CN110108818B - High performance liquid chromatography for high performance separation detection of phthalide derivatives and application thereof - Google Patents
High performance liquid chromatography for high performance separation detection of phthalide derivatives and application thereof Download PDFInfo
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Abstract
A high performance liquid chromatography for high performance separation detection of phthalide derivatives adopts a phenyl-hexyl chromatographic column or a phenyl column, the detection wavelength is 226-230nm or 280nm, the flow rate is 0.8-1.2ml/min, the column temperature is 20-35 ℃, the sample injection amount is 10-100 mu l, the mobile phase consists of an A phase and a B phase, and the volume ratio of the A phase to the B phase in the mobile phase is 60: 40-80:20, wherein the phase A is an aqueous solution with the pH value of 3-5, and the volume ratio of acetonitrile to methanol in the phase B is 1: 0.8-1.2. The method has the advantages of good specificity, high separation degree, excellent sensitivity and the like, can realize effective separation of more than ten phthalide derivatives, and can be used for separation, content determination or monitoring and detection, separation and purification of related substances of the phthalide derivatives so as to better control the quality of the raw materials and the preparation thereof, ensure the effectiveness and safety of the medicine and realize controllable medicine quality.
Description
Technical Field
The invention belongs to the field of pharmaceutical analysis, and particularly relates to a high performance liquid chromatography for high performance separation and detection of phthalide derivatives and application thereof.
Background
The phthalide is also called o-hydroxymethylbenzoic acid lactone, the structural characteristic is that the dicyclic fusion of gamma-lactone (ring A) and benzene (ring B) is formed by that gamma-hydroxycarboxylic acid loses 1 molecule of water, and the research shows that many phthalides have pharmacological activity and can be applied to the prevention or treatment of cardiovascular and cerebrovascular diseases, the phthalide is the third new chemical entity drug which is independently researched and developed in China, 2- (α -hydroxypentyl) benzoic acid medicinal salt enters a clinical development stage, and other documents report that 3-butenyl phthalide has the activity of inhibiting thromboxane synthesis.
The national drug standard WS1- (X-124) -2005Z of butylphthalide discloses the content of butylphthalide and a detection method of related substances. The method adopts octadecylsilane chemically bonded silica as a filler, methanol and water (65:35) as a mobile phase, the detection wavelength is 280nm, and the resolution test solution is a methanol solution containing butylphthalide and propylphthalide.
The impurity spectra of butylphthalide raw material medicines are analyzed by adopting an ultra-high performance liquid phase-triple quadrupole mass spectrometry combined technology, and the like of the Jie and the like, and the method discloses that butylphthalide generates a large amount of impurity 1(2- (1-hydroxypentyl) -benzoic acid under an alkali condition, the impurity 1 is further degraded to obtain a small amount of impurity 3(2- (1-oxopentyl) -benzoic acid), and the impurity 2 is a process impurity introduced by a phthalic anhydride method. The high performance liquid fraction adopts C18 chromatographic column, and the mobile phase is methanol-0.1% formic acid solution (60: 40).
Chinese patent application CN201610395891.4 discloses a method for separating and measuring butylphthalide and related substances thereof by high performance liquid chromatography. The method comprises the step of performing gradient elution by using a high performance liquid chromatograph, octadecylsilane chemically bonded silica as a filling agent and 0.2% disodium hydrogen phosphate and acetonitrile as mobile phases, wherein the detection wavelength is 226-230 nm. The method solves the problem that impurities and main components of phthalic acid, an intermediate I, an intermediate II, propylphthalide, butylidenephthalide and dibutylphthalide are difficult to separate. The related substances in the patent do not comprise phthalide and 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone, and the separation and measurement of the related substances of the phthalide and the 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone cannot be realized. In addition, the measurement method described in this patent cannot effectively separate and detect phthalide derivative impurities other than the above six impurities.
Disclosure of Invention
The invention aims to provide a high performance liquid detection method, which realizes effective separation of more than ten phthalide derivatives by optimizing chromatographic conditions and gradient elution conditions, has the advantages of good specificity, high sensitivity and the like, and can be used for separation, content determination, preparation and purification of the phthalide derivatives or monitoring of related substances thereof.
The invention aims to provide a high performance liquid chromatography for high performance separation and detection of phthalide derivatives, which is characterized in that a phenyl-hexyl chromatographic column or a phenyl column is adopted, the detection wavelength is 226-230nm or 280nm, the flow rate is 0.8-1.2ml/min, the column temperature is 20-35 ℃, the sample injection amount is 10-100 mu l, and a mobile phase consists of an A phase and a B phase, wherein the volume ratio of the A phase to the B phase in the mobile phase is 60: 40-80:20, wherein the phase A is an aqueous solution with the pH value of 3-5, the phase B is a mixed solvent consisting of acetonitrile and methanol, and the volume ratio of the acetonitrile to the methanol in the phase B is 1: 0.8-1.2.
In the preferred technical scheme of the invention, the mixing volume ratio of the phase A to the phase B in the mobile phase is 62: 38-75:25.
In the preferable technical scheme of the invention, the mixing volume ratio of the phase A to the phase B in the mobile phase is 60: 40-80:20, and preferably the mixing volume ratio of the phase A to the phase B in the mobile phase is 62: 38-75:25.
In the preferred technical scheme of the invention, the mixing volume ratio of the phase A to the phase B in the mobile phase is 40:60-55:45 when the time is 40-50min, and the mixing volume ratio of the phase A to the phase B in the mobile phase is preferably 45:55-49: 51.
In a preferred embodiment of the present invention, the phase a is 0.08-0.12% by volume of an aqueous acetic acid solution, and preferably the volume ratio of the phase a to the aqueous acetic acid solution is selected from any one of 0.09%, 0.10%, and 0.11%.
In a preferred embodiment of the present invention, the volume ratio of acetonitrile to methanol in the phase B is selected from any one of 1:0.8, 1:0.9, 1:1, 1:1.1, and 1: 1.2.
In a preferred embodiment of the present invention, the dilution solvent is used to prepare a test solution or a reference solution, the dilution solvent is a 20-40% by volume aqueous acetonitrile solution, and preferably the volume ratio of the aqueous acetonitrile solution is selected from any one of 20%, 25%, 30%, and 40%.
In a preferred embodiment of the present invention, the column temperature of the column is selected from any one of 20 ℃, 25 ℃, 30 ℃ and 35 ℃.
