CN110118835B - Method for determining related substances of lipoic acid injection by high performance liquid chromatography - Google Patents
Method for determining related substances of lipoic acid injection by high performance liquid chromatography Download PDFInfo
- Publication number
- CN110118835B CN110118835B CN201910455592.9A CN201910455592A CN110118835B CN 110118835 B CN110118835 B CN 110118835B CN 201910455592 A CN201910455592 A CN 201910455592A CN 110118835 B CN110118835 B CN 110118835B
- Authority
- CN
- China
- Prior art keywords
- impurities
- mobile phase
- lipoic acid
- volume
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention belongs to the field of drug detection, and particularly relates to a method for determining related substances of a lipoic acid injection by using a high performance liquid chromatography. The mobile phase of the method comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a potassium dihydrogen phosphate A solution, the mobile phase B is a mixed solution of methanol and acetonitrile, and related substances (including lipoic acid and at least 11 common impurities) in the lipoic acid injection can be separated in a high performance liquid chromatogram by adopting a specific gradient elution mode; and the sensitivity of detecting each component and the accuracy of the content thereof are further improved by optimizing the conditions. The method has the advantages of good specificity, high analysis speed and high reproducibility, is convenient for quality detection and monitoring of lipoic acid injection, and is beneficial to safe popularization and application of lipoic acid.
Description
Technical Field
The invention relates to the field of medicine detection, and in particular relates to a method for determining related substances of a lipoic acid injection by using a high performance liquid chromatography.
Background
Lipoic Acid (Alpha Lipoic Acid) is a coenzyme existing in mitochondria, has high free radical reaction capability, can eliminate free radicals in vivo, and has the effects of diminishing inflammation and resisting aging. Is widely applied to preventing and treating heart disease, diabetes, liver disease and senile dementia at present. The thioctic acid injection (Oribo) from German Standad corporation was imported and marketed in China in 2000.
The lipoic acid sample is unstable and easily generates impurities, and mainly comprises oxidation impurities, 6, 8-cyclotrithiooctanoic acid, dihydrolipoic acid and the like. The adjuvant used in the prescription process of the lipoic acid injection is ethylenediamine, and the lipoic acid and the adjuvant ethylenediamine are easy to generate mono-substituted and di-substituted impurities of lipoic acid amide, so that the two impurities must be controlled. There is no method for detecting the above impurities (especially mono-substituted and di-substituted lipoic acid amide impurities) disclosed at present, so that a method for providing related substances of lipoic acid injection is needed.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for determining related substances of a lipoic acid injection by using a high performance liquid chromatography, which comprises the following steps:
a method for determining related substances of lipoic acid injection by high performance liquid chromatography comprises the steps of enabling a mobile phase to comprise a mobile phase A and a mobile phase B, enabling the mobile phase A to be a potassium dihydrogen phosphate A solution, enabling the mobile phase B to be a mixed solution of methanol and acetonitrile, adopting gradient elution, and counting by taking the total volume of the mobile phase as 100%,
in 0-40 min, the volume of the mobile phase A is decreased from 80% to 40%, and the volume of the mobile phase B is increased from 20% to 60%;
in the 40-70 min, the volume of the mobile phase A is 40%, and the volume of the mobile phase B is 60%;
at 70-72min, the volume of the mobile phase A is gradually increased from 40% to 80%, and the volume of the mobile phase B is gradually decreased from 60% to 20%.
The invention discovers that when the high performance liquid chromatography is adopted to detect the impurities which are easy to appear in the lipoic acid injection, the selection of the mobile phase has great influence on the separation of the impurities, when the mobile phase A and the mobile phase B with fixed proportion are adopted to detect, the impurities are difficult to be well separated, and a great deal of attempts discover that when the method is adopted to carry out gradient elution, the impurities which are easy to appear in the lipoic acid injection can be well distinguished.
The related substances of the lipoic acid injection comprise lipoic acid and impurities, wherein the impurities are one or a mixture of more than one selected from lipoic acid amide mono-substituted impurities (formula 1), lipoic acid amide di-substituted impurities (formula 2), oxidation impurities (formula 3-formula 6), lipoic acid ethyl ester (formula 7), 6, 8-dichloro octanoic acid ethyl ester (formula 8), impurities A (formula 9), impurities C (formula 10) and impurities D (formula 11).
The 11 known impurities can be simultaneously separated from other unknown impurities by the method of the invention.
In order to further improve the accuracy and sensitivity of detection, the invention optimizes other conditions of the high performance liquid chromatography to obtain the following preferred schemes (combining the preferred schemes can obtain the preferred embodiment of the invention):
preferably, the concentration of the potassium dihydrogen phosphate A solution is 0.004-0.006 mol/L, and preferably 0.005 mol/L.
Preferably, the pH value of the potassium dihydrogen phosphate A solution is 2.9-3.3, and preferably 3.1.
Preferably, the volume ratio of the methanol to the acetonitrile is 40: 10-45: 5, and preferably 42: 8.
Preferably, octadecylsilane bonded silica is used as the filler.
Preferably, the column has a size of 4.6mm × 250mm, 5 μm, and Waters is preferred
T3，4.6mm×250mm，5μm。
Preferably, the flow rate of the mobile phase is 0.8-1.2 mL/min, preferably 1.0 mL/min.
