Determination method of 4-hydroxy-7-phenoxyisoquinoline-3-carboxylic acid methyl ester and its related substances

Abstract

The invention discloses a method for determining 4-hydroxy-7-phenoxyisoquinoline-3-methyl formate and related substances, and belongs to the technical field of drug analysis. The method adopts reverse phase liquid chromatography to detect and analyze 4-hydroxy-7-phenoxyisoquinoline-3-methyl formate and related substances impurities A, impurity B, impurity C, impurity D, and impurity E, and uses octadecylsilane bonded silica gel as a chromatographic column as a filler, mobile phase A is trifluoroacetic acid aqueous solution, mobile phase B is acetonitrile, and gradient elution is adopted. The method has the advantages of high sensitivity, accurate impurity content determination results, good specificity, repeatability and linearity.

Description

Technical Field

[0001] The present invention relates to a method for determining a substance, in particular to a method for determining 4-hydroxy-7-phenoxyisoquinoline-3-carboxylic acid methyl ester and related substances. Background Art

[0002] Roxadustat, chemically known as N-[(4-hydroxy-1-methyl-7-phenoxy-3-isoquinoline)carbonyl]glycine, is primarily used to treat anemia associated with chronic kidney disease and end-stage renal disease. Developed by Phibrogen, Inc. in the United States, it is a hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI). It can inhibit HIF-prolyl hydroxylase under normal oxygen tension, stabilize HIF-α, promote the production of endogenous erythropoietin (Erythropoietin), improve iron absorption and utilization, and comprehensively regulate erythropoiesis. Two Phase III clinical trials conducted in China for the treatment of anemia in patients with dialysis-dependent (DD-CKD) and non-dialysis-dependent (NDD-CKD) chronic kidney disease both met their primary efficacy endpoints.

[0003] Methyl 4-hydroxy-7-phenoxyisoquinoline-3-carboxylate is used as the starting material of rosuvastatin, and its molecular formula is C 17 H 13 NO4, molecular weight is 295.08, structural formula is:

[0004]

[0005] The analysis and detection of the starting material plays an important role in reaction control and yield improvement, and also directly affects the quality of the final product. By analyzing the synthetic process of the raw material of this product and sorting out the impurity spectrum, this product involves the following 5 impurities:

[0006] Impurity A: Impurity B: Impurity C: Impurity D: And impurity E:

[0007] The above impurities are introduced into raw materials or preparations, directly affecting their quality, safety and effectiveness. There are no reports in the prior art on the determination method of 4-hydroxy-7-phenoxyisoquinoline-3-carboxylic acid methyl ester and the above-mentioned related substances. Summary of the Invention

[0008] Purpose of the invention: The present invention aims to provide a stable, effective and simple method for the determination of 4-hydroxy-7-phenoxyisoquinoline-3-carboxylic acid methyl ester and its related substances, which can effectively separate 4-hydroxy-7-phenoxyisoquinoline-3-carboxylic acid methyl ester and multiple impurities with good peak shape and meet the required resolution.

[0009] Technical solution: The method for determining 4-hydroxy-7-phenoxyisoquinoline-3-carboxylic acid methyl ester and its related substances of the present invention comprises the following steps:

[0010] (1) Prepare test solution, control solution, resolution solution, and system suitability solution;

[0011] (2) Reverse-phase liquid chromatography (RP-HPLC) was used, with mobile phase A being a 0.08% to 0.12% trifluoroacetic acid aqueous solution and mobile phase B being acetonitrile. Gradient elution was used, with the initial volume percentage of mobile phase B being 20% ​​to 30%. Detection and analysis were performed using a high performance liquid chromatograph.

[0012] The relevant substances include:

[0013] Impurity A: Impurity B: Impurity C: Impurity D: And impurity E:

[0014] Preferably, the gradient elution program is: from 0 to 30 min, the volume percentage of mobile phase A is reduced from 70% to 80% to 5% to 15%, and the volume percentage of mobile phase B is increased from 20% to 30% to 85% to 95%; from 30 to 35 min, the volume percentage of mobile phase A is 5% to 15%, and the volume percentage of mobile phase B is 85% to 95%.

