CN110108818A - A kind of high performance liquid chromatography and its application efficiently separating detection phthalide derivant - Google Patents
A kind of high performance liquid chromatography and its application efficiently separating detection phthalide derivant Download PDFInfo
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- CN110108818A CN110108818A CN201910466351.4A CN201910466351A CN110108818A CN 110108818 A CN110108818 A CN 110108818A CN 201910466351 A CN201910466351 A CN 201910466351A CN 110108818 A CN110108818 A CN 110108818A
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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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
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
- G01N30/8634—Peak quality criteria
Abstract
A kind of high performance liquid chromatography efficiently separating detection phthalide analog derivative, using phenyl-hexyl chromatographic column or phenyl column, Detection wavelength is 226-230nm or 280nm, flow velocity is 0.8-1.2ml/min, and column temperature is 20-35 DEG C, and sample volume is 10-100 μ l, mobile phase is by A phase and B phase composition, A phase in mobile phase: the volume ratio that B is mixed is 60:40-80:20, and A phase is the aqueous solution of pH value 3-5, and the volume ratio of acetonitrile and methanol is 1:0.8-1.2 in B phase.Method of the invention has many advantages, such as that specificity is good, separating degree is high, sensitivity is excellent, efficiently separating for more than ten phthalide derivants may be implemented, it can be used for separation, assay or its monitoring and detection in relation to substance of phthalide derivant, isolate and purify, preferably to control the quality of these raw materials and its preparation, to ensure the validity of drug, safety and realize that drug quality is controllable.
Description
Technical field
The invention belongs to Pharmaceutical Analysis fields, and in particular to a kind of high-efficient liquid phase color for efficiently separating detection phthalide derivant
Spectrometry and its application.
Background technique
Phthalide is also referred to as phthalanone, and structure feature is bicyclic the melting of gamma lactone (A ring) and benzene (B ring)
It closes, is that γ-hydroxycarboxylic acid loses 1 molecular water and the lactone that is formed.Existing research shows that many phthalide analog compounds have pharmacology
Activity can be applied to the prevention or treatment of cardiovascular and cerebrovascular disease.Butylphenyl phthaleine is third newization that China independently researchs and develops
Learn entity medicine.2- (Alpha-hydroxy amyl) benzoic acid pharmaceutical salts have entered clinical development.Separately there is document report 3- cyclobutenyl
Phthalide has the activity for inhibiting thromboxane synthesis.In addition, 3- (3 '-hydroxyl)-butylphenyl phthaleine, (also known as 3- is just for 3- hydroxyl -3- butylphenyl phthaleine
- 1 (3H)-isobenzofuranone of butyl -3- hydroxyl) it is the butylbenzene phthalide derivative for having pharmacological activity.Aforementioned phthalide-type chemical combination
Object structure proximate, can choose identical starting material in being synthetically prepared and intermediate is synthesized.Phthalide analog compound is not
Stablize, vulnerable to the factors such as illumination, temperature influence and change and mutually convert.And in process of production, starting material, in
Mesosome, condensate, side reaction product, catabolite in storage etc., may all be brought into finished product as impurity shadow
Ring product quality.Therefore, scientific and reasonable method for separating and detecting measurement phthalide-type medicament contg and its related substance are studied and defined,
For ensureing that phthalide analog derivative drug quality has a very important significance, to ensure the validity and safety of drug, into
And realize that drug quality is controllable.
National drug standards WS1- (X-124) -2005Z of butylphenyl phthaleine discloses the content of butylphenyl phthaleine and the inspection in relation to substance
Survey method.This method uses octadecylsilane chemically bonded silica for filler, methanol: water (65:35) is mobile phase, Detection wavelength
280nm, it is the methanol solution containing butylphenyl phthaleine and propyl benzene phthalein that separating degree, which tests solution,.
Li Jie etc. is composed using impurity of the ultra high efficiency liquid phase-triple level four bars mass spectrometric hyphenated technique to butylphenyl phthaleine bulk pharmaceutical chemicals and is carried out
Analysis discloses butylphenyl phthaleine and generates a large amount of impurity 1 (2- (1- hydroxyl amyl)-benzoic acid) under alkaline conditions, and impurity 1 further drops
A small amount of impurity 3 (2- (1- oxopentyl)-benzoic acid) of solution, impurity 2 are the process impurity that phthalic anhydride method introduces.Efficiently
Liquid phase part uses C18 chromatographic column, and mobile phase is -0.1% formic acid solution of methanol (60: 40).
Chinese patent application CN201610395891.4 disclose a kind of high efficiency liquid chromatography for separating and determining butylphenyl phthaleine and
Its method in relation to substance.This method is by high performance liquid chromatograph, using octadecylsilane chemically bonded silica as filler,
0.2% disodium hydrogen phosphate and acetonitrile are that mobile phase carries out gradient elution, and Detection wavelength is 226~230nm.This method solve neighbours
What phthalic acid, intermediate compound I, intermediate II, propyl benzene phthalein, butylidene phthalide, double butylphthalide impurity and principal component were difficult to separate
Problem.It does not include -1 (3H)-isobenzofuranone of phthalide and 3- normal-butyl -3- hydroxyl in the related object of the patent, it can not be to having
It closes substance phthalide and -1 (3H)-isobenzofuranone of 3- normal-butyl -3- hydroxyl realizes separation determination.In addition to this, which remembers
The measuring method of load cannot be efficiently separated and be detected to other phthalide derivant impurity except above six kinds of impurity.
Summary of the invention
The purpose of the present invention is to provide a kind of efficient liquid phase detection method, this method passes through optimization chromatographic condition and gradient
Elution requirement realizes efficiently separating for more than ten phthalide derivants, and has many advantages, such as that specificity is good, and high sensitivity can be used for
Separation, assay, preparation purifying or its monitoring in relation to substance of phthalide derivant.
One of the objects of the present invention is to provide it is a kind of efficiently separate detection phthalide analog derivative high performance liquid chromatography,
It is characterized in that, this method uses phenyl-hexyl chromatographic column or phenyl column, Detection wavelength is 226-230nm or 280nm, flow velocity
For 0.8-1.2ml/min, column temperature is 20-35 DEG C, and sample volume is 10-100 μ l, and mobile phase is by A phase and B phase composition, wherein flowing
A phase in phase: the volume ratio that B is mixed is 60:40-80:20, and A phase is the aqueous solution of pH value 3-5, the B phase be by acetonitrile and
The mixed solvent of methanol composition, the volume ratio of acetonitrile and methanol is 1:0.8-1.2 in B phase.
In the preferred technical solution of the present invention, A phase in the mobile phase: the mixed volume ratio of B phase is 62:38-75:
25。
In the preferred technical solution of the present invention, when 0-40min, A phase in the mobile phase: the mixed volume ratio of B phase is
60:40-80:20, A phase in preferable mobile phase: the mixed volume ratio of B phase is 62:38-75:25.