As a preferable embodiment of the present invention, the following conditions are employed for the measurement:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; the specification of the chromatographic column is Waters
T3，4.6mm×250mm，5μm；
Mobile phase A: a potassium dihydrogen phosphate A solution with a concentration of 0.004-0.006 mol/L, pH value of 2.9-3.3;
mobile phase B: a mixed solution of methanol and acetonitrile in a volume ratio of 40: 10-45: 5;
column temperature: 25-35 ℃;
detection wavelength: 210nm to 220 nm;
flow rate: 0.8-1.2 mL/min;
adopting gradient elution, wherein the total volume of the mobile phase is 100 percent,
in 0-40 min, the volume of the mobile phase A is decreased from 80% to 40%, and the volume of the mobile phase B is increased from 20% to 60%;
in the 40-70 min, the volume of the mobile phase A is 40%, and the volume of the mobile phase B is 60%;
at 70-72min, the volume of the mobile phase A is gradually increased from 40% to 80%, and the volume of the mobile phase B is gradually decreased from 60% to 20%.
Preferably, the lipoic acid injection is diluted to 1.0-2.0 mg/mL by using a diluent as a test solution.
Preferably, the lipoic acid and the impurity reference substance are diluted by the diluent to be used as a mixed reference solution,
wherein the concentration of the lipoic acid is 1.0-2.0 mg/mL;
the impurity reference substance is one or more of lipoic acid amide mono-substituted impurities, lipoic acid amide di-substituted impurities, oxidation impurities, lipoic acid ethyl ester, 6, 8-dichloro octanoic acid ethyl ester, impurities A, impurities C and impurities D;
the concentration of the lipoic acid amide disubstituted impurities is 0.002-0.004 mg/mL, the concentration of the oxidized impurities is 0.1-0.2 mg/mL, and the concentrations of other impurities are 0.02-0.04 mg/mL respectively.
The content of each component in the test sample can be clearly and accurately measured by adopting the test sample solution and the mixed control solution within the concentration range, and the concentration of the test sample solution and the concentration of each component in the mixed control solution can be proportionally set within the range for convenient calculation in practical application. If the content of the lipoic acid injection in the test solution is set to be 1.5mg/mL, the concentration of the lipoic acid in the mixed control solution is 1.5 mg/mL; the concentration of the lipoic acid amide disubstituted impurity is 0.003mg/mL, the concentration of the oxidized impurity is 0.15mg/mL, and the concentrations of other impurities are 0.03mg/mL respectively, so that the accuracy can be ensured, and the calculation of the content of each component is facilitated. In order to achieve the above-mentioned objects at the same time, the concentrations of the components may be set in various proportions within the scope of the present invention, and are not further limited herein.
In practical use, the sample solution and the mixed control solution are preferably filtered and then injected into the liquid chromatograph.
Preferably, the diluent is a mixture of a diluent and a diluent in a volume ratio of 35: 65-45: 55 of a mixture of a solution of monopotassium phosphate B and ethanol;
preferably, the volume ratio is 40:60, adding a solvent to the mixture;
preferably, the concentration of the potassium dihydrogen phosphate B solution is 6.8 +/-0.1 g/L;
preferably, the pH value of the potassium dihydrogen phosphate B solution is 3.5 +/-0.1.
Preferably, the pH values of the potassium dihydrogen phosphate a solution and the potassium dihydrogen phosphate B solution described herein are adjusted by phosphoric acid.
The invention has the following beneficial effects: the method provided by the invention can separate related substances (including lipoic acid and at least 11 common impurities) in the lipoic acid injection in a high performance liquid chromatogram; and the sensitivity of detecting each component and the accuracy of the content thereof are further improved by optimizing the conditions. The method has the advantages of good specificity, high analysis speed and high reproducibility, is convenient for quality detection and monitoring of lipoic acid injection, and is beneficial to safe popularization and application of lipoic acid.
Drawings
FIG. 1 is a chromatogram of mixed impurities in a lipoic acid injection.
FIG. 2 is a chromatogram of a blank diluent of a lipoic acid injection.
FIG. 3 is a chromatogram of a blank adjuvant of a lipoic acid injection.
FIG. 4 is a mixed impurity chromatogram of a lipoic acid injection according to the isocratic method of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
A method for measuring related substances of lipoic acid injection by high performance liquid chromatography adopts the following conditions for measurement:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent;
mobile phase A: potassium dihydrogen phosphate A solution with a concentration of 0.005mol/L, pH value of 3.1;
mobile phase B: a mixed solution of methanol and acetonitrile in a volume ratio of 42: 8;
column temperature: 30 ℃;
detection wavelength: 215 nm;
flow rate: 1.0 mL/min;
adopting gradient elution, wherein the total volume of the mobile phase is 100 percent,
in 0-40 min, the volume of the mobile phase A is decreased from 80% to 40%, and the volume of the mobile phase B is increased from 20% to 60%;
in the 40-70 min, the volume of the mobile phase A is 40%, and the volume of the mobile phase B is 60%;
at 70-72min, the volume of the mobile phase A is gradually increased from 40% to 80%, and the volume of the mobile phase B is gradually decreased from 60% to 20%.
Diluting the solution by using a diluent until the content of the lipoic acid injection in the test solution is 1.5mg/mL, and mixing the lipoic acid injection in a control solution until the concentration of the lipoic acid is 1.5 mg/mL; the concentration of the lipoic acid amide disubstituted impurity is 0.003mg/mL, the concentration of the oxidized impurity is 0.15mg/mL, and the concentrations of other impurities are 0.03mg/mL respectively.
The volume ratio of the diluent is 40:60 of a mixture of potassium dihydrogen phosphate B solution and ethanol; the concentration of the potassium dihydrogen phosphate B solution is 6.8g/L, and the pH value is 3.5.
The specific preparation process of each solution and the test verification process and the result of the method are shown in experimental examples 1-4.
Experimental example 1 System suitability test
Diluting liquid: 0.05mol/L potassium dihydrogen phosphate solution-ethanol (40:60), adjusted to pH 3.5 with phosphoric acid.
