Method for detecting impurity F1 in coenzyme Q10 raw material or preparation

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting an impurity F1 in a coenzyme Q10 raw material or preparation, and the method comprises the following steps: a test solution and a reference solution are prepared, the test solution is the coenzyme Q10 raw material or preparation, and the reference solution is the impurity F1; the solvent in the test solution and the reference solution is absolute ethyl alcohol; the test solution and the reference solution are detected by high performance liquid chromatography, and chromatographic conditions are as follows: the flow velocity is 1.5-2.0 mL / min, the mobile phase A is methanol, the mobile phase B is absolute ethyl alcohol, and the column temperature is 32-42 DEG C; the detection wavelength is 270-280 nm, a gradient elution procedure is adopted for elution, after gradient elution, the content of the impurity F1 is detected, and the structural formula of the impurity F1 is shown in the specification; the method has the advantages of high separation degree, high accuracy, high response and strong anti-interference capability.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for detecting impurity F1 in coenzyme Q10 raw materials or preparations. Background Technology

[0002] Coenzyme Q 10 (Coenzyme Q) 10 The system name is 2,3-dimethoxy-5-methyl-6-polyisoprenyl-1,4-benzoquinone, and its international common name is Ubidecarenone. It is a lipid-soluble benzoquinone compound with important physiological functions. Its chemical structure contains a quinone ring core and a side chain composed of 10 isopentenyl units (all-E configuration 3,7,11,15,19,23,27,31,35,39-decamethyl-2,6,10,14,18,22,26,30,34,38-tetradecenyl). This unique structure gives it both lipophilic and redox properties. It is a yellow crystalline powder at room temperature and is lipid-soluble and photosensitizing. Clinical studies have confirmed that coenzyme Q10... 10 It has multiple protective mechanisms in the cardiovascular system: ① It improves cardiac pumping function by enhancing myocardial cell energy metabolism; ② It inhibits low-density lipoprotein oxidation and slows down the process of atherosclerosis; ③ It regulates intracellular calcium ion balance and alleviates myocardial ischemia-reperfusion injury.

[0003] Coenzyme Q 10 Impurity F1 is an impurity that degrades from the raw materials and formulations, and its structural formula is as follows: . Under the chromatographic conditions specified in the Chinese Pharmacopoeia, this impurity exhibits a low response. Blank excipients and baseline fluctuations can interfere with its detection, leading to low accuracy of the results.

[0004] Patent application CN 110231425 A discloses a method for extracting coenzyme Q10 from blood and a high-performance liquid chromatography (HPLC) method for its detection. The chromatographic column used is a C8 column at 30℃, and the mobile phase flow rate is 0.5 mL / min. This patent application has certain shortcomings and cannot meet the specific requirements for determination, specifically: ① Isocratic elution occurs, and under these chromatographic conditions, the blank excipient peak and known impurities overlap; impurity F1 exhibits significant tailing. ② At a flow rate of 0.5 mL / min, the main peak in the test solution elutes late, prolonging sample collection time and significantly increasing testing costs. ③ The method has poor feasibility; testing samples according to the method in patent application CN 110231425 A revealed poor feasibility and the inability to obtain accurate results. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for detecting impurity F1 in coenzyme Q10 raw materials or preparations, which has high separation, high accuracy, high response and strong anti-interference ability.

[0006] This invention provides a method for detecting impurity F1 in coenzyme Q10 raw materials or preparations, comprising the following steps: A test solution and a control solution are prepared, wherein the test sample is a coenzyme Q10 raw material or preparation, and the control is impurity F1; the solvent in the test solution and the control solution is anhydrous ethanol; The test solution and reference solution were analyzed by high-performance liquid chromatography (HPLC). The chromatographic conditions were: flow rate 1.5–2.0 mL / min, mobile phase A: methanol, mobile phase B: anhydrous ethanol, column temperature 32–42 °C; detection wavelength 270–280 nm; and a gradient elution program.

[0007] After gradient elution, the content of impurity F1 is detected. The structural formula of impurity F1 is as follows: .

[0008] Preferably, the flow rate is 1.8 mL / min.

[0009] Preferably, the column temperature is 37°C.

[0010] Preferably, the detection wavelength is 275nm.

[0011] Preferably, the chromatographic column used in the high performance liquid chromatography is C18.

[0012] Preferably, the concentration of the test solution is 0.2~0.4 mg / mL.

[0013] Preferably, the concentration of the test solution is 0.3 mg / mL.

[0014] Preferably, the concentration of the reference solution is 0.8~2 μg / mL.

[0015] Preferably, the concentration of the reference solution is 1.5 μg / mL.

[0016] Preferably, the injection volume used in high performance liquid chromatography is 20~80μL, and more preferably 50μL.

[0017] The beneficial effects of this invention are that the method is simple to operate, efficient, sensitive and specific, and has strong durability. It fully complies with the Chinese Pharmacopoeia method validation guidelines and can simultaneously detect impurity F1 in coenzyme Q10 raw materials and injection solutions, while reducing the influence of baseline and blank excipients on the detection of this impurity.

