Method for detecting evocamide related substances

Abstract

The invention relates to a method for detecting related substances of EvoCASE. The method comprises the following steps: step 1, preparing a system applicable solution; step 2, preparing a test solution; step 3, preparing a contrast solution; step 4, carrying out liquid chromatography detection on the evocamide related substances; according to the detection method of the evocamide related substances, the five known impurities of the evocamide are determined by adopting a self-contrast method with correction factors, the impurity separation and analysis of the evocamide related substances are realized, and the detection method is good in separation degree, strong in specificity, high in sensitivity, high in accuracy, simple and easy to implement. The determination method has important practical significance for quality control of the evocamide bulk drug.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, and in particular to a method for detecting evokine related substances. Background Technology

[0002] Secondary hyperparathyroidism (SHPT) is a syndrome caused by excessive secretion of parathyroid hormone by the parathyroid glands due to hypocalcemia or hyperphosphatemia caused by various reasons.

[0003] Evocasserole, chemically named (4-{(3S)-3-[(1R)-1-(naphth-1-yl)ethylamino]pyrrolidine-1-yl}phenylacetic acid, has the following chemical structural formula: Evocarce is a novel arylalkylamine CaR agonist indicated for hemodialysis and peritoneal dialysis patients with secondary hyperparathyroidism (SHPT). It works by inhibiting parathyroid hormone (PTH) secretion through the expression of Ca receptors on the surface of parathyroid cells, thereby reducing the concentration of PTH in the blood and alleviating symptoms. Furthermore, evokarce can also regulate PTH biosynthesis and parathyroid cell proliferation, controlling PTH production.

[0004] Among the factors affecting drug safety, the quality and control of related substances are particularly important. Therefore, effective methods for controlling and detecting related substances are essential and can fundamentally address drug safety issues. Evocarbate has multiple synthetic routes, and different production routes, storage conditions, and formulation processes result in varying related substances. Currently, pharmacopoeias of various countries lack methods for controlling related substances in evokcarbate, and no other literature reports methods for detecting related substances in evokcarbate. Therefore, developing detection methods for evokcarbate related substances is necessary and has profound significance for its quality control.

[0005] Therefore, this invention proposes a method for detecting evokalse-related substances to solve the above-mentioned problems. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for detecting evokcarbose-related substances using high performance liquid chromatography that is durable, easy and quick to operate, and highly adaptable to various systems.

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is: a method for detecting evokinetic related substances, the innovation of which lies in: including the following steps: Step 1, Preparation of system suitability solutions: Take 7.5 mg each of impurities A, B, C, D and E, and place them in 100 ml volumetric flasks respectively. Dilute to the mark with solvent to prepare stock solutions for the impurities. Weigh 5 mg of evokort and place it in a 50 ml volumetric flask. Add 0.1 ml of each impurity stock solution, dilute to the mark with solvent, and shake well to prepare system suitability solutions. Step 2, Preparation of test solution: Protect from light, accurately weigh the evocalcet sample to be tested, dilute with solvent to prepare a solution containing 1 mg of evocalcet sample per ml, and use it as the test solution. Prepare fresh before use. Step 3, Preparation of control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well to prepare the control solution; Step 4: HPLC detection of evokorticide related substances: Inject 15 μl of the system suitability solution into the HPLC system and record the chromatogram. Impurities A, B, C, evokorticide, D, and E in the system suitability solution will elute in sequence. When the resolution between evokorticide and the adjacent impurity peak is greater than 1.5, accurately measure 15 μl each of the reference solution and the test solution and inject them into the HPLC system, and record the chromatogram.

[0008] Furthermore, the impurity A is named 2-[4-(1H-pyrrolo-1-yl)]phenylacetic acid, with the following structural formula: Impurity B is named (R)-1-(1-naphthyl)ethylamine, and its structural formula is: Impurity C is named 2-[4-(carboxymethyl)phenyl]-2-[4-[(S)3-[[(R)-1-(naphth-1-yl)ethyl]amino]pyrrolidine-1-yl]phenyl]acetic acid, with the following structural formula: Impurity D is named 4-[(S)-3-[[(R)-1-(1-naphthyl)ethyl]amino]-1-pyrrolidinyl]benzoic acid, with the following structural formula: Impurity E is named 4–[(S)-3–[[[(R)-1-(naphthyl)-1-yl]ethyl]amino]pyrrolidine-1-yl]benzaldehyde, with the following structural formula: .

