High performance liquid detection method for letemovir content

Abstract

The invention discloses a high performance liquid detection method for letemovir content, and belongs to the technical field of pharmaceutical chemicals. The method comprises the following steps: (1) preparing a sample; (2) detection: carrying out isocratic elution by adopting a chromatographic column taking octadecyl bonded silica gel as a filler, taking an ammonium dihydrogen phosphate solution with the concentration of 20mmol / L as a mobile phase A and taking acetonitrile-methanol (80: 20) as a mobile phase B; and (3) analysis: recording a chromatogram, and calculating the content of the letemovir component by a peak area according to an external standard method. According to the method, an isocratic elution detection method is selected, a base line is smooth, the requirement on an instrument is low, the detection time is short, the detection efficiency can be improved, and consumables and reagents are saved.

Description

Technical Field

[0001] The invention relates to a high-performance liquid phase detection method for the content of letermovir, and belongs to the technical field of pharmaceutical chemical industry. Background Art

[0002] Letermovir is used to prevent and treat adult cytomegalovirus (CMV)-positive patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT). From January 12, 2011 to March 15, 2017, it was qualified as a rare drug for the prevention and treatment of adult CMV-positive patients undergoing (HSCT). On November 1 and November 8, 2017, it was approved for marketing by the Canadian Drug Administration (CDA) and the US FDA, respectively, with the trade name Prevymis. However, the standards and methods for Letermovir are not included in the pharmacopoeias of various countries.

[0003] The content method in the current import registration standard of Letermovir Injection (standard number: JX20220051) is consistent with the relevant method. The specific operation is: using octadecylsilane bonded silica gel as a filler and gradient elution. However, due to the gradient elution, the baseline is not flat and the instrument requirements are high. In addition, the content detection method takes a long time, which increases the working time of personnel and the loss of reagents and consumables. Summary of the invention

[0004] In view of the deficiencies in the prior art, the object of the present invention is to provide a high-performance liquid phase detection method for the content of letermovir, which shortens the detection time, improves the detection efficiency, and saves the use of consumables and reagents.

[0005] In order to solve the problems of the prior art, the technical solution provided by the present invention is: A high performance liquid phase detection method for the content of letermovir comprises the following steps: (1) Prepare samples; (2) Detection: A chromatographic column with octadecyl bonded silica as filler, ammonium dihydrogen phosphate solution as mobile phase A, and acetonitrile-methanol as mobile phase B was used for isocratic elution; (3) Analysis: Record the chromatogram and calculate the content of the letermovir component based on the peak area using the external standard method.

[0006] Furthermore, in step (2), the inner diameter of the chromatographic column is 4.6 mm, the length of the chromatographic column is 150 mm, and the inner diameter of the filler is 2.7 μm.

[0007] Furthermore, in step (2), the chromatographic column is HALO 90A C18 or a chromatographic column with equivalent performance.

[0008] Furthermore, in step (2), the detection wavelength is 220 nm, the column temperature of the chromatographic column is 53-57° C., the flow rate is 0.75-0.85 mL / min, and the injection volume is 5 μL.

[0009] Furthermore, in step (2), the detection wavelength is 220 nm, the column temperature of the chromatographic column is 55° C., the flow rate is 0.80 mL / min, and the injection volume is 5 μL.

[0010] Furthermore, in step (2), the concentration of the diammonium phosphate solution is 20 mmol / L and the pH is 3.3.

[0011] Furthermore, in step (2), the volume ratio of acetonitrile to methanol is 80:20.

[0012] Furthermore, in step (2), the volume ratio of the mobile phase A to the mobile phase B is (50-58): (42-50).

[0013] Furthermore, in step (2), the running time of the isocratic elution is 20 min.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention refers to the method in the standard for imported injections. According to the intermediates and impurities in the synthesis process of our company's products, the gradient program of the method is optimized to ensure the separation of impurities and the universality of the method. Isocratic elution is selected to achieve effective detection of letermovir. The method is durable, efficient and ensures the specificity, sensitivity and accuracy of the detection. It is suitable for production process monitoring and finished product quality control of raw materials and related preparations.

[0015] (2) The method of the present invention uses isocratic elution. Under the premise of ensuring the separation degree, the baseline is flat, the requirements for the instrument are low, the detection time is shortened, the detection efficiency is improved, the working time of personnel and the loss of reagents and consumables are reduced, and at the same time, the separation degree between the letermovir peak and the 5F letermovir peak of the relevant substance item should be no less than 1.5, and it has been methodologically verified and is suitable for the detection of the letermovir content. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the test chart of gradient elution and isocratic elution; Figure 2 This is the linear regression equation diagram of Experimental Example 2. DETAILED DESCRIPTION

[0017] Example 1 (1) Sample preparation: Diluent: acetonitrile-water-phosphoric acid (75:25:0.1).

[0018] Blank solution: diluent.

[0019] Test solution: Take about 7.5 mg of Letermovir API, weigh accurately, place in a 50 mL volumetric flask, add diluent to dissolve and dilute to volume to make a solution containing about 0.15 mg of Letermovir API per 1 mL, shake well. Prepare 2 portions in parallel.