[0015] Preferably, the gradient elution program is: from 0 to 30 min, the volume percentage of mobile phase A decreases from 70% to 80% to 5% to 15%, and the volume percentage of mobile phase B increases from 20% to 30% to 85% to 95%; from 30 to 35 min, the volume percentage of mobile phase A is 5% to 15%, and the volume percentage of mobile phase B is 85% to 95%; from 35 to 35.1 min, the volume percentage of mobile phase A increases from 5% to 15% to 70% to 80%, and the volume percentage of mobile phase B decreases from 85% to 95% to 20% to 30%; from 35.1 to 45 min, the volume percentage of mobile phase A is 70% to 80%, and the volume percentage of mobile phase B is 20% to 30%.

[0016] Preferably, the gradient elution program is: from 0 to 30 min, the volume percentage of mobile phase A decreases from 75% to 10%, and the volume percentage of mobile phase B increases from 25% to 90%; from 30 to 35 min, the volume percentage of mobile phase A is 10%, and the volume percentage of mobile phase B is 90%; from 35 to 35.1 min, the volume percentage of mobile phase A increases from 10% to 75%, and the volume percentage of mobile phase B decreases from 90% to 25%; from 35.1 to 45 min, the volume percentage of mobile phase A is 75%, and the volume percentage of mobile phase B is 25%.

[0017] Preferably, the detector used in the liquid phase detection is an ultraviolet absorption detector, and its detection wavelength is 255-265 nm and 225-235 nm.

[0018] Preferably, the chromatographic column used for the liquid phase detection uses octadecylsilane bonded silica gel as a filler, and the column temperature is 30-40°C.

[0019] Preferably, the flow rate of the mobile phase is 0.8-1.2 ml / min.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) the detection method is stable, effective and simple to operate, and can effectively separate 4-hydroxy-7-phenoxyisoquinoline-3-carboxylic acid methyl ester and multiple impurities, with strong sample component retention ability and good separation degree; (2) the sensitivity is high, the impurity content determination result is accurate, and the relevant known impurities can be accurately detected under the chromatographic conditions of the present invention, and the main peak and the impurity peak can be effectively separated; (3) through methodological verification, the detection method is accurate and feasible, and has good specificity, repeatability and linearity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the chromatogram of the blank solution (260 nm) in Example 1;

[0022] Figure 2 is the chromatogram of the blank solution (230 nm) in Example 1;

[0023] Figure 3 is the chromatogram of the separation solution (260 nm) in Example 1;

[0024] Figure 4 is the chromatogram of the separation solution (230 nm) in Example 1;

[0025] Figure 5 This is the chromatogram of the test solution (260 nm) in Example 1;

[0026] Figure 6 It is the chromatogram of the test solution (230nm) in Example 1. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0028] Example 1

[0029] Separation investigation

[0030] HPLC conditions: The chromatographic column was an octadecylsilane bonded silica gel column, model Agilent Eclipse XDB-C18 column (size: 4.6 mm × 150 mm, particle size: 3.5 μm), with 0.1% trifluoroacetic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, with gradient elution according to Table 1 below at a flow rate of 1.0 ml / min; the detection wavelengths were 260 nm and 230 nm; and the column temperature was 35°C.

[0031] Table 1 Gradient elution

[0032] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0.0 75 25 30.0 10 90 35.0 10 90 35.1 75 25 45.0 75 25

[0033] Solution preparation:

[0034] Test solution: Take an appropriate amount of 4-hydroxy-7-phenoxyisoquinoline-3-carboxylic acid methyl ester, weigh accurately, add diluent acetonitrile, sonicate to dissolve and dilute to make a solution containing approximately 0.5 mg of 4-hydroxy-7-phenoxyisoquinoline-3-carboxylic acid methyl ester per 1 ml.