[0009] Furthermore, the solvent is a solution of acetonitrile and water in a volume ratio of 50:50.

[0010] Furthermore, the chromatographic conditions of the liquid chromatograph in step 4 are as follows: an ACE Excel 3 C18-PFP column with dimensions of 150 mm * 4.6 mm and a packing particle size of 3.0 μm is used; the column temperature is 35 °C, gradient elution is used, and the flow rate is 0.6 ml / min; an ultraviolet detector is used with a detection wavelength of 220 nm.

[0011] Furthermore, the gradient elution uses a buffer salt solution-acetonitrile-methanol mobile phase I and an acetonitrile-methanol mobile phase II, wherein the volume ratio of buffer salt solution to acetonitrile and methanol in mobile phase I is 90:5:5, and the volume ratio of methanol to acetonitrile in mobile phase II is 1:1.

[0012] Further, the buffer salt includes one or more of sodium formate, potassium formate, sodium acetate, potassium acetate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and potassium hydroxide, with a concentration of 10~100 mmol / L.

[0013] Furthermore, the gradient elution procedure is as follows: First gradient elution time 0 min, mobile phase I 100%, mobile phase II 0%; The second gradient elution time was 5 min, with mobile phase I accounting for 72% and mobile phase II accounting for 28%. The third gradient elution time is 30 min, with mobile phase I accounting for 28% and mobile phase II accounting for 72%. The fourth gradient elution time was 40 min, with mobile phase I accounting for 28% and mobile phase II accounting for 72%. The fifth gradient elution time was 41 min, with mobile phase I comprising 100% and mobile phase II comprising 0%. The sixth gradient elution time was 50 min, with mobile phase I accounting for 100% and mobile phase II accounting for 0%.

[0014] Furthermore, after recording the chromatogram in step 4, if impurity peaks appear in the chromatogram of the test solution, the impurity content is calculated according to the following formula: in, The peak area of ​​impurities in the test solution. The peak area of ​​the main peak in the control solution; Impurities B, C, and A are all calculated based on the corrected peak area, which is the peak area of ​​the impurity multiplied by the correction factor.

[0015] Furthermore, the correction factors for impurities B, C, and A are 0.46, 1.3, and 2.3, respectively.

[0016] The advantages of the present invention are: (1) The method for detecting evokcarbide-related substances of the present invention uses a self-comparison method with correction factor to determine five known impurities of evokcarbide, thereby realizing the separation and analysis of impurities of evokcarbide-related substances. It has good separation, strong specificity, high sensitivity, high accuracy and simple method. This method has important practical significance for the quality control of evokcarbide raw material.

[0017] (2) This invention creatively explored the mobile phase system for the five impurities of evokcarse related substances, used buffer salt solution to enhance the separation effect of impurities, adjusted the mobile phase ratio and programmed temperature rise process, optimized the sample processing method, and finally established a high performance liquid chromatography system suitable for the detection of evokcarse related substances. Attached Figure Description

[0018] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0019] Figure 1 This is a chromatogram showing the system suitability of the evokorheic related substances of the present invention. Detailed Implementation

[0020] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0021] Example The chemical names and structural formulas of the evokine-related substances detected in this embodiment are as follows: Impurity A: Named 2-[4-(1H-pyrrolo-1-yl)]phenylacetic acid, with the following structural formula: Impurity B: Named (R)-1-(1-naphthyl)ethylamine, with the following structural formula: Impurity C: Named 2-[4-(carboxymethyl)phenyl]-2-[4-[(S)3-[[(R)-1-(naphth-1-yl)ethyl]amino]pyrrolidine-1-yl]phenyl]acetic acid, with the following structural formula: Impurity D: Named 4-[(S)-3-[[(R)-1-(1-naphthyl)ethyl]amino]-1-pyrrolidinyl]benzoic acid, with the following structural formula: Impurity E: Named 4–[(S)-3–[[[(R)-1-(naphthyl)-1-yl]ethyl]amino]pyrrolidine-1-yl]benzaldehyde, with the following structural formula: To separate the above impurities, this invention creatively explored a mobile phase system, using a buffer salt solution to enhance the separation effect of impurities, adjusting the mobile phase ratio and the programmed temperature rise process, and optimizing the sample processing method, ultimately establishing the high-performance liquid chromatography system described in this invention.