In a preferred embodiment of the present invention, the amount of the sample is selected from the group consisting of 10. mu.l, 20. mu.l, 50. mu.l and 100. mu.l.
In a preferred embodiment of the present invention, the gradient elution conditions are selected from the group consisting of the volume ratio of the phase a to the phase B as shown in any one of tables 1, 2 and 3.
In the preferred technical scheme of the invention, the organic phase with high proportion is used for washing the micromolecular polar substance within 50-50.1min, and the reduction chromatographic column is eluted within 50.1-60 min.
TABLE 1
| Time (minutes) | 0 | 5 | 40 | 50 | 50.1 | 60 |
| A％ | 62 | 62 | 45 | 30 | 62 | 62 |
| B％ | 38 | 38 | 55 | 70 | 38 | 38 |
TABLE 2
| Time (minutes) | 0 | 5 | 40 | 50 | 50.1 | 60 |
| A％ | 65 | 65 | 45 | 30 | 30 | 65 |
| B％ | 35 | 35 | 55 | 70 | 70 | 35 |
TABLE 3
| Time (minutes) | 0 | 5 | 40 | 50 | 50.1 | 60 |
| A％ | 75 | 70 | 49 | 35 | 75 | 75 |
| B％ | 25 | 30 | 51 | 65 | 25 | 25 |
In a preferred embodiment of the present invention, the phthalide derivative is selected from phthalide derivatives, and the phthalide derivative is selected from o-carboxybenzyl alcohol, o-carboxybenzaldehyde, phthalide, methyl phthalide, ethyl phthalide, propyl phthalide, pentyl phthalide, isobutyl phthalide, sec-butyl phthalide, tert-butyl phthalide, 2- (α -carbonyl amyl) benzoic acid, 2- (α -hydroxypentyl) benzoic acid, or a pharmaceutically acceptable salt or an acid residue thereof, any one or a combination of 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone, butyl phthalide, and butenyl phthalide, and preferably 2- (α -hydroxypentyl) benzoic acid, or a pharmaceutically acceptable salt or an acid residue thereof, 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone, or a mixture of these two or a, Any one of or a combination of butenyl phthalide and butylphthalide.
In a preferred embodiment of the present invention, the pharmaceutically acceptable salt of 2- (α -hydroxypentyl) benzoic acid is selected from any one of sodium salt, potassium salt, lithium salt, calcium salt, magnesium salt, and zinc salt thereof.
In a preferred embodiment of the present invention, the Phenyl-Hexyl column or Phenyl column is commercially available, preferably Phenyl-Hexyl column or Phenyl column manufactured by Agilent or Kinetex, more preferably any one of Zorbax Eclipse plus XDB-Phenyl, Zorbax SB-Phenyl, urate XB-Phenyl, Agilent zoax rbax plus Phenyl-Phenyl or a combination thereof.
In the preferred technical scheme of the invention, the column length of the phenyl-hexyl chromatographic column or the phenyl column is 4.6mm multiplied by 250mm, and the particle size of the filler is preferably 5 μm.
The invention also aims to provide application of the high performance liquid chromatography for efficiently separating and detecting the phthalide derivatives in the method for separating and detecting the related substances of the phthalide derivatives, which is characterized in that the method adopts a phenyl-hexyl chromatographic column or a phenyl column, the detection wavelength is 226-230nm or 280nm, the flow rate is 0.8-1.2ml/min, the column temperature is 20-35 ℃, the sample injection amount is 10-100 mu l, and the mobile phase consists of an A phase and a B phase, wherein the volume ratio of the A phase to the B phase in the mobile phase is 60: 40-80:20, wherein the phase A is an aqueous solution with the pH value of 3-5, the phase B is a mixed solvent consisting of acetonitrile and methanol, and the volume ratio of the acetonitrile to the methanol in the phase B is 1: 0.8-1.2.
In a preferred technical scheme for measuring related substances, the mixing volume ratio of the phase A to the phase B in the mobile phase is 62: 38-75:25.
In the preferable technical scheme for measuring the related substances, the mixing volume ratio of the phase A to the phase B in the mobile phase is 60: 40-80:20, and preferably the mixing volume ratio of the phase A to the phase B in the mobile phase is 62: 38-75:25.
In a preferred technical scheme for measuring related substances, the mixing volume ratio of the phase A to the phase B in the mobile phase is 40:60-55:45 when the time is 40-50min, and the mixing volume ratio of the phase A to the phase B in the mobile phase is preferably 45:55-49: 51.
In a preferred embodiment of the substance measurement related to the present invention, the phase a is an aqueous acetic acid solution of 0.08-0.12% by volume, and preferably the volume ratio of the aqueous acetic acid solution of the phase a is selected from any one of 0.09%, 0.10%, and 0.11%.
In a preferred embodiment of the substance measurement related to the present invention, the volume ratio of acetonitrile to methanol in phase B is selected from any one of 1:0.8, 1:0.9, 1:1, 1:1.1, and 1: 1.2.
In a preferred embodiment of the substance measurement related to the present invention, a dilution solvent is used to prepare a sample solution or a reference solution, the dilution solvent is a 20-40% by volume acetonitrile aqueous solution, and preferably the volume ratio of the acetonitrile aqueous solution is selected from any one of 20%, 25%, 30%, and 40%.
In a preferred embodiment of the method for measuring a substance according to the present invention, the column temperature of the column is selected from any one of 20 ℃, 25 ℃, 30 ℃ and 35 ℃.
In a preferred embodiment of the substance measurement method of the present invention, the amount of the sample is selected from the group consisting of 10. mu.l, 20. mu.l, 50. mu.l and 100. mu.l.
In a preferred embodiment of the substance measurement according to the present invention, the gradient elution conditions are selected from the group consisting of the volume ratio of the phase a to the phase B as shown in any one of tables 1, 2 and 3.
In a preferred embodiment of the substance determination method of the present invention, the phthalide derivative is selected from any one of 2- (α -hydroxypentyl) benzoic acid or a pharmaceutically acceptable salt thereof, 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone, butenyl phthalide, butyl phthalide, or a combination thereof.
In a preferred embodiment of the substance measurement related to the present invention, the pharmaceutically acceptable salt of 2- (α -hydroxypentyl) benzoic acid is selected from any one of a sodium salt, a potassium salt, a lithium salt, a calcium salt, a magnesium salt, and a zinc salt.
