CN114280173B - Method for detecting lipoic acid injection polymer - Google Patents
Method for detecting lipoic acid injection polymer Download PDFInfo
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- CN114280173B CN114280173B CN202111484938.1A CN202111484938A CN114280173B CN 114280173 B CN114280173 B CN 114280173B CN 202111484938 A CN202111484938 A CN 202111484938A CN 114280173 B CN114280173 B CN 114280173B
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Abstract
The invention provides a lipoic acid injection polymer detection method, which belongs to the technical field of medicine analysis and comprises the following steps: (1) preparation of test solution and control solution: test solution: precisely measuring a proper amount of lipoic acid injection, and diluting with a mobile phase to prepare a lipoic acid solution; control solution: precisely measuring the solution of the sample, placing the solution in a 100ml measuring flask, diluting the solution to a scale with a mobile phase, and shaking the solution uniformly; (2) chromatographic conditions: chromatographic column: the filler is octadecylsilane chemically bonded silica gel, and the detection wavelength is as follows: 200nm-240nm, mobile phase: 0.1% (V/V) phosphoric acid solution-acetonitrile=80:10-100:10, flow rate: 0.1-0.9ml/min, column temperature: 20-40 ℃, sample injection amount: 5-15 μl; (3) measurement method: precisely measuring 5-15 μl of each of the control solution and the sample solution, and injecting into a liquid chromatograph. The method is simple and convenient to operate and has good reproducibility; the specificity is strong, and the content of the lipoic acid polymer can be accurately measured.
Description
Technical Field
The invention relates to a method for detecting lipoic acid injection polymer, belonging to the technical field of drug analysis.
Technical Field
The lipoic acid contains a disulfide five-membered ring structure, has high electron density and very high free radical reaction capacity, is known as a universal antioxidant, can remove free radicals of organisms, can promote the organisms to synthesize vitamin C by utilizing glucose, effectively removes melanin, and has the effects of resisting inflammation and aging.
Is widely used for preventing and treating heart diseases, diabetes, liver diseases and senile dementia at present. Clinically, the current lipoic acid variety is mainly used for treating paresthesia caused by diabetic peripheral neuropathy (diabetic peripheralneuropathy, DPN). The lipoic acid injection (Oribao) of the original German Cheddar company is approved in 2000 in China to be imported and marketed, and some domestic imitated preparations related to lipoic acid liquid or solid are also approved by domestic SFDA to be marketed successively.
Compared with mixed lipoic acid and levolipoic acid, the lipoic acid has more active advantages in the aspects of treating type II diabetes, promoting the uptake of glucose by skeletal muscle, reducing the level of plasma insulin and free fatty acid, improving the synthesis of glycogen under the action of insulin, oxidizing glucose, increasing the oxygen content of animal blood flow and the like.
Therefore, the dextrorotatory lipoic acid has wider prospect in preventing and treating heart diseases, diabetes, liver diseases, senile dementia and other diseases. However, dextrorotatory lipoic acid has a major drawback in terms of its physical properties, namely, it is very unstable; and the melting point of the dextrorotatory lipoic acid is lower than that of a racemate, and the dextrorotatory lipoic acid can be polymerized at a lower temperature only at about 47 ℃, so that the polymerization is aggravated under the conditions of acid catalysis and illumination, and the drug effect and the safety of clinical treatment are affected.
The extremely polymeric nature of lipoic acid has long been discovered, explored and utilized by researchers. As early as 1956, in the paper "Disulfide Polymers of DL-Lipoic Acid" by Thomas et al in Journal of the American Chemical Society, a reaction of DL-Lipoic Acid thermal polymerization was reported for the first time, which is to heat and melt Lipoic Acid at 65 ℃, keep the temperature for 15min, cool down to room temperature, continuously extract the reaction product with benzene solvent until the color of the extraction solvent becomes colorless, concentrate the benzene solvent, and finally obtain a viscous pale yellow polymer. However, the degree of polymerization and molecular weight of the product are not described in the report, and highly toxic benzene solvents are used for extraction, making the preparation method difficult to popularize.
