Method for detecting specific impurities in enalastat S1 intermediate

Abstract

The invention discloses a method for detecting specific impurities in an enalastat S1 intermediate in the technical field of medical analysis, a 2-amino-4, 6-dichloropyridine-3-methyl formate test solution is detected by HPLC (high performance liquid chromatography), according to a liquid chromatography method, an octadecylsilane chemically bonded silica chromatographic column is selected, and the specific impurities in the enalastat S1 intermediate can be detected by using the octadecylsilane chemically bonded silica chromatographic column to obtain the specific impurities in the enalastat S1 intermediate. The chromatographic column has very good peak shapes for alkaline, acidic and neutral compounds, and a mobile phase is a reverse solvent: trifluoroacetic acid aqueous solution-trifluoroacetic acid acetonitrile solution. An ultraviolet detector is adopted in the liquid chromatography. The method is high in response value, capable of effectively separating products and impurities, simple in detection operation and high in accuracy, the applicability, the specificity, the linearity and the range of the system all meet the requirements, and the problems of purity detection and impurity control of the substance are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis, and in particular to a method for detecting specific impurities in an enastat S1 intermediate. Background Art

[0002] The intermediate of Ennastat S1, chemical name: 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester, CAS number: 1044872-40-3, structural formula is as follows:

[0003]

[0004] The current process route of Ennastat S1 is as follows:

[0005]

[0006] Based on the control strategy of raw material impurity research, possible impurities in Enfastat S1 can be traced back to the key intermediate 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester used.

[0007] 2-Amino-4,6-dichloropyridine-3-carboxylic acid methyl ester is an intermediate of Ennastat S1. Combined with the synthetic route of 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester, there may be impurities such as 2-amino-4-hydroxy-6-oxo-1,6-dihydropyridine-3-carboxylic acid methyl ester, 2-amino-4-chloro-6-oxopiperidine-3-carboxylic acid methyl ester and 2-amino-4-chloro-6-hydroxy-3-picolinic acid methyl ester. The presence of impurities will affect the quality of the product, and its quality also determines the quality of the modified drug. In particular, the content of certain specific impurities will have a significant impact on the purity of subsequent products. The existing technology currently has no means for detecting specific impurities in 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester. Therefore, in order to study the control problem of specific impurities in this type of substance, a series of studies on the detection of specific impurities in 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester were carried out. Summary of the invention

[0008] The purpose of the present invention is to provide a method for detecting specific impurities in an enastat S1 intermediate, so as to overcome the current lack of exclusive detection methods and means to detect and control its specific impurities. The method uses liquid chromatography to detect specific impurities in 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester, has a high response value, can effectively separate products and impurities, has simple detection operation, high accuracy, and the system applicability and specificity, linearity and range all meet the requirements, thereby solving the problems of purity detection and impurity control of the substance.

[0009] The technical solution adopted by the present invention is as follows: a method for detecting specific impurities in an enastat S1 intermediate, wherein a test sample is dissolved and detected by liquid chromatography;

[0010] The liquid chromatography column is an octadecylsilane bonded silica gel column;

[0011] Mobile phase A is an aqueous solution containing 0.1 wt % trifluoroacetic acid and mobile phase B is an acetonitrile solution containing 0.1 wt % trifluoroacetic acid;

[0012] The column temperature of liquid chromatography was 20°C to 40°C and the detection wavelength was 270nm;

[0013] The flow rate of the mobile phase was 0.8-1.2 ml / min, and the elution method was gradient elution;

[0014] The injection volume for liquid chromatography is 5 to 15 μl;

[0015] The specific impurities are 2-amino-4-hydroxy-6-oxo-1,6-dihydropyridine-3-carboxylic acid methyl ester, 2-amino-4-chloro-6-oxopiperidine-3-carboxylic acid methyl ester and 2-amino-4-chloro-6-hydroxy-3-picolinic acid methyl ester;

[0016] The liquid chromatography method adopts an ultraviolet detector;

[0017] The liquid chromatography method adopts a gradient elution mode;

[0018] The detection steps of the liquid chromatography method are:

[0019] Set the liquid chromatograph according to the chromatographic conditions until the baseline is stable and wait for injection;

[0020] Preparation of impurity stock solution: Take 10 mg each of 2-amino-4-hydroxy-6-oxo-1,6-dihydropyridine-3-carboxylic acid methyl ester, 2-amino-4-chloro-6-oxopiperidine-3-carboxylic acid methyl ester and 2-amino-4-chloro-6-hydroxy-3-picolinic acid methyl ester, accurately weigh, place in a 100 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well;

[0021] Test solution: Take about 10 mg of 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester, accurately weigh it, put it in a 10 ml volumetric flask, add dimethyl sulfoxide to dissolve it, dilute it to the scale, and shake it well;

[0022] Separation solution: Take about 10 mg of 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester, accurately weigh it, put it in a 10 ml volumetric flask, add 1 ml of impurity stock solution, add dimethyl sulfoxide to dissolve it, dilute to the scale, and shake well;

[0023] According to the above chromatographic conditions, the separation solution was sampled and the separation between the impurity 2-amino-4-chloro-6-oxopiperidine-3-carboxylic acid methyl ester, the impurity 2-amino-4-chloro-6-hydroxy-3-pyridinecarboxylic acid methyl ester, the impurity 2-amino-4-hydroxy-6-oxo-1,6-dihydropyridine-3-carboxylic acid methyl ester and the product 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester was examined.

[0024] According to the above chromatographic conditions, the test solution was sampled and the peak area was recorded. The impurity content was calculated according to the area normalization method. The calculation formula is as follows:

[0025]

[0026] Where:

[0027] A 杂 —The peak area of ​​impurities in the test solution;

[0028] A 总 —The sum of the peak areas of all chromatographic peaks in the test solution.

[0029] The liquid chromatography method adopts a gradient elution mode.

[0030] The liquid chromatography gradient elution procedure:

[0031] Time, min Mobile phase A, % Mobile phase B, % 0 85 15 15 60 40 20 30 70 25 30 70 30 85 15 40 85 15

[0032] In the present invention, the structural formulas of 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester (1), 2-amino-4-hydroxy-6-oxo-1,6-dihydropyridine-3-carboxylic acid methyl ester (2), 2-amino-4-chloro-6-oxopiperidine-3-carboxylic acid methyl ester (3) and 2-amino-4-chloro-6-hydroxy-3-picolinic acid methyl ester (4) are as follows:

[0033]

[0034]

[0035] The chromatographic column is a ZORBAX Eclipse XDB-C18 chromatographic column with a specification of (4.6×150 mm×5 μm).

[0036] Furthermore, the liquid chromatography column temperature is 30°C.

[0037] Furthermore, the flow rate of the mobile phase of the liquid chromatography method is 0.8-1.2 ml / min, including but not limited to 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min.

[0038] Furthermore, the liquid chromatography injection volume is 10-20 μl, including but not limited to 10 μl, 15 μl, and 20 μl.

[0039] Beneficial effects: The instrument provided by the present invention has low operating cost, simple and convenient detection process, good repeatability, and can be used to evaluate product quality. The present invention meets the requirements in terms of system applicability and specificity, linearity and range. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the chromatogram of the test solution of methyl 2-amino-4,6-dichloropyridine-3-carboxylate.

[0041] Figure 2 This is the sensitive solution chromatogram of methyl 2-amino-4,6-dichloropyridine-3-carboxylate.

[0042] Figure 3 This is the linear relationship diagram of 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester.

[0043] Figure 4 This is the linear relationship diagram of 2-amino-4-hydroxy-6-oxo-1,6-dihydropyridine-3-carboxylic acid methyl ester.

[0044] Figure 5 This is the linear relationship diagram of methyl 2-amino-4-chloro-6-oxopiperidine-3-carboxylate.

[0045] Figure 6 The linear relationship diagram of 2-amino-4-chloro-6-hydroxy-3-picolinic acid methyl ester. DETAILED DESCRIPTION

[0046] The present invention is further described below in conjunction with embodiments and drawings, but the present invention is not limited to the following embodiments.

[0047] Detection of 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester by high performance liquid chromatography

[0048] The instruments and chemicals used in the examples include: liquid chromatograph (Agilent 1260, Agilent); electronic balance (XSR105, Mettler); ZORBAX Eclipse XDB-C18 chromatographic column (4.6×150 mm×5 μm, Agilent); and formic acid of chromatographic grade.

[0049] Example 1

[0050] 1. HPLC analysis conditions:

[0051] Chromatographic column: ZORBAX Eclipse XDB-C18 column (4.6×150mm×5μm, Agilent)

[0052] Mobile phase A: 0.1% trifluoroacetic acid in water

[0053] Mobile phase B: 0.1% trifluoroacetic acid in acetonitrile;

[0054] The detection wavelength of liquid chromatography was 270 nm;

[0055] The column temperature for liquid chromatography was 30°C;

[0056] The flow rate of the mobile phase was 1.0 ml / min, and the elution method was gradient elution. The gradient elution conditions were as follows:

[0057] Time, min Mobile phase A, % Mobile phase B, % 0 85 15 15 60 40 20 30 70 25 30 70 30 85 15 40 85 15

[0058] The injection volume for liquid chromatography was 10 μl.

[0059] 2. Solution preparation:

[0060] Mobile phase preparation:

[0061] Mobile phase A (0.1% trifluoroacetic acid in water): Take 1.0 ml of trifluoroacetic acid, add water to 1000 ml, and mix well;

[0062] Mobile phase B (acetonitrile solution containing 0.1% trifluoroacetic acid): Take 1.0 ml of trifluoroacetic acid, add acetonitrile to 1000 ml, and mix well;

[0063] Preparation of test solution: Weigh 10 mg of 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester, accurately weigh it into a 10 ml volumetric flask, add dimethyl sulfoxide to dissolve it, dilute it to the scale, shake well, and obtain a test solution with a concentration of 1 mg / ml.

[0064] Blank solution: dimethyl sulfoxide.

[0065] 3. Determination

[0066] HPLC was used for detection, and the detection conditions were as described above.

[0067] Take blank and test solution and inject them into liquid chromatograph, record the chromatogram, the injection sequence is as follows:

[0068] name Number of injection needles blank ≥2, until the two needles completely overlap without interference Test solution 1

[0069] Example 2

[0070] 1. Exclusivity

[0071] 1.1 Solution preparation:

[0072] Blank: Dimethyl sulfoxide

[0073] Specific impurity solution: Take 10 mg each of 2-amino-4-hydroxy-6-oxo-1,6-dihydropyridine-3-carboxylic acid methyl ester, 2-amino-4-chloro-6-oxopiperidine-3-carboxylic acid methyl ester and 2-amino-4-chloro-6-hydroxy-3-picolinic acid methyl ester into a 100 ml volumetric flask and dissolve them to the mark with dimethyl sulfoxide.

[0074] Sensitivity solution: Take 100 mg of methyl 2-amino-4,6-dichloropyridine-3-carboxylate into a 100 ml volumetric flask, then transfer 1 ml of the specific impurity solution into the same volumetric flask, add dimethyl sulfoxide to dissolve and dilute to the scale.

[0075] Test solution: Take 10 mg of 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester, accurately weigh it, put it into a 10 ml volumetric flask, add dimethyl sulfoxide to dissolve it, dilute it to the scale, shake well, and you will get a 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester solution with a concentration of 1 mg / ml.

[0076] 1.2 Determination method

[0077] Inject blank, system suitability solution and test solution in the following order and record the chromatogram.

