CN117368378B - Method for detecting content of auxiliary materials in levocarnitine oral solution - Google Patents
Method for detecting content of auxiliary materials in levocarnitine oral solution Download PDFInfo
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- 229940057738 levocarnitine oral solution Drugs 0.000 title claims abstract description 31
- 239000000463 material Substances 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 27
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 108
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 claims abstract description 36
- FTLYMKDSHNWQKD-UHFFFAOYSA-N (2,4,5-trichlorophenyl)boronic acid Chemical compound OB(O)C1=CC(Cl)=C(Cl)C=C1Cl FTLYMKDSHNWQKD-UHFFFAOYSA-N 0.000 claims abstract description 34
- 229940085605 saccharin sodium Drugs 0.000 claims abstract description 34
- 238000010828 elution Methods 0.000 claims abstract description 33
- -1 organic acid salt Chemical class 0.000 claims abstract description 26
- 238000001514 detection method Methods 0.000 claims abstract description 23
- 238000007865 diluting Methods 0.000 claims abstract description 21
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 claims abstract description 20
- 235000011090 malic acid Nutrition 0.000 claims abstract description 20
- 239000001630 malic acid Substances 0.000 claims abstract description 20
- PHIQHXFUZVPYII-ZCFIWIBFSA-N (R)-carnitine Chemical compound C[N+](C)(C)C[C@H](O)CC([O-])=O PHIQHXFUZVPYII-ZCFIWIBFSA-N 0.000 claims abstract description 19
- 229960001518 levocarnitine Drugs 0.000 claims abstract description 18
- 239000000872 buffer Substances 0.000 claims abstract description 15
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 13
- 238000002347 injection Methods 0.000 claims abstract description 10
- 239000007924 injection Substances 0.000 claims abstract description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000000945 filler Substances 0.000 claims abstract description 9
- FPLYNRPOIZEADP-UHFFFAOYSA-N octylsilane Chemical group CCCCCCCC[SiH3] FPLYNRPOIZEADP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000000741 silica gel Substances 0.000 claims abstract description 9
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 9
- 239000000243 solution Substances 0.000 claims description 39
- 239000013558 reference substance Substances 0.000 claims description 38
- 239000002904 solvent Substances 0.000 claims description 32
- 239000007853 buffer solution Substances 0.000 claims description 13
- 239000012488 sample solution Substances 0.000 claims description 13
- 239000000523 sample Substances 0.000 claims description 12
- 239000011259 mixed solution Substances 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 10
- 239000008055 phosphate buffer solution Substances 0.000 claims description 9
- 238000005303 weighing Methods 0.000 claims description 8
- 239000008351 acetate buffer Substances 0.000 claims description 3
- 238000010812 external standard method Methods 0.000 claims description 2
- 239000002671 adjuvant Substances 0.000 claims 1
- 239000012535 impurity Substances 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 3
- 238000012360 testing method Methods 0.000 description 15
- 239000012085 test solution Substances 0.000 description 9
- CAAULPUQFIIOTL-UHFFFAOYSA-N methyl dihydrogen phosphate Chemical group COP(O)(O)=O CAAULPUQFIIOTL-UHFFFAOYSA-N 0.000 description 6
- 239000008363 phosphate buffer Substances 0.000 description 6
- 229910019142 PO4 Inorganic materials 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 5
- 239000010452 phosphate Substances 0.000 description 5
- 229940100688 oral solution Drugs 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102000004420 Creatine Kinase Human genes 0.000 description 1
- 108010042126 Creatine kinase Proteins 0.000 description 1
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- 201000009623 Myopathy Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
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- 210000004027 cell Anatomy 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
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- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 208000010706 fatty liver disease Diseases 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 239000005445 natural material Substances 0.000 description 1
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- 229940126585 therapeutic drug Drugs 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to the field of medicine quality detection, in particular to a method for detecting the content of auxiliary materials in a levocarnitine oral solution. After diluting the sample to be detected, detecting by adopting a high performance liquid chromatography, wherein the chromatographic conditions are as follows: mobile phase: organic acid salt buffer and methanol; flow rate: 1.0mL/min; column temperature: 25-40 ℃; chromatographic column: using octyl silane bonded silica gel as a filler; sample injection volume: 10. Mu.L; detection wavelength: 200-210 nm; elution mode: gradient elution; the auxiliary materials comprise malic acid and saccharin sodium. The method can realize the effective separation of the levocarnitine, the malic acid and the saccharin sodium; meanwhile, the interference of other relevant impurities of the levocarnitine on auxiliary material detection is avoided, and the content of the auxiliary material of the levocarnitine oral solution can be accurately detected.
