CN110221009B - Method for measuring butylphthalide related substances by HPLC - Google Patents
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Abstract
A method for measuring butylphthalide related substances by using a high performance liquid phase adopts a phenyl-hexyl chromatographic column or a phenyl column, the detection wavelength is 227nm 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, a 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, 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 method has the advantages of good specificity, high separation degree, excellent sensitivity and the like, can realize effective separation of more than ten kinds, can be used for simultaneously detecting various related substances in the butylphthalide, is beneficial to the quality control of the butylphthalide medicine, ensures the effectiveness and safety of the medicine and realizes the quality control of the medicine.
Description
Technical Field
The invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for determining butylphthalide related substances by using a high performance liquid phase.
Background
The butylphthalide belongs to a C-3 substituted phthalide compound, is the 3 rd new chemical entity medicine independently researched and developed in China, and the biological activity of the butylphthalide is deeply researched. The phthalide compounds are unstable and are easily changed and mutually transformed under the influence of factors such as illumination, temperature, acid, alkali and the like. The content of related substances in the product is influenced by the starting materials, intermediates, polymers, side reaction products brought in during the production process, degradation products in the storage process and the like. Therefore, the development of a detection method for related substances is an important part in the research of the quality of medicines, and the safety, effectiveness and quality controllability of the medicines can be improved.
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.
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 relevant substances in the patent do not include phthalide and 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone, the relevant substances of phthalide and 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone cannot be separated and measured, and other phthalide derivative impurities except the above six impurities cannot be effectively separated and detected.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a method for measuring butylphthalide related substances by using a high-efficiency liquid phase, which can efficiently separate a plurality of related substances in butylphthalide and realize the simultaneous detection of the plurality of related substances in the butylphthalide.
The invention aims to provide a method for measuring butylphthalide related substances by using a high-performance liquid phase, which is characterized in that a phenyl-hexyl chromatographic column or a phenyl column is adopted, the detection wavelength is 227nm 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, and the gradient elution condition is that the mixing volume ratio of A to B in the mobile phase is 60: 40-80:20, and the mixing volume ratio of A to B in the mobile phase is 40:60-55:45 at 40-50 min.
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 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 45:55-49:51 at 40-50 min.
In a preferred embodiment of the present invention, the phase a is an aqueous acetic acid solution with a volume ratio of 0.08-0.12%, 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%, and more preferably 0.10%.
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 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 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. Washing the polar small molecular substances with organic phase with high proportion at 50-50.1min, and eluting the reduction chromatographic column at 50.1-60 min.
TABLE 1
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 related substance is selected from any one of phthalide, 2- (α -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 present invention, the related substance 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 methyl phthalide, ethyl phthalide, amyl phthalide, sec-butyl phthalide, tert-butyl phthalide, 2- (a-hydroxypentyl) benzoic acid, or a potassium salt, a sodium salt, or an acid radical thereof, or butenyl phthalide, or a combination 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 preferable 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 grain diameter of the filler is 5 mu m.
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 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 the 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, can be used for simultaneously detecting various related substances in butylphthalide, and has the following unexpected technical effects:
1. effectively separating the coincident peak of phthalide and 2- (alpha-carbonyl amyl) benzoic acid, realizing the separation and determination of butyl phthalide irreversible degradation product 2- (alpha-carbonyl amyl) benzoic acid, and being beneficial to the quality control of raw materials and preparations;
2. 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;
3. 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;
4. effectively improves the separation degree of the o-carboxybenzyl alcohol and the o-carboxybenzaldehyde, and is beneficial to the separation and detection of the o-carboxybenzyl alcohol and the o-carboxybenzaldehyde and the content control.
The high performance liquid chromatography for efficiently separating and detecting the phthalide derivatives can be used for separating and detecting the butylphthalide and related substances thereof so as to better control the quality of the medicines, ensure the effectiveness and the safety of the medicines and further realize the controllability of the quality of the medicines.