[0035] Control solution: Accurately measure 1.0 ml of the test solution and dilute it with acetonitrile to make a solution containing approximately 0.5 μg of methyl 4-hydroxy-7-phenoxyisoquinoline-3-carboxylate per 1 ml.

[0036] Stock solution of impurity reference substances: Take appropriate amounts of impurity A reference substance, impurity B reference substance, impurity C reference substance, impurity D reference substance, and impurity E reference substance, dissolve them in acetonitrile and dilute them to make a solution with approximately 10 μg of impurity D and 5 μg of each of the other impurities per 1 ml.

[0037] Separation solution: Take an appropriate amount of 4-hydroxy-7-phenoxyisoquinoline-3-carboxylic acid methyl ester test sample, accurately weigh it, add acetonitrile and sonicate to dissolve it. Take an appropriate amount of the above-mentioned impurity reference stock solution to prepare a mixed solution with about 1 μg of impurity D, 0.5 μg of the other impurities, and 0.5 mg of 4-hydroxy-7-phenoxyisoquinoline-3-carboxylic acid methyl ester per 1 ml.

[0038] System suitability solution: Take an appropriate amount of methyl 4-hydroxy-7-phenoxyisoquinoline-3-carboxylate reference substance, accurately weigh it, add acetonitrile and sonicate to dissolve it. Take an appropriate amount of the above-mentioned impurity reference substance stock solution to prepare a mixed solution with approximately 1 μg of impurity D, 0.5 μg of the remaining impurities, and 0.5 mg of methyl 4-hydroxy-7-phenoxyisoquinoline-3-carboxylate per 1 ml.

[0039] Blank solution: acetonitrile

[0040] Accurately measure blank solution, separation solution, and test solution for determination. Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 、 Figure 6 The test results are shown in Table 2. The results showed that the blank solution had no interference, and the main component and impurities as well as the impurities could be effectively separated, indicating that the method had good specificity.

[0041] Table 2 Separation test results

[0042]

[0043] Example 2

[0044] Investigation of limit of quantification and limit of detection

[0045] Following the method described in Example 1, a reference substance (methyl 4-hydroxy-7-phenoxyisoquinoline-3-carboxylate) and an impurity reference substance stock solution were prepared. Appropriate amounts of each impurity reference substance stock solution were accurately measured and gradually diluted with acetonitrile for determination. The signal-to-noise ratio of approximately 10:1 was used as the limit of quantification, and the signal-to-noise ratio of approximately 3:1 was used as the limit of detection. The solution at the limit of quantification concentration was injected six times consecutively to assess the injection precision of the solution. The results are shown in Tables 3 and 4. The results demonstrate the good sensitivity of this method.

[0046] Table 3 Detection limit and quantification limit

[0047]

[0048] Table 4 Results of the quantitation limit injection precision test

[0049] S / N Impurity A Impurity D Impurity B Impurity C Impurity E principal component 1 25.3 39.3 17.8 12.1 13.6 35.3 2 31.7 47.5 21.6 14.4 21.8 42.9 3 30.1 46.1 20.5 14.8 16.1 41.7 4 44.7 66.8 30.8 20.9 17.9 59.8 5 25.1 39.1 18.0 11.1 14.6 35.4 6 39.3 58.8 26.2 17.9 18.3 52.3 mean 32.7 49.6 22.5 15.2 17.1 44.6 Retention time Impurity A Impurity D Impurity B Impurity C Impurity E principal component 1 14.093 14.850 22.630 23.767 7.690 17.800 2 14.093 14.850 22.630 23.777 7.690 17.800 3 14.097 14.850 22.633 23.770 7.673 17.800 4 14.097 14.850 22.630 23.773 7.673 17.800 5 14.093 14.850 22.630 23.773 7.673 17.797 6 14.097 14.850 22.633 23.767 7.667 17.803 mean 14.095 14.850 22.631 23.771 7.677 17.800 RSD (%) 0.02 0.00 0.01 0.02 0.13 0.01 Peak area Impurity A Impurity D Impurity B Impurity C Impurity E principal component 1 0.0308 0.0464 0.0241 0.0139 0.0339 0.0477 2 0.0316 0.0458 0.0247 0.0136 0.0417 0.0468 3 0.0292 0.0450 0.0224 0.0156 0.0372 0.0468 4 0.0324 0.0482 0.0242 0.0148 0.0428 0.0454 5 0.0311 0.0471 0.0250 0.0126 0.0384 0.0490 6 0.0321 0.0463 0.0250 0.0146 0.0365 0.0458 mean 0.0312 0.0465 0.0242 0.0142 0.0384 0.0469 RSD (%) 3.68 2.36 4.03 7.39 8.68 2.78