[0020] Reference solution: Take about 7.5 mg of Letermovir working reference substance, weigh accurately, place in a 50 mL volumetric flask, add diluent to dissolve and dilute to volume to make a solution containing about 0.15 mg of Letermovir working reference substance per 1 mL, shake well. Prepare 2 copies in parallel.

[0021] (2) Testing: High performance liquid chromatography was used for detection. The detection conditions were as follows: Chromatographic column model: HALO 90A C18; chromatographic column specifications: 4.6mm×150mm, 2.7μm; mobile phase A: 20mmol / L diammonium phosphate solution (pH adjusted to 3.3 with phosphoric acid), mobile phase B: acetonitrile-methanol (80:20), mobile phase A-mobile phase B (55:45); isocratic elution, running time: 20 minutes; column temperature, 55℃; detection wavelength, 220nm; flow rate, 0.8mL / min; injection volume, 5μL.

[0022] Accurately measure the test solution and reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0023] (3) Calculation: Calculate the content of levitra based on the peak area using the external standard method.

[0024] Experimental Example 1 The same letermovir solution was tested by the gradient elution method in the prior art and the isocratic elution method of the present invention. The test results are shown in Table 1 and Attached Figure 1 .

[0025] Table 1. Run times for isocratic and gradient elution From Table 1, we can see that the running time of isocratic elution is 20 min, the running time of gradient elution is 40 min, and Figure 1 It can be seen that compared with gradient elution, the baseline drift of isocratic elution is improved. It can be seen that the method of the present invention selects isocratic elution, which has a flat baseline, short detection time and high detection efficiency.

[0026] Experimental Example 2 Linear Relationship Accurately weigh the letermovir working reference substance, and prepare solutions with concentrations of about 75 μg / mL, 90 μg / mL, 120 μg / mL, 150 μg / mL, and 180 μg / mL, respectively, which are recorded as linear solutions 1-5. Take the above linear solutions 1-5 and inject them respectively, and record the chromatograms. Perform linear regression calculation with the solution concentration as the horizontal axis and the peak area as the vertical axis to obtain the linearity.

[0027] Table 2. Linear relationship verification results From Table 2 and Appendix Figure 2 It can be seen that in the future, the temovir working reference substance will be prepared into linear solutions of different concentrations, and the linear regression calculation will be performed with the solution concentration as the horizontal axis and the peak area as the vertical axis. The regression curve is y=0.4494x+0.5377, R 2 =0.9999, and the correlation coefficient R is greater than 0.999, indicating that the solution concentration has a good linear relationship with the main peak area.

[0028] Experimental Example 3 Repeatability Accurately weigh 7.5 mg of Letermovir API, place in a 50 mL volumetric flask, add diluent to dissolve and dilute to volume to make a solution containing about 0.15 mg of Letermovir API per 1 mL, shake well. Take 6 portions of 10 mL of sample solution respectively, record as repeatable solutions 1-6. Test repeatable solutions 1-6. Record chromatogram. Calculate the content of Letermovir, and the calculation results are listed in Table 3.

[0029] Table 3. Repeatability verification results Note: RSD is relative standard deviation.

[0030] As shown in Table 3, the RSD of the letermovir content calculated by detection is 0.3%, and it can be seen that the RSD is less than 2.0%, indicating that the detection method of the present invention has good repeatability and good precision.

[0031] Experimental Example 4 Intermediate Precision Accurately weigh 7.5 mg of Letermovir API, place in a 50 mL volumetric flask, add diluent to dissolve and dilute to volume to make a solution containing about 0.15 mg of Letermovir API per 1 mL, shake well. Take 6 portions of 10 mL of sample solution respectively, record as intermediate precision solution 1-6. Detect intermediate precision solution 1-6. Record chromatogram. Calculate the content of Letermovir, and the calculation results are listed in Table 4.

[0032] Table 4. Intermediate precision verification results As can be seen from Table 4, different analysts selected different equipment to detect intermediate precision solutions 1-6 at different times, calculated the content of letermovir, and compared with the repeatability results, the RDS was 0.6%, and it can be seen that the RSD was less than 2.0%, indicating that the detection method of the present invention has good intermediate precision.

[0033] Experimental Example 5 Accuracy Accurately weigh the letermovir working reference substance, prepare solutions with concentrations of 75 μg / mL, 150 μg / mL, and 180 μg / mL, respectively, and record them as accuracy test solution 1, accuracy test solution 2, and accuracy test solution 3. Take accuracy test solution 1, accuracy test solution 2, and accuracy test solution 3 and inject them three times in succession, and record the chromatogram. Calculate the recovery rate of the test solution, and the calculation results are listed in Table 5.

[0034] Table 5. Accuracy verification results As shown in Table 5, the average recovery rate of the accuracy test solution is 101.2%, and the relative standard deviation RSD of the recovery rate is 0.6%. The recovery rate can be used to characterize the accuracy, which shows that the method of the present invention has high recovery rate and high accuracy.