Blank adjuvant solution: accurately weighing about 33mg of ethylenediamine, placing the ethylenediamine in a 5mL brown measuring flask, diluting the ethylenediamine with water to a scale, shaking up, placing 3mL of ethylenediamine in a 50mL brown measuring flask, diluting the ethylenediamine with a solvent to a scale, and shaking up to obtain a blank auxiliary material solution.
Preparation of mixed control solution: taking A, C, D impurities, oxidized impurities, thioctic acid amide mono-substituted impurities, thioctic acid amide di-substituted impurities, thioctic acid ethyl ester, 6, 8-dichloro ethyl caprylate and appropriate amount of thioctic acid, adding a diluent (the diluent is a mixture of a monopotassium phosphate B solution and ethanol, the volume ratio is 40:60 (400 mL of the monopotassium phosphate B solution and 600mL of ethanol are measured after being dissolved and diluted to 1000mL by adding water), and dissolving and diluting to prepare a solution containing 1.5mg of thioctic acid, 0.003mg of di-substituted impurities and 0.15mg of oxidized impurities and 0.03mg of other impurities in each 1mL as a mixed control solution.
Preparing an impurity positioning solution: taking an appropriate amount of impurity A, C, D, oxidized impurity, lipoic acid amide mono-substituted impurity, lipoic acid amide di-substituted impurity, lipoic acid ethyl ester and 6, 8-dichloro ethyl caprylate, respectively adding diluent to dissolve and dilute to prepare a solution containing 0.003mg of di-substituted impurity and 0.03mg of oxidized impurity and 0.15mg of other impurities in each 1mL as an impurity positioning solution.
Preparing a test solution: taking 3mL of lipoic acid injection, placing the lipoic acid injection in a 50mL measuring flask, adding a diluent to dilute the lipoic acid injection until the diluent is scaled and shaken uniformly, filtering the mixture, and taking a subsequent filtrate as a test solution.
Control solution: precisely measuring 5mL of the test solution, placing the test solution into a 100mL measuring flask, adding the diluent to dilute to a scale, and shaking up. Precisely measuring 1mL, placing in a 25mL measuring flask, adding the diluent to dilute to scale, shaking, filtering, and taking the subsequent filtrate as a control solution.
And (3) determination: the chromatographic column adopts octadecylsilane chemically bonded silica as a filler, the mobile phase A is 0.005mol/L potassium dihydrogen phosphate A solution (the pH value is adjusted to 3.1 by phosphoric acid), and the mobile phase B is methanol: acetonitrile (42: 8), column temperature 30 ℃, detection wavelength 215nm, flow rate 1.0mL/min, gradient elution, the ratio change process of the operation is shown in Table 1.
TABLE 1 related substance gradient elution procedure
Precisely measuring 20 μ l of each solution, injecting into high performance liquid chromatograph, and recording chromatogram. The results are shown in Table 2, the chromatogram of the mixed impurities is shown in figure 1, the chromatogram of the blank diluent is shown in figure 2, and the chromatogram of the blank auxiliary materials is shown in figure 3.
TABLE 2 materials methodology-specificity-mix control results
And (4) conclusion: the blank diluent and the blank auxiliary materials do not interfere the detection of related substances in the test solution, and the separation degree between each peak and adjacent chromatographic peaks in the mixed control solution meets the specification, so that the specificity of the method for determining related substances in the lipoic acid injection by using the high performance liquid chromatography provided by the invention is good.
Experimental example 2 Linear and Range testing
Stock solutions of various impurity solutions: precisely weighing A, C, D impurities, thioctic acid amide mono-substituted impurities, thioctic acid amide di-substituted impurities, thioctic acid ethyl ester and 6, 8-dichloro ethyl caprylate, respectively placing the weighed materials in different 10mL measuring bottles, respectively placing 75mg of oxidized impurities in 50mL measuring bottles, placing 3mg of di-substituted impurities in 100mL measuring bottles, adding diluent to dilute to a scale, shaking up, and respectively using the diluted solution as stock solutions of all impurity solutions.
Lipoic acid control stock solution: precisely weighing 35mg of lipoic acid reference substance, placing the lipoic acid reference substance in a 50mL measuring flask, adding the diluent to dilute to a scale, and shaking up to obtain the lipoic acid reference substance.
Linear solution: precisely measuring the lipoic acid reference substance stock solution and 1mL of each impurity stock solution, placing the lipoic acid reference substance stock solution and each impurity stock solution into the same 10mL measuring flask, adding a diluent to dilute to a scale, and shaking up to obtain a linear stock solution. Precisely measuring the linear stock solutions 2mL, 1.5mL, 1mL, 0.5mL, 0.2mL and the like in different 10mL measuring bottles respectively, adding the diluent to dilute to the scales, and shaking up. The solution was defined as 200% linear, 150% linear, 100% linear, 50% linear, 20% linear, etc.
Precisely measuring 20 μ l of each solution, injecting into a liquid chromatograph, and recording chromatogram. The results are shown in Table 3.
TABLE 3 materials methodology-Linear results
| Composition (I) | Standard curve | Linearity and range | r |
| Lipoic acid | y＝9199.1228x+2143.4630 | 0.3858～15.4320ug/mL | 0.9992 |
| Disubstituted | y＝664340.6814x+3537.7873 | 0.0075～0.7500ug/mL | 0.9997 |
| Single substitution | y＝3953.8692x-73.6585 | 0.6214～6.2140ug/mL | 1.0000 |
| Oxidizing impurities | y＝8309.1641x-786.5122 | 2.1628～21.6280ug/mL | 0.9999 |
| Impurity C | y＝7287.5040x+744.1220 | 0.7194～7.1940ug/mL | 0.9999 |
| Impurity D | y＝3118.5209x+354.7805 | 0.6806～6.8060ug/mL | 0.9997 |
| Impurity A | y＝21531.2704x-1096.1220 | 0.6546～6.5460ug/mL | 0.9999 |
| Lipoic acid ethyl ester | y＝3351.6871x-2018.5514 | 0.6382～6.3820ug/mL | 0.9994 |
| 6, 8-Dichlorooctanoic acid ethyl ester | y＝7729.1341x-320.5417 | 0.5788～5.57881ug/mL | 0.9996 |
And (4) conclusion: the lipoic acid is in the concentration range of 0.3858-15.4320 ug/mL (equivalent to 5% -200% of the concentration of the self control), the linear equation is that y is 9199.1228x +2143.4630, the correlation coefficient r is 0.9992 > 0.9990, and the linear relation between the peak area and the concentration is good.