[0022] The method for detecting evokorbutal-related substances in this embodiment includes the following steps: Step 1: Preparation of system suitability solutions: Take 7.5 mg each of impurities A, B, C, D and E, and place them in 100 ml volumetric flasks respectively. Dilute to the mark with solvent to prepare the stock solutions for the impurities. Weigh 5 mg of evokort and place it in a 50 ml volumetric flask. Add 0.1 ml of each impurity stock solution, dilute to the mark with solvent, and shake well to prepare the system suitability solutions. The solvent is a 50:50 volume ratio solution of acetonitrile and water. Step 2, Preparation of test solution: Protect from light, accurately weigh the evocalcet sample to be tested, dilute with solvent to prepare a solution containing 1 mg of evocalcet sample per ml, and use it as the test solution. Prepare fresh before use. Step 3, Preparation of control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well to prepare the control solution; Step 4: HPLC detection of evokorticide related substances: Inject 15 μl of the system suitability solution into the HPLC system and record the chromatogram. Impurities A, B, C, evokorticide, D, and E in the system suitability solution will elute in sequence. When the resolution between evokorticide and the adjacent impurity peak is greater than 1.5, accurately measure 15 μl each of the reference solution and the test solution and inject them into the HPLC system, and record the chromatogram.

[0023] Chromatographic conditions and system suitability: The chromatographic conditions of the liquid chromatograph were as follows: ACE Excel 3 C18-PFP column (octadecyl pentafluorophenyl mixed bonded silica gel as packing material), 150mm*4.6mm in size, 3.0μm particle size of packing material, column temperature 35℃, and ultraviolet detector with a detection wavelength of 220nm.

[0024] Gradient elution was performed at a flow rate of 0.6 ml / min. Mobile phase I consisted of buffer salt solution-acetonitrile-methanol, and mobile phase II consisted of acetonitrile-methanol. In mobile phase I, the volume ratio of buffer salt solution to acetonitrile and methanol was 90:5:5, and in mobile phase II, the volume ratio of methanol to acetonitrile was 1:1. The buffer salt included one or more of sodium formate, potassium formate, sodium acetate, potassium acetate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and potassium hydroxide, with a concentration of 10–100 mmol / L.

[0025] The gradient elution procedure is as follows: Time (minutes) Mobile phase I (%) Mobile phase II (%) 0 100 0 5 72 28 30 28 72 40 28 72 41 100 0 50 100 0 After recording the chromatogram, if impurity peaks appear in the chromatogram of the test solution, calculate the impurity content according to the following formula: in, The peak area of ​​impurities in the test solution. The peak area of ​​the main peak in the control solution; Impurity B is calculated based on the corrected peak area, which is obtained by multiplying the peak area of ​​impurity B by a correction factor of 0.46. Impurity C is calculated based on the corrected peak area, which is obtained by multiplying the peak area of ​​impurity C by a correction factor of 1.3. Impurity A is calculated based on the corrected peak area, which is obtained by multiplying the peak area of ​​impurity A by a correction factor of 2.3. Impurities D, E, and other individual impurities are calculated based on the measured peak area.

[0026] In accordance with internationally accepted guidelines, the methodology of this invention was validated. The results of the methodology validation of related substances are as follows: (1) Exclusivity Acceptable criteria: The blank solution does not interfere with the determination of known impurities and the main peak; new impurities generated under various destructive conditions do not interfere with the detection of the main peak and known impurities; and the purity of the main peak is greater than 990.

[0027] Verification results: The blank solution showed no interference, the separation between the main peak and adjacent impurities was good, and under the destructive test conditions, the separation between the main peak and adjacent impurity peaks was not less than 1.5, and the peak purity met the requirements.

[0028] (2) System applicability Acceptable standard: The separation degree between the main peak and adjacent impurities is greater than 1.0.

[0029] Verification results: such as Figure 1 As shown, the system suitability is good. Impurities A, B, C, evokort, D and E in the solution eluted in sequence, and the separation degree between the main peak and adjacent impurities was greater than 1.5.

[0030] (3) Linearity and Range Acceptable criteria: Linearity is calculated by using the least squares method to perform linear regression on the measured response signal (peak area) against the analyte concentration, requiring r ≥ 0.990.

[0031] Verification results: The peak area of ​​impurity A showed a good linear relationship with the concentration in the range of 0.0462~0.7692 μg / ml (r=0.9993, n=6).