In 2010, japanese scholars KISANUKI et al, published under Journal of Polymer Science:PartA: polymer Chemistry in the paper "Ring-Opening Polymerization of Lipoic Acidand Characterization of the Polymer," incorporated lipoic acid into classical polymer reaction conditions for polymerization. One of them is to carry out bulk melt tube-sealing polymerization without adding any initiator, and the molecular weight of the obtained polymer is determined to be in the range of 105 to 106 by GPC analysis, and the degree of polymerization is very high.
German European patent CN200780029055.3 discloses an oligomeric lipoic acid derivative, the polymerization degree n of which is 4-6, particularly preferably in the form of alkaline earth metal salt, organic amine salt or amino acid salt, and claims the use of a composition containing the oligomeric lipoic acid for preparing a medicament for stabilizing carotenoid or carotenoid derivative, which is characterized in that a base is reacted with lipoic acid in an aqueous solution and reacted at high temperature for 1-5 hours, and then a composition of the oligomeric lipoic acid and a base counter ion is precipitated by adding a poor solvent.
The existing lipoic acid injection polymer detection method has the defects of complex operation, poor reproducibility, poor specificity, poor separation degree, low chromatographic column efficiency and inaccurate content measurement.
Therefore, the improved lipoic acid injection polymer detection method is simple and convenient to operate and has better reproducibility; the specificity is strong, the separation degree is good, the column efficiency of the chromatographic column is high, and the content of the lipoic acid polymer can be accurately measured.
Disclosure of Invention
The invention aims to provide an improved lipoic acid injection polymer detection method, which is simple and convenient to operate, good in reproducibility, strong in specificity, good in separation degree, high in chromatographic column efficiency and capable of accurately measuring the content of lipoic acid polymer.
The above object of the present invention is achieved by the following technical solutions:
a lipoic acid injection polymer detection method comprises the following steps:
(1) Preparation of test solution and control solution
Test solution: precisely measuring a proper amount of lipoic acid injection, and diluting with a mobile phase to prepare a lipoic acid solution;
control solution: precisely measuring the solution of the sample, placing the solution in a container, diluting the solution to a scale with a mobile phase, and shaking the solution uniformly;
(2) Chromatographic conditions
Chromatographic column: the filler is octadecylsilane chemically bonded silica gel,
detection wavelength: 200nm to 240nm of the nanometer silicon dioxide,
mobile phase: 0.1% (V/V) phosphoric acid solution-acetonitrile=80:10-100:10,
flow rate: 0.1-0.9ml/min,
column temperature: 20-40 ℃,
sample injection amount: 5-15 μl;
(3) Measurement method
Precisely measuring 5-15 μl of each of the control solution and the sample solution, and injecting into a liquid chromatograph.
Preferably, in step (1), the mobile phase is: 0.1% (V/V) phosphoric acid solution-acetonitrile=80:10-100:10.
Preferably, in step (1), the mobile phase is: 0.1% (V/V) phosphoric acid solution-acetonitrile=90:10.
Preferably, in step (2), octadecylsilane chemically bonded silica filler (250 mm. Times.4.6 mm,5 μm) is present in the column.
Preferably, in step (2), the detection wavelength is: 200nm, flow rate: 0.5ml/min, column temperature: 30 ℃, sample injection amount: 10 μl.
Preferably, in step (3), the liquid chromatograph is agilent 1260 in the united states.
Preferably, in step (3), the number of theoretical plates of the chromatographic column is greater than 5000.
Preferably, in step (3), the lipoic acid has a separation degree from adjacent impurity peaks of greater than 1.5.
The beneficial effects are that:
the lipoic acid injection polymer detection method is simple and convenient to operate and has good reproducibility; the separation degree between the main component and the polymer and between the main component and the impurities of the polymers is more than 1.5, the number of theoretical plates of the chromatographic column is more than 5000, the specificity is strong, and the content of the lipoic acid polymer can be accurately measured.
The invention is further illustrated by the drawings and the specific examples, which are not meant to limit the scope of the invention.
Drawings
FIG. 1 is a graph of a lipoic acid and polymer impurity mixed solution of example 1 of the present invention.
FIG. 2 is a graph of a mixed solution of lipoic acid and polymer impurities according to comparative example 1 of the present invention.