In the preferred technical solution of the present invention, when 40-50min, A phase in the mobile phase: the mixed volume ratio of B phase is
40:60-55:45, A phase in preferable mobile phase: the mixed volume ratio of B phase is 45:55-49:51.
In the preferred technical solution of the present invention, the A phase is the acetic acid aqueous solution of 0.08-0.12% volume ratio, preferably A
The volume ratio of phase acetic acid aqueous solution be selected from 0.09%, 0.10%, 0.11% it is any.
In the preferred technical solution of the present invention, the volume ratio of acetonitrile and methanol is selected from 1:0.8,1:0.9,1 in the B phase:
1,1:1.1,1:1.2's is any.
In the preferred technical solution of the present invention, retarder thinner is described for configuring test solution or reference substance solution
Retarder thinner be 20%-40% volume ratio acetonitrile solution, preferably the volume ratio of acetonitrile solution be selected from 20%, 25%,
30%, 40% it is any.
In the preferred technical solution of the present invention, the column temperature of chromatographic column is selected from 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C any.
In the preferred technical solution of the present invention, sample volume be selected from 10 μ l, 20 μ l, 50 μ l, 100 μ l it is any.
In the preferred technical solution of the present invention, condition of gradient elution is selected from table 1, table 2, table 3 any the A phase and B phase
Volume ratio composition.
In the preferred technical solution of the present invention, in 50-50.1min, small molecule pole is rinsed using a high proportion of organic phase
Property substance, elution reduction chromatographic column when 50.1-60min.
Table 1
| Time (minute) | 0 | 5 | 40 | 50 | 50.1 | 60 |
| A% | 62 | 62 | 45 | 30 | 62 | 62 |
| B% | 38 | 38 | 55 | 70 | 38 | 38 |
Table 2
| Time (minute) | 0 | 5 | 40 | 50 | 50.1 | 60 |
| A% | 65 | 65 | 45 | 30 | 30 | 65 |
| B% | 35 | 35 | 55 | 70 | 70 | 35 |
Table 3
| Time (minute) | 0 | 5 | 40 | 50 | 50.1 | 60 |
| A% | 75 | 70 | 49 | 35 | 75 | 75 |
| B% | 25 | 30 | 51 | 65 | 25 | 25 |
In the preferred technical solution of the present invention, the phthalide analog derivative is selected from phthalide-type described in phthalide analog derivative
Derivative be selected from adjacent carboxyl benzyl alcohol, 2-carboxybenzaldehyde, phthalide, methyl phthalide, ethyl phthalide, propyl phthalide, amyl phthalide,
Isobutyl group phthalide, sec-butyl phthalide, tert-butyl phthalide, 2- (ɑ-carbonyl amyl) benzoic acid, 2- (ɑ-Hydroxy pentyl) benzoic acid or
Its pharmaceutical salts or its acid group, -1 (3H)-isobenzofuranone of 3- normal-butyl -3- hydroxyl, butylphenyl phthaleine, butylidene phthalide it is any
Or combinations thereof, preferably 2- (ɑ-Hydroxy pentyl) benzoic acid or its officinal salt or its acid group, 3- normal-butyl -3- hydroxyl -1
(3H)-isobenzofuranone, butylidene phthalide, butylphenyl phthaleine it is any or combinations thereof.
In the preferred technical solution of the present invention, the officinal salt of the 2- (ɑ-Hydroxy pentyl) benzoic acid is selected from its sodium
Salt, sylvite, lithium salts, calcium salt, magnesium salts, zinc salt it is any.
In the preferred technical solution of the present invention, the phenyl-hexyl chromatographic column or phenyl column be it is commercially available, preferably
The phenyl-hexyl chromatographic column or phenyl column of Agilent or Kinetex company production, more preferably Zorbax Eclipse
Plus、Kinetex Phenyl-Hexyl、ZORBAX Phenyl、ZORBAX Eclipse XDB-Phenyl、ZORBAX SB-
Phenyl, Ultimate XB-Phenyl, Agilent ZORBAX plus Pheny-Hexyl's is any or combinations thereof.
In the preferred technical solution of the present invention, the column length of the phenyl-hexyl chromatographic column or phenyl column be 4.6mm ×
250mm, preferably 5 μm of packing material size.
Another object of the present invention is to provide the efficient liquid of the present invention for efficiently separating detection phthalide analog derivative
Phase chromatography is for separation detection phthalide analog derivative in relation to the application in the method for substance, which is characterized in that this method uses
Phenyl-hexyl chromatographic column or phenyl column, Detection wavelength are 226-230nm or 280nm, flow velocity 0.8-1.2ml/min, and column temperature is
20-35 DEG C, sample volume is 10-100 μ l, and mobile phase is by A phase and B phase composition, wherein A phase in mobile phase: the volume that B is mixed
Than for 60:40-80:20, A phase is the aqueous solution of pH value 3-5, and the B phase is the mixed solvent being made of acetonitrile and methanol, B
The volume ratio of acetonitrile and methanol is 1:0.8-1.2 in phase.
The present invention is in relation in the optimal technical scheme of substance-measuring, A phase in the mobile phase: the mixed volume ratio of B phase
For 62:38-75:25.
The present invention is in relation in the optimal technical scheme of substance-measuring, when 0-40min, A phase in the mobile phase: B phase
Mixed volume ratio is 60:40-80:20, A phase in preferable mobile phase: the mixed volume ratio of B phase is 62:38-75:25.
The present invention is in relation in the optimal technical scheme of substance-measuring, when 40-50min, A phase in the mobile phase: B phase
Mixed volume ratio is 40:60-55:45, A phase in preferable mobile phase: the mixed volume ratio of B phase is 45:55-49:51.
The present invention is in relation in the optimal technical scheme of substance-measuring, the A phase is the acetic acid of 0.08-0.12% volume ratio
Aqueous solution, the preferably volume ratio of A phase acetic acid aqueous solution be selected from 0.09%, 0.10%, 0.11% it is any.
The present invention is in relation in the optimal technical scheme of substance-measuring, the volume ratio of acetonitrile and methanol is selected from the B phase
1:0.8,1:0.9,1:1,1:1.1,1:1.2's is any.
The present invention is in relation in the optimal technical scheme of substance-measuring, retarder thinner is for configuring test solution or reference substance
Solution, the retarder thinner are the acetonitrile solution of 20%-40% volume ratio, and the volume ratio of preferably acetonitrile solution is selected from
20%, 25%, 30%, 40% it is any.
The present invention is in relation in the optimal technical scheme of substance-measuring, the column temperature of chromatographic column is selected from 20 DEG C, 25 DEG C, 30 DEG C, 35
DEG C it is any.