The double substitution is in the concentration range of 0.0075-0.7500 ug/mL (equivalent to 2% -200% of the limit concentration), the linear equation is that y is 664340.6814x +3537.7873, the correlation coefficient r is 0.9997 > 0.9990, and the linear relation between the peak area and the concentration is good.
The monosubstitution is in the concentration range of 0.6214-6.2140 ug/mL (equivalent to 20% -200% of the limit concentration), the linear equation is that y is 3953.8692x-73.6585, the correlation coefficient r is 1.0000 > 0.9990, and the linear relation between the peak area and the concentration is good.
The concentration of the oxidized impurities is 2.1628-21.6280 ug/mL (equivalent to 20% -200% of the limit concentration), the linear equation is 8309.1641x-786.5122, the correlation coefficient r is 0.9999 > 0.9990, and the linear relation between the peak area and the concentration is good;
the concentration of the impurity C is 0.7194-7.1940 ug/mL (equivalent to 20% -200% of the limit concentration), the linear equation is 7287.5040x +744.1220, the correlation coefficient r is 0.9999 > 0.9990, and the linear relation between the peak area and the concentration is good.
The concentration of the impurity D is 0.6806-6.8060 ug/mL (equivalent to 20% -200% of the limit concentration), the linear equation is 3118.5209x +354.7805, the correlation coefficient r is 0.9997 > 0.9990, and the linear relation between the peak area and the concentration is good.
The concentration of the impurity A is in the range of 0.6546-6.5460 ug/mL (equivalent to 20% -200% of the limit concentration), the linear equation is that y is 21531.2704x-1096.1220, the correlation coefficient r is 0.9999 > 0.9990, and the linear relation between the peak area and the concentration is good.
The lipoic acid ethyl ester has a linear equation of y being 3351.6871x-2018.5514, a correlation coefficient r being 0.9994 being more than 0.9990, and a linear relation between a peak area and concentration is good in a concentration range of 0.6382-6.3820 ug/mL (equivalent to 20% -200% of the limit concentration).
The concentration of 6, 8-dichloro ethyl caprylate is 0.5788-5.57881 ug/mL (equivalent to 20% -200% of the limit concentration), the linear equation is 7729.1341x-320.5417, the correlation coefficient r is 0.9996 > 0.9990, and the linear relation between the peak area and the concentration is good.
Experimental example 3 recovery test
Test solution: taking 3mL of lipoic acid injection, placing the lipoic acid injection in a 50mL measuring flask, adding a diluent to dilute the lipoic acid injection until the diluent is scaled and shaken uniformly, filtering the mixture, and taking a subsequent filtrate as a test solution.
Stock solutions of various impurity solutions: precisely weighing A, C, D impurities, thioctic acid amide mono-substituted impurities, thioctic acid amide di-substituted impurities, thioctic acid ethyl ester and 6, 8-dichloro ethyl caprylate, respectively placing the weighed materials in different 10mL measuring bottles, respectively placing 75mg of oxidized impurities in 50mL measuring bottles, placing 3mg of di-substituted impurities in 100mL measuring bottles, adding diluent to dilute to a scale, shaking up, and respectively using the diluted solution as stock solutions of all impurity solutions.
Recovery control solution stock solution: precisely measuring 2mL of each impurity stock solution, placing the impurity stock solutions into the same 20mL measuring flask, adding the diluent to dilute to a scale, and shaking up to obtain the product.
Recovery control solution: precisely measuring the recovery ratio reference solution stock solution 1mL, placing in a 10mL measuring flask, adding diluent to dilute to scale, and shaking.
Recovery of test solution: precisely measuring 0.6mL of lipoic acid injection, putting the lipoic acid injection into a 10mL measuring flask, parallelly measuring 9 parts, averagely dividing the lipoic acid injection into 3 groups, precisely adding 0.5mL, 1.0mL and 1.5mL of the recovery rate reference solution stock solution respectively, adding diluent respectively to dilute to a scale, shaking uniformly, filtering, and taking subsequent filtrate as sample solutions with recovery rates of 50%, 100% and 150% respectively.
The solutions were measured precisely at 20. mu.l each and injected into a liquid chromatograph, and the results are shown in Table 4.
TABLE 4 materials methodology-recovery results
And (4) conclusion: under the concentration of 50%, 100% and 150%, the average recovery rate in each group and the average recovery rate between groups of each impurity are both between 90% and 108%, which shows that the method is used for detecting the impurities and has good accuracy.
Experimental example 4 durability test
Diluting liquid: 0.05mol/L potassium dihydrogen phosphate solution-ethanol (40:60), adjusted to pH 3.5 with phosphoric acid.
Blank adjuvant solution: accurately weighing about 33mg of ethylenediamine, placing the ethylenediamine in a 5mL brown measuring flask, diluting the ethylenediamine with water to a scale, shaking up, placing 3mL of ethylenediamine in a 50mL brown measuring flask, diluting the ethylenediamine with a solvent to a scale, and shaking up to obtain a blank auxiliary material solution.