Detailed Description
Unless otherwise specified, the materials, adjuvants or equipment used in the examples of the present invention are commercially available products of the type conventional in the art; the test methods used are all conventional in the art.
Example 1
A lipoic acid injection polymer detection method comprises the following steps:
(1) Preparation of test solution and control solution
Test solution: precisely measuring a proper amount of lipoic acid injection (25 mg/ml), diluting with mobile phase (mobile phase: 0.1% (V/V) phosphoric acid solution-acetonitrile (90:10), and preparing 1ml of lipoic acid solution of 0.6 mg;
control solution: precisely measuring 1ml of the sample solution, placing in a 100ml measuring flask, diluting to scale with mobile phase (mobile phase: 0.1% (V/V) phosphoric acid solution-acetonitrile (90:10), and shaking;
(2) Chromatographic conditions
Chromatographic column: octadecylsilane chemically bonded silica is used as filler (250 mm×4.6mm,5 μm or chromatographic column with equivalent performance),
mobile phase: 0.1% (V/V) phosphoric acid solution-acetonitrile (90:10),
detection wavelength: the wavelength of the light is 200nm,
flow rate: the volume of the solution is 0.5ml/min,
column temperature: 30 ℃,
sample injection amount: 10 μl;
(3) Measurement method
Precisely measuring 10 μl of each of the control solution and the sample solution, injecting into a liquid chromatograph (Agilent 1260 in the United states), wherein the number of theoretical plates of a chromatographic column is greater than 5000, recording a chromatogram, as shown in FIG. 1, which is a spectrum of a mixed solution of lipoic acid and polymer impurities in the embodiment 1 of the invention, and sequentially taking out peaks: lipoic acid, dimer I, dimer II, dimer III, trimer I, trimer II, trimer III, under the following test conditions: the chromatographic condition of example 1 of the present invention, the test instrument was agilent 1260 in the united states; the separation degree of lipoic acid and adjacent impurity peaks is more than 1.5; in the chromatogram of the sample solution, the sum of the areas of the polymers (relative retention times of 1.62, 1.68, 1.74, 2.10, 2.18, 2.24) should not be greater than the peak area (1.0%) of the control solution.
Comparative example 1
The existing method for detecting the lipoic acid injection polymer is a molecular exclusion chromatography method, and comprises the following steps:
(1) Preparation of test solution and control solution
Test solution: precisely measuring a proper amount of lipoic acid injection (25 mg/ml), and diluting with a mobile phase to prepare 1ml of solution containing 0.6mg of lipoic acid;
control solution: precisely measuring 1ml of the sample solution, placing in a 100ml measuring flask, diluting to scale with mobile phase, and shaking;
(2) Chromatographic conditions
Chromatographic column: spherical hydrophilic modified silica gel is used as a filler;
mobile phase: phosphate buffer [0.005mol/L disodium hydrogen phosphate solution-0.005 mol/L sodium dihydrogen phosphate solution (60:40) ] -acetonitrile (93:7)
Detection wavelength: the wavelength of the light is 240nm,
flow rate: the volume of the solution is 0.5ml/min,
column temperature: 25 c,
sample injection amount: 10 μl;
(3) Measurement method
Precisely measuring 10 μl of each of the control solution and the sample solution, injecting into a liquid chromatograph (the test instrument is Shimadzu 20A), recording a chromatogram, which is shown in FIG. 2, of the mixed solution of lipoic acid and polymer impurities of comparative example 1 of the present invention, wherein the test conditions are those of comparative example 1, and the test instrument is Shimadzu 20A; the main peak in the chromatogram of the test solution is preceded by a polymer peak, and the sum of the areas of the polymer peaks is not greater than the area of the peak of the control solution.
In the invention, the embodiment 1 is mainly different from the comparative example 1 in that the chromatographic column and the mobile phase are different, and the embodiment 1 of the invention meets the requirements of specificity and accuracy of a detection method by improving the separation principle of each substance.
As can be seen from the comparison of the mixed solution patterns of the lipoic acid and the polymer impurities, the separation of the polymer impurities cannot be realized in the polymers in the comparative example 1, and the detection method of the embodiment 1 of the invention has the advantages of simple operation, better reproducibility, strong specificity, good separation degree and high column efficiency of the chromatographic column, and can accurately determine the content of the lipoic acid polymer.