Description
Technical Field
The invention relates to the field of medicine quality detection, in particular to a method for detecting the content of auxiliary materials in a levocarnitine oral solution.
Background
Levocarnitine, also known as l-carnitine or l-carnitine, is a natural substance in the body that is essential for mammalian energy metabolism, and has the main function of promoting lipid metabolism. The lack of levocarnitine can cause hypoglycemia and ketone body reduction, and insufficient energy supply in tissue cells, resulting in cell damage; and the fatty utilization is reduced, and the fatty acid is accumulated in the liver, skeletal muscles and cardiac muscles, so that liver enzymes and creatine kinase are increased, and liver cell fatty degeneration and myopathy are caused. Supplementing levocarnitine may treat primary carnitine deficiency. In addition, the levocarnitine can be used as an auxiliary therapeutic drug for cardiovascular diseases, fatty liver, diabetes, immune dysfunction, renal dialysis, blood lipid reduction and the like. Meanwhile, the health-care food has the effects of resisting fatigue, resisting hypoxia, improving physical performance and the like, and can be used as a health-care product.
The domestic market demand of the levocarnitine preparation product is larger, and many enterprises develop the research of the levocarnitine related preparation. Compared with tablets and injections, the oral solution is more convenient to take, and can be used by infants and adults. In order to mask the special smell of the levocarnitine, adjust the taste of the preparation and improve the crowd acceptance, auxiliary materials such as regulator, corrective and the like are required to be added into the oral solution. According to investigation, malic acid and saccharin sodium are added into the imported levocarnitine oral solution, wherein the malic acid belongs to a pH regulator/antioxidant, and is beneficial to maintaining the stability of the preparation; saccharin sodium is used as a flavoring agent, so that the taste can be effectively improved. The dosage of the two auxiliary materials is particularly important in order to ensure the safety, stability and effectiveness of the medicine. At present, the literature, the patent and the like have no load of the method for detecting the relevant auxiliary materials of the levocarnitine oral solution, so that the method for detecting the content of the auxiliary materials of the levocarnitine oral solution is developed and established.
Disclosure of Invention
The invention aims to provide a method for detecting the content of auxiliary materials in a levocarnitine oral solution, which can accurately detect the content of the auxiliary materials in the levocarnitine oral solution, is simple and easy to operate, has high sensitivity, has good specificity, system applicability, solution stability and chromatographic condition durability, and provides an effective prescription analysis method for drug development.
According to the method for detecting the content of auxiliary materials in the levocarnitine oral solution, a sample to be detected is diluted by a solvent and then is detected by a high performance liquid chromatography, and chromatographic conditions are as follows:
mobile phase: organic acid salt buffer and methanol;
flow rate: 1.0mL/min;
column temperature: 25-40 ℃, preferably 30 ℃;
chromatographic column: using octyl silane bonded silica gel as a filler;
sample injection volume: 10. Mu.L;
detection wavelength: 200 to 210nm, preferably 205nm;
elution mode: gradient elution;
the gradient elution conditions were:
the first gradient elution time is 0, the organic acid salt buffer solution accounts for 95+/-2%, and the methanol accounts for 5+/-2%;
the second gradient elution time is 6+/-2 min, the organic acid salt buffer solution accounts for 90+/-2%, and the methanol accounts for 10+/-2%;
the third gradient elution time is 20+/-2 min, the organic acid salt buffer accounts for 90+/-2%, and the methanol accounts for 10+/-2%;
the fourth gradient elution time is 25+/-2 min, the organic acid salt buffer solution accounts for 70+/-2%, and the methanol accounts for 30+/-2%;
fifth gradient elution time is 45+/-2 min, organic acid salt buffer accounts for 70+/-2%, and methanol accounts for 30+/-2%;
the sixth gradient elution time is 48+/-2 min, the organic acid salt buffer accounts for 95+/-2%, and the methanol accounts for 5+/-2%;
wherein the auxiliary materials are malic acid and saccharin sodium; the solvent is methanol-organic acid salt buffer solution, the volume content of methanol is 3-10%, the organic acid salt buffer solution is phosphate buffer solution or acetate buffer solution, and the pH is 2.2-2.7.