In a preferred embodiment of the present invention, the related substance is selected from any one of phthalide, 2- (a-carbonylpentyl) benzoic acid, 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone, propylphthalide, o-carboxybenzyl alcohol, o-carboxybenzaldehyde, and isobutylphthalide, or a combination thereof.
In a preferred embodiment of the method for measuring a substance related to the present invention, the substance related to the present invention is selected from any one of a combination of phthalide and 2- (α -carbonylpentyl) benzoic acid, a combination of 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone and propylphthalide, a combination of o-carboxybenzyl alcohol and o-carboxybenzaldehyde, and a combination of isobutyl phthalide and butylphthalide.
In a preferred embodiment of the present invention, the related substance further includes any one of or a combination of methyl phthalide, ethyl phthalide, amyl phthalide, sec-butyl phthalide, tert-butyl phthalide, a pharmaceutically acceptable salt or acid group of 2- (a-hydroxypentyl) benzoic acid, and butenyl phthalide.
In a preferred embodiment of the present invention, when the phthalide derivative is selected from 2- (α -hydroxypentyl) benzoic acid or a pharmaceutically acceptable salt thereof, the related substance does not include 2- (α -hydroxypentyl) benzoic acid or a pharmaceutically acceptable salt thereof or an acid group thereof.
In a preferred technical scheme for measuring related substances, when the phthalide derivative is selected from 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone, the related substances do not include 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone.
In the preferable technical scheme for measuring the related substances, when the phthalide derivatives are selected from butylphthalide, the related substances do not include butylphthalide.
In the preferable technical scheme for measuring the related substances, when the phthalide derivatives are selected from butenyl phthalide, the related substances do not comprise the butenyl phthalide.
The invention also aims to provide application of the high performance liquid chromatography for efficiently separating and detecting the phthalide derivatives in the method for separating and detecting the content of the phthalide derivatives, which is characterized in that the method adopts a phenyl-hexyl chromatographic column or a phenyl column, the detection wavelength is 226-230nm or 280nm, the flow rate is 0.8-1.2ml/min, the column temperature is 20-35 ℃, the sample injection amount is 10-100 mu l, and a mobile phase consists of an A phase and a B phase, wherein the volume ratio of the A phase to the B phase in the mobile phase is 60: 40-80:20, wherein the phase A is an aqueous solution with the pH value of 3-5, the phase B is a mixed solvent consisting of acetonitrile and methanol, and the volume ratio of the acetonitrile to the methanol in the phase B is 1: 0.8-1.2.
In the preferable technical scheme of the content determination, the mixing volume ratio of the phase A to the phase B in the mobile phase is 62: 38-75:25.
In the preferable technical scheme of the content determination, when the time is 0-40min, the mixing volume ratio of the phase A to the phase B in the mobile phase is 60: 40-80:20, and preferably the mixing volume ratio of the phase A to the phase B in the mobile phase is 62: 38-75:25.
In the preferable technical scheme of the content determination, the mixing volume ratio of the phase A to the phase B in the mobile phase is 40:60-55:45 when the time is 40-50min, and the mixing volume ratio of the phase A to the phase B in the mobile phase is preferably 45:55-49: 51.
In a preferred embodiment of the content measurement of the present invention, the phase a is an acetic acid aqueous solution with a volume ratio of 0.08-0.12%, and preferably the volume ratio of the phase a acetic acid aqueous solution is selected from any one of 0.09%, 0.10%, and 0.11%.
In a preferable technical scheme of the content determination, the volume ratio of the acetonitrile to the methanol in the phase B is selected from any one of 1:0.8, 1:0.9, 1:1, 1:1.1 and 1: 1.2.
In a preferred technical scheme of the content determination of the invention, a dilution solvent is used for preparing a test sample solution or a reference sample solution, the dilution solvent is an acetonitrile aqueous solution with a volume ratio of 20-40%, and preferably the volume ratio of the acetonitrile aqueous solution is selected from any one of 20%, 25%, 30% and 40%.
In the preferable technical scheme of the content determination of the invention, the column temperature of the chromatographic column is selected from any one of 20 ℃, 25 ℃, 30 ℃ and 35 ℃.
In a preferable embodiment of the content measurement of the present invention, the sample amount is selected from any one of 10. mu.l, 20. mu.l, 50. mu.l, and 100. mu.l.
In a preferred embodiment of the content measurement of the present invention, the gradient elution conditions are selected from the group consisting of the volume ratio of the phase a to the phase B as shown in any one of tables 1, 2 and 3.
In a preferred embodiment of the content measurement, the phthalide derivative is selected from any one of o-carboxybenzyl alcohol, o-carboxybenzaldehyde, phthalide, methyl phthalide, ethyl phthalide, propyl phthalide, pentyl phthalide, isobutyl phthalide, sec-butyl phthalide, tert-butyl phthalide, 2- (a-carbonyl pentyl) benzoic acid, 2- (a-hydroxy pentyl) benzoic acid, or a pharmaceutically acceptable salt or an acid group thereof, and 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone, butyl phthalide, butenyl phthalide, or a combination thereof.
In a preferred embodiment of the content measurement, the pharmaceutically acceptable salt of 2- (α -hydroxypentyl) benzoic acid is selected from any one of sodium salt, potassium salt, lithium salt, calcium salt, magnesium salt, and zinc salt thereof.
In the preferable technical scheme of the invention for content measurement, the concentration of a test sample in the measurement of the content of the phthalide derivative is 0.25-3mg/ml, and preferably 0.5-2 mg/ml.
More than ten phthalide derivatives can be effectively separated under the chromatographic condition. Thus, the stationary and mobile phase selections of the present invention may also be applied in preparative chromatography. The invention also aims to provide a preparation method of the phthalide derivatives, which is characterized in that the method uses a phenyl-hexyl chromatographic column or a phenyl column as a stationary phase, and a mobile phase is formed by mixing an A phase and a B phase, wherein the mixing volume ratio of A to B is 60: 40-80:20, wherein the phase A is an aqueous solution with the pH value of 3-5, the phase B is a mixed solvent consisting of acetonitrile and methanol, and the volume ratio of the acetonitrile to the methanol in the phase B is 1: 0.8-1.2, the flow rate of the mobile phase is 0.8-1.2ml/min, the column temperature: 20-35 ℃.
