, it shakes up, is filtered through 0.45 μm of miillpore filter, take subsequent filtrate spare；Step 3: compound determination: reference substance solution, test solution are detected by the positive and negative total ion chromatogram of HPLC-TOF/MS, compares and analyzes and obtains a result.Chromatogram of Radix Isatidis fingerprint spectrum method stabilization, the reliable, favorable reproducibility that this research is established, can provide foundation for the quality evaluation of field pennycress medicinal material.Guarantee is provided for the stable uniformity and curative effect of dispelling wind detoxicating capsule quality.
Description
Technical field
The present invention relates to pharmaceutical chemistry composition detection technical field more particularly to a kind of method of quality control of Radix Isatidis.
Background technique
The dry root of cruciferae isatis Isatis indigoticaFort..Autumn excavation, removes silt, shines
It is dry.The ground such as the Inner Mongol, Shaanxi, Gansu, Hebei, Shandong, Jiangsu, Zhejiang, Anhui, Guizhou are distributed in, are often cultivation.With heat-clearing
The effect of removing toxic substances, cool blood relieving sore-throat.It is malicious when for warm epidemic disease, pharyngalgia of generating heat, febrile virulent maculae, mumps, scarlet fever, major part pestilence, pellet
Poison, carbuncle swells.Modern research shows that Radix Isatidis has the effects that antibacterial, antiviral, anti-inflammatory, section is immune.Its antiviral, anti-inflammatory activity
Ingredient is amino acid, and antibacterial, Antiendotoxic active components are organic acid；Banlangen Polysaccharide specificity, nospecific immunity,
There is facilitation in terms of humoral immunity and cellular immunity." Chinese Pharmacopoeia " (version in 2010) accuses (R, S)-in chromatogram of Radix Isatidis
It is controlled according to the content in spring.There is document report to measure in Radix Isatidis using HPLC method to accuse according to spring, indigo red, gland (R, S)-
The research of glycosides, amino acid equal size.
Reported Radix Isatidis chemical component has: organic acid, lignanoids, alkaloids, sterols, mustard seed glycoside,
Sulfur-bearing class compound, a variety of amino acid, nucleosides and polysaccharide.In addition class compound also containing alkaloids includes indoles biology
Alkali, such as hydroxyl indigo red, isaindigodione；Quinazolone Alkaloid and other types of biological alkali.Radix Isatidis is also dispelling wind removing toxic substances
Important composition medicinal material in capsule, therefore accurately determine the medical substance basis of the medicinal material, quality control, to compound dispelling wind solution
The drug effect contribution of toxic capsule lays the foundation and data supporting.
Summary of the invention
The purpose of the present invention is to solve the disadvantages of Radix Isatidis compound test inaccuracy in the prior art, and propose
A kind of method of quality control of Radix Isatidis.
To achieve the goals above, present invention employs following technical solutions:
A kind of method of quality control of Radix Isatidis, comprising the following steps:
Step 1: reference substance solution preparation:
Reference substance includes: arginine, and cytidine, uridine, adenosine, guanosine, phenylalanine, announcement is according to spring, different scoparin；
The above-mentioned reference substance of precise 2.00mg, is respectively placed in 10mL measuring bottle, and appropriate 50% methanol dissolution is added, and
With 50% methanol constant volume, obtaining mass concentration is 200 μ g/mL reference substance stock solutions, by reference substance stock solution with 50% before sample introduction
10 times of methanol dilution；
Step 2: test solution preparation:
Isatis Root 1g is taken, 30% methanol 20mL is added and sets in round-bottomed flask, weighed weight, ultrasonic extraction 45 minutes,
It lets cool, weighed weight, and mends weight with 70% methanol, shake up, filtered through 0.45 μm of miillpore filter, take subsequent filtrate spare；
Step 3: compound determination:
Reference substance solution, test solution are detected by the positive and negative total ion chromatogram of HPLC-TOF/MS, is compared point
Analysis is obtained a result.