Preferably, the detection is performed by high performance liquid chromatography, and the gradient elution procedure is as follows:
| time (min) | Organic acid salt buffer (%) | Methanol (%) |
| 0 | 95 | 5 |
| 6 | 90 | 10 |
| 20 | 90 | 10 |
| 25 | 70 | 30 |
| 42 | 70 | 30 |
| 48 | 95 | 5 |
Preferably, the organic acid salt buffer is a phosphate buffer.
The invention relates to a method for detecting the content of auxiliary materials in a levocarnitine oral solution, which specifically comprises the following steps:
(1) Preparing a sample solution;
(2) Preparing a system applicability solution;
(3) Preparing a reference substance solution;
(4) The detection method comprises the following steps: and (3) separating by adopting high performance liquid chromatography and utilizing a gradient elution program, and controlling the content of auxiliary materials by using an external standard method.
The preferable technical scheme is as follows:
in the step (1), the preparation method of the sample solution comprises the following steps: taking 2ml of levocarnitine oral solution, placing in a 20ml measuring flask, diluting to scale with solvent, and shaking uniformly to obtain the test solution.
In the step (2), the preparation method of the system applicability solution comprises the following steps: dissolving and diluting the levocarnitine reference substance, the malic acid reference substance and the saccharin sodium reference substance with a solvent to prepare a mixed solution containing 1mg/mL of levocarnitine, 0.3mg/mL of malic acid and 0.05mg/mL of saccharin sodium, thereby obtaining a system applicability solution.
In the step (3), the preparation method of the reference substance solution comprises the following steps: respectively precisely weighing 3mg of DL-malic acid and 3mg of saccharin sodium reference substance, dissolving in solvent, and diluting to obtain mixed solution containing about 0.3mg/ml of DL-malic acid and about 0.05mg/ml of saccharin sodium as reference substance solution.
Compared with the prior art, the invention has the following beneficial effects:
1. the method can realize the effective separation of the levocarnitine, the malic acid and the saccharin sodium; meanwhile, the interference of other relevant impurities of the levocarnitine on the detection of malic acid and saccharin sodium is avoided, and the content of the malic acid and the saccharin sodium in the auxiliary materials of the levocarnitine oral solution can be accurately detected.
2. The method for detecting the content of the auxiliary materials in the levocarnitine oral solution is simple to operate, easy to control, high in sensitivity, good in specificity, system applicability, solution stability and chromatographic condition durability, and an effective detection method is provided for analyzing the dosage of the auxiliary materials of a sample.
Drawings
FIG. 1 is a chromatogram of a blank solvent for a levocarnitine oral solution;
FIG. 2 is a chromatogram of a test solution of a levocarnitine oral solution;
FIG. 3 is a chromatogram of a control solution.
Detailed Description
In order to make the objects, technical solutions and effects of the present application clearer and more specific, the present invention is further described below with reference to examples. And are intended to be illustrative of the invention only and are not intended to limit the scope of the invention.
Example 1
The chromatographic column uses octyl silane bonded silica gel as filler, the specification is 250 multiplied by 4.6mm,5 μm, and the Xtime series; column temperature: 30 ℃; flow rate: 1.0mL/min; a UV detector with a detection wavelength of 200nm; sample injection volume: 10 mu L.
Wherein the solvent is methanol-phosphate buffer solution, the volume content of methanol is 3%, and the pH value of phosphate is 2.5; the mobile phase of the high performance liquid chromatography comprises: mobile phase a, 0.02M phosphate buffer, pH 2.5; mobile phase B, methanol; gradient elution was performed using the following procedure:
| time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 97 | 3 |
| 8 | 92 | 8 |
| 20 | 88 | 12 |
| 23 | 70 | 30 |
| 40 | 72 | 28 |
| 48 | 95 | 5 |
Preparing a reference substance solution: and respectively precisely weighing appropriate amounts of DL-malic acid and saccharin sodium reference substances, dissolving in a solvent, and diluting to obtain a mixed solution containing about 0.3mg/ml DL-malic acid and about 0.05mg/ml saccharin sodium as reference substance solution.
Preparing a sample solution: taking 2ml of levocarnitine oral solution, placing in a 20ml measuring flask, diluting to scale with solvent, and shaking uniformly to obtain the test solution.
Good specificity:
proved by verification, the solvent and each impurity do not interfere with the detection of the levocarnitine peak and the malic acid peak and the saccharin sodium peak; (2) The degree of separation between the main peak and the adjacent impurities and between the adjacent impurities is not less than 1.5.