In a preferred embodiment of the separation and purification of phthalide derivatives, the mobile phase is preferably the mobile phase used in the high performance liquid chromatography of the present invention.
In the preferable technical scheme for separating and purifying the phthalide derivatives, the mixing volume ratio of the phase A to the phase B in the mobile phase is 62: 38-75:25.
In a preferred embodiment of the separation and purification of the phthalide derivatives, the phase A is 0.08-0.12% by volume of acetic acid aqueous solution, preferably the volume ratio of the phase A acetic acid aqueous solution is selected from any one of 0.09%, 0.10% and 0.11%.
In a preferable technical scheme for separating and purifying the phthalide derivatives, the volume ratio of the acetonitrile to the methanol in the phase B is selected from any one of 1:0.8, 1:0.9, 1:1, 1:1.1 and 1: 1.2.
In a preferred embodiment of the separation and purification for preparing phthalide derivatives, the phthalide derivatives are selected from any one of or a combination of o-carboxybenzyl alcohol, o-carboxybenzaldehyde, phthalide, methyl phthalide, ethyl phthalide, propyl phthalide, pentyl phthalide, isobutyl phthalide, sec-butyl phthalide, tert-butyl phthalide, 2- (alpha-carbonyl amyl) benzoic acid, 2- (alpha-hydroxypentyl) benzoic acid or a pharmaceutically acceptable salt or an acid group thereof, and 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone, butyl phthalide, butenyl phthalide.
In a preferred embodiment of the separation and purification for preparing the phthalide derivative, the phthalide derivative is a substance with a relatively high chromatographic resolution, and is preferably selected from any one or a combination of phthalide, methylphthalide, 2- (a-carbonylpentyl) benzoic acid, pentylphthalide, sec-butylphthalide, and tert-butylphthalide, and more preferably, any one or a combination of phthalide, methylphthalide, and 2- (a-carbonylpentyl) benzoic acid with a maximum chromatographic resolution.
In the preferable technical scheme of separation and purification of the phthalide derivatives, after the elution solution dissolving the target phthalide substances is obtained by separation and collection, the target phthalide substances with high purity are prepared by the steps of evaporation, separation, washing and drying.
In a preferred technical scheme for separating and purifying the phthalide derivatives, the evaporation is selected from any one of reduced pressure evaporation and rotary evaporation or a combination thereof.
In a preferred embodiment of the separation and purification for preparing phthalide derivatives, the separation is selected from any one or a combination of filtration, centrifugation and standing separation.
In the preferable technical scheme of separation and purification of the phthalide derivatives, the washing is solvent washing.
In a preferred technical scheme for separating and purifying the phthalide derivatives, the drying is selected from any one or combination of reduced pressure drying, freeze drying, vacuum drying and spray drying.
In a preferred embodiment of the separation and purification of the phthalide derivative, the pharmaceutically acceptable salt of 2- (a-hydroxypentyl) benzoic acid is selected from any one of sodium salt, potassium salt, lithium salt, calcium salt, magnesium salt and zinc salt thereof.
The invention also aims to provide the application of the high-purity target phthalein substance prepared by separation and purification in the invention as a standard substance or a reference substance.
In a preferred technical scheme of the invention, the purity of the target phthalide substance is not less than 99.0%, preferably not less than 99.5%, and more preferably not less than 99.9%.
In order to clearly convey the scope of the invention, the invention defines the following terms:
the acid radical is an anion part of the phthalide derivative or the pharmaceutically acceptable salt thereof in an ionized form in the solution.
The diluting solvent of the present invention is used to dissolve and dilute a reference solution or a test solution.
Unless otherwise indicated, when the present invention relates to percentages between liquids, said percentages are volume/volume percentages; the invention relates to the percentage between liquid and solid, said percentage being volume/weight percentage; the invention relates to the percentages between solid and liquid, said percentages being weight/volume percentages; the balance being weight/weight percent.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention researches and screens the chromatographic conditions and gradient elution conditions of the high performance liquid chromatography to obtain the high performance liquid chromatography for high performance separation and detection of phthalide derivatives. The method has the advantages of good specificity, high separation degree, excellent sensitivity and the like, can realize effective separation of more than ten phthalide derivatives, and can be used for separation, content determination or monitoring and detection of related substances of the phthalide derivatives so as to better control the quality of the raw materials and the preparations thereof, ensure the effectiveness and safety of the medicine and further realize controllable medicine quality.
2. The high performance liquid chromatography for researching and efficiently separating and detecting the phthalide derivatives has the unexpected technical effects that firstly, the separation degree of phthalide and 2- (alpha-carbonyl amyl) benzoic acid is effectively improved, and separation detection and content control of phthalide and 2- (alpha-carbonyl amyl) benzoic acid are facilitated; secondly, the separation degree of 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone and propylphthalide is effectively improved, and the separation, detection and content control of 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone and propylphthalide are facilitated; thirdly, the separation degree of the isobutylphthalide and the butylphthalide is effectively improved, and the separation, the detection and the content control of the isobutylphthalide and the butylphthalide are facilitated; and fourthly, the separation degree of the o-carboxybenzyl alcohol and the o-carboxybenzaldehyde is effectively improved, and the separation, detection and content control of the o-carboxybenzyl alcohol and the o-carboxybenzaldehyde are facilitated. Therefore, the high performance liquid chromatography for high performance separation detection of phthalide derivatives can be used for quality control of phthalide raw materials and preparations thereof, and provides powerful technical guarantee for ensuring the quality, safety and effectiveness of medicaments.
3. The high performance liquid chromatography for high performance separation detection of phthalide derivatives of the invention realizes effective separation of more than ten phthalide derivatives, and can also be used for preparing high purity target phthalide derivatives, and the prepared high purity target phthalide substances are used as standard substances or reference substances.
Drawings
FIG. 1 is a chromatogram of a test sample of comparative example 1;
FIG. 2 is a chromatogram of a test sample of comparative example 2;
FIG. 3 is a chromatogram of a test sample of comparative example 3;
FIG. 4 chromatogram of the test sample of example 1;
FIG. 5 chromatogram of the test sample of example 2;
FIG. 6 chromatogram of the test sample of example 3;
FIG. 7 chromatogram of the test sample of example 4.
Detailed Description
The present invention will be specifically described with reference to examples. The embodiments of the present invention are only for illustrating the technical solutions of the present invention, and do not limit the essence of the present invention.