Mixing the control solution: taking an appropriate amount of A, C, D impurities, oxidized impurities, thioctic acid amide mono-substituted impurities, thioctic acid amide di-substituted impurities, thioctic acid ethyl ester, 6, 8-dichloro octanoic acid ethyl ester and thioctic acid, and adding a diluent to dilute to prepare a solution containing 1.5mg of thioctic acid, 0.003mg of di-substituted impurities and 0.15mg of oxidized impurities and 0.03mg of other impurities in each 1mL of the solution to serve as a mixed control solution.
Test solution: and (3) taking 3mL of lipoic acid injection, placing the lipoic acid injection in a 50mL measuring flask, adding diluent to dilute the lipoic acid injection until scales are evenly shaken, and filtering the mixture to obtain the compound.
Control solution: precisely measuring 5mL of the test solution, placing the test solution into a 100mL measuring flask, adding the diluent to dilute to a scale, and shaking up. Precisely measuring 1mL, placing in a 25mL measuring flask, adding the diluent to dilute to scale, shaking up, and filtering to obtain the final product.
The solution was measured precisely at 20. mu.l and injected into a liquid chromatograph under various chromatographic conditions, and the results are shown in tables 5 to 6.
TABLE 5 materials methodology-durability-Mixed control results
TABLE 6 methodology of matter-durability-detection of impurities
And (4) conclusion: when the column temperature changes by +/-5 ℃, the phosphate concentration changes by +/-0.001 mol/L, the pH value of the mobile phase changes by +/-0.2, and chromatographic columns of the same manufacturer, the same model and different batches are replaced, compared with the normal condition, the mixed contrast and the impurity detection amount have no obvious difference. The results show that the chromatographic conditions have good durability when the chromatographic conditions are slightly changed (the temperature of the column changes by +/-5 ℃, the phosphate concentration changes by +/-0.001 mol/L, the pH value of the mobile phase changes by +/-0.2, and chromatographic columns of the same manufacturer, the same model and different batches are replaced).
Comparative example 1
This comparative example differs from example 1 in that: gradient elution is not adopted, and the volume ratio of the whole process is 50: 50 of the mixed solution of the mobile phase A and the mobile phase B is used as a mobile phase for elution. The spectrum of the mixed impurities is shown in FIG. 4, and the mixed impurities comprise (mono-substitution, oxidation impurities, di-substitution, impurities C, impurities D and impurities A). It can be seen from the graph that the peak-appearance time of the mono-substituted impurity, the oxidized impurity, the disubstituted impurity and the solvent is within 2-5 minutes, the separation degree between the impurities is less than 1.5, the peak-appearance time of the impurities is relatively late under the chromatographic condition, and the operation time is relatively long.
From the results, it was found that each impurity can be efficiently detected using the gradient method, the degree of separation is more than 1.5, and the sample running time is short.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (38)
1. A method for determining related substances of a lipoic acid injection by using a high performance liquid chromatography method is characterized in that octadecylsilane chemically bonded silica is used as a filling agent, a mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A is a potassium dihydrogen phosphate A solution with a pH value of 2.9-3.3, the mobile phase B is a mixed solution of methanol and acetonitrile in a volume ratio of 40: 10-45: 5, gradient elution is adopted, and the total volume of the mobile phase is 100%,
in 0-40 min, the volume of the mobile phase A is decreased from 80% to 40%, and the volume of the mobile phase B is increased from 20% to 60%;
in the 40-70 min, the volume of the mobile phase A is 40%, and the volume of the mobile phase B is 60%;
at 70-72min, the volume of the mobile phase A is gradually increased from 40% to 80%, and the volume of the mobile phase B is gradually decreased from 60% to 20%;
the related substances of the lipoic acid injection comprise lipoic acid amide mono-substituted impurities shown in formula 1, lipoic acid amide di-substituted impurities shown in formula 2, oxidation impurities shown in formulas 3-6, lipoic acid ethyl ester shown in formula 7, 6, 8-dichloro ethyl octanoate shown in formula 8, impurities A shown in formula 9, impurities C shown in formula 10 and impurities D shown in formula 11;
2. the method according to claim 1, wherein the concentration of the potassium dihydrogen phosphate A solution is 0.004-0.006 mol/L; and/or the pH value of the potassium dihydrogen phosphate A solution is 3.1.
3. The method according to claim 2, wherein the concentration of the potassium dihydrogen phosphate A solution is 0.005 mol/L.
4. A process according to any one of claims 1 to 3, wherein the volume ratio of methanol to acetonitrile is 42: 8.
5. A method according to any one of claims 1 to 3, wherein the chromatographic column has a size of 4.6mm x 250mm, 5 μm.
6. The method of claim 4, wherein the chromatography column is Waters
T3，4.6mm×250mm，5μm。
7. The method according to any one of claims 1 to 3 and 6, wherein the flow rate of the mobile phase is 0.8 to 1.2 mL/min.
8. The method according to claim 4, wherein the flow rate of the mobile phase is 0.8 to 1.2 mL/min.
9. The method according to claim 5, wherein the flow rate of the mobile phase is 0.8 to 1.2 mL/min.
10. The method of claim 7, wherein the flow rate of the mobile phase is 1.0 mL/min.
11. The method according to claim 8 or 9, wherein the flow rate of the mobile phase is 1.0 mL/min.
12. The method according to any one of claims 1 to 3, 6 and 8 to 10, wherein the measurement is performed under the following conditions:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; mobile phase A: a potassium dihydrogen phosphate A solution with a concentration of 0.004-0.006 mol/L, pH value of 2.9-3.3;
mobile phase B: a mixed solution of methanol and acetonitrile in a volume ratio of 40: 10-45: 5;
column temperature: 25-35 ℃;
detection wavelength: 210nm to 220 nm;
flow rate: 0.8-1.2 mL/min;
adopting gradient elution, wherein the total volume of the mobile phase is 100 percent,
in 0-40 min, the volume of the mobile phase A is decreased from 80% to 40%, and the volume of the mobile phase B is increased from 20% to 60%;
in the 40-70 min, the volume of the mobile phase A is 40%, and the volume of the mobile phase B is 60%;
at 70-72min, the volume of the mobile phase A is gradually increased from 40% to 80%, and the volume of the mobile phase B is gradually decreased from 60% to 20%.