TABLE 1 results of specific solution testing
Example 2
The chromatographic column uses octyl silane bonded silica gel as filler, the specification is 250 multiplied by 4.6mm,5 μm, and the Xtime series; column temperature: 25 ℃; flow rate: 1.0mL/min; a UV detector with a detection wavelength of 210nm; sample injection volume: 10 mu L.
Wherein the solvent is methanol-phosphate buffer solution, the volume content of methanol is 5%, and the pH value of acetate is 2.2; the mobile phase of the high performance liquid chromatography comprises: mobile phase a, acetate buffer, pH 2.2; mobile phase B, methanol; gradient elution was performed using the following procedure:
| time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 93 | 7 |
| 6 | 88 | 12 |
| 22 | 90 | 10 |
| 25 | 72 | 28 |
| 42 | 70 | 30 |
| 46 | 95 | 5 |
Preparing a reference substance solution: and respectively precisely weighing appropriate amounts of DL-malic acid and saccharin sodium reference substances, dissolving in a solvent, and diluting to obtain a mixed solution containing about 0.3mg/ml DL-malic acid and about 0.05mg/ml saccharin sodium as reference substance solution.
Preparing a sample solution: taking 2ml of levocarnitine oral solution, placing in a 20ml measuring flask, diluting to scale with solvent, and shaking uniformly to obtain the test solution.
Good system applicability:
the solvent does not interfere with the detection of each peak, the number of theoretical plates of the malic acid peak is not less than 7000, and the tailing factor is 1.1; the number of saccharin sodium theoretical plates is not lower than 29000, and the tailing factor is 1.5; and continuously sampling the reference substance solution for 6 times, wherein the RSD of the peak areas of the malic acid and the saccharin sodium is less than 0.5%. The results showed good systemicity.
Example 3
The chromatographic column uses octyl silane bonded silica gel as filler, the specification is 250 multiplied by 4.6mm,5 μm, and the Xtime series; column temperature: 35 ℃; flow rate: 1.0mL/min; a UV detector with a detection wavelength of 205nm; sample injection volume: 10 mu L.
Wherein the solvent is methanol-phosphate buffer solution, the volume content of methanol is 7%, and the pH value of phosphate is 2.7; the mobile phase of the high performance liquid chromatography comprises: mobile phase a, 0.02M phosphate buffer, pH 2.7; mobile phase B, methanol; gradient elution was performed using the following procedure:
| time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 95 | 5 |
| 6 | 90 | 10 |
| 18 | 90 | 10 |
| 27 | 70 | 30 |
| 44 | 68 | 32 |
| 48 | 93 | 7 |
Preparing a reference substance solution: and respectively precisely weighing appropriate amounts of DL-malic acid and saccharin sodium reference substances, dissolving in a solvent, and diluting to obtain a mixed solution containing about 0.3mg/ml DL-malic acid and about 0.05mg/ml saccharin sodium as reference substance solution.
Preparing a sample solution: taking 2ml of levocarnitine oral solution, placing in a 20ml measuring flask, diluting to scale with solvent, and shaking uniformly to obtain the test solution.
Good linearity:
and gradually diluting the malic acid and saccharin sodium reference substance solution with a proper amount of solvent to prepare a series of linear solutions. The test results are shown in Table 2.
TABLE 2 Linear results
Example 4
The chromatographic column uses octyl silane bonded silica gel as filler, the specification is 250 multiplied by 4.6mm,5 μm, and the Xtime series; column temperature: 40 ℃; flow rate: 1.0mL/min; a UV detector with a detection wavelength of 205nm; sample injection volume: 10 mu L.
Wherein the solvent is methanol-phosphate buffer solution, the volume content of methanol is 10%, and the pH value of phosphate is 2.5; the mobile phase of the high performance liquid chromatography comprises: mobile phase a, 0.02M phosphate buffer, pH 2.5; mobile phase B, methanol; gradient elution was performed using the following procedure:
| time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 95 | 5 |
| 6 | 90 | 10 |
| 20 | 90 | 10 |
| 25 | 70 | 30 |
| 42 | 70 | 30 |
| 48 | 95 | 5 |
Preparing a reference substance solution: and respectively precisely weighing appropriate amounts of DL-malic acid and saccharin sodium reference substances, dissolving in a solvent, and diluting to obtain a mixed solution containing about 0.3mg/ml DL-malic acid and about 0.05mg/ml saccharin sodium as reference substance solution.
Preparing a sample solution: taking 2ml of levocarnitine oral solution, placing in a 20ml measuring flask, diluting to scale with solvent, and shaking uniformly to obtain the test solution.
Good accuracy:
taking the L-carnitine oral solution, a DL-malic acid reference substance and a saccharin sodium reference substance, preparing samples with 80%, 100% and 120% of different concentrations, measuring, preparing 3 samples for each concentration, and evaluating the recovery rate of 9 samples. The test results are shown in tables 3 and 4.
TABLE 3 DL malic acid accuracy test results
Table 4 sodium saccharin accuracy test results
Example 5
The chromatographic column uses octyl silane bonded silica gel as filler, the specification is 250 multiplied by 4.6mm,5 μm, and the Xtime series; column temperature: 30 ℃; flow rate: 1.0mL/min; a UV detector with a detection wavelength of 205nm; sample injection volume: 10 mu L.
Wherein the solvent is methanol-phosphate buffer solution, the volume content of methanol is 5%, and the pH value of phosphate is 2.5; the mobile phase of the high performance liquid chromatography comprises: mobile phase a, 0.02M phosphate buffer, pH 2.5; mobile phase B, methanol; gradient elution was performed using the following procedure:
| time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 95 | 5 |
| 4 | 90 | 10 |
| 20 | 90 | 10 |
| 25 | 68 | 32 |
| 42 | 70 | 30 |
| 50 | 95 | 5 |
Preparing a reference substance solution: and respectively precisely weighing appropriate amounts of DL-malic acid and saccharin sodium reference substances, dissolving in a solvent, and diluting to obtain a mixed solution containing about 0.3mg/ml DL-malic acid and about 0.05mg/ml saccharin sodium as reference substance solution.
Preparing a sample solution: taking 2ml of levocarnitine oral solution, placing in a 20ml measuring flask, diluting to scale with solvent, and shaking uniformly to obtain the test solution.
Good stability:
taking reference substance solution and test substance solution, respectively sampling at different times, and testing the sample solution, wherein the test results are shown in tables 5 and 6. The RSD of each chromatographic peak area is less than 1% within 48 hours of the reference substance solution and the test sample solution, and the stability is good.
TABLE 5 stability test results for control
TABLE 6 test sample stability test results
Example 6
The chromatographic column uses octyl silane bonded silica gel as filler, the specification is 250 multiplied by 4.6mm,5 μm, and the Xtime series; column temperature: 30 ℃; flow rate: 1.0mL/min; a UV detector with a detection wavelength of 205nm; sample injection volume: 10 mu L.
Wherein the solvent is methanol-phosphate buffer solution, the volume content of methanol is 5%, and the pH value of phosphate is 2.5; the mobile phase of the high performance liquid chromatography comprises: mobile phase a, 0.02M phosphate buffer, pH 2.5; mobile phase B, methanol; gradient elution was performed using the following procedure:
| time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 95 | 5 |
| 6 | 90 | 10 |
| 20 | 90 | 10 |
| 25 | 70 | 30 |
| 42 | 70 | 30 |
| 48 | 97 | 3 |
Preparing a reference substance solution: and respectively precisely weighing appropriate amounts of DL-malic acid and saccharin sodium reference substances, dissolving in a solvent, and diluting to obtain a mixed solution containing about 0.3mg/ml DL-malic acid and about 0.05mg/ml saccharin sodium as reference substance solution.
Preparing a sample solution: taking 2ml of levocarnitine oral solution, placing in a 20ml measuring flask, diluting to scale with solvent, and shaking uniformly to obtain the test solution.
Good durability:
the degree of influence on the detection result was tested by making small changes to each of the chromatographic conditions. In the implementation test, the column temperature of the chromatographic column is +/-5 ℃, the flow rate is +/-0.2 mL/min, the pH of the mobile phase is +/-0.2, and different chromatographic columns are respectively verified. The control solution and the test solution were respectively taken and tested under each condition, and the impurity content was statistically analyzed, and the test results are shown in tables 7 and 8.
TABLE 7 malic acid durability test results
Table 8 sodium saccharin durability test results
According to the test results of the embodiment, the method for detecting the content of the auxiliary materials of the levocarnitine oral solution is simple to operate, easy to control, high in sensitivity, good in specificity, system applicability, linearity, accuracy, solution stability and chromatographic condition durability, and an effective detection method is provided.
Claims (6)
1. A method for detecting the content of auxiliary materials in a levocarnitine oral solution is characterized by comprising the following steps of: after diluting a sample to be detected by a solvent, detecting by a high performance liquid chromatography, wherein the method specifically comprises the following steps of:
(1) Preparing a sample solution;
(2) Preparing a system applicability solution;
(3) Preparing a reference substance solution;
(4) The detection method comprises the following steps: adopting high performance liquid chromatography, separating by gradient elution program, and controlling adjuvant content by external standard method;
the chromatographic conditions were as follows:
mobile phase: organic acid salt buffer and methanol;
flow rate: 1.0mL/min;
column temperature: 25-40 ℃;
chromatographic column: using octyl silane bonded silica gel as a filler;
sample injection volume: 10. Mu.L;
detection wavelength: 200-210 nm;
elution mode: gradient elution;
the gradient elution conditions were:
the first gradient elution time is 0, the organic acid salt buffer accounts for 95 percent, and the methanol accounts for 5 percent;
the second gradient elution time is 6+/-2 min, the organic acid salt buffer solution accounts for 90 percent, and the methanol accounts for 10 percent;
the third gradient elution time is 20+/-2 min, the organic acid salt buffer solution accounts for 90 percent, and the methanol accounts for 10 percent;
fourth gradient elution time 25+ -2 min, organic acid salt buffer 70% and methanol 30%;
fifth gradient elution time is 45+/-2 min, organic acid salt buffer accounts for 70% and methanol accounts for 30%;
the sixth gradient elution time is 48+/-2 min, the organic acid salt buffer accounts for 95 percent, and the methanol accounts for 5 percent;
wherein the auxiliary materials are malic acid and saccharin sodium; the solvent is methanol-organic acid salt buffer solution, the volume content of methanol is 3-10%, the organic acid salt buffer solution is phosphate buffer solution or acetate buffer solution, and the pH is 2.2-2.7.
2. The method for detecting the content of auxiliary materials in the levocarnitine oral solution according to claim 1, which is characterized in that: the detection is carried out by adopting a high performance liquid chromatography, and the gradient elution program is as follows:
the first gradient elution time is 0, the organic acid salt buffer accounts for 95 percent, and the methanol accounts for 5 percent;
the second gradient elution time is 6min, the organic acid salt buffer solution accounts for 90 percent, and the methanol accounts for 10 percent;
the third gradient elution time is 20min, the organic acid salt buffer solution accounts for 90 percent, and the methanol accounts for 10 percent;
the fourth gradient elution time is 25min, the organic acid salt buffer solution accounts for 70 percent, and the methanol accounts for 30 percent;
fifth gradient elution time is 45min, organic acid salt buffer accounts for 70% and methanol accounts for 30%;
the sixth gradient elution time was 48min, organic acid salt buffer at 95% and methanol at 5%.
3. The method for detecting the content of auxiliary materials in the levocarnitine oral solution according to claim 1, which is characterized in that: the organic acid salt buffer solution is phosphate buffer solution.
4. The method for detecting the content of auxiliary materials in the levocarnitine oral solution according to claim 1, which is characterized in that: the column temperature was 30 ℃.
5. The method for detecting the content of auxiliary materials in the levocarnitine oral solution according to claim 1, which is characterized in that: the detection wavelength is 205nm.
6. The method for detecting the content of auxiliary materials in the levocarnitine oral solution according to claim 1, which is characterized in that: the said
In the step (1), the preparation method of the sample solution comprises the following steps: taking 2ml of levocarnitine oral solution, placing in a 20ml measuring flask, diluting to scale with solvent, and shaking uniformly to obtain a sample solution; in the step (2), the preparation method of the system applicability solution comprises the following steps: dissolving and diluting a levocarnitine reference substance, a malic acid reference substance and a saccharin sodium reference substance with a solvent to prepare a mixed solution containing 1mg/mL of levocarnitine, 0.3mg/mL of malic acid and 0.05mg/mL of saccharin sodium, thereby obtaining a system applicability solution; in the step (3), the preparation method of the reference substance solution comprises the following steps: respectively precisely weighing 3mg of malic acid and 3mg of saccharin sodium reference substance, dissolving in solvent, and diluting to obtain mixed solution containing 0.3mg/ml malic acid and 0.05mg/ml saccharin sodium as reference substance solution.
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