The compounds tested and the purity according to the invention are shown in Table 4.
TABLE 4
HPLC equipment: shimadzu LC-20A high performance liquid chromatograph, SPD-20A UV detector.
A chromatographic column: agilent ZORBAX plus phenyl-Hexyl 4.6mm × 250mm, 5 μm; or Firmor Titank C18, 4.6mm × 250mm, 5 μm; or GL-C18, 4.6 mm. times.250 mm, 5 μm.
Comparative example 1
According to the national butylphthalide drug standard WS1- (X-124) -2005Z, butylphthalide and eight related substances are separated and detected by an HPLC method.
A chromatographic column: firmometer Titan C18, 4.6mm × 250mm, 5 μm;
mobile phase: methanol-water (65: 35);
detection wavelength 280nm, flow rate: 1.0ml/min, column temperature: 30 ℃, sample introduction: 20 μ l.
Diluting the solvent: methanol.
Solution preparation and assay: precisely weighing appropriate amounts of butylphthalide, potassium 2- (alpha-hydroxypentyl) benzoate, phthalide, 2- (alpha-carbonylpentyl) benzoic acid, ethylphthalide, 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone, propylphthalide, pentylphthalide, and butenyl phthalide, and preparing a mixed solution containing butylphthalide at 0.5mg/ml and potassium 2- (alpha-hydroxypentyl) benzoate, phthalide, alpha-carbonylpentyl) benzoic acid, ethylphthalide, 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone, propylphthalide, pentylphthalide, and butenyl phthalide at 5 μ g/ml by using methanol as a solvent to serve as a sample solution; precisely measuring 1ml of the test solution, placing the test solution in a 100ml measuring flask, adding methanol to dilute to the scale, and shaking up to obtain a control solution. Precisely measuring the sample solution and the control solution by 20 μ l each according to the above chromatographic conditions, injecting into a liquid chromatograph, and recording chromatogram. The chromatogram of the test sample is shown in FIG. 1, and the experimental data of the test sample is shown in Table 5.
Under the chromatographic conditions, positioning sample solutions of phthalide and 2- (alpha-carbonyl amyl) benzoic acid are prepared respectively. The results showed that the retention time of phthalide localized peak was 4.668min, and the retention time of 2- (alpha-carbonylpentyl) benzoic acid localized peak was 4.847 min.
TABLE 5
Based on the peak locations and retention times of phthalide and 2- (alpha-carbonyl amyl) benzoic acid, the chromatographic peak with retention time of 4.627min in chromatogram 1 and table 6 of the sample was determined to be the coincidence peak of phthalide and 2- (alpha-carbonyl amyl) benzoic acid.
As can be seen from fig. 1 and table 6, phthalide and 2- (α -carbonylpentyl) benzoic acid cannot be effectively separated under the chromatographic conditions, and cannot be used for separation and detection of a pharmaceutical product containing phthalide and 2- (α -carbonylpentyl) benzoic acid among related substances and for quality control of the pharmaceutical product. Under the chromatographic condition, the separation degree of the 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone and the propylphthalide is only 1.00, which does not meet the requirement that the separation degree is more than 1.5, and the separation condition needs to be optimized to better control the quality of the medicine.
Comparative example 2
Chromatographic conditions are as follows:
a chromatographic column: agilent ZORBAX plus phenyl-Hexyl, 4.6mm X250 mm, 5 μm.
Mobile phase: phase A is 0.1% acetic acid water solution (volume ratio); and the phase B is acetonitrile.
Detection wavelength 280nm, flow rate: 1.0ml/min, column temperature: 30 ℃, sample introduction: 10 μ l.
Solvent: 30% acetonitrile in water (by volume).
The gradient elution conditions are shown in Table 6.
TABLE 6
| Time (minutes) | 0 | 5 | 40 | 50 | 50.1 | 60 |
| A％ | 75 | 70 | 49 | 35 | 75 | 75 |
| B％ | 25 | 30 | 51 | 65 | 25 | 25 |
Solution preparation and assay:
accurately weighing appropriate amounts of butylphthalide, 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone and propylphthalide, and preparing a mixed solution containing 2mg/ml of butylphthalide, impurities of 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone and propylphthalide by using a 30% acetonitrile solution (volume ratio) to be used as a test solution, wherein the mixed solution contains 20 mu g/ml of butylphthalide and 20 mu g/ml of each of the prophthalide; according to the chromatographic conditions, 10. mu.l of the test solution is taken and injected into a liquid chromatograph, and the chromatogram is recorded. The chromatogram of the test solution is shown in FIG. 2, and the experimental data of the test solution is shown in Table 7.
TABLE 7
As can be seen from FIG. 2 and Table 7, under the chromatographic conditions, the degree of separation of 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone and propylphthalide was only 0.78, which does not meet the requirement of high performance liquid phase for a degree of separation of greater than 1.5.
Comparative example 3
Chromatographic conditions are as follows: the column chromatography was GL-C18, 4.6mm X250 mm, 5 μm.
Mobile phase: the phase A is 20mmol/L K2HPO4 solution, and the pH is adjusted to 7.5 by phosphoric acid; and the phase B is acetonitrile.
Flow rate at detection wavelength 280 nm: 1.0ml/min, column temperature: 30 ℃, sample introduction: 20 μ l.
Diluting the solvent: phase A-phase B (80:20) (volume ratio).
The gradient elution conditions are shown in Table 8.
TABLE 8
| Time (minutes) | 0 | 5 | 30 | 45 | 45.1 | 55 |
| A％ | 20 | 25 | 63 | 63 | 20 | 20 |
| B％ | 80 | 75 | 37 | 37 | 80 | 80 |
Solution preparation and assay:
precisely weighing appropriate amounts of butylphthalide, potassium 2- (alpha-hydroxypentyl) benzoate, phthalide, 2- (alpha-carbonylpentyl) benzoic acid, methylphthalide, ethylphthalide, propylphthalide, pentylphthalide, o-carboxybenzyl alcohol, o-carboxybenzaldehyde, isobutylphthalide, sec-butylphthalide and isobutylphthalide, and preparing a mixed solution containing about 0.5mg/ml of butylphthalide, potassium 2- (alpha-hydroxypentyl) benzoate, phthalide, alpha-carbonylpentyl) benzoic acid, methylphthalide, ethylphthalide, propylphthalide, pentylphthalide, o-carboxybenzyl alcohol, o-carboxybenzaldehyde, isobutylphthalide, sec-butylphthalide and isobutylphthalide, each of which is about 1 μ g/ml, by using a diluent solvent as a sample solution; the concentration of the isobutylphthalide single site solution was 5. mu.g/ml. Precisely measuring the sample solution and the isobutylphthalide positioning solution by 20 μ l each according to the chromatographic conditions, respectively injecting into a liquid chromatograph, and recording the chromatogram. The chromatogram of the test solution is shown in FIG. 3, and the experimental data of the test solution is shown in Table 9.