13. The method according to claim 4, characterized in that the following conditions are used for the determination:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; mobile phase A: a potassium dihydrogen phosphate A solution with a concentration of 0.004-0.006 mol/L, pH value of 2.9-3.3;
mobile phase B: a mixed solution of methanol and acetonitrile in a volume ratio of 40: 10-45: 5;
column temperature: 25-35 ℃;
detection wavelength: 210nm to 220 nm;
flow rate: 0.8-1.2 mL/min;
adopting gradient elution, wherein the total volume of the mobile phase is 100 percent,
in 0-40 min, the volume of the mobile phase A is decreased from 80% to 40%, and the volume of the mobile phase B is increased from 20% to 60%;
in the 40-70 min, the volume of the mobile phase A is 40%, and the volume of the mobile phase B is 60%;
at 70-72min, the volume of the mobile phase A is gradually increased from 40% to 80%, and the volume of the mobile phase B is gradually decreased from 60% to 20%.
14. The method according to claim 5, characterized in that the following conditions are used for the determination:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; mobile phase A: a potassium dihydrogen phosphate A solution with a concentration of 0.004-0.006 mol/L, pH value of 2.9-3.3;
mobile phase B: a mixed solution of methanol and acetonitrile in a volume ratio of 40: 10-45: 5;
column temperature: 25-35 ℃;
detection wavelength: 210nm to 220 nm;
flow rate: 0.8-1.2 mL/min;
adopting gradient elution, wherein the total volume of the mobile phase is 100 percent,
in 0-40 min, the volume of the mobile phase A is decreased from 80% to 40%, and the volume of the mobile phase B is increased from 20% to 60%;
in the 40-70 min, the volume of the mobile phase A is 40%, and the volume of the mobile phase B is 60%;
at 70-72min, the volume of the mobile phase A is gradually increased from 40% to 80%, and the volume of the mobile phase B is gradually decreased from 60% to 20%.
15. The method according to claim 7, characterized in that the following conditions are used for the determination:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; mobile phase A: a potassium dihydrogen phosphate A solution with a concentration of 0.004-0.006 mol/L, pH value of 2.9-3.3;
mobile phase B: a mixed solution of methanol and acetonitrile in a volume ratio of 40: 10-45: 5;
column temperature: 25-35 ℃;
detection wavelength: 210nm to 220 nm;
flow rate: 0.8-1.2 mL/min;
adopting gradient elution, wherein the total volume of the mobile phase is 100 percent,
in 0-40 min, the volume of the mobile phase A is decreased from 80% to 40%, and the volume of the mobile phase B is increased from 20% to 60%;
in the 40-70 min, the volume of the mobile phase A is 40%, and the volume of the mobile phase B is 60%;
at 70-72min, the volume of the mobile phase A is gradually increased from 40% to 80%, and the volume of the mobile phase B is gradually decreased from 60% to 20%.
16. The method according to claim 11, characterized in that the following conditions are used for the determination:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; mobile phase A: a potassium dihydrogen phosphate A solution with a concentration of 0.004-0.006 mol/L, pH value of 2.9-3.3;
mobile phase B: a mixed solution of methanol and acetonitrile in a volume ratio of 40: 10-45: 5;
column temperature: 25-35 ℃;
detection wavelength: 210nm to 220 nm;
flow rate: 0.8-1.2 mL/min;
adopting gradient elution, wherein the total volume of the mobile phase is 100 percent,
in 0-40 min, the volume of the mobile phase A is decreased from 80% to 40%, and the volume of the mobile phase B is increased from 20% to 60%;
in the 40-70 min, the volume of the mobile phase A is 40%, and the volume of the mobile phase B is 60%;
at 70-72min, the volume of the mobile phase A is gradually increased from 40% to 80%, and the volume of the mobile phase B is gradually decreased from 60% to 20%.
17. The method according to any one of claims 1 to 3, 6, 8 to 10 and 13 to 16, wherein the lipoic acid injection is diluted to 1.0 to 2.0mg/mL by a diluent as a test solution.
18. The method of claim 4, wherein the lipoic acid injection is diluted to 1.0-2.0 mg/mL by a diluent as a test solution.
19. The method of claim 5, wherein the lipoic acid injection is diluted to 1.0-2.0 mg/mL by a diluent as a test solution.
20. The method of claim 7, wherein the lipoic acid injection is diluted to 1.0-2.0 mg/mL by a diluent as a test solution.
21. The method of claim 11, wherein the lipoic acid injection is diluted to 1.0-2.0 mg/mL by a diluent as a test solution.
22. The method of claim 12, wherein the lipoic acid injection is diluted to 1.0-2.0 mg/mL by a diluent as a test solution.
23. The method according to any one of claims 1 to 3, 6, 8 to 10, 13 to 16, and 18 to 22, wherein the lipoic acid and the impurity control are diluted with a diluent as a mixed control solution,
wherein the concentration of the lipoic acid is 1.0-2.0 mg/mL;
the impurity reference substance is one or more of lipoic acid amide mono-substituted impurities, lipoic acid amide di-substituted impurities, oxidation impurities, lipoic acid ethyl ester, 6, 8-dichloro octanoic acid ethyl ester, impurities A, impurities C and impurities D;
the concentration of the lipoic acid amide disubstituted impurities is 0.002-0.004 mg/mL, the concentration of the oxidized impurities is 0.1-0.2 mg/mL, and the concentrations of other impurities are 0.02-0.04 mg/mL respectively.