[0018] This invention can avoid interference from impurities and excipients that may exist in raw materials and preparations on the detection of impurity F1 in coenzyme Q10. At the same time, through the study of the specificity, linearity and range, accuracy, precision, detection limit and quantitation limit of the method, it is proved that the method provided by this invention is suitable for the detection requirements of impurity F1 in coenzyme Q10 raw materials and coenzyme Q10 injection.

[0019] This invention optimizes the peak shape in high-performance liquid chromatography (HPLC), improves the response and detection accuracy of impurity F1, and reduces interference from blank excipients and blank solvents. It exhibits high response and strong anti-interference capability. Attached Figure Description

[0020] Figure 1 This is a blank solvent spectrum.

[0021] Figure 2 This is a blank excipient diagram.

[0022] Figure 3 This is a spectrum of impurity mixtures.

[0023] Figure 4 The spectrum is that of the test solution of Comparative Example 1.

[0024] Figure 5 The image shows the chromatogram of the test solution from Example 1. Detailed Implementation

[0025] A method for detecting impurity F1 in coenzyme Q10 raw materials or preparations, comprising the following steps: A test solution and a control solution are prepared, wherein the test sample is a coenzyme Q10 raw material or preparation, and the control is impurity F1; the solvent in the test solution and the control solution is anhydrous ethanol; The test solution (concentration 0.3 mg / mL) and the reference solution (concentration 1.5 μg / mL) were detected by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: C18 column, injection volume 50 μL, flow rate 1.8 mL / min, mobile phase A: methanol, mobile phase B: anhydrous ethanol, column temperature 37℃; detection wavelength 275 nm; and gradient elution program. The gradient elution program was as follows:

[0026] After gradient elution, the content of impurity F1 is detected. The structural formula of impurity F1 is as follows: .

[0027] Example 1: Specificity (1) Chromatographic conditions: Testing instrument: Agilent Column: Agilent ZORBAX Eclipse Plus C18 Detection wavelength: 275nm Injection volume: 50 μl Flow rate: 1.8 ml / min Mobile phase A: Methanol Mobile phase B: Anhydrous ethanol.

[0028] Gradient elution procedure:

[0029] (2) Test method: Inject 50 μl each of blank solvent, blank excipient solution, localization solutions of each impurity, test solution, reference solution, and mixed solution of impurities and sample into the liquid chromatograph and record the chromatogram.

[0030] (3) Experimental results: (1) The blank solvent and blank excipient have no interference at the retention time of the impurity F1 peak; (2) Coenzyme Q 10 Other impurities do not interfere with the retention time of impurity F1 peak, and the separation degree between impurity F1 and the preceding and following impurity peaks is greater than 1.5.

[0031] Specific results are shown in Table 1. The graphs are shown below. Figures 1-3 .

[0032] Table 1 Specificity Results

[0033] Example 2: Repeatability (1) Chromatographic conditions: Same as in Example 1.

[0034] (2) Test method: Prepare 6 test solutions in parallel, inject 50 μl of each solution into the liquid chromatograph, and record the chromatogram.

[0035] (3) Experimental results: As shown in Table 2, for the 6 repeatable solutions, the content of impurity F1 calculated by the external standard method was 0.11% with an RSD of 5.75%, and the content of impurity F1 calculated by the self-control method was 0.12% with an RSD of 7.00%. Both were less than 20.0%, and the detection amounts of the two methods were basically consistent with each other, with no significant difference. The repeatability was good. See Table 2 for specific results.

[0036] Table 2 Repeatability Results

[0037] Example 3: Intermediate Precision (1) Chromatographic conditions: Similar to Example 1, the 6 test solution samples were repeatedly measured by different analysts on different dates using different equipment.

[0038] (2) Test method: Prepare 6 test solutions in parallel, inject 50 μl of each solution into the liquid chromatograph, and record the chromatogram.

[0039] (3) Test results: As shown in Tables 3-4, under the external standard method, the average content of impurity F1 in the 6 intermediate precision solutions was 0.12%, and the RSD was 4.19%; in the 12 test solutions for repeatability, the average content of impurity F1 was 0.12%, and the RSD was 7.61%; under the self-control method, the average content of impurity F1 in the 6 intermediate precision solutions was 0.13%, and the RSD was 2.87%; in the 12 test solutions for repeatability, the average content of impurity F1 was 0.12%, and the RSD was 8.70%. The intermediate precision was good. Specific results are shown in Tables 3-4.

[0040] Table 3. Intermediate precision test results (external standard method)

[0041] Table 4. Intermediate precision test results (self-control method)

[0042] Example 4: Accuracy (1) Chromatographic conditions: Same as Example 1.

[0043] (2) Test method: Take 9 samples and prepare 50%, 100% and 150% spiked test sample mixed solutions according to the limit concentration. Inject the solutions into the liquid chromatograph, record the chromatogram, and calculate the recovery rate of impurity F1.

[0044] (3) Test results: As shown in Table 5, the recovery rates of impurity F1 in the accuracy solutions at low, medium and high concentration levels were between 97.97% and 100.14%, with an average recovery rate of 98.9%, ranging from 80% to 120%. The RSD of the recovery rate for the nine samples was 1.04%, which meets the requirements. See Table 5 for specific results.