TABLE 9
Under the chromatographic conditions, the retention time of the positioning peak of the positioning solution of the isobutylphthalide is separately determined to be 32.649 min.
As can be seen in FIG. 3, sec-butylbenzene phthalein exhibits overlapping doublets with retention times of 31.798 and 31.991 due to the two optical isomers. And determining that the chromatographic peak with the retention time of 32.914min in the chart 3 is the coincident peak of the isobutylphthalide and the butylphthalide by combining the retention time of the localized peak of the isobutylphthalide with 32.649 min. And the degree of separation of o-carboxybenzyl alcohol from o-carboxybenzaldehyde in FIG. 3 is 0.333, which does not meet the requirement that the degree of separation is greater than 1.5. Therefore, the chromatographic conditions need to be optimized, so that the o-carboxybenzyl alcohol, the o-carboxybenzaldehyde, the isobutyl phthalide and the butyl phthalide can be effectively separated, the requirement that the high performance liquid phase separation degree is more than 1.5 is met, the related substances and the content thereof in the medicine can be better separated and monitored, and the quality of the medicine can be controlled.
Example 1
The same material as in comparative example 1 was measured using HPLC, the C18 column was adjusted to a phenylhexyl column, the flow phase was adjusted and gradient elution was performed.
A chromatographic column: agilent ZORBAX plus phenyl-Hexyl, 4.6mm X250 mm, 5 μm.
Mobile phase: phase A is 0.1% acetic acid water solution (volume ratio); phase B is acetonitrile: methanol (1: 1, volume ratio).
Detection wavelength 227nm, flow rate: 1.0ml/min, column temperature: 30 ℃, sample introduction: 50 μ l.
Diluting the solvent: 30% acetonitrile in water (by volume).
The gradient elution conditions are shown in Table 10.
Watch 10
Solution formulation and assay
Precisely weighing appropriate amounts of butylphthalide, potassium 2- (alpha-hydroxypentyl) benzoate, phthalide, 2- (alpha-carbonylpentyl) benzoic acid, ethylphthalide, 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone, propylphthalide, pentylphthalide and butenyl phthalide, using 30% acetonitrile aqueous solution as a dilution solvent, preparing a mixed solution containing 0.25mg/ml butylphthalide and 2.5 μ g/ml each of 2- (alpha-hydroxypentyl) potassium benzoate, phthalide, 2- (alpha-carbonylpentyl) benzoic acid, ethylphthalide, 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone, propylphthalide, pentylphthalide, and butenyl phthalide as a sample solution; precisely measuring 1ml of the test solution, preparing the test solution into a solution containing butylphthalide 2.5 mu g/ml by using a 30% acetonitrile solution, and shaking up to obtain a control solution. Precisely measuring the sample solution and the control solution by 50 μ l each according to the above chromatographic conditions, injecting into a liquid chromatograph, and recording chromatogram. The chromatogram of the test sample is shown in FIG. 4, and the experimental data of the test sample is shown in Table 11.
TABLE 11
As can be seen from fig. 4 and table 11, under the chromatographic conditions, the retention time of phthalide was 7.232 and the retention time of 2- (a-carbonylpentyl) benzoic acid was 21.096. Compared with comparative example 1, example 1 can effectively separate the coincident peaks of phthalide and 2- (alpha-carbonyl amyl) benzoic acid, and the separation degree of 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone and propyl phthalide is 2.14, so that the related substances and the content thereof in the medicine can be separated and monitored, and the quality of the medicine can be controlled.
Example 2
A chromatographic column: agilent ZORBAX plus phenyl-Hexyl, 4.6mm X250 mm, 5 μm.
Mobile phase: phase A is 0.1% acetic acid water solution; phase B is acetonitrile: methanol (1: 1) solution.
Detection wavelength of 280nm, flow rate: 1.0ml/min, column temperature: 30 ℃, sample introduction: 10 μ l.
Solvent: 35% acetonitrile water solution (volume ratio);
the concentration of the test sample: 3 mg/ml.
The gradient elution conditions are shown in Table 12.
TABLE 12
| Time (minutes) | 0 | 5 | 40 | 50 | 50.1 | 60 |
| A％ | 75 | 70 | 49 | 35 | 75 | 75 |
| B％ | 25 | 30 | 51 | 65 | 25 | 25 |
Solution preparation and assay:
precisely weighing appropriate amounts of butylphthalide, potassium 2- (alpha-hydroxypentyl) benzoate, phthalide, 2- (alpha-carbonylpentyl) benzoic acid, methylphthalide, ethylphthalide, 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone, propylphthalide, pentylphthalide, o-carboxybenzyl alcohol, o-carboxybenzaldehyde, isobutylphthalide, sec-butylphthalide and isobutylphthalide, and preparing into a solution containing butylphthalide 3mg/ml, potassium 2- (alpha-hydroxypentyl) benzoate, phthalide, alpha-carbonylpentyl) benzoic acid, methylphthalide, ethylphthalide and 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone by using a 35% acetonitrile aqueous solution (volume ratio) as a diluent solvent, A mixed solution of 3 mu g/ml of each of propyl phthalide, amyl phthalide, o-carboxybenzyl alcohol, o-carboxybenzaldehyde, isobutyl phthalide, sec-butyl phthalide and isobutyl phthalide is used as a test solution; precisely measuring 10 μ l of the sample solution according to the above chromatographic conditions, injecting into a liquid chromatograph, and recording chromatogram. The chromatogram of the test sample is shown in FIG. 5, and the experimental data of the test sample is shown in Table 13.