Preferably, in step 3:
Chromatographic condition: chromatographic column is 18 chromatographic column of Diamonsil II C (250mm × 4.6mm, 5 μm)；Mobile phase is second
- 0.05% formic acid solution of nitrile, eluent gradient elution, volume flow 1.0mL/min；35 DEG C of column temperature；Carry out 200~600nm
It sweeps entirely；5 μ L of sample volume；
Mass Spectrometry Conditions: split ratio is set as 1:4, TOF-MS experiment condition: negative ions carry out full scan (ESI) respectively, sweep
Retouch 50~1200Da of mass range；The volume flow 6L/min of dry gas, dry 180 DEG C, atomization air pressure 0.8Bar of temperature degree, sun
Under ion mode, capillary voltage 4500V, capillary voltage 2600V, fragmentation voltage 200Vpp under ion mode, selection
Sodium formate solution is internal standard correction.
A kind of method of quality control of Radix Isatidis, comprising the following steps:
Step 1: reference substance solution preparation:
Reference substance includes: arginine, and cytidine, uridine, adenosine, guanosine, phenylalanine, announcement is according to spring, different scoparin；
The above-mentioned reference substance of precise 2.00mg, is respectively placed in 10mL measuring bottle, and appropriate 50% methanol dissolution is added, and
With 50% methanol constant volume, obtaining mass concentration is 200 μ g/mL reference substance stock solutions, by reference substance stock solution with 50% before sample introduction
10 times of methanol dilution；
Step 2: test solution preparation:
It takes chromatogram of Radix Isatidis to crushed 40 meshes, takes powder 1g, it is accurately weighed, 50% methanol 20mL is added, sets tool plug cone
In shape bottle, weighed weight ultrasonic extraction 45 minutes, is cooled to room temperature, then weighed weight, and the weight of less loss is supplied with 50% methanol
Amount, shake up filtration, take subsequent filtrate to get.
Step 3: compound determination:
Reference substance solution, test solution classes of compounds are detected by the positive and negative total ion chromatogram of HPLC-TOF/MS,
It compares and analyzes and obtains a result.
Preferably, in step 3:
Chromatographic condition are as follows: chromatographic column: 18 5 μ 250*4.6mm of Orca C；Detection wavelength: 220nm；Column temperature: 35 DEG C；Stream
Speed: 1mL/min；Mobile phase: A- acetonitrile, B-0.05% phosphoric acid；Type of elution: gradient elution 0~20min, 0%~12%A, 20
~70min, 12%~35%A；Sample volume: 20 μ L；
Mass Spectrometry Conditions: split ratio is set as 1:4, TOF-MS experiment condition: negative ions carry out full scan (ESI) respectively, sweep
Retouch 50~1200Da of mass range；The volume flow 6L/min of dry gas, dry 180 DEG C, atomization air pressure 0.8Bar of temperature degree, sun
Under ion mode, capillary voltage 4500V, capillary voltage 2600V, fragmentation voltage 200Vpp under ion mode, selection
Sodium formate solution is internal standard correction.
A kind of method of quality control of Radix Isatidis proposed by the present invention, beneficial effect are: the Radix Isatidis that this research is established
Medicinal materials fingerprint method stabilization, reliable, favorable reproducibility, can provide foundation for the quality evaluation of field pennycress medicinal material.For dispelling wind
The stable uniformity and curative effect of detoxicating capsule quality provide guarantee.
Detailed description of the invention
Fig. 1 is the HPLC chromatogram stacking chart of precision test of the present invention；
Fig. 2 is the HPLC chromatogram of stability test of the present invention；
Fig. 3 is the HPLC chromatogram stacking chart of repetitive test of the present invention.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.