[0035] Experimental Example 6 Durability Accurately weigh 7.5 mg of Letermovir API, place in a 50 mL volumetric flask, add diluent to dissolve and dilute to a constant volume to make a solution containing about 0.15 mg of Letermovir API per 1 mL, shake well. Test the solution at a column temperature of 55°C and a flow rate of 0.8 mL / min to obtain a standard value, and then test the solution under the conditions of changing the column temperature to 55°C ± 2°C and the flow rate to 0.8 ± 0.05 mL / min. The test results are listed in Table 6.

[0036] Table 6. Durability verification results As can be seen from Table 6, when the test column temperature or injection flow rate is changed, the content of letermovir is basically consistent with that tested under standard conditions, and the recovery rate is also good, indicating that the method of the present invention has good durability.

[0037] Experimental Example 7 Stability Accurately weigh 7.5 mg of Letermovir API, place in a 50 mL volumetric flask, add diluent to dissolve and dilute to a constant volume to make a solution containing about 0.15 mg of Letermovir API per 1 mL, shake well. The solution was tested at a column temperature of 55°C and a flow rate of 0.8 mL / min at different time points, and the test results are listed in Table 7.

[0038] Table 7. Stability verification results As can be seen from Table 7, the solutions were tested at different time points at a column temperature of 55°C and a flow rate of 0.8 mL / min. The recoveries of the reference solution and the test solution were both good, indicating that the method of the present invention has good stability.

[0039] Experimental Example 8 The system suitability requirement under the relevant substance detection item is that the resolution between the peaks of letermovir and 5F letermovir should be no less than 1.5. The content method needs to explore different ratios to make the resolution meet the requirements. Use 20mmol / L diammonium phosphate solution (pH adjusted to 3.3 with phosphoric acid) as mobile phase A and acetonitrile-methanol (80:20) as mobile phase B; explore different ratios of mobile phases. 5F letermovir and letermovir peak in turn.

[0040] Table 8. Mobile phase ratio After exploration, it was found that as the proportion of mobile phase B decreased, the retention time increased, the separation decreased, and the asymmetry increased. Therefore, a volume ratio of mobile phase A: mobile phase B of 55:45 was selected as the mobile phase, which had sufficient retention time and good separation, meeting the detection requirements.

[0041] The instruments and reagents used in the specific implementation of the present invention are as follows: Main instruments: high performance liquid chromatograph (model: Thermo U3000, manufacturer: Thermo; model: Vanquish Core, manufacturer: Thermo Fisher), pH meter (model: FE28 meter, manufacturer: Mettler), 1 / 10,000 electronic balance (model: MCA3.6P-2CCN-M, manufacturer: Sartorius).

[0042] Main samples and reagents: Letermovir API (self-made), Letermovir working reference substance (Guangzhou Aqixi New Drug Research and Development Co., Ltd.), 5F Letermovir reference substance (Guangzhou Aqixi Pharmaceutical Technology Co., Ltd.).

Claims

1. A high performance liquid phase detection method for the content of letermovir, characterized in that: The following steps are involved: (1) Prepare samples; (2) Detection: A chromatographic column with octadecyl bonded silica as filler, ammonium dihydrogen phosphate solution as mobile phase A, and acetonitrile-methanol as mobile phase B was used for isocratic elution; (3) Analysis: Record the chromatogram and calculate the content of the letermovir component based on the peak area using the external standard method.

2. The high performance liquid phase detection method for the content of Letermovir according to claim 1, characterized in that: In step (2), the inner diameter of the chromatographic column is 4.6 mm, the length of the chromatographic column is 150 mm, and the inner diameter of the filler is 2.7 μm.

3. The high performance liquid phase detection method for the content of Letermovir according to claim 1 or 2, characterized in that: In step (2), the chromatographic column is HALO 90A C18 or a chromatographic column with equivalent performance.

4. The high performance liquid phase detection method for the content of Letermovir according to claim 3, characterized in that: In step (2), the detection wavelength is 220 nm, the column temperature of the chromatographic column is 53-57° C., the flow rate is 0.75-0.85 mL / min, and the injection volume is 5 μL.

5. The high performance liquid phase detection method for the content of Letermovir according to claim 4, characterized in that: In step (2), the detection wavelength is 220 nm, the column temperature of the chromatographic column is 55° C., the flow rate is 0.8 mL / min, and the injection volume is 5 μL.

6. The high performance liquid phase detection method for the content of Letermovir according to claim 1, characterized in that: In step (2), the concentration of the diammonium phosphate solution is 20 mmol / L and the pH is 3.

3.

7. The high performance liquid phase detection method for the content of Letermovir according to claim 5 or 6, characterized in that: In step (2), the volume ratio of acetonitrile to methanol is 80:

20.

8. The high performance liquid phase detection method for the content of Letermovir according to claim 7, characterized in that: In step (2), the volume ratio of mobile phase A to mobile phase B is (50-58): (42-50).

9. The high performance liquid phase detection method for the content of Letermovir according to claim 8, characterized in that: In step (2), the running time of the isocratic elution is 20 min.

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