The present invention is in relation in the optimal technical scheme of substance-measuring, the sample volume is selected from 10 μ l, 20 μ l, 50 μ l, 100 μ l
It is any.
The present invention is in relation in the optimal technical scheme of substance-measuring, condition of gradient elution is selected from table 1, table 2, any institute of table 3
The volume ratio of the A phase stated and B phase forms.
The present invention is in relation in the optimal technical scheme of substance-measuring, the phthalide derivant is selected from 2- (ɑ-Hydroxy pentyl) benzene
Formic acid or its officinal salt, -1 (3H)-isobenzofuranone of 3- normal-butyl -3- hydroxyl, butylidene phthalide, butylphenyl phthaleine it is any
Or combinations thereof.
The present invention in relation in the optimal technical scheme of substance-measuring, the 2- (ɑ-Hydroxy pentyl) benzoic acid it is pharmaceutically acceptable
Salt is selected from any of sodium salt, sylvite, lithium salts, calcium salt, magnesium salts or zinc salt.
The present invention is in relation in the optimal technical scheme of substance-measuring, the related substance is selected from phthalide, 2- (ɑ-carbonyl penta
Base) benzoic acid, -1 (3H)-isobenzofuranone of 3- normal-butyl -3- hydroxyl, propyl phthalide, adjacent carboxyl benzyl alcohol, adjacent carboxyl benzene first
Aldehyde, isobutyl group phthalide it is any or combinations thereof.
The present invention is in relation in the optimal technical scheme of substance-measuring, the related substance is selected from phthalide and 2- (ɑ-carbonyl
Amyl) combination of benzoic acid, the combination of -1 (3H)-isobenzofuranone of 3- normal-butyl -3- hydroxyl and propyl phthalide, adjacent carboxyl benzene
The combination of methanol and 2-carboxybenzaldehyde, the combination of isobutyl group phthalide and butylphenyl phthaleine it is any.
The present invention in relation in the optimal technical scheme of substance-measuring, the related substance further comprise methyl phthalide,
Ethyl phthalide, amyl phthalide, sec-butyl phthalide, tert-butyl phthalide, 2- (ɑ-Hydroxy pentyl) benzoic acid officinal salt or acid group, fourth
Any of or a combination of alkenyl phthalide.
The present invention is in relation in the optimal technical scheme of substance-measuring, the phthalide derivant is selected from 2- (ɑ-Hydroxy pentyl)
When benzoic acid or its officinal salt, related substance does not include 2- (ɑ-Hydroxy pentyl) benzoic acid or its officinal salt or its acid group.
The present invention is in relation in the optimal technical scheme of substance-measuring, the phthalide derivant is selected from 3- normal-butyl -3- hydroxyl
When base -1 (3H)-isobenzofuranone, the related substance does not include -1 (3H)-isobenzofuranone of 3- normal-butyl -3- hydroxyl.
The present invention is in relation in the optimal technical scheme of substance-measuring, when the phthalide analog derivative is selected from butylphenyl phthaleine, institute
Stating related substance does not include butylphenyl phthaleine.
The present invention is in relation in the optimal technical scheme of substance-measuring, the phthalide analog derivative is selected from butylidene phthalide
When, the related substance does not include butylidene phthalide.
Another object of the present invention is to provide the efficient liquid of the present invention for efficiently separating detection phthalide analog derivative
Phase chromatography is for the application in separation detection phthalide-type derivative content method, which is characterized in that this method using phenyl-oneself
Base chromatographic column or phenyl column, Detection wavelength are 226-230nm or 280nm, flow velocity 0.8-1.2ml/min, column temperature 20-35
DEG C, sample volume is 10-100 μ l, and mobile phase is by A phase and B phase composition, wherein A phase in mobile phase: the volume ratio that B is mixed is
60:40-80:20, A phase are the aqueous solution of pH value 3-5, and the B phase is the mixed solvent being made of acetonitrile and methanol, in B phase
The volume ratio of acetonitrile and methanol is 1:0.8-1.2.
In the optimal technical scheme of assay of the present invention, A phase in the mobile phase: the mixed volume ratio of B phase is 62:
38-75:25。
In the optimal technical scheme of assay of the present invention, when 0-40min, A phase in the mobile phase: the mixing of B phase
Volume ratio is 60:40-80:20, A phase in preferable mobile phase: the mixed volume ratio of B phase is 62:38-75:25.
In the optimal technical scheme of assay of the present invention, when 40-50min, A phase in the mobile phase: the mixing of B phase
Volume ratio is 40:60-55:45, A phase in preferable mobile phase: the mixed volume ratio of B phase is 45:55-49:51.
In the optimal technical scheme of assay of the present invention, the A phase is that the acetic acid of 0.08-0.12% volume ratio is water-soluble
Liquid, the preferably volume ratio of A phase acetic acid aqueous solution be selected from 0.09%, 0.10%, 0.11% it is any.
In the optimal technical scheme of assay of the present invention, the volume ratio of acetonitrile and methanol is selected from 1 in the B phase:
0.8,1:0.9,1:1,1:1.1,1:1.2's is any.
In the optimal technical scheme of assay of the present invention, retarder thinner is used to configure test solution or reference substance is molten
Liquid, the retarder thinner are the acetonitrile solution of 20%-40% volume ratio, and the volume ratio of preferably acetonitrile solution is selected from
20%, 25%, 30%, 40% it is any.
In the optimal technical scheme of assay of the present invention, the column temperature of chromatographic column is selected from 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C
It is any.
In the optimal technical scheme of assay of the present invention, the sample volume is selected from 10 μ l, 20 μ l, 50 μ l, 100 μ l
It is any.
In the optimal technical scheme of assay of the present invention, condition of gradient elution is selected from any A of table 1, table 2, table 3
Mutually formed with the volume ratio of B phase.
In the optimal technical scheme of assay of the present invention, the phthalide analog derivative is selected from adjacent carboxyl benzyl alcohol, neighbour
Carboxyl benzaldehyde, phthalide, methyl phthalide, ethyl phthalide, propyl phthalide, amyl phthalide, isobutyl group phthalide, sec-butyl phthalide, uncle
Butylphthalide, 2- (ɑ-carbonyl amyl) benzoic acid, 2- (ɑ-Hydroxy pentyl) benzoic acid or its pharmaceutical salts or the positive fourth of its acid group, 3-
- 1 (3H)-isobenzofuranone of base -3- hydroxyl, butylphenyl phthaleine, butylidene phthalide it is any or combinations thereof.
In the optimal technical scheme of assay of the present invention, the officinal salt of 2- (ɑ-Hydroxy pentyl) benzoic acid is selected from
Its sodium salt, sylvite, lithium salts, calcium salt, magnesium salts or zinc salt it is any.