[0050] Example 3

[0051] Linear Investigation

[0052] Reference solutions of the main ingredient (methyl 4-hydroxy-7-phenoxyisoquinoline-3-carboxylate) and impurity reference solutions were prepared according to the method described in Example 1. Appropriate amounts of each impurity reference solution were accurately measured and diluted with acetonitrile to prepare a series of concentrations to serve as linear solutions for each component. The test results are shown in Table 5 below. Correction factors were determined using the standard curve method, and the correction factors obtained from the linearity results are shown in Table 6. The results demonstrate that, using the chromatographic conditions of the present invention, the components tested exhibited good linear relationships.

[0053] Table 5 Linearity test results

[0054]

[0055] Table 6 Correction Factors

[0056] name Slope Correction Factor Main component - 260nm 0.4762 / Main component - 230nm 0.4102 / Impurity A 0.3172 1.5 Impurity B 0.2379 2.0 Impurity C 0.1555 3.1 Impurity D 0.5961 0.80 Impurity E-230nm 0.2543 1.6

[0057] Example 4

[0058] Solution stability investigation

[0059] At different times, 5 μl of the system suitability solution, test solution, and control solution were injected into a liquid chromatograph, and the chromatograms were recorded. The results are shown in Tables 7 and 8. The results show that after the system suitability solution and the control solution were stored at room temperature for 51 hours, the ratios of the peak areas of each impurity and the main peak compared to those at 0 hours were between 90.0% and 110.0%. When the test solution was stored at room temperature for 51 hours, the impurity content at each time point compared to 0 hours showed no impurities above the reporting limit. This indicates that the system suitability solution, test solution, and control solution are stable at room temperature for at least 51 hours.

[0060] Table 7 Stability test of related substance solutions - system suitability solution

[0061]

[0062] Table 8 Stability test of related substance solution - control solution

[0063] time Peak area ratio 0h 0.2630 / 7h 0.2572 97.8％ 14h 0.2608 99.2％ 21h 0.2654 100.9％ 28h 0.2687 102.2％ 36h 0.2718 103.3％ 44h 0.2744 104.3％ 51h 0.2778 105.6％

[0064] Example 5

[0065] Repeatability study

[0066] Accurately weigh 6 samples of methyl 4-hydroxy-7-phenoxyisoquinoline-3-carboxylate, add 100% impurity reference solution to each sample, dissolve in acetonitrile and dilute to the mark to serve as the test solution. Accurately measure each test solution and dilute 1000 times to serve as the control solution. Accurately measure 5μl of the above test solution and control solution and inject them into the liquid chromatograph. Record the chromatogram and calculate the recovery of each impurity as [(measured amount - original amount) / added amount]. The results are shown in Table 9. The results show that the recovery rates of each impurity are between 80% and 120%, with RSDs less than 10.0%, indicating that this method has good reproducibility.

[0067] Table 9 Repeatability calculation table

[0068] Impurity name Average recovery rate Recovery RSD Impurity A 100.8％ 1.0％ Impurity B 102.3％ 1.5％ Impurity C 101.3％ 1.9％ Impurity D 99.1％ 1.4％ Impurity E 103.5％ 7.9％

[0069] Example 6

[0070] Accuracy inspection

[0071] Accurately weigh 12 portions of methyl 4-hydroxy-7-phenoxyisoquinoline-3-carboxylate sample, add impurity reference solution with impurity limits of 50%, 100%, and 150%, dissolve in diluent and dilute to the mark to serve as the test solution. Accurately measure each test solution and dilute 1000 times to serve as the control solution. Accurately measure 5 μl of the above test solution and control solution and inject them into the liquid chromatograph, record the chromatogram, and calculate the recovery rate of each impurity [(measured amount - original amount) / added amount]. The results are shown in Table 10. The results show that the recovery rate of each impurity is between 80% and 120%, and the RSD is less than 10.0%, indicating that this method has good accuracy.