[0032] Impurity B showed a good linear relationship between peak area and concentration in the range of 0.0162~0.8087 μg / ml (r=0.9996, n=6).

[0033] Impurity C showed a good linear relationship between peak area and concentration in the range of 0.0467~0.7778 μg / ml (r=0.9998, n=6).

[0034] Impurity D showed a good linear relationship between peak area and concentration in the range of 0.0456~0.7592 μg / ml (r=0.9998, n=6).

[0035] Impurity E showed a good linear relationship between peak area and concentration in the range of 0.0461 to 0.7690 μg / ml (r = 0.9993, n = 6).

[0036] (4) Limit of detection and limit of quantitation Acceptable criteria: Signal-to-noise ratio for the detection limit ≥3; Signal-to-noise ratio for the quantitation limit ≥10.

[0037] Verification results: The limit of detection for impurity A is 0.35 ng (230 ppm) and the limit of quantification is 0.69 ng (460 ppm). The limit of detection for impurity B is 0.12 ng (80 ppm) and the limit of quantitation is 0.69 ng (160 ppm). The limit of detection for impurity C is 0.35 ng (230 ppm) and the limit of quantitation is 0.70 ng (470 ppm). The limit of detection for impurity D is 0.34 ng (230 ppm) and the limit of quantitation is 0.68 ng (460 ppm). The limit of detection for impurity E is 0.35 ng (230 ppm) and the limit of quantification is 0.69 ng (460 ppm).

[0038] The detection sensitivity is good.

[0039] (5) Sample injection precision Acceptable criteria: Retention time RSD not greater than 1.0%, peak area RSD not greater than 5.0%.

[0040] Verification results: The peak area and retention time of each impurity peak met the standards, and the injection precision was good.

[0041] (6) Precision Acceptable standard: RSD not greater than 5.0%.

[0042] Validation results: Repeated testing of 6 samples and samples with the same amount of impurities showed that, calculated using the self-comparison method with correction factors, the repeatability RSD% for impurity A was 3.1 (n=6); for impurity B, the repeatability RSD% for 6 samples was 3.8 (n=6); for impurity C, the repeatability RSD% for 6 samples was 3.4 (n=6); for impurity D, the repeatability RSD% was 3.5 (n=6); and for impurity E, the repeatability RSD% was 3.3. This method showed good repeatability for each impurity.

[0043] (7) Accuracy Acceptable criteria: Recovery rate should be between 85.0% and 115.0%, with an RSD of no more than 5%.

[0044] Validation results: Recovery tests at three different levels (low, medium, and high) from the limit of quantitation to 120% showed that: The recovery rate of impurity A was 96.0%, with an RSD% of 3.9 (n=9). The recovery rate of impurity B was 99.4%, with an RSD% of 2.3 (n=9). The recovery rate of impurity C was 92.4%, with an RSD% of 3.8 (n=9). The recovery rate of impurity D was 102.6%, and the RSD% was 4.3 (n=9). The recovery rate of impurity E was 97.5%, with an RSD of 3.7 (n=9). The recovery rates of all impurities detected by this method were good, ranging from 80.0% to 120.0%.

[0045] This method has good accuracy.

[0046] (8) Solution stability Acceptable standard: The solution remains stable during the testing period.

[0047] Verification results: The system suitability solution was stable for 120 hours at 4°C; the control solution was stable for 120 hours at 4°C; and the test solution was stable for 48 hours at 4°C.

[0048] (9) Durability Acceptable standard: The test results are not affected when the testing conditions change slightly.

[0049] Validation results: The robustness of the analytical method was examined under the following conditions: wavelength ±2 nm, relative flow rate change ±0.1 ml / min, relative change of initial mobile phase ratio ±2%, column temperature change ±2℃, and mobile phase pH ±0.2. The results showed that the separation degree of each impurity peak from other component peaks met the standard under each condition. The absolute deviation of the content of each known impurity was no more than 20% of the limit, the absolute deviation of the largest unknown single impurity was no more than 0.05%, and the absolute deviation of the total impurities was no more than 5% (0.08%) of the limit. The method has good robustness.