[0045] Table 5 Accuracy Results - Impurity F1

[0046] Example 5: Linear (1) Chromatographic conditions: Same as in Example 1.

[0047] (2) Experimental method: Take impurity F1 and coenzyme Q 10 For the reference standard, prepare a series of solutions with six different concentrations, inject them into the liquid chromatograph, record the chromatograms, plot the function curves of the peak area of ​​each impurity at different concentrations and its concentration, and calculate the intercept and correlation coefficient r.

[0048] (3) Experimental results: As shown in Tables 6-7, impurity F1 showed good linearity in the concentration range of 0.12 μg / ml to 3.35 μg / ml, with a linear equation of y = 14.8877x + 1.3159, a correlation coefficient (r) of 0.9995, and an absolute value of the intercept ratio of 5.45%. Coenzyme Q 10 Within the concentration range of 0.12 μg / ml to 3.44 μg / ml, the linear equation was y = 25.9491x + 1.3753, the correlation coefficient (r) was 1.0000, and the absolute value of the intercept ratio was 3.76%, indicating good linearity. Specific results are shown in Tables 6-7.

[0049] Table 6. Results of linear determination of impurity F1 (Part 1)

[0050] Table 7 Coenzyme Q 10 Linearity determination results of the reference standard (I)

[0051] Example 6: Limit of Detection and Limit of Quantification (1) Chromatographic conditions: Same as in Example 1.

[0052] (2) Test method: The impurity F1 reference solution was diluted and then measured. The chromatogram was recorded. When the S / N (signal-to-noise ratio) was ≥3, the concentration was the limit of detection; when the S / N (signal-to-noise ratio) was ≥10, the concentration was the limit of quantitation. The limit of quantitation solution was injected 6 times consecutively, the peak area was recorded, and the relative standard deviation of the peak area was calculated.

[0053] (3) Test results: As shown in Tables 7-8, the conclusion is that the limit of quantitation (LOQ) concentration of impurity F1 is 0.145 μg / ml, which is equivalent to 0.037% of the test sample concentration and 7.643% of the limit concentration, with a minimum S / N of 12.53; the limit of detection (LOD) concentration is 0.0387 μg / ml, with an S / N of 3.54, which meets the requirements. In the LOQ precision solution, the S / N of impurity F1 peaks are all greater than 10, the RSD of retention time is 0.57, and the RSD of peak area is 5.73%, which meets the requirements. Specific results are shown in Tables 8-9.

[0054] Table 8 Results of Limit of Detection and Limit of Quantification

[0055] Table 9 Precision results for the limit of quantitation

[0056] Comparative Example 1 The difference between this application and Example 1 lies in the elution procedure. The gradient elution procedure of Comparative Example 1 is as follows:

[0057] Everything else is the same as in Example 1.

[0058] The experimental results are as follows: Under the chromatographic conditions of Example 1, the baseline of impurity F1 peak was relatively flat, and the separation from adjacent impurity peaks was good. To ensure the detection capability of the impurity, we performed a limit of quantitation (LOQ) determination on impurity F1. The baseline in Example 1 was even flatter, and the LOQ concentration of impurity F1 was less than 0.05% of the main component concentration. See the detailed chromatograms below. Figures 4-5 .

[0059] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; within the framework of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of one or more embodiments of this application as described above, which are not provided in detail for the sake of brevity.

[0060] One or more embodiments in this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of one or more embodiments in this application should be included within the protection scope of this application.

Claims

1. A method for detecting impurity F1 in coenzyme Q10 raw materials or preparations, characterized in that, Includes the following steps, A test solution and a control solution are prepared, wherein the test sample is a coenzyme Q10 raw material or preparation, and the control is impurity F1; the solvent in the test solution and the control solution is anhydrous ethanol; The test solution and reference solution were analyzed by high-performance liquid chromatography (HPLC). The chromatographic conditions were: flow rate 1.5–2.0 mL / min, mobile phase A: methanol, mobile phase B: anhydrous ethanol, column temperature 32–42 °C; detection wavelength 270–280 nm; and a gradient elution program. After gradient elution, the content of impurity F1 is detected. The structural formula of impurity F1 is as follows: 。 2. The detection method as described in claim 1, characterized in that, The flow rate is 1.8 mL / min.

3. The detection method as described in claim 1, characterized in that, The column temperature is 37°C.

4. The detection method as described in claim 1, characterized in that, The detection wavelength is 275nm.

5. The detection method as described in claim 1, characterized in that, The chromatographic column used in the high performance liquid chromatography method is C18.

6. The detection method as described in claim 1, characterized in that, The concentration of the test solution is 0.2~0.4 mg / mL.

7. The detection method as described in claim 6, characterized in that, The concentration of the test solution is 0.3 mg / mL.

8. The detection method as described in claim 1, characterized in that, The concentration of the reference solution is 0.8~2 μg / mL.

9. The detection method as described in claim 8, characterized in that, The concentration of the reference solution was 1.5 μg / mL.

10. The detection method as described in claim 1, characterized in that, The injection volume used in high performance liquid chromatography is 20~80μL.

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