In the optimal technical scheme of assay of the present invention in the preferred technical solution of the present invention, the phthalide derivant contains
Measuring the test sample concentration in fixed is 0.25-3mg/ml, preferably 0.5-2mg/ml.
Under chromatographic condition of the invention, more than ten phthalide analog derivatives can be efficiently separated.Therefore, stationary phase of the invention
It can also be applied in preparation chromatography with mobile phase selection.Another object of the present invention is to provide a kind of phthalide analog derivatives
Preparation method, which is characterized in that this method uses phenyl-hexyl chromatographic column or phenyl column as stationary phase, mobile phase by A phase and
B is mixed, wherein the aqueous solution that A:B mixed volume ratio 60:40-80:20, A phase is pH value 3-5, the B phase are served as reasons
The mixed solvent of acetonitrile and methanol composition, the volume ratio of acetonitrile and methanol is 1:0.8-1.2 in B phase, and the flow velocity of mobile phase is
0.8-1.2ml/min, column temperature: 20-35 DEG C.
In preparing the optimal technical scheme that phthalide analog derivative isolates and purifies, the mobile phase is more preferably this
Mobile phase in the invention high performance liquid chromatography.
In preparing the optimal technical scheme that phthalide analog derivative isolates and purifies, A phase in the mobile phase: B phase is mixed
Conjunction volume ratio is 62:38-75:25.
In preparing the optimal technical scheme that phthalide analog derivative isolates and purifies, A phase is the second of 0.08-0.12% volume ratio
Aqueous acid, the preferably volume ratio of A phase acetic acid aqueous solution be selected from 0.09%, 0.10%, 0.11% it is any.
In preparing the optimal technical scheme that phthalide analog derivative isolates and purifies, the body of acetonitrile and methanol in the B phase
Product is than any selected from 1:0.8,1:0.9,1:1,1:1.1,1:1.2.
In preparing the optimal technical scheme that phthalide analog derivative isolates and purifies, phthalide analog derivative is selected from adjacent carboxyl benzene first
Alcohol, 2-carboxybenzaldehyde, phthalide, methyl phthalide, ethyl phthalide, propyl phthalide, amyl phthalide, isobutyl group phthalide, sec-butylbenzene
Phthalein, tert-butyl phthalide, 2- (ɑ-carbonyl amyl) benzoic acid, 2- (ɑ-Hydroxy pentyl) benzoic acid or its pharmaceutical salts or its acid group, 3-
- 1 (3H)-isobenzofuranone of normal-butyl -3- hydroxyl, butylphenyl phthaleine, butylidene phthalide it is any or combinations thereof.
In preparing the optimal technical scheme that phthalide analog derivative isolates and purifies, phthalide analog derivative be chromatographic isolation degree compared with
Big substance is preferably selected from phthalide, methyl phthalide, 2- (ɑ-carbonyl amyl) benzoic acid, amyl phthalide, sec-butyl phthalide, tertiary fourth
Any or combinations thereof, the more preferably maximum phthalide of chromatographic isolation degree, methyl phthalide, 2- (ɑ-carbonyl amyl) benzene of base phthalide
Formic acid it is any or combinations thereof.
In preparing the optimal technical scheme that phthalide analog derivative isolates and purifies, separates and collects and obtain solubilized target phthalide-type
After the elution solution of substance, through evaporation, separation, washing, drying steps, the target phthalide substance of high-purity is made.
In preparing the optimal technical scheme that phthalide analog derivative isolates and purifies, the evaporation, which is selected from, to be evaporated under reduced pressure, revolves
Turn any or combinations thereof of evaporation.
In preparing the optimal technical scheme that phthalide analog derivative isolates and purifies, the separation be selected from be separated by filtration, from
Heart separation, settle and separate it is any or combinations thereof.
In preparing the optimal technical scheme that phthalide analog derivative isolates and purifies, the washing is solvent washing.
In preparing the optimal technical scheme that phthalide analog derivative isolates and purifies, the drying is selected from and is dried under reduced pressure, is cold
Be lyophilized dry, vacuum drying, spray drying it is any or combinations thereof.
In preparing the optimal technical scheme that phthalide analog derivative isolates and purifies, 2- (ɑ-Hydroxy pentyl) benzoic acid can
Pharmaceutical salts be selected from its sodium salt, sylvite, lithium salts, calcium salt, magnesium salts, zinc salt it is any.
High-purity target phthalide substance obtained is isolated and purified another object of the present invention is to provide the present invention to be used as
Application in standard items or reference substance.
In the preferred technical solution of the present invention, the purity of the target phthalide substance is not less than 99.0%, preferably not
Lower than 99.5%, more desirably not less than 99.9%.
In order to clearly state protection scope of the present invention, the present invention is bound following terms:
Acid group of the present invention is phthalide analog derivative of the present invention or its officinal salt in the solution with ionization
Anion part existing for form.
Retarder thinner of the invention is for dissolving and diluting reference substance solution or test solution.
Unless otherwise indicated, the present invention relates to when the percentage between liquid and liquid, the percentage is volume/body
Product percentage；The present invention relates to when percentage between liquid and solid, the percentage is volume/weight percentage；This hair
When the bright percentage being related between solid and liquid, the percentage is weight/volume percent；Remaining is w/w hundred
Divide ratio.
Compared with prior art, the present invention have it is following the utility model has the advantages that
1, the chromatographic condition and condition of gradient elution of high performance liquid chromatography have been screened in present invention research, obtain a kind of efficient
The high performance liquid chromatography of separation detection phthalide analog derivative.This method has specificity is good, separating degree is high, sensitivity is excellent etc.
Efficiently separating for more than ten phthalide derivants may be implemented in advantage, can be used for the separation of phthalide derivant, assay or its have
Close substance monitoring and detection, preferably to control the quality of these raw materials and its preparation, with ensure drug validity and
Safety, and then realize that drug quality is controllable.
2, present invention research efficiently separates the high performance liquid chromatography of detection phthalide analog derivative, has following unexpected
Technical effect be conducive to phthalide and 2- (ɑ-carbonyl first is that effectively improve the separating degree of phthalide and 2- (ɑ-carbonyl amyl) benzoic acid
Base amyl) benzoic acid separation detection and content control；Second is that effectively improving 3- normal-butyl -3- hydroxyl -1 (3H)-different benzo
The separating degree of furanone and propyl phthalide is conducive to point of -1 (3H)-isobenzofuranone of 3- normal-butyl -3- hydroxyl and propyl phthalide
It is controlled from detection and content；Third is that effectively improve the separating degree of isobutyl group phthalide and butylphenyl phthaleine, be conducive to isobutyl group phthalide with
The separation and detection of butylphenyl phthaleine and content control；Fourth is that effectively improving the separation of adjacent carboxyl benzyl alcohol and 2-carboxybenzaldehyde
Degree controls conducive to the separation and detection of adjacent carboxyl benzyl alcohol and 2-carboxybenzaldehyde and content.Therefore, of the invention to efficiently separate
The high performance liquid chromatography of detection phthalide analog derivative can be used for the quality control of phthalide-type raw material and its preparation, to ensure drug
Quality, safety and validity strong technical guarantee is provided.