[0050] The above verification results show that all indicators of this method meet the requirements of the 2025 edition of the Chinese Pharmacopoeia and are suitable for the detection of related substances in evokinetics.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for detecting evokalse-related substances, characterized in that: Includes the following steps: Step 1, Preparation of system suitability solutions: Take 7.5 mg each of impurities A, B, C, D and E, and place them in 100 ml volumetric flasks respectively. Dilute to the mark with solvent to prepare stock solutions for the impurities. Weigh 5 mg of evokort and place it in a 50 ml volumetric flask. Add 0.1 ml of each impurity stock solution, dilute to the mark with solvent, and shake well to prepare system suitability solutions. Step 2, Preparation of test solution: Protect from light, accurately weigh the evocalcet sample to be tested, dilute with solvent to prepare a solution containing 1 mg of evocalcet sample per ml, and use it as the test solution. Prepare fresh before use. Step 3, Preparation of control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well to prepare the control solution; Step 4: HPLC detection of evokorticide related substances: Inject 15 μl of the system suitability solution into the HPLC system and record the chromatogram. Impurities A, B, C, evokorticide, D, and E in the system suitability solution will elute in sequence. When the resolution between evokorticide and the adjacent impurity peak is greater than 1.5, accurately measure 15 μl each of the reference solution and the test solution and inject them into the HPLC system, and record the chromatogram.

2. The method for detecting evokalse related substances according to claim 1, characterized in that: The impurity A is named 2-[4-(1H-pyrrolo-1-yl)]phenylacetic acid, and its structural formula is: Impurity B is named (R)-1-(1-naphthyl)ethylamine, and its structural formula is: Impurity C is named 2-[4-(carboxymethyl)phenyl]-2-[4-[(S)3-[[(R)-1-(naphth-1-yl)ethyl]amino]pyrrolidine-1-yl]phenyl]acetic acid, with the following structural formula: Impurity D is named 4-[(S)-3-[[(R)-1-(1-naphthyl)ethyl]amino]-1-pyrrolidinyl]benzoic acid, with the following structural formula: Impurity E is named 4–[(S)-3–[[[(R)-1-(naphthyl)-1-yl]ethyl]amino]pyrrolidine-1-yl]benzaldehyde, with the following structural formula: 。 3. The method for detecting evokalse related substances according to claim 2, characterized in that: The solvent is a solution of acetonitrile and water in a mass ratio of 50:

50.

4. The method for detecting evokalse related substances according to claim 2, characterized in that: The chromatographic conditions of the liquid chromatograph in step 4 are as follows: ACE Excel 3 C18-PFP column, 150mm*4.6mm in size, with a packing particle size of 3.0μm; column temperature 35℃, gradient elution, flow rate 0.6ml / min; and ultraviolet detector with a detection wavelength of 220nm.

5. The method for detecting evokalse related substances according to claim 4, characterized in that: The gradient elution uses a buffer salt solution-acetonitrile-methanol mobile phase I and an acetonitrile-methanol mobile phase II. The volume ratio of buffer salt solution to acetonitrile and methanol in mobile phase I is 90:5:5, and the volume ratio of methanol to acetonitrile in mobile phase II is 1:

1.

6. The method for detecting evokalse related substances according to claim 5, characterized in that: The buffer salt includes one or more of sodium formate, potassium formate, sodium acetate, potassium acetate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and potassium hydroxide, with a concentration of 10~100 mmol / L.

7. The method for detecting evokalse related substances according to claim 6, characterized in that: The gradient elution procedure is as follows: First gradient elution time 0 min, mobile phase I 100%, mobile phase II 0%; The second gradient elution time was 5 min, with mobile phase I accounting for 72% and mobile phase II accounting for 28%. The third gradient elution time is 30 min, with mobile phase I accounting for 28% and mobile phase II accounting for 72%. The fourth gradient elution time was 40 min, with mobile phase I accounting for 28% and mobile phase II accounting for 72%. The fifth gradient elution time was 41 min, with mobile phase I comprising 100% and mobile phase II comprising 0%. The sixth gradient elution time was 50 min, with mobile phase I accounting for 100% and mobile phase II accounting for 0%.

8. The method for detecting evokalse related substances according to claim 1, characterized in that: After recording the chromatogram in step 4, if impurity peaks appear in the chromatogram of the test solution, the impurity content shall be calculated according to the following formula: in, The peak area of ​​impurities in the test solution. The peak area of ​​the main peak in the control solution; Impurities B, C, and A are all calculated based on the corrected peak area, which is the peak area of ​​the impurity multiplied by the correction factor.

9. The method for detecting evokalse related substances according to claim 8, characterized in that: The correction factors for impurities B, C, and A are 0.46, 1.3, and 2.3, respectively.

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