Embodiment one:
A kind of method of quality control of Radix Isatidis, comprising the following steps:
Step 1: reference substance solution preparation:
Reference substance includes: arginine, and cytidine, uridine, adenosine, guanosine, phenylalanine, announcement is according to spring, different scoparin；
The above-mentioned reference substance of precise 2.00mg, is respectively placed in 10mL measuring bottle, and appropriate 50% methanol dissolution is added, and
With 50% methanol constant volume, obtaining mass concentration is 200 μ g/mL reference substance stock solutions, by reference substance stock solution with 50% before sample introduction
10 times of methanol dilution；
Step 2: test solution preparation:
Isatis Root 1g is taken, 30% methanol 20mL is added and sets in round-bottomed flask, weighed weight, ultrasonic extraction 45 minutes,
It lets cool, weighed weight, and mends weight with 70% methanol, shake up, filtered through 0.45 μm of miillpore filter, take subsequent filtrate spare；
Step 3: compound determination:
Reference substance solution, test solution are detected by the positive and negative total ion chromatogram of HPLC-TOF/MS, is compared point
Analysis is obtained a result.
Chromatographic condition: chromatographic column is 18 chromatographic column of Diamonsil II C (250mm × 4.6mm, 5 μm)；Mobile phase is second
- 0.05% formic acid solution of nitrile, eluent gradient elution, volume flow 1.0mL/min；35 DEG C of column temperature；Carry out 200~600nm
It sweeps entirely；5 μ L of sample volume；
Mass Spectrometry Conditions: split ratio is set as 1:4, TOF-MS experiment condition: negative ions carry out full scan (ESI) respectively, sweep
Retouch 50~1200Da of mass range；The volume flow 6L/min of dry gas, dry 180 DEG C, atomization air pressure 0.8Bar of temperature degree, sun
Under ion mode, capillary voltage 4500V, capillary voltage 2600V, fragmentation voltage 200Vpp under ion mode, selection
Sodium formate solution is internal standard correction.
By the positive and negative total ion chromatogram of HPLC-TOF/MS, 23 ion stream peaks are detected altogether, identify wherein 22
Compound.Including 8 Amino acids, 1 composition of alkaloids, 3 carbohydrate contents, 3 lignin ingredients, 3 Huangs
Ketones component and 4 small molecule liposoluble ingredients.Wherein 3 kinds of compounds are confirmed using reference substance.Wherein in finger-print
Principal character peak is adenosine, hypoxanthine, phenylalanine, guanine derivatives etc..Accurate molecular quality, the multistage of each compound
Mass spectrum and PDA spectral information see the table below 1
1 Radix Isatidis liquid matter of table points out compound structure information table
Radix Isatidis liquid matter points out compound structure information table
Embodiment two:
A kind of method of quality control of Radix Isatidis, comprising the following steps:
Step 1: reference substance solution preparation:
Reference substance includes: arginine, and cytidine, uridine, adenosine, guanosine, phenylalanine, announcement is according to spring, different scoparin；
The above-mentioned reference substance of precise 2.00mg, is respectively placed in 10mL measuring bottle, and appropriate 50% methanol dissolution is added, and
With 50% methanol constant volume, obtaining mass concentration is 200 μ g/mL reference substance stock solutions, by reference substance stock solution with 50% before sample introduction
10 times of methanol dilution；
Step 2: test solution preparation:
It takes chromatogram of Radix Isatidis to crushed 40 meshes, takes powder 1g, it is accurately weighed, 50% methanol 20mL is added, sets tool plug cone
In shape bottle, weighed weight ultrasonic extraction 45 minutes, is cooled to room temperature, then weighed weight, and the weight of less loss is supplied with 50% methanol
Amount, shake up filtration, take subsequent filtrate to get.
Step 3: compound determination:
Reference substance solution, test solution classes of compounds are detected by the positive and negative total ion chromatogram of HPLC-TOF/MS,
It compares and analyzes and obtains a result.