Watch 13
Example 2 the mobile phase composition of comparative example 2 was optimized by adding methanol to the organic B phase. Relative to comparative example 1, the coincident peaks of phthalide and 2- (a-carbonylpentyl) benzoic acid can be efficiently separated. Compared with the comparative examples 1-2, the separation degree of the propylphthalide and the 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone reaches 2.97, and the requirement of high performance liquid phase on the separation degree is met. Relative to comparative example 3, the separation degree of o-carboxybenzyl alcohol and o-carboxybenzaldehyde was 1.95, the separation degree of isobutylphthalide and butylphthalide was 2.10, and the separation degree of other impurities and impurities, impurities and main components all met the specification.
Example 3
The chromatographic conditions were as follows:
a chromatographic column: agilent ZORBAX plus phenyl-Hexyl, 4.6mm X250 mm, 5 μm.
Mobile phase: phase a is 0.1% acetic acid in water, phase B is acetonitrile: methanol (1: 1).
Detection wavelength 227nm, flow rate: 1.0ml/min, column temperature: 30 ℃, sample introduction: 20 μ l.
Solvent: 30% aqueous acetonitrile.
The concentration of the test sample: 0.25 mg/ml.
The gradient elution conditions are shown in Table 14.
TABLE 14
| Time (minutes) | 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 preparation and assay:
precisely weighing appropriate amounts of butylphthalide, potassium 2- (alpha-hydroxypentyl) benzoate, phthalide, 2- (alpha-carbonylpentyl) benzoic acid, methylphthalide, ethylphthalide, 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone, propylphthalide, pentylphthalide, o-carboxybenzyl alcohol, o-carboxybenzaldehyde, isobutylphthalide, sec-butylphthalide and isobutylphthalide, and preparing into solution containing butylphthalide at an amount of about 0.25mg/ml, potassium 2- (alpha-hydroxypentyl) benzoate, phthalide, 2- (alpha-carbonylpentyl) benzoic acid, methylphthalide, ethylphthalide, 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone, propylphthalide, pentylphthalide, the test solutions were prepared from a mixed solution of o-carboxybenzyl alcohol, o-carboxybenzaldehyde, isobutyl phthalide, sec-butylphthalide and isobutyl phthalide each at a concentration of about 0.25. mu.g/ml. Precisely measuring 50 μ l of the sample solution according to the above chromatographic conditions, injecting into a liquid chromatograph, and recording chromatogram. The chromatogram of the test sample is shown in FIG. 6, and the experimental data of the test sample is shown in Table 15.
Watch 15
Under the chromatographic conditions, the degree of separation between the fourteen species was greater than 1.5. The coincident peaks of phthalide and 2- (a-carbonylpentyl) benzoic acid were efficiently separated relative to comparative example 1. The degree of separation of propylphthalide from 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone reached 3.01, relative to comparative example 1-2. Relative to comparative example 3, the degree of separation of o-carboxybenzyl alcohol and o-carboxybenzaldehyde was 1.89, and the degree of separation of isobutylphthalide from butylphthalide was 2.28.
Example 4
A chromatographic column: agilent ZORBAX plus phenyl-Hexyl, 4.6mm X250 mm, 5 μm.
Mobile phase: phase a is 0.1% acetic acid in water, phase B is acetonitrile: methanol (1: 1).
Detection wavelength 227nm, flow rate: 1.0ml/min, column temperature: 30 ℃, sample introduction: 50 μ l.
Solvent: 30% acetonitrile in water (by volume).
The concentration of the test sample: 0.25 mg/ml.
The gradient elution conditions are shown in Table 16.
TABLE 16
| Time (minutes) | 0 | 5 | 40 | 50 | 50.1 | 60 |
| A％ | 62 | 62 | 45 | 30 | 62 | 62 |
| B％ | 38 | 38 | 55 | 70 | 38 | 38 |
The solution preparation method and concentration are the same as those of example 3, and the chromatogram of the sample of example 4 is shown in FIG. 7.
1. Quantitative limit and detection limit
The detection limit of fourteen substances is 0.55-1.05ng, and the quantification limit is 1.05-2.10 ng.
2. Linearity and range
In the concentration range of 0.021 mu g/ml to 1.145 mu g/ml, the concentrations of fourteen substances and the peak area have good linear relation, and r is more than or equal to 0.9999.
3. Good precision and repeatability
Repeatability: the measured results of the same batch of samples which are repeatedly measured for 6 times have no obvious difference, which shows that the method has good repeatability.
4. The recovery rate and the accuracy are good
The method measures the high, medium and low concentrations (50%, 100% and 150% of limit concentration) of each impurity in the product, the recovery rates of 9 samples are all in the range of 90-110%, and the RSD (remote site resolution) is less than 5.0%. The detection result is in accordance with the verification requirement, and the method has good accuracy.
5. The solution stability is good
The related substance mixed sample solution is subjected to sample injection detection for 0h, 5h, 10h, 15h, 20h and 25h respectively at room temperature, and the RSD of each impurity peak area is less than 2.0%; the sample injection detection of the related substance test solution is carried out for 0h, 5h, 10h, 15h, 20h and 25h at room temperature, the detection amount of each impurity has no obvious change, and no new impurity is detected, which shows that the product is placed for 25h at room temperature, and the solution stability is good.
6. Better durability
The method changes the column temperature, flow rate, buffer salt concentration and organic phase ratio, and changes chromatographic columns of different manufacturers, and the detected amount of impurities and the number of the detected impurities are not obviously changed.
In the system applicability solution, the separation degree between impurities and main components and the durability are good.
Claims (24)
1. The high performance liquid chromatography for high performance separation detection of phthalide derivatives is characterized in that a phenyl-hexyl chromatographic column is adopted, the detection wavelength is 226-230nm or 280nm, the flow rate is 0.8-1.2mL/min, the column temperature is 20-35 ℃, the sample injection amount is 10-100 mu L, and the mobile phase consists of an A phase and a B phase, wherein the A phase is an acid solution with the pH value of 3-5, the B phase is a mixed solvent consisting of acetonitrile and methanol, and the volume ratio of the acetonitrile to the methanol in the B phase is 1: 0.8-1.2;
the phthalide derivatives are selected from more than two of o-carboxybenzyl alcohol, o-carboxybenzaldehyde, phthalide, methyl phthalide, ethyl phthalide, propyl phthalide, amyl phthalide, isobutyl phthalide, sec-butyl phthalide, tert-butyl phthalide, 2- (alpha-carbonyl amyl) benzoic acid, 2- (alpha-hydroxyl amyl) benzoic acid or medicinal salt of 2- (alpha-hydroxyl amyl) benzoic acid, 3-n-butyl-3-hydroxyl-1 (3H) -isobenzofuranone, butyl phthalide and butenyl phthalide;
the volume composition of phase a and phase B in the gradient elution conditions was:
。
2. The HPLC method according to claim 1, wherein the phase A is 0.08-0.12 vol.% aqueous acetic acid.
3. The HPLC method for separating and detecting phthalide derivatives according to any one of claims 1-2, wherein the phase A is 0.09%, 0.10% or 0.11% by volume of aqueous acetic acid.