[0072] Table 10 Accuracy calculation table

[0073] Impurity name Recovery rate range Average recovery rate RSD Impurity A 99.80％～105.62％ 102.5％ 3.0％ Impurity B 100.99％～109.20％ 104.2％ 2.2％ Impurity C 95.44％～104.46％ 100.5％ 2.6％ Impurity D 97.04％～103.38％ 100.1％ 3.0％ Impurity E 90.74％～111.63％ 100.8％ 8.4％

[0074] In summary, the method of the present invention has strong specificity, high sensitivity, good repeatability and linearity, and high accuracy for the determination of methyl 4-hydroxy-7-phenoxyisoquinoline-3-carboxylate and its related substances.

Claims

1. A method for determining 4-hydroxy-7-phenoxyisoquinoline-3-carboxylic acid methyl ester and its related substances, characterized in that: The method comprises the following steps: (1) Prepare test solution, control solution, resolution solution and system suitability solution; (2) using reverse phase liquid chromatography, mobile phase A is a 0.08% to 0.12% trifluoroacetic acid aqueous solution by volume, mobile phase B is acetonitrile, gradient elution is used, the initial volume percentage of mobile phase B is 20% to 30%, and high performance liquid chromatography is used for detection and analysis; The relevant substances include: Impurity A: Impurity B: Impurity C: Impurity D: And impurity E: The gradient elution program is as follows: from 0 to 30 minutes, the volume percentage of mobile phase A decreases from 70% to 80% to 5% to 15%, and the volume percentage of mobile phase B increases from 20% to 30% to 85% to 95%; from 30 to 35 minutes, the volume percentage of mobile phase A is 5% to 15%, and the volume percentage of mobile phase B is 85% to 95%; from 35 to 35.1 minutes, the volume percentage of mobile phase A increases from 5% to 15% to 70% to 80%, and the volume percentage of mobile phase B decreases from 85% to 95% to 20% to 30%; from 35.1 to 45 minutes, the volume percentage of mobile phase A is 70% to 80%, and the volume percentage of mobile phase B is 20% to 30%; The detector used in the reversed-phase liquid chromatography detection is an ultraviolet absorption detector, and its detection wavelength is 255nm-265nm and 225nm-235nm; the chromatographic column used in the reversed-phase liquid chromatography detection uses octadecylsilane bonded silica gel as a filler.

2. The method for determining 4-hydroxy-7-phenoxyisoquinoline-3-carboxylic acid methyl ester and its related substances according to claim 1, characterized in that: The gradient elution program is: from 0 to 30 minutes, the volume percentage of mobile phase A decreases from 75% to 10%, and the volume percentage of mobile phase B increases from 25% to 90%; from 30 to 35 minutes, the volume percentage of mobile phase A is 10%, and the volume percentage of mobile phase B is 90%; from 35 to 35.1 minutes, the volume percentage of mobile phase A increases from 10% to 75%, and the volume percentage of mobile phase B decreases from 90% to 25%; from 35.1 to 45 minutes, the volume percentage of mobile phase A is 75%, and the volume percentage of mobile phase B is 25%.

3. The method for determining 4-hydroxy-7-phenoxyisoquinoline-3-carboxylic acid methyl ester and its related substances according to claim 1, characterized in that: The column temperature of the chromatographic column is 30°C to 40°C.

4. The method for determining 4-hydroxy-7-phenoxyisoquinoline-3-carboxylic acid methyl ester and its related substances according to claim 1, characterized in that: The flow rate of the mobile phase is 0.8 ml / min to 1.2 ml / min.

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