24. The method of claim 4, wherein the lipoic acid and the contaminant control are diluted with a diluent as a mixed control solution,
wherein the concentration of the lipoic acid is 1.0-2.0 mg/mL;
the impurity reference substance is one or more of lipoic acid amide mono-substituted impurities, lipoic acid amide di-substituted impurities, oxidation impurities, lipoic acid ethyl ester, 6, 8-dichloro octanoic acid ethyl ester, impurities A, impurities C and impurities D;
the concentration of the lipoic acid amide disubstituted impurities is 0.002-0.004 mg/mL, the concentration of the oxidized impurities is 0.1-0.2 mg/mL, and the concentrations of other impurities are 0.02-0.04 mg/mL respectively.
25. The method of claim 5, wherein the lipoic acid and the contaminant control are diluted with a diluent as a mixed control solution,
wherein the concentration of the lipoic acid is 1.0-2.0 mg/mL;
the impurity reference substance is one or more of lipoic acid amide mono-substituted impurities, lipoic acid amide di-substituted impurities, oxidation impurities, lipoic acid ethyl ester, 6, 8-dichloro octanoic acid ethyl ester, impurities A, impurities C and impurities D;
the concentration of the lipoic acid amide disubstituted impurities is 0.002-0.004 mg/mL, the concentration of the oxidized impurities is 0.1-0.2 mg/mL, and the concentrations of other impurities are 0.02-0.04 mg/mL respectively.
26. The method of claim 7, wherein the lipoic acid and the contaminant control are diluted with a diluent to form a mixed control solution,
wherein the concentration of the lipoic acid is 1.0-2.0 mg/mL;
the impurity reference substance is one or more of lipoic acid amide mono-substituted impurities, lipoic acid amide di-substituted impurities, oxidation impurities, lipoic acid ethyl ester, 6, 8-dichloro octanoic acid ethyl ester, impurities A, impurities C and impurities D;
the concentration of the lipoic acid amide disubstituted impurities is 0.002-0.004 mg/mL, the concentration of the oxidized impurities is 0.1-0.2 mg/mL, and the concentrations of other impurities are 0.02-0.04 mg/mL respectively.
27. The method of claim 11, wherein the lipoic acid and contaminant control are diluted with a diluent as a mixed control solution,
wherein the concentration of the lipoic acid is 1.0-2.0 mg/mL;
the impurity reference substance is one or more of lipoic acid amide mono-substituted impurities, lipoic acid amide di-substituted impurities, oxidation impurities, lipoic acid ethyl ester, 6, 8-dichloro octanoic acid ethyl ester, impurities A, impurities C and impurities D;
the concentration of the lipoic acid amide disubstituted impurities is 0.002-0.004 mg/mL, the concentration of the oxidized impurities is 0.1-0.2 mg/mL, and the concentrations of other impurities are 0.02-0.04 mg/mL respectively.
28. The method of claim 12, wherein the lipoic acid and contaminant control are diluted with a diluent as a mixed control solution,
wherein the concentration of the lipoic acid is 1.0-2.0 mg/mL;
the impurity reference substance is one or more of lipoic acid amide mono-substituted impurities, lipoic acid amide di-substituted impurities, oxidation impurities, lipoic acid ethyl ester, 6, 8-dichloro octanoic acid ethyl ester, impurities A, impurities C and impurities D;
the concentration of the lipoic acid amide disubstituted impurities is 0.002-0.004 mg/mL, the concentration of the oxidized impurities is 0.1-0.2 mg/mL, and the concentrations of other impurities are 0.02-0.04 mg/mL respectively.
29. The method of claim 17, wherein the lipoic acid and contaminant control are diluted with a diluent as a mixed control solution,
wherein the concentration of the lipoic acid is 1.0-2.0 mg/mL;
the impurity reference substance is one or more of lipoic acid amide mono-substituted impurities, lipoic acid amide di-substituted impurities, oxidation impurities, lipoic acid ethyl ester, 6, 8-dichloro octanoic acid ethyl ester, impurities A, impurities C and impurities D;
the concentration of the lipoic acid amide disubstituted impurities is 0.002-0.004 mg/mL, the concentration of the oxidized impurities is 0.1-0.2 mg/mL, and the concentrations of other impurities are 0.02-0.04 mg/mL respectively.
30. The method of claim 17, wherein the diluent is a mixture of 35: 65-45: 55 of potassium dihydrogen phosphate B solution and ethanol.
31. The method of any one of claims 18 to 22 and 24 to 29, wherein the diluent is a mixture of 35: 65-45: 55 of potassium dihydrogen phosphate B solution and ethanol.
32. The method of claim 23, wherein the diluent is a mixture of 35: 65-45: 55 of potassium dihydrogen phosphate B solution and ethanol.
33. The method of claim 30 or 32, wherein the diluent is a mixture of 40:60 of potassium dihydrogen phosphate B solution and ethanol.