3, the high performance liquid chromatography for efficiently separating detection phthalide analog derivative of the invention realizes more than ten phthalides and spreads out
Biology efficiently separates, it may also be used for the target phthalide derivant for preparing high-purity prepares resulting high-purity target phthalide-type
Substance is used as the application in standard items or reference substance.
Detailed description of the invention
1 sample chromatogram figure of Fig. 1 comparative example；
2 sample chromatogram figure of Fig. 2 comparative example；
3 sample chromatogram figure of Fig. 3 comparative example；
1 sample chromatogram figure of Fig. 4 embodiment；
2 sample chromatogram figure of Fig. 5 embodiment；
3 sample chromatogram figure of Fig. 6 embodiment；
4 sample chromatogram figure of Fig. 7 embodiment.
Specific embodiment
The present invention is illustrated below with reference to embodiment.The embodiment of the present invention is merely to illustrate technical side of the invention
Case, and non-limiting essence of the invention.
Test compound and purity of the invention is shown in Table 4.
Table 4
Hplc device: Shimadzu LC-20A high performance liquid chromatograph, SPD-20A UV detector.
Chromatographic column: Agilent ZORBAX plus Pheny-Hexyl 4.6mm × 250mm, 5 μm；Or Féraud door
Titank C18,4.6mm × 250mm, 5 μm；Or GL-C18,4.6mm × 250mm, 5 μm.
Comparative example 1
Referring to butylphenyl phthaleine national drug standards WS1- (X-124) -2005Z, using HPLC method separation detection butylphenyl phthaleine and eight
The related substance of kind.
Chromatographic column: Féraud door Titank C18,4.6mm × 250mm, 5 μm；
Mobile phase: methanol-water (65:35)；
Detection wavelength 280nm, flow velocity: 1.0ml/min, column temperature: 30 DEG C, sample volume: 20 μ l.
Retarder thinner: methanol.
Solution is prepared and measuring method: precision weighs butylphenyl phthaleine, 2- (ɑ-Hydroxy pentyl) Potassium Benzoate, phthalide, 2- (ɑ-carbonyl
Amyl) benzoic acid, ethyl phthalide, -1 (3H)-isobenzofuranone of 3- normal-butyl -3- hydroxyl, propyl phthalide, amyl phthalide and fourth
Alkenyl phthalide is each appropriate, the use of methanol is solvent, is configured to 0.5mg/ml containing butylphenyl phthaleine, contains 2- (ɑ-Hydroxy pentyl) benzene first
Sour potassium, phthalide, 2- (ɑ-carbonyl amyl) benzoic acid, ethyl phthalide, -1 (3H)-isobenzofuranone of 3- normal-butyl -3- hydroxyl, third
The mixed solution of base phthalide, amyl phthalide and each 5 μ g/ml of butylidene phthalide, as test solution；It is molten that precision measures test sample
Liquid 1ml is placed it in 100ml measuring bottle, is added methanol dilution to scale, is shaken up, as contrast solution.According to above-mentioned chromatographic condition,
Precision measures test solution and each 20 μ l of contrast solution, is injected separately into liquid chromatograph, records chromatogram.Sample chromatogram figure
See Fig. 1, the experimental data of test sample is shown in Table 5.
Under the chromatographic condition, the localizing sample solution of phthalide, 2- (ɑ-carbonyl amyl) benzoic acid is respectively configured.As a result it shows
Show, the retention time that phthalide positions peak is 4.668min, and the retention time at 2- (ɑ-carbonyl amyl) benzoic acid positioning peak is
4.847min。
Table 5
Positioning peak and its retention time based on aforementioned phthalide, 2- (ɑ-carbonyl amyl) benzoic acid, determine sample chromatogram
The chromatographic peak that retention time is 4.627min in Fig. 1 and table 6 is the coincidence peak of phthalide and 2- (ɑ-carbonyl amyl) benzoic acid.
By Fig. 1 and table 6 as it can be seen that phthalide and 2- (ɑ-carbonyl amyl) benzoic acid can not effectively be divided under the chromatographic condition
From, it is impossible to be used in separation detection and its drug matter in relation to containing the drug of phthalide and 2- (ɑ-carbonyl amyl) benzoic acid in substance
Amount control.And under the chromatographic condition, the separating degree of 3- normal-butyl -3- hydroxyl -1 (3H)-isobenzofuranone and propyl phthalide
Only 1.00, the requirement that separating degree is greater than 1.5 is not met, needs to optimize its separation condition, preferably control drug quality.
Comparative example 2
Chromatographic condition:
Chromatographic column: Agilent ZORBAX plus Pheny-Hexyl, 4.6mm × 250mm, 5 μm.
Mobile phase: A phase is 0.1% acetic acid aqueous solution (volume ratio)；B phase is acetonitrile.
Detection wavelength 280nm, flow velocity: 1.0ml/min, column temperature: 30 DEG C, sample volume: 10 μ l.
Solvent: 30% acetonitrile solution (volume ratio).
Condition of gradient elution is shown in Table 6.
Table 6
| Time (minute) | 0 | 5 | 40 | 50 | 50.1 | 60 |
| A% | 75 | 70 | 49 | 35 | 75 | 75 |
| B% | 25 | 30 | 51 | 65 | 25 | 25 |
Solution is prepared and measuring method:
It is appropriate that precision weighs butylphenyl phthaleine, -1 (3H)-isobenzofuranone of 3- normal-butyl -3- hydroxyl, propyl phthalide, uses
30% acetonitrile solution (volume ratio) is configured to 2mg/ml containing butylphenyl phthaleine, impure 3- normal-butyl -3- hydroxyl -1 (3H)-different benzo furan
It mutters the mixed solution of each 20 μ g/ml of ketone, propyl phthalide, as test solution；According to above-mentioned chromatographic condition, test solution 10 is taken
μ l injects liquid chromatograph, records chromatogram.Test solution chromatogram is shown in Fig. 2, and the experimental data of test sample is shown in Table 7.
Table 7
By Fig. 2 and table 7 as it can be seen that under the chromatographic condition, -1 (3H)-isobenzofuranone of 3- normal-butyl -3- hydroxyl and third
The separating degree of base phthalide is only 0.78, does not meet the requirement that efficient liquid phase is greater than 1.5 about separating degree.
Comparative example 3
Chromatographic condition: chromatographic column GL-C18,4.6mm × 250mm, 5 μm.
Mobile phase: A phase is 20mmol/L K2HPO4 solution, phosphoric acid tune pH to 7.5；B phase is acetonitrile.
Detection wavelength 280nm flow velocity: 1.0ml/min, column temperature: 30 DEG C, sample volume: 20 μ l.
Retarder thinner: A phase-B phase (80:20) (volume ratio).
Condition of gradient elution is shown in Table 8.
Table 8
| Time (minute) | 0 | 5 | 30 | 45 | 45.1 | 55 |
| A% | 20 | 25 | 63 | 63 | 20 | 20 |
| B% | 80 | 75 | 37 | 37 | 80 | 80 |
Solution is prepared and measuring method:
Precision weighs butylphenyl phthaleine, 2- (ɑ-Hydroxy pentyl) Potassium Benzoate, phthalide, 2- (ɑ-carbonyl amyl) benzoic acid, methyl
Phthalide, ethyl phthalide, propyl phthalide, amyl phthalide, adjacent carboxyl benzyl alcohol, 2-carboxybenzaldehyde, isobutyl group phthalide, sec-butylbenzene
Phthalein, isobutyl group phthalide are each appropriate, are configured to using retarder thinner containing butylphenyl phthaleine about 0.5mg/ml, 2- (ɑ-Hydroxy pentyl) benzene
Potassium formate, phthalide, 2- (ɑ-carbonyl amyl) benzoic acid, methyl phthalide, ethyl phthalide, propyl phthalide, amyl phthalide, adjacent carboxyl benzene
The mixed solution of each about 1 μ g/ml of methanol, 2-carboxybenzaldehyde, isobutyl group phthalide, sec-butyl phthalide, isobutyl group phthalide, as confession
Test sample solution；The concentration that isobutyl group phthalide individually positions solution is 5 μ g/ml.According to above-mentioned chromatographic condition, it is molten that precision measures test sample
Liquid and isobutyl group phthalide position each 20 μ l of solution, are injected separately into liquid chromatograph, record chromatogram.Test solution chromatogram is shown in
The experimental data of Fig. 3, test sample are shown in Table 9.
Table 9
Under above-mentioned chromatographic condition, the retention time for individually measuring isobutyl group phthalide positioning solution positioning peak is
32.649min。
As seen from Figure 3, the weight that sec-butyl phthalide is 31.798 and 31.991 because of two optical isomers presentation retention times
It folds bimodal.Retention time in conjunction with isobutyl group phthalide positioning peak is 32.649min, determines that retention time is 32.914min in Fig. 3
Chromatographic peak be that isobutyl group phthalide and butylphenyl phthaleine are overlapped peak.And in Fig. 3 adjacent carboxyl benzyl alcohol and 2-carboxybenzaldehyde separation
Degree is 0.333, does not meet the requirement that separating degree is greater than 1.5.Therefore, it is necessary to optimize chromatographic condition, adjacent carboxyl benzene first is efficiently separated
Alcohol and 2-carboxybenzaldehyde, isobutyl group phthalide and butylphenyl phthaleine make it meet the requirement that efficient liquid phase separating degree is greater than 1.5, in turn
Aforementioned related substance and its content in drug are preferably separated and monitored, it is controllable to ensure that drug quality is realized.
Embodiment 1
Using identical substance in HPLC measurement comparative example 1, C18 column is adjusted to Phenylhexyl column, adjustment mobile phase is simultaneously
Carry out gradient elution.
Chromatographic column: Agilent ZORBAX plus Pheny-Hexyl, 4.6mm × 250mm, 5 μm.
Mobile phase: A phase is 0.1% acetic acid aqueous solution (volume ratio)；B phase is acetonitrile: methanol (1:1, volume ratio).
Detection wavelength 227nm, flow velocity: 1.0ml/min, column temperature: 30 DEG C, sample volume: 50 μ l.
Retarder thinner: 30% acetonitrile solution (volume ratio).
Condition of gradient elution is shown in Table 10.
Table 10
Solution is prepared and measuring method
Precision weighs butylphenyl phthaleine, 2- (ɑ-Hydroxy pentyl) Potassium Benzoate, phthalide, 2- (ɑ-carbonyl amyl) benzoic acid, ethyl
Phthalide, -1 (3H)-isobenzofuranone of 3- normal-butyl -3- hydroxyl, propyl phthalide, amyl phthalide and butylidene phthalide are each appropriate,
The use of 30% acetonitrile solution is retarder thinner, is configured to 0.25mg/ml containing butylphenyl phthaleine, impure 2- (ɑ-Hydroxy pentyl)
Potassium Benzoate, phthalide, 2- (ɑ-carbonyl amyl) benzoic acid, ethyl phthalide, -1 (3H)-isobenzofuran of 3- normal-butyl -3- hydroxyl
Ketone, propyl phthalide, amyl phthalide and each 2.5 μ g/ml of butylidene phthalide mixed solution, as test solution；Precision measures
Test solution 1ml is configured to the solution of the 2.5 μ g/ml containing butylphenyl phthaleine with 30% acetonitrile solution, is shaken up, molten as compareing
Liquid.According to above-mentioned chromatographic condition, precision measures test solution and each 50 μ l of contrast solution, is injected separately into liquid chromatograph, records
Chromatogram.Sample chromatogram figure is shown in Fig. 4, and the experimental data of test sample is shown in Table 11.
Table 11
By Fig. 4 and table 11 as it can be seen that under the chromatographic condition, the retention time of phthalide is 7.232,2- (ɑ-carbonyl amyl) benzene
The retention time of formic acid is 21.096.For comparative example 1, embodiment 1 can be by phthalide and 2- (ɑ-carbonyl amyl) benzene
The coincidence peak of formic acid is effectively separated, and the separation of 3- normal-butyl -3- hydroxyl -1 (3H)-isobenzofuranone and propyl phthalide
Degree is 2.14, conducive to separating and monitoring aforementioned related substance and its content in drug, realizes that drug quality is controllable.
Embodiment 2
Chromatographic column: Agilent ZORBAX plus Pheny-Hexyl, 4.6mm × 250mm, 5 μm.
Mobile phase: A phase is 0.1% acetic acid aqueous solution；B phase is acetonitrile: methanol (1:1) solution.
Detection wavelength is 280nm, flow velocity: 1.0ml/min, column temperature: 30 DEG C, sample volume: 10 μ l.
Solvent: 35% acetonitrile solution (volume ratio)；
Test sample concentration: 3mg/ml.
Condition of gradient elution is shown in Table 12.
Table 12
| Time (minute) | 0 | 5 | 40 | 50 | 50.1 | 60 |
| A% | 75 | 70 | 49 | 35 | 75 | 75 |
| B% | 25 | 30 | 51 | 65 | 25 | 25 |
Solution is prepared and measuring method:
Precision weighs butylphenyl phthaleine, 2- (ɑ-Hydroxy pentyl) Potassium Benzoate, phthalide, 2- (ɑ-carbonyl amyl) benzoic acid, methyl
Phthalide, ethyl phthalide, -1 (3H)-isobenzofuranone of 3- normal-butyl -3- hydroxyl, propyl phthalide, amyl phthalide, adjacent carboxyl benzene first
Alcohol, 2-carboxybenzaldehyde, isobutyl group phthalide, sec-butyl phthalide, isobutyl group phthalide are each appropriate, use 35% acetonitrile solution (body
Product ratio) it is retarder thinner, it is configured to 3mg/ml containing butylphenyl phthaleine, 2- (ɑ-Hydroxy pentyl) Potassium Benzoate, phthalide, 2- (ɑ-carbonyl
Base amyl) benzoic acid, methyl phthalide, ethyl phthalide, -1 (3H)-isobenzofuranone of 3- normal-butyl -3- hydroxyl, propyl phthalide,
Amyl phthalide, adjacent carboxyl benzyl alcohol, 2-carboxybenzaldehyde, isobutyl group phthalide, sec-butyl phthalide, each 3 μ g/ml of isobutyl group phthalide
Mixed solution, as test solution；According to above-mentioned chromatographic condition, precision measures 10 μ l of test solution, injects liquid chromatograph,
Record chromatogram.Sample chromatogram figure is shown in Fig. 5, and the experimental data of test sample is shown in Table 13.
Table 13
Embodiment 2 optimizes the flowing phase composition of comparative example 2, i.e., adds methanol in organic B phase.Relative to comparative example 1
For, the coincidence peak of phthalide and 2- (ɑ-carbonyl amyl) benzoic acid can be efficiently separated.For comparative example 1-2, propyl
The separating degree of phthalide and -1 (3H)-isobenzofuranone of 3- normal-butyl -3- hydroxyl reaches 2.97, meets the related separation of efficient liquid phase
The requirement of degree.For comparative example 3, the separating degree of adjacent carboxyl benzyl alcohol and 2-carboxybenzaldehyde is 1.95, isobutyl-benzene
The separating degree of phthalein and butylphenyl phthaleine is 2.10, and other impurities and impurity, and separating degree all meets regulation between impurity and principal component.
Embodiment 3
Chromatographic condition is as follows:
Chromatographic column: Agilent ZORBAX plus Pheny-Hexyl, 4.6mm × 250mm, 5 μm.
Mobile phase: A phase is 0.1% acetic acid aqueous solution, and B phase is acetonitrile: methanol (1:1).
Detection wavelength 227nm, flow velocity: 1.0ml/min, column temperature: 30 DEG C, sample volume: 20 μ l.
Solvent: 30% acetonitrile solution.
Test sample concentration: 0.25mg/ml.
Condition of gradient elution is shown in Table 14.
Table 14
| Time (minute) | 0 | 5 | 40 | 50 | 50.1 | 60 | 70 |
| A% | 65 | 65 | 45 | 30 | 30 | 65 | 65 |
| B% | 35 | 35 | 55 | 70 | 70 | 35 | 35 |
Solution is prepared and measuring method:
Precision weighs butylphenyl phthaleine, 2- (ɑ-Hydroxy pentyl) Potassium Benzoate, phthalide, 2- (ɑ-carbonyl amyl) benzoic acid, methyl
Phthalide, ethyl phthalide, -1 (3H)-isobenzofuranone of 3- normal-butyl -3- hydroxyl, propyl phthalide, amyl phthalide, adjacent carboxyl benzene first
Alcohol, 2-carboxybenzaldehyde, isobutyl group phthalide, sec-butyl phthalide, isobutyl group phthalide are each appropriate, will using 30% acetonitrile solution
It is configured to containing butylphenyl phthaleine about 0.25mg/ml, containing 2- (ɑ-Hydroxy pentyl) Potassium Benzoate, phthalide, 2- (ɑ-carbonyl amyl) benzene first
Acid, methyl phthalide, ethyl phthalide, -1 (3H)-isobenzofuranone of 3- normal-butyl -3- hydroxyl, propyl phthalide, amyl phthalide, neighbour
The mixing of each about 0.25 μ g/ml of carboxyl benzyl alcohol, 2-carboxybenzaldehyde, isobutyl group phthalide, sec-butyl phthalide, isobutyl group phthalide is molten
Liquid, as test solution.According to above-mentioned chromatographic condition, precision measures 50 μ l of test solution, injects liquid chromatograph, records color
Spectrogram.Sample chromatogram figure is shown in Fig. 6, and the experimental data of test sample is shown in Table 15.
Table 15
Under the chromatographic condition, the separating degree between 14 substances is greater than 1.5.For comparative example 1, phthalide and
The coincidence peak energy of 2- (ɑ-carbonyl amyl) benzoic acid enough efficiently separates.For comparative example 1-2, propyl phthalide and the positive fourth of 3-
The separating degree of -1 (3H)-isobenzofuranone of base -3- hydroxyl reaches 3.01.For comparative example 3, adjacent carboxyl benzyl alcohol and
The separating degree of 2-carboxybenzaldehyde is 1.89, and the separating degree of isobutyl group phthalide and butylphenyl phthaleine is 2.28.
Embodiment 4
Chromatographic column: Agilent ZORBAX plus Pheny-Hexyl, 4.6mm × 250mm, 5 μm.
Mobile phase: A phase is 0.1% acetic acid aqueous solution, and B phase is acetonitrile: methanol (1:1).
Detection wavelength 227nm, flow velocity: 1.0ml/min, column temperature: 30 DEG C, sample volume: 50 μ l.
Solvent: 30% acetonitrile solution (volume ratio).
Test sample concentration: 0.25mg/ml.
Condition of gradient elution is shown in Table 16.
Table 16
| Time (minute) | 0 | 5 | 40 | 50 | 50.1 | 60 |
| A% | 62 | 62 | 45 | 30 | 62 | 62 |
| B% | 38 | 38 | 55 | 70 | 38 | 38 |
With embodiment 3,4 sample chromatogram figure of embodiment is shown in Fig. 7 for solution preparation method and concentration.
1, quantitative limit and detection limit
The detection of 14 substances is limited to 0.55-1.05ng, is quantitatively limited to 1.05-2.10ng.
2, linearity and range
In 0.021 μ g/ml-1.145 μ g/ml concentration range, the concentration and peak area of 14 substances are in good line
Sexual intercourse, r are all larger than equal to 0.9999.
3, precision and repeatability are good
Repeatability: same batch sample measures result no significant difference replication 6 times, shows that this law repeatability is good
It is good.
4, the rate of recovery and accuracy are good
This method measure each impurity in this product high, normal, basic 3 concentration (50%, 100%, the 150% of limit concentration it is molten
Liquid), the rate of recovery of 9 parts of samples is in 90%~110% range, and its rate of recovery RSD is respectively less than 5.0%.Show testing result
Meet verifying to require, the accuracy of this method is good.
5, stability of solution is good
Related material mixing sample solution at room temperature respectively at 0h, 5h, 10h, 15h, 20h, 25h sample detection,
The RSD of each impurity peak area is respectively less than 2.0%；Related substance test solution at room temperature respectively at 0h, 5h, 10h,
15h, 20h, 25h sample detection, each impurity detected level are not detected new impurity without significant change, illustrate this product at room temperature
25h is placed, stability of solution is good.
6, durability is preferable
After this method is changed column temperature, flow velocity, buffer salinity, organic Phase Proportion, change different manufacturers chromatographic column, impurity
Detected level and the impurity number of detection have no significant change.
In system suitability solution, the separating degree between impurity and impurity, impurity and principal component is good, and durability is preferable.
Claims (10)
1. a kind of high performance liquid chromatography for efficiently separating detection phthalide analog derivative, which is characterized in that this method uses phenyl-
Hexyl chromatographic column or phenyl column, Detection wavelength are 226-230nm or 280nm, flow velocity 0.8-1.2ml/min, column temperature 20-35
DEG C, sample volume is 10-100 μ l, and mobile phase is by A phase and B phase composition, wherein A phase in mobile phase: the volume ratio that B is mixed is
60:40-80:20, A phase are the aqueous solution of pH value 3-5, and the B phase is the mixed solvent being made of acetonitrile and methanol, in B phase
The volume ratio of acetonitrile and methanol is 1:0.8-1.2.
2. high performance liquid chromatography according to claim 1, A phase in the mobile phase: the mixed volume ratio of B phase is
62:38-75:25.
3. -2 described in any item high performance liquid chromatographies according to claim 1, in 0-40min, A in the mobile phase
Phase: the mixed volume ratio of B phase is 60:40-80:20, and A phase in preferable mobile phase: the mixed volume ratio of B phase is 62:38-75:25.
4. high performance liquid chromatography according to claim 1-3, in 40-50min, A in the mobile phase
Phase: the mixed volume ratio of B phase is 40:60-55:45, and A phase in preferable mobile phase: the mixed volume ratio of B phase is 45:55-49:51.
5. high performance liquid chromatography according to claim 1-4, the A phase is 0.08-0.12% volume ratio
Acetic acid aqueous solution, preferably the volume ratio of A phase acetic acid aqueous solution be selected from 0.09%, 0.10%, 0.11% it is any.
6. high performance liquid chromatography according to claim 1-5, the volume ratio of acetonitrile and methanol choosing in the B phase
From any of 1:0.8,1:0.9,1:1,1:1.1,1:1.2.
7. high performance liquid chromatography according to claim 1-6, the phthalide analog derivative is selected from adjacent carboxyl
Benzyl alcohol, 2-carboxybenzaldehyde, phthalide, methyl phthalide, ethyl phthalide, propyl phthalide, amyl phthalide, isobutyl group phthalide, Zhong Ding
Base phthalide, tert-butyl phthalide, 2- (ɑ-carbonyl amyl) benzoic acid, 2- (ɑ-Hydroxy pentyl) benzoic acid or its pharmaceutical salts or its acid
Root, -1 (3H)-isobenzofuranone of 3- normal-butyl -3- hydroxyl, butylphenyl phthaleine, butylidene phthalide it is any or combinations thereof, preferably
For 2- (ɑ-Hydroxy pentyl) benzoic acid or its officinal salt or its acid group, -1 (3H)-isobenzofuran of 3- normal-butyl -3- hydroxyl
Ketone, butylidene phthalide, butylphenyl phthaleine it is any or combinations thereof.
8. the high performance liquid chromatography of the described in any item phthalide analog derivatives of claim 1-7 is spread out for separation detection phthalide-type
Biology is in relation to the application in substance method, and preferably the phthalide derivant is selected from 2- (ɑ-Hydroxy pentyl) benzoic acid or it can medicine
With salt, -1 (3H)-isobenzofuranone of 3- normal-butyl -3- hydroxyl, butylidene phthalide, butylphenyl phthaleine it is any or combinations thereof.
9. the high performance liquid chromatography of the described in any item phthalide analog derivatives of claim 1-7 is for detecting phthalide analog derivative
Application in content method, the phthalide analog derivative are selected from adjacent carboxyl benzyl alcohol, 2-carboxybenzaldehyde, phthalide, methylbenzene
Phthalein, ethyl phthalide, propyl phthalide, amyl phthalide, isobutyl group phthalide, sec-butyl phthalide, tert-butyl phthalide, 2- (ɑ-carbonyl amyl)
Benzoic acid, 2- (ɑ-Hydroxy pentyl) benzoic acid or its pharmaceutical salts or its acid group, 3- normal-butyl -3- hydroxyl -1 (3H)-different benzo furan
Mutter ketone, butylphenyl phthaleine, butylidene phthalide it is any or combinations thereof.
10. a kind of method for preparing phthalide analog derivative, which is characterized in that it is characterized in that, this method uses phenyl-hexyl color
Column or phenyl column are composed as stationary phase, mobile phase is mixed by A phase and B, wherein A:B mixed volume ratio 60:40-80:
20, A phases are the aqueous solution of pH value 3-5, and the B phase is the mixed solvent being made of acetonitrile and methanol, acetonitrile and methanol in B phase
Volume ratio be 1:0.8-1.2, the flow velocity of mobile phase is 0.8-1.2ml/min, column temperature: 20-35 DEG C.
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| CN113740451A (en) * | 2021-08-04 | 2021-12-03 | 京博农化科技有限公司 | Content analysis method for detecting phthalide by high performance liquid chromatography |
| CN115453014A (en) * | 2022-10-28 | 2022-12-09 | 成都施贝康生物医药科技有限公司 | Detection method of butylphthalide and related substances thereof |
| CN115469039A (en) * | 2022-10-28 | 2022-12-13 | 成都施贝康生物医药科技有限公司 | Butylphthalide and related substance detection method thereof |
| CN115453014B (en) * | 2022-10-28 | 2024-04-19 | 成都施贝康生物医药科技有限公司 | Detection method of butylphthalide and related substances thereof |
| CN115469039B (en) * | 2022-10-28 | 2024-04-26 | 成都施贝康生物医药科技有限公司 | Butylphthalide and detection method of related substances thereof |
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| CN110108818B (en) | 2020-03-17 |
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