Chromatographic condition are as follows: chromatographic column: 18 5 μ 250*4.6mm of Orca C；Detection wavelength: 220nm；Column temperature: 35 DEG C；Stream
Speed: 1mL/min；Mobile phase: A- acetonitrile, B-0.05% phosphoric acid；Type of elution: gradient elution 0~20min, 0%~12%A, 20
~70min, 12%~35%A；Sample volume: 20 μ L；
Mass Spectrometry Conditions: split ratio is set as 1:4, TOF-MS experiment condition: negative ions carry out full scan (ESI) respectively, sweep
Retouch 50~1200Da of mass range；The volume flow 6L/min of dry gas, dry 180 DEG C, atomization air pressure 0.8Bar of temperature degree, sun
Under ion mode, capillary voltage 4500V, capillary voltage 2600V, fragmentation voltage 200Vpp under ion mode, selection
Sodium formate solution is internal standard correction.
1, precision test
Prepare chromatogram of Radix Isatidis test solution by sample preparation methods, measured under determining chromatographic condition, continuously into
Sample 6 times, HPLC chromatogram is measured, using the retention time at No. 3 peaks and chromatographic peak area as reference, calculates the opposite of each chromatographic peak
Retention time and relative peak area.The HPLC chromatogram of precision test is shown in Fig. 1, and precision test data are shown in Table 2,3.As it can be seen that
The relative retention time of each chromatographic peak and the RSD value of relative peak area are respectively less than 5%, meet the requirement of finger-print.
2 precision test relative retention time data of table
3 precision test relative peak area data of table
2, stability test
The test solution under precision is taken, it is closed, it is placed in room temperature, respectively 0,3,6,9,12, for 24 hours under time interval
Finger-print is detected, using the retention time at No. 3 peaks and chromatographic peak area as reference, calculates the relative retention time of each chromatographic peak
And relative peak area.The HPLC chromatogram of stability test is shown in Fig. 2, by table 4,5.As it can be seen that the relative retention time of each chromatographic peak
And the RSD value of relative peak area is respectively less than 5%, meets the requirement of finger-print.Illustrate that test solution is stablized interior for 24 hours.
4 stability test relative retention time result of table
5 stability test relative peak area result of table
3, repetitive test
6 parts of test solutions are prepared by determining sample solution preparation method, are measured under determining chromatographic condition, are remembered
Chromatogram is recorded, using the retention time at No. 3 peaks and chromatographic peak area as reference, calculates the relative retention time and phase of each chromatographic peak
To peak area.The HPLC chromatogram of repetitive test is shown in that Fig. 3, repetitive test the results are shown in Table 6,7, it is seen then that the phase of each chromatographic peak
5% is respectively less than to the RSD value of retention time and relative peak area, meets the requirement of finger-print.
6 repetitive test relative retention time result of table
7 repetitive test relative peak area result of table
In conclusion chromatogram of Radix Isatidis fingerprint spectrum method stabilization, reliable, favorable reproducibility that this research is established, can be to lose
The quality evaluation of sauce herbal medicine material provides foundation.Guarantee is provided for the stable uniformity and curative effect of dispelling wind detoxicating capsule quality.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to technical solution, design obtained from equivalent substitution or change, design, should all cover in protection model of the invention
Within enclosing.
Claims (4)
1. a kind of method of quality control of Radix Isatidis, which comprises the following steps:
Step 1: reference substance solution preparation:
Reference substance includes: arginine, and cytidine, uridine, adenosine, guanosine, phenylalanine, announcement is according to spring, different scoparin；
The above-mentioned reference substance of precise 2.00mg, is respectively placed in 10mL measuring bottle, and appropriate 50% methanol is added and dissolves, and with
50% methanol constant volume obtains mass concentration for 200 μ g/mL reference substance stock solutions, by 50% first of reference substance stock solution before sample introduction
Alcohol dilutes 10 times；
Step 2: test solution preparation:
Isatis Root 1g is taken, 30% methanol 20mL is added and sets in round-bottomed flask, weighed weight ultrasonic extraction 45 minutes, is let cool,
Weighed weight, and weight is mended with 70% methanol, it shakes up, is filtered through 0.45 μm of miillpore filter, take subsequent filtrate spare；
Step 3: compound determination:
Reference substance solution, test solution are detected by the positive and negative total ion chromatogram of HPLC-TOF/MS, is compared and analyzed
Result out.