34. The method of claim 31, wherein the diluent is a 40:60 of potassium dihydrogen phosphate B solution and ethanol.
35. The method of claim 30 or 32, wherein the concentration of the monopotassium phosphate B solution is 6.8 ± 0.1 g/L.
36. The method of claim 31, wherein the concentration of the monopotassium phosphate B solution is 6.8 ± 0.1 g/L.
37. The method of claim 30 or 32, wherein the solution of monopotassium phosphate B has a pH of 3.5 ± 0.1.
38. The method of claim 31, wherein the solution of monopotassium phosphate B has a pH of 3.5 ± 0.1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910455592.9A CN110118835B (en) | 2019-05-29 | 2019-05-29 | Method for determining related substances of lipoic acid injection by high performance liquid chromatography |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910455592.9A CN110118835B (en) | 2019-05-29 | 2019-05-29 | Method for determining related substances of lipoic acid injection by high performance liquid chromatography |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110118835A CN110118835A (en) | 2019-08-13 |
| CN110118835B true CN110118835B (en) | 2022-02-18 |
Family
ID=67523510
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910455592.9A Active CN110118835B (en) | 2019-05-29 | 2019-05-29 | Method for determining related substances of lipoic acid injection by high performance liquid chromatography |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110118835B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112034060A (en) * | 2020-08-25 | 2020-12-04 | 开封康诺药业有限公司 | Analysis method of pantoprazole sodium related substance for injection |
| CN114397376A (en) * | 2021-12-10 | 2022-04-26 | 山东齐都药业有限公司 | Method for determining related substances in lipoic acid raw material and preparation by high performance liquid chromatography |
| CN115015439B (en) * | 2022-06-30 | 2023-11-03 | 瑞玞生物医学(深圳)有限公司 | Separation detection method for antioxidant in composite solution |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101607955A (en) * | 2008-06-18 | 2009-12-23 | 上海津力化工有限公司 | A kind of preparation method of low residual lipoic acid |
| CN102603709A (en) * | 2012-03-02 | 2012-07-25 | 海南灵康制药有限公司 | Thioctic acid compound and preparation method thereof |
| CN103058989A (en) * | 2013-01-30 | 2013-04-24 | 山东齐都药业有限公司 | Method for preparing alpha-lipoic acid |
| CN107226804A (en) * | 2016-03-25 | 2017-10-03 | 江苏奥赛康药业股份有限公司 | The impurity and preparation method of a kind of R-DHLA or its amino butanetriol salt and their detection method |
| CN107991403A (en) * | 2017-11-22 | 2018-05-04 | 山东省分析测试中心 | The analysis method of genotoxicity impurity 6,8- dicloro caprylate ethyl esters in a kind of lipoic acid |
-
2019
- 2019-05-29 CN CN201910455592.9A patent/CN110118835B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101607955A (en) * | 2008-06-18 | 2009-12-23 | 上海津力化工有限公司 | A kind of preparation method of low residual lipoic acid |
| CN102603709A (en) * | 2012-03-02 | 2012-07-25 | 海南灵康制药有限公司 | Thioctic acid compound and preparation method thereof |
| CN103058989A (en) * | 2013-01-30 | 2013-04-24 | 山东齐都药业有限公司 | Method for preparing alpha-lipoic acid |
| CN107226804A (en) * | 2016-03-25 | 2017-10-03 | 江苏奥赛康药业股份有限公司 | The impurity and preparation method of a kind of R-DHLA or its amino butanetriol salt and their detection method |
| CN107991403A (en) * | 2017-11-22 | 2018-05-04 | 山东省分析测试中心 | The analysis method of genotoxicity impurity 6,8- dicloro caprylate ethyl esters in a kind of lipoic acid |
Non-Patent Citations (3)
| Title |
|---|
| Chromatographic analysis of lipoic acid and related compounds;Hiroyuki Kataoka;《Journal of Chromatography B》;19981231;247-262 * |
| High-performance liquid chromatography of lipoic acid and analogues;STEWART C. HOWARD et al;《Journal of chromarography》;19811231;129-131 * |
| HP LC 法测定硫辛酸注射液中的有关物质;夏天水等;《中国药房》;20161231;第27卷(第21期);2980-2982 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110118835A (en) | 2019-08-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110118835B (en) | Method for determining related substances of lipoic acid injection by high performance liquid chromatography | |
| CN118777480B (en) | Method for detecting content of genotoxic impurities in indobufen bulk drug | |
| CN113125611B (en) | Method for detecting content of impurity 6-formyl pterin folic acid | |
| CN118150745B (en) | Method for simultaneously determining multiple genotoxic impurities in raw material medicine of lebsiella | |
| CN111965273B (en) | HPLC (high Performance liquid chromatography) method for detecting genotoxic impurities in candesartan cilexetil | |
| CN109374778B (en) | Method for determining organic impurities in 2-mercaptobenzimidazole | |
| CN108414636A (en) | A kind of detection method of the cinnarizine in relation to substance | |
| CN109765316B (en) | Method for detecting levetiracetam from medicine | |
| CN113933424B (en) | Method for detecting impurities in apixaban starting material | |
| CN113702514A (en) | Method for determining atorvastatin calcium related impurity I | |
| CN115656364B (en) | Detection method of related substances in rifampicin and application of detection method in MNP monitoring | |
| CN114397376A (en) | Method for determining related substances in lipoic acid raw material and preparation by high performance liquid chromatography | |
| CN110095554B (en) | Method for analyzing milrinone related substances by high performance liquid chromatography | |
| CN113504317A (en) | Detection method and application of genotoxic impurities in apixaban | |
| CN114594168A (en) | Method for detecting indobufen impurity | |
| CN114609277B (en) | Method for measuring content of compound acetylsalicylic acid tablet | |
| CN115754069B (en) | Detection method and application of larotaxel content and its related substances | |
| CN114354794B (en) | A quality control method for pazufloxacin mesylate injection | |
| CN114660213B (en) | Component content determination method of compound reserpine hydrochlorothiazide tablet | |
| CN115453014B (en) | Detection method of butylphthalide and related substances thereof | |
| CN111257440A (en) | GC-HS-based method for determining potential genotoxic impurities in sodium valproate | |
| CN115469039B (en) | Butylphthalide and detection method of related substances thereof | |
| CN116930368B (en) | A method for detecting stiripentol isomers | |
| CN118225952B (en) | A method for detecting impurities in N-(3-chloropropyl)dibutylamine | |
| CN115326999B (en) | Detection method of oseltamivir epoxy intermediate and isomer thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |