Detection method of coenzyme Q10 injection
By using methanol and ethanol as the mobile phase in liquid chromatography, gradient elution, and octadecylsilane-bonded silica gel packing material under specific conditions, the interference problem in the detection of vitamin E in coenzyme Q10 injection was solved, achieving efficient and accurate detection results.
Patent Information
- Application Number
- CN202411202375.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-29
- Publication Date
- 2026-03-10
AI Technical Summary
Existing technologies cannot effectively avoid the interference of excipients in coenzyme Q10 injection on vitamin E detection, resulting in inaccurate and unstable test results.
Methanol and ethanol were used as the mobile phase in a gradient elution liquid chromatography method with octadecylsilane-bonded silica gel packing. The detection wavelength was 281-291 nm, the column temperature was 25-37 °C, the flow rate was 0.8-1.2 ml/min, and the injection volume was 10-30 μl to ensure the separation of vitamin E and coenzyme Q10.
This method enables accurate and rapid detection of vitamin E in coenzyme Q10 injection, avoiding interference from excipients and improving the specificity, precision, and stability of the detection.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of chemical medicine detection, and particularly relates to a detection method of vitamin E in coenzyme Q10 injection. BACKGROUND
[0002] Coenzyme Q10 is a kind of fat-soluble antioxidant, which can activate human cells and cell energy nutrition, has the functions of improving human immunity, enhancing antioxidant, delaying aging and enhancing human vitality, and has the effect of activating cell metabolism. The structure of coenzyme Q10 is similar to that of vitamin E, vitamin K and plastoquinone. In the human body, coenzyme Q10 mainly participates in the activation and manufacture of energy in cells, and can effectively prevent atherosclerosis. Coenzyme Q10 has the effect of strengthening heart skills, and can effectively relieve the condition of heart hypoxia by supplementing the missing coenzyme Q10 for the heart. In addition, coenzyme Q10 can promote oxidative phosphorylation reaction, effectively protect the integrity of biological membranes, improve the immune function of the body, activate human cells and cell energy nutrition, enhance antioxidant, delay aging and enhance human vitality. In clinical application, coenzyme Q10 injection is mainly used for the adjuvant treatment of cardiovascular diseases, duodenal ulcers and cancer patients, and is also widely used in the fields of food additives and nutritional health products.
[0003] Coenzyme Q10 is a yellow to orange yellow crystalline powder, odorless and tasteless, insoluble in water and methanol, slightly soluble in ethanol, soluble in diethyl ether, acetone, easily soluble in chloroform, carbon tetrachloride and benzene, and contains quinone group in the molecular structure. Coenzyme Q10 is easily decomposed by light and oxygen, and the preparation sample has the problem of poor stability. The coenzyme Q10 dosage forms developed at home and abroad include tablets, lyophilized agents and injections. Since coenzyme Q10 belongs to the second class of difficult-to-dissolve drugs in the biological pharmaceutical classification system, its solubility is low and its stability is poor, which greatly increases the difficulty of preparation of its preparation. In the preparation of coenzyme Q10 injection, some antioxidants such as vitamin E are added to effectively improve the stability of coenzyme Q10 and the efficacy and safety of the drug.
[0004] At present, in the second part of the People's Republic of China Pharmacopoeia 2020 edition, the content of vitamin E is detected by gas chromatography, and the solvent is n-hexane. N-hexane and coenzyme Q10 injection cannot be mutually soluble. Through literature review, the content of vitamin E in compound nutritional suspension is determined by liquid chromatography. The chromatographic conditions are as follows: using Merck Hibar C18 (4.6 mm*250 mm, 5 mu m) chromatographic column, using methanol as mobile phase, flow rate is 1.0 ml / min, detection wavelength is 280 nm; The content of vitamin E in natural vitamin E soft capsules is determined, and the chromatographic conditions are as follows: Alltima C18 chromatographic column (150 mm*4.6 mm, 5 mu m), methanol as mobile phase, flow rate is 1.0 ml / min, detection wavelength is 285 nm. The above literatures are all reversed C18 chromatographic column, and the mobile phase does not contain water phase, which is similar to the content determination method of coenzyme Q10 raw material, but the above methods are not suitable for the content determination of vitamin E in coenzyme Q10 injection. The content of auxiliary material interferes with the detection of vitamin E.
[0005] The method provided by the application can avoid the interference of the auxiliary material in the injection on the detection of vitamin E, and through the research on the specificity, accuracy, precision, detection limit, quantitative limit, linear range and durability of the method, it is proved that the method provided by the application is suitable for the detection requirements of vitamin E in coenzyme Q10 injection. SUMMARY
[0006] The application aims to provide a liquid phase detection method of vitamin E with simple operation, short detection time and good method reproducibility.
[0007] To achieve the above application purposes, the application provides a detection method of coenzyme Q10 injection, and the specific implementation scheme is as follows: The detection method of coenzyme Q10 injection provided by the application is a detection method of vitamin E in coenzyme Q10 injection, methanol is used as mobile phase A, ethanol is used as mobile phase B, gradient elution, and the gradient elution parameters are as follows: .
[0008] The detection method of coenzyme Q10 injection provided by the application is a detection method of vitamin E in coenzyme Q10 injection, methanol is used as mobile phase A, ethanol is used as mobile phase B, gradient elution, and the gradient elution parameters are as follows:
[0009] The detection method of coenzyme Q10 injection provided by the application is a detection method of vitamin E in coenzyme Q10 injection, methanol is used as mobile phase A, ethanol is used as mobile phase B, gradient elution, and the gradient elution parameters are as follows:
[0010] The application discloses a detection method of coenzyme Q10 injection, and the method uses octadecylsilane bonded silica gel as a filler, and the detection wavelength is 286±2nm, the column temperature is 30±5 DEG C, the flow rate is 1.0±0.2ml / min, and the injection volume is 20ul.
[0011] The application discloses a detection method of coenzyme Q10 injection, and the method uses octadecylsilane bonded silica gel as a filler, and the detection wavelength is 286±2nm, the column temperature is 30±5 DEG C, the flow rate is 1.0±0.2ml / min, and the injection volume is 20ul. 1) blank solvent: anhydrous ethanol; 2) blank adjuvant solution: accurately measuring the blank adjuvant, and diluting to the scale in a certain volume of a measuring flask with anhydrous ethanol, and shaking uniformly; 3) control solution: accurately measuring the vitamin E control, and dissolving and quantitatively diluting to the scale with anhydrous ethanol, and shaking uniformly; 4) test solution: accurately measuring the coenzyme Q10 injection, and diluting to the scale in a certain volume of a measuring flask with anhydrous ethanol, and shaking uniformly; 5) accurately measuring the blank solvent, the blank adjuvant solution, the test solution and the control solution, and injecting into a liquid chromatograph respectively, and recording a chromatogram.
[0012] The application discloses a detection method of coenzyme Q10 injection, and the method uses octadecylsilane bonded silica gel as a filler, and the detection wavelength is 286±2nm, the column temperature is 30±5 DEG C, the flow rate is 1.0±0.2ml / min, and the injection volume is 20ul.
[0013] The application discloses a detection method of coenzyme Q10 injection, and the method uses octadecylsilane bonded silica gel as a filler, and the detection wavelength is 286±2nm, the column temperature is 30±5 DEG C, the flow rate is 1.0±0.2ml / min, and the injection volume is 20ul.
[0014] Beneficial effect analysis of the application: The method provided by the application can avoid the interference of the adjuvant existing in the injection on the detection of vitamin E, and meanwhile, through the research on the specificity, accuracy, precision, detection limit, quantitative limit, linear range and durability of the method, it is proved that the method is suitable for the detection requirements of vitamin E in coenzyme Q10 injection.
[0015] The application mainly aims at determining the content of vitamin E in coenzyme Q10 injection, can effectively separate vitamin E from other adjuvants and coenzyme Q10, and ensures that the reference material is stable and reliable in quality. The application makes quantitative analysis on the vitamin in coenzyme Q10 injection, makes up for the deficiency of the existing data on the quality control method, and has important significance for improving the quality control technology of vitamin E in coenzyme Q10 injection.
[0016] Brief description of drawings: Figure 1 It is a linear relationship diagram of vitamin E. Specific implementation
[0017] The following examples are only for further illustration of the present invention and do not limit the scope of the present invention in any way. Example
[0018] 1. Solution preparation: Reference solution: Dissolve an appropriate amount of vitamin E reference standard in anhydrous ethanol and dilute to 14 μg / ml. Test solution: Accurately measure 1 ml of this product into a 50 ml volumetric flask, dilute to the mark with anhydrous ethanol, and shake well.
[0019] 2. Chromatographic conditions 2.1. Mobile phase and detection wavelength Octadecylsilane-bonded silica gel is used as the packing material (recommended column size: 4.6*150mm, 5μm). Mobile phase: Methanol as mobile phase A, ethanol as mobile phase B, gradient elution according to the table below; Detection wavelength: 286 nm, injection volume: 20 μl 2.2 Column temperature, flow rate, and injection volume Column temperature: 35℃; flow rate: 1.0 ml / min; injection volume: 20 μl;
[0020] 3. Methodological Examination 3.1 Exclusivity A vitamin E-deficient negative control solution was prepared according to the method for preparing the test solution, and the interference of the negative test sample and the blank solvent (anhydrous ethanol) was investigated. HPLC analysis was performed under the planned chromatographic conditions, and the chromatograms were recorded. The results are as follows.
[0021] Table 1 Specificity Detection Results
[0022] The data above show that the blank solvent and blank excipient do not interfere with the detection of vitamin E; the theoretical plate number of the reference solution, calculated based on the vitamin E peak, is greater than 2000, indicating that this method has good specificity.
[0023] 3.2 System Precision Accurately measure 20 µl of the reference solution and inject it into the liquid chromatograph. Repeat the injection 6 times and record the chromatograms. The results are shown in Table 2.
[0024] Table 2 System precision test results
[0025] The data above show that when the reference solution was injected six times consecutively, the RSD of vitamin E retention time was less than 1.0% and the RSD of peak area was less than 2.0%, indicating that the system has good precision.
[0026] 3.3 Solution stability Take the control solution and test solution, sealed at room temperature, respectively at different time points, sample detection, record chromatogram, the results are shown in table 3~4.
[0027] Table 3 stability of control solution
[0028] Table 4 stability of test solution
[0029] From the above data, the control solution, the control solution is placed at room temperature for 27 hours, the RSD value of peak area is less than 2.0%, indicating that the control solution is placed at room temperature for 27 hours, which is relatively stable.
[0030] 3.4 Linearity and range Linear stock solution: about 14 mg of vitamin E was accurately measured and dissolved in 20 ml of anhydrous ethanol, and then diluted to the mark. Shake well. (About 700 μg / ml of vitamin E) Limit of quantification solution: 1 ml of control solution was accurately measured and placed in a 200 ml volumetric flask, and then diluted to the mark with solvent. Shake well.
[0031] 50% linear solution: 1 ml of linear stock solution was accurately measured and placed in a 100 ml volumetric flask, and then diluted to the mark with solvent. Shake well.
[0032] 80% linear solution: 0.8 ml of linear stock solution was accurately measured and placed in a 25 ml volumetric flask, and then diluted to the mark with solvent. Shake well.
[0033] 100% linear solution: 1 ml of linear stock solution was accurately measured and placed in a 50 ml volumetric flask, and then diluted to the mark with solvent. Shake well.
[0034] 150% linear solution: 1.5 ml of linear stock solution was accurately measured and placed in a 50 ml volumetric flask, and then diluted to the mark with solvent. Shake well.
[0035] 200% linear solution: 1 ml of linear stock solution was accurately measured and placed in a 25 ml volumetric flask, and then diluted to the mark with solvent. Shake well.
[0036] Accurately measure 20 μl of each linear solution and inject it into the liquid chromatograph, respectively, and record the chromatogram, Figure 1 The linear relationship diagram of vitamin E, and the test results are shown in table 5.
[0037] Table 5 results of linearity and range experiment
[0038] From the above data, it can be seen that vitamin E has a good linear relationship with peak area in the concentration range of 200% limit concentration, the correlation coefficient r is greater than 0.9990, the absolute value of the intercept is less than 2.0% of the response value, and the RSD value of the response factor is less than 2%, indicating good linear relationship.
[0039] 3.5 Accuracy Recovery stock solution: accurately measure about 14 mg of vitamin E into a 20 ml, dissolve and dilute to the mark with absolute ethanol, shake well. (About 700 μg / ml of vitamin E) 80% recovery solution: accurately measure 0.8 ml of the recovery stock solution into a 50 ml volumetric flask, dilute to the mark with blank excipients, shake well to obtain. Prepare 3 parallel.
[0040] 100% recovery solution: accurately measure 1 ml of the recovery stock solution into a 50 ml volumetric flask, dilute to the mark with blank excipients 2, shake well to obtain. Prepare 3 parallel.
[0041] 120% recovery solution: accurately measure 1.2 ml of the recovery stock solution into a 50 ml volumetric flask, dilute to the mark with blank excipients 2, shake well to obtain. Prepare 3 parallel.
[0042] Accurately measure 20 μl of the reference solution and the recovery solution, inject into the liquid chromatograph, record the chromatogram, the results are shown in Table 6.
[0043] Table 6 Accuracy test results
[0044] From the above data, it can be seen that the recovery rate calculated by external standard method at different concentration levels of high (120%), medium (100%) and low (80%) is between 98%~102%, and the RSD is less than 2.0%, indicating that the method has good accuracy.
[0045] 3.6 Reproducibility Accurately measure 20 μl of the reference solution and the test solution (6 parallel preparations), respectively, inject into the liquid chromatograph, record the chromatogram, the results are shown in Table 7.