Drawings
FIG. 1 chromatogram of comparative example 1 sample
FIG. 2 chromatogram of comparative example 2 test sample
FIG. 3 chromatogram of comparative example 3 test sample
FIG. 4 chromatogram of test sample of example 1
FIG. 5 example 2 chromatogram of test sample
FIG. 6 chromatogram of example 3 sample
FIG. 7 example 4 chromatogram of test sample
Detailed Description
The present invention will be described in detail with reference to examples, which are provided only for illustrating the technical solutions of the present invention and are not intended to limit the spirit 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.
| Time (minutes) | 0 | 5 | 40 | 50 | 50.1 | 60 |
| A％ | 62 | 62 | 45 | 30 | 62 | 62 |
| B％ | 38 | 38 | 55 | 70 | 38 | 38 |
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 (12)
1. A method for measuring butylphthalide related substances by using a high performance liquid phase is characterized in that a phenyl-hexyl chromatographic column or a phenyl column is adopted, the detection wavelength is 227nm 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, and the volume ratio of acetonitrile to methanol in the phase B is 1: 0.8-1.2;
the phase A is 0.08-0.12% of acetic acid aqueous solution by volume ratio;
the related substances are selected from any one or combination of phthalide, 2- (alpha-carbonyl amyl) benzoic acid, 3-n-butyl-3-hydroxy-1 (3H) -isobenzofuranone, propyl phthalide, o-carboxyl benzyl alcohol, o-carboxyl benzaldehyde, isobutyl phthalide, methyl phthalide, ethyl phthalide, amyl phthalide, sec-butyl phthalide, tert-butyl phthalide, 2- (alpha-hydroxyl amyl) benzoic acid or potassium salt, sodium salt or acid radical thereof, butenyl phthalide, and the related substances do not include any one or combination of 2- (alpha-carbonyl amyl) benzoic acid, propyl phthalide, 2- (alpha-hydroxyl amyl) benzoic acid or potassium salt, sodium salt or acid radical thereof, butenyl phthalide;
the gradient elution conditions were:
。
2. The method for determining butylphthalide-related substance according to claim 1, wherein the volume ratio of the aqueous solution of phase A acetic acid is selected from any one of 0.09%, 0.10%, and 0.11%.
3. The method for determining butylphthalide-related substances according to claim 2, wherein the volume ratio of the A-phase acetic acid aqueous solution is 0.10%.
4. The method for determining butylphthalide-related substances according to claim 1, wherein 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.
5. The method for determining butylphthalide-related substances according to claim 1, wherein a dilution solvent is used to prepare a sample solution or a reference solution, and the dilution solvent is a 20-40% by volume aqueous acetonitrile solution.
6. The method for determining butylphthalide-related substance according to claim 5, wherein the volume ratio of the acetonitrile aqueous solution is selected from any one of 20%, 25%, 30% and 40%.
7. The high performance liquid chromatography-based method for assaying butylphthalide-related substance according to any one of claims 1 to 6, wherein the column temperature of the column is selected from any one of 20 ℃, 25 ℃, 30 ℃ and 35 ℃.
8. The method for determining butylphthalide-related substance according to any one of claims 1 to 6, 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 high performance liquid phase assay method for butylphthalide-related substances according to any one of claims 1 to 6, wherein said related substances are selected from any one of a combination of phthalide and 2- (alpha-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 isobutylphthalide and butylphthalide.
10. The high performance liquid chromatography-based method for assaying butylphthalide-related substance according to any one of claims 1 to 6, wherein said phenyl-hexyl column or phenyl column is a phenyl-hexyl column or phenyl column manufactured by Agilent or Kinetex.
11. The method for high performance liquid chromatography of butylphthalide-related substance according to claim 10, wherein said Phenyl-Hexyl column or Phenyl column is any one of Zorbax Eclipse Plus, Kinetex Phenyl-Hexyl, ZORBAX Phenyl, ZORBAX Eclipse XDB-Phenyl, ZORBAX SB-Phenyl, Ultimate XB-Phenyl, Agilent ZORBAX Plus Phenyl-Hexyl, or a combination thereof.
12. The method for measuring butylphthalide-related substance according to claim 10, wherein the length of the phenyl-hexyl chromatographic column or the phenyl column is 4.6mm x 250mm, and the particle size of the packing is 5 μm.
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