Claims (6)
1. A lipoic acid injection polymer detection method comprises the following steps:
(1) Preparation of test solution and control solution
Test solution: precisely measuring a proper amount of lipoic acid injection, and diluting with a mobile phase to prepare a lipoic acid solution;
control solution: precisely measuring the solution of the sample, placing the solution in a container, diluting the solution to a scale with a mobile phase, and shaking the solution uniformly;
(2) Chromatographic conditions
Chromatographic column: the filler is octadecylsilane chemically bonded silica gel,
detection wavelength: 200nm to 240nm of the nanometer silicon dioxide,
mobile phase: phosphoric acid solution-acetonitrile=90:10 with volume percentage of 0.1%,
flow rate: 0.1-0.9ml/min,
column temperature: 20-40 ℃,
sample injection amount: 5-15 μl;
(3) Measurement method
Precisely measuring 5-15 μl of each of the control solution and the sample solution, and injecting into a liquid chromatograph.
2. The method for detecting lipoic acid injection polymer according to claim 1, characterized in that: in the step (2), the size of the octadecylsilane chemically bonded silica filler in the column is 250mm×4.6mm,5 μm.
3. The method for detecting lipoic acid injection polymer according to claim 2, characterized in that: in step (2), the detection wavelength: 200nm, flow rate: 0.5ml/min, column temperature: 30 ℃, sample injection amount: 10 μl.
4. The method for detecting lipoic acid injection polymer according to claim 3, characterized in that: in step (3), the liquid chromatograph is Agilent 1260 in the United states.
5. The method for detecting lipoic acid injection polymer according to claim 4, wherein the method comprises the following steps: in the step (3), the number of theoretical plates of the chromatographic column is more than 5000.
6. The method for detecting lipoic acid injection polymer according to claim 5, wherein the method comprises the steps of: in the step (3), the separation degree of the lipoic acid and the adjacent impurity peaks is more than 1.5.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5621117A (en) * | 1994-07-30 | 1997-04-15 | Asta Medica Aktiengesellschaft | Method for the racemization of enantiomers of α-lipoic acid |
| JP2006306825A (en) * | 2005-04-30 | 2006-11-09 | Bhn Kk | STABILIZED alpha-LIPOIC ACID COMPOSITION AND ITS USE |
| CN105439925A (en) * | 2015-10-29 | 2016-03-30 | 南京海融医药科技有限公司 | Preparation method for lipoic acid polymer impurities and detection method for lipoic acid polymer impurities |
| CN113292533A (en) * | 2021-05-25 | 2021-08-24 | 四川智强医药科技开发有限公司 | Method for purifying polymer impurities in lipoic acid |
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- 2021-12-07 CN CN202111484938.1A patent/CN114280173B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5621117A (en) * | 1994-07-30 | 1997-04-15 | Asta Medica Aktiengesellschaft | Method for the racemization of enantiomers of α-lipoic acid |
| JP2006306825A (en) * | 2005-04-30 | 2006-11-09 | Bhn Kk | STABILIZED alpha-LIPOIC ACID COMPOSITION AND ITS USE |
| CN105439925A (en) * | 2015-10-29 | 2016-03-30 | 南京海融医药科技有限公司 | Preparation method for lipoic acid polymer impurities and detection method for lipoic acid polymer impurities |
| CN113292533A (en) * | 2021-05-25 | 2021-08-24 | 四川智强医药科技开发有限公司 | Method for purifying polymer impurities in lipoic acid |
Non-Patent Citations (3)
| Title |
|---|
| E. S. Nazarova et al.Analytical procurement of pharmaceutical development of preparation with thioctic acid in the form of injectable solution.International Journal of Green Pharmacy.2017,第11卷S774-783. * |
| Steven A. Kates et al.Lipoic acid analogs with enhanced pharmacological activity.Bioorganicamp Medicinal Chemistry.2013,第22卷505-512. * |
| 刘刚等.水相中无溶剂α-硫辛酸的制备.广州化工.2008,第36卷(第01期),1-3. * |
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