4. The HPLC method according to claim 1, wherein the volume ratio of acetonitrile to methanol in phase B is selected from any one of 1:0.8, 1:0.9, 1:1, 1:1.1, and 1: 1.2.
5. The HPLC method according to claim 1, wherein the diluent solvent is used to prepare a sample solution or a reference solution, and the diluent solvent is 20-40% acetonitrile in water.
6. The HPLC method according to claim 5, wherein the dilution solvent is a 20%, 25%, 30% or 40% aqueous acetonitrile solution.
7. The high performance liquid chromatography for separating and detecting phthalide derivatives according to claim 1, wherein the column temperature of the column is selected from any one of 20 ℃, 25 ℃, 30 ℃ and 35 ℃.
8. The HPLC method according to claim 1, wherein the sample amount is selected from any one of 10. mu.L, 20. mu.L, 50. mu.L and 100. mu.L.
9. The HPLC method for detecting phthalide derivatives according to claim 1, wherein the gradient elution condition is selected from the group consisting of the volume composition of phase A and phase B in any one of tables 1, 2 and 3:
TABLE 1
TABLE 2
TABLE 3
。
10. The high performance liquid chromatography for high performance separation and detection of phthalide derivatives as claimed in claim 1, wherein the phthalide derivatives are selected from more than two of 2- (alpha-hydroxypentyl) benzoic acid or pharmaceutically acceptable salts of 2- (alpha-hydroxypentyl) benzoic acid, 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone, butenyl phthalide, and butylbenzene phthalide.
11. The high performance liquid chromatography for high performance separation and detection of phthalide derivatives according to claim 10, wherein the pharmaceutically acceptable salt of 2- (a-hydroxypentyl) benzoic acid is selected from any one of sodium salt, potassium salt, lithium salt, calcium salt, magnesium salt, and zinc salt thereof.
12. The HPLC method according to claim 1, wherein the phenyl-hexyl column is commercially available.
13. The HPLC method according to claim 12, wherein the phenyl-hexyl column is a phenyl-hexyl column manufactured by Agilent or Kinetex.
14. The HPLC method for separating and detecting phthalide derivatives according to any one of claims 12 to 13, wherein the Phenyl-Hexyl column is any one of Zorbax Eclipse Plus, Kinetex Phenyl-Hexyl, Agilent ZORBAX Plus Phenyl-Hexyl.
15. The HPLC method according to claim 1, wherein the size of the phenyl-hexyl column is 4.6mm x 250mm, and the particle size of the packing is 5 μm.
16. The high performance liquid chromatography for high performance separation and detection of phthalide derivatives as claimed in claim 1, wherein the phthalide derivatives are o-carboxybenzyl alcohol, o-carboxybenzaldehyde, phthalide, methyl phthalide, ethyl phthalide, propyl phthalide, pentyl phthalide, isobutyl phthalide, sec-butyl phthalide, tert-butyl phthalide, 2- (alpha-carbonyl pentyl) benzoic acid, pharmaceutically acceptable salts of 2- (alpha-hydroxypentyl) benzoic acid or 2- (alpha-hydroxypentyl) benzoic acid, 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone, butyl phthalide and butenyl phthalide.
17. The method for preparing the high-purity phthalide derivative is characterized in that a phenyl-hexyl chromatographic column is used as a stationary phase, a mobile phase is formed by mixing a phase A and a phase B, the phase A is an acid solution with a pH value of 3-5, the phase B is a mixed solvent consisting of acetonitrile and methanol, and the volume ratio of the acetonitrile to the methanol in the phase B is 1: 0.8-1.2, the flow rate of the mobile phase is 0.8-1.2mL/min, the column temperature: 20-35 ℃;
the phthalide derivatives are selected from more than two of o-carboxybenzyl alcohol, o-carboxybenzaldehyde, phthalide, methyl phthalide, ethyl phthalide, propyl phthalide, amyl phthalide, isobutyl phthalide, sec-butyl phthalide, tert-butyl phthalide, 2- (alpha-carbonyl amyl) benzoic acid, 2- (alpha-hydroxyl amyl) benzoic acid or medicinal salt of 2- (alpha-hydroxyl amyl) benzoic acid, 3-n-butyl-3-hydroxyl-1 (3H) -isobenzofuranone, butyl phthalide and butenyl phthalide;
the volume ratio of the phase A to the phase B in the gradient elution condition is as follows:
。
18. The method of claim 17, wherein phase a is 0.08-0.12% by volume aqueous acetic acid.
19. The method of claim 17, wherein phase a is 0.09%, 0.10%, or 0.11% by volume aqueous acetic acid.
20. The process of claim 17, wherein the volume ratio of acetonitrile to methanol in phase B is selected from any one of 1:0.8, 1:0.9, 1:1, 1:1.1, 1: 1.2.
21. The method of claim 17, wherein the phthalide derivative is selected from the group consisting of 2- (α -hydroxypentyl) benzoic acid or a pharmaceutically acceptable salt of 2- (α -hydroxypentyl) benzoic acid, 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone, butenyl phthalide, and butyl phthalide.
22. The method of claim 17, further comprising the steps of:
separating and collecting to obtain an elution solution for dissolving the target phthalide derivative, and evaporating, separating, washing and drying the collected elution solution to obtain the high-purity target phthalide derivative.
23. The method according to claim 22, wherein drying is selected from any one or a combination of reduced pressure drying, freeze drying, vacuum drying, spray drying.
24. The process of claim 21, wherein the pharmaceutically acceptable salt of 2- (a-hydroxypentyl) benzoic acid is selected from any one of a sodium salt, a potassium salt, a lithium salt, a calcium salt, a magnesium salt, and a zinc salt thereof.
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