2. a kind of method of quality control of Radix Isatidis according to claim 1, which is characterized in that in step 3:
Chromatographic condition: chromatographic column is Diamonsil II C18 chromatographic column (250mm × 4.6mm, 5 μm)；Mobile phase is acetonitrile-
0.05% formic acid solution, eluent gradient elution, volume flow 1.0mL/min；35 DEG C of column temperature；It is complete to carry out 200~600nm
It sweeps；5 μ L of sample volume；
Mass Spectrometry Conditions: split ratio is set as 1:4, TOF-MS experiment condition: negative ions carry out full scan (ESI) respectively, scan matter
Measure 50~1200Da of range；The volume flow 6L/min of dry gas, dry 180 DEG C, atomization air pressure 0.8Bar of temperature degree, cation
Under mode, capillary voltage 4500V, capillary voltage 2600V, fragmentation voltage 200Vpp under ion mode select formic acid
Sodium solution is internal standard correction.
3. a kind of method of quality control of Radix Isatidis, which comprises the following steps:
Step 1: reference substance solution preparation:
Reference substance includes: arginine, and cytidine, uridine, adenosine, guanosine, phenylalanine, announcement is according to spring, different scoparin；
The above-mentioned reference substance of precise 2.00mg, is respectively placed in 10mL measuring bottle, and appropriate 50% methanol is added and dissolves, and with
50% methanol constant volume obtains mass concentration for 200 μ g/mL reference substance stock solutions, by 50% first of reference substance stock solution before sample introduction
Alcohol dilutes 10 times；
Step 2: test solution preparation:
It takes chromatogram of Radix Isatidis to crushed 40 meshes, takes powder 1g, it is accurately weighed, 50% methanol 20mL is added, sets stuffed conical flask
In, weighed weight ultrasonic extraction 45 minutes, is cooled to room temperature, then weighed weight, and the weight of less loss is supplied with 50% methanol, is shaken
Even filtration, take subsequent filtrate to get.
Step 3: compound determination:
Reference substance solution, test solution classes of compounds are detected by the positive and negative total ion chromatogram of HPLC-TOF/MS, is carried out
Comparative analysis is obtained a result.
4. a kind of method of quality control of Radix Isatidis according to claim 3, which is characterized in that in step 3:
Chromatographic condition are as follows: chromatographic column: 18 5 μ 250*4.6mm of Orca C；Detection wavelength: 220nm；Column temperature: 35 DEG C；Flow velocity:
1mL/min；Mobile phase: A- acetonitrile, B-0.05% phosphoric acid；Type of elution: gradient elution 0~20min, 0%~12%A, 20~
70min, 12%~35%A；Sample volume: 20 μ L；
Mass Spectrometry Conditions: split ratio is set as 1:4, TOF-MS experiment condition: negative ions carry out full scan (ESI) respectively, scan matter
Measure 50~1200Da of range；The volume flow 6L/min of dry gas, dry 180 DEG C, atomization air pressure 0.8Bar of temperature degree, cation
Under mode, capillary voltage 4500V, capillary voltage 2600V, fragmentation voltage 200Vpp under ion mode select formic acid
Sodium solution is internal standard correction.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111208253A (en) * | 2020-03-19 | 2020-05-29 | 安徽济人药业有限公司 | Method for detecting effective components of patrinia and evaluating quality |
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2019
- 2019-06-24 CN CN201910546826.0A patent/CN110261505A/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111208253A (en) * | 2020-03-19 | 2020-05-29 | 安徽济人药业有限公司 | Method for detecting effective components of patrinia and evaluating quality |
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