[0046] Table 7 Reproducibility test results
[0047] From the above data, it can be seen that the content of vitamin E in each 1 ml of the 6 test samples is between 560~840 μg, the average is 700.52 μg / ml, and the RSD value of the content result is less than 2%, indicating that the method has good reproducibility.
[0048] 3.7 Intermediate precision Different personnel, at different times, with different instruments, the same homogeneous sample (solution preparation same reproducibility test) preparation of 6 copies in parallel, in accordance with the law of detection, to investigate the method of intermediate precision. The first group of test results are detailed in reproducibility test.
[0049] Determination: accurately measure the blank solvent, blank excipient solution, reference solution, test solution 20 μl, respectively, into the liquid chromatograph, record the chromatogram, the results are shown in Tables 8 ~ 9.
[0050] Table 8 Intermediate precision test results (1)
[0051] Table 9 Intermediate precision test results (2)
[0052] From the above data, different experimenters on different dates using different instruments for determination of 6 test samples, with 6 test solution of 12 test solution, each 1 ml contains vitamin E in 560 ~ 840 μg, the average is 699.55 μg / ml, the RSD value of the content of 12 test solution is less than 2%, indicating that the method of intermediate precision is good.
[0053] 3.8 Durability The column temperature (± 5 ℃), flow rate (± 0.2 ml / min), mobile phase ratio (± 5%), detection wavelength (± 2 nm) are changed, and the rest of the chromatographic conditions remain unchanged. The blank solvent, blank excipient solution, reference solution and test solution are detected according to the law, and the results are shown in Tables 10 ~ 11.
[0054] Table 10 Durability test results (1)
[0055] Table 11 Durability test results (2)
[0056] From the above data, fine tuning of chromatographic conditions: blank solvent and blank excipient do not interfere with the detection of vitamin E; in the reference solution, the theoretical plate number of vitamin E is greater than 2000; the RSD value of the test solution is less than 2%; indicating that the chromatographic condition is good in durability.
[0057] The present application mainly aims at determining the content of vitamin E in coenzyme Q10 injection, can effectively separate vitamin E from other excipients and coenzyme Q10, and ensure that the reference material quality is stable and reliable. The present application makes up for the deficiency of the existing data on the quality control method by quantitatively analyzing the vitamin in coenzyme Q10 injection, and has important significance for improving the quality control technology of vitamin E in coenzyme Q10 injection.
Claims
1. A method for detecting coenzyme Q10 injection, characterized by, The method is a detection method of vitamin E in coenzyme Q10 injection, methanol is used as mobile phase A, ethanol is used as mobile phase B, gradient elution, and the gradient elution parameters are as follows: 。 2. The detection method for coenzyme Q10 injection according to claim 1, characterized in that, The ratio of the mobile phase A to the mobile phase B is 0-85:100-15.
3. The detection method for coenzyme Q10 injection according to claim 1, characterized in that, The method uses octadecylsilane-bonded silica gel as the filler, the detection wavelength is 281-291 nm, the column temperature is 25-37 DEG C, the flow rate is 0.8-1.2 ml / min, and the injection volume is 10-30 μl.
4. The detection method for coenzyme Q10 injection according to claim 4, characterized in that, The method uses octadecylsilane-bonded silica gel as the filler, the detection wavelength is 286±2 nm, the column temperature is 30±5 DEG C, the flow rate is 1.0±0.2 ml / min, and the injection volume is 20 μl.
5. The method for detecting coenzyme Q10 injection according to any one of claims 1 to 4, wherein The method comprises the following steps: 1) blank solvent: anhydrous ethanol; 2) blank excipient solution: accurately measure the appropriate amount of blank excipient into a certain volume of a measuring flask, dilute to the scale with anhydrous ethanol, and shake well; 3) control solution: take the appropriate amount of vitamin E control, accurately weigh, dissolve and quantitatively dilute to the scale with anhydrous ethanol, and shake well; 4) test solution: accurately measure the appropriate amount of coenzyme Q10 injection into a certain volume of a measuring flask, dilute to the scale with anhydrous ethanol, and shake well; 5) accurately measure the blank solvent, blank excipient solution, test solution and control solution, respectively, inject into the liquid chromatograph, and record the chromatogram.
6. The detection method for coenzyme Q10 injection according to claim 5, characterized in that, The concentration of vitamin E control in the solution is 20-40 μg / ml, and the concentration of coenzyme Q10 in the solution is 0.8-1.5 mg / ml.
7. The detection method for coenzyme Q10 injection according to claim 6, characterized in that, The concentration of coenzyme Q10 in the solution is 0.1 mg / ml.