[0078] Injection order:

[0079]

[0080]

[0081] 1.3 Results: No interference in blank; The peak separation of each component in the sensitivity solution is ≥1.5, and the signal-to-noise ratio (S / N) of specific impurities is greater than 10.

[0082]

[0083] 2. Linearity and range

[0084] 2.1 Solution preparation:

[0085] Stock solution: Accurately weigh 10 mg each of 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester, 2-amino-4-hydroxy-6-oxo-1,6-dihydropyridine-3-carboxylic acid methyl ester, 2-amino-4-chloro-6-oxopiperidine-3-carboxylic acid methyl ester and 2-amino-4-chloro-6-hydroxy-3-picolinic acid methyl ester into a 10 ml volumetric flask, and dissolve to volume with dimethyl sulfoxide; then transfer 1 ml of the above solution to a 100 ml volumetric flask, dissolve to volume with dimethyl sulfoxide to prepare a single standard solution of 10 μg / ml;

[0086] Use a pipette to accurately pipette 2.5ml, 4.0ml, 5.0ml, 6.0ml, 8.0ml and 10.0ml into a 50ml volumetric flask, and then dilute to the scale with mobile phase to obtain 0.5μg / ml, 0.8μg / ml, 1.0μg / ml, 1.2μg / ml, 1.6μg / ml and 2.0μg / ml standard curve solutions.

[0087] Linear solution ① (0.5 μg / ml, 40%): Accurately measure 2.5 ml of the stock solution, place it in a 50 ml volumetric flask, dilute to the scale with the mobile phase, shake well, and the solution is ready.

[0088] Linear solution ② (0.8 μg / ml, 80%): Accurately measure 4.0 ml of the stock solution, place it in a 50 ml volumetric flask, dilute to the scale with the mobile phase, shake well, and the solution is ready.

[0089] Linear solution ③ (1.0 μg / ml, 100%): Accurately measure 5.0 ml of the stock solution, place it in a 50 ml volumetric flask, dilute to the scale with the mobile phase, shake well, and obtain.

[0090] Linear solution ④ (1.2 μg / ml, 120%): Accurately measure 6.0 ml of the stock solution, place it in a 50 ml volumetric flask, dilute to the scale with the mobile phase, shake well, and obtain.

[0091] Linear solution ⑤ (1.6 μg / ml, 160%): Accurately measure 8.0 ml of the stock solution, place it in a 50 ml volumetric flask, dilute to the scale with the mobile phase, shake well, and obtain.

[0092] Linear solution ⑥ (2.0 μg / ml, 200%): Accurately measure 10.0 ml of the stock solution, place it in a 50 ml volumetric flask, dilute to the scale with the mobile phase, shake well, and obtain.

[0093] 2.2 Determination method:

[0094] Take linear solution ① to linear solution ⑥, inject 2 injections each, record the chromatogram, perform linear regression based on the concentration and peak area of ​​the linear solution, and draw a standard curve.

[0095] 2.3 Results:

[0096] Table 1: Linear data table of methyl 2-amino-4,6-dichloropyridine-3-carboxylate

[0097] Limit concentration ratio / % Concentration / μg / ml Peak area / mAU 50 0.50 14.99 80 0.81 23.85 100 1.01 30.42 120 1.21 36.72 160 1.61 50.22 200 2.02 62.41

[0098] Table 2: Linear data table of methyl 2-amino-4-hydroxy-6-oxo-1,6-dihydropyridine-3-carboxylate

[0099] Limit concentration ratio / % Concentration / μg / ml Peak area / mAU 50 0.50 26.20 80 0.81 42.24 100 1.01 49.68 120 1.21 63.98 160 1.61 83.73 200 2.02 101.21

[0100] Table 3: Linear data table of methyl 2-amino-4-chloro-6-oxopiperidine-3-carboxylate

[0101]

[0102]

[0103] Table 4: Linear data table of methyl 2-amino-4-chloro-6-hydroxy-3-picolinate

[0104] Limit concentration ratio / % Concentration / μg / ml Peak area / mAU 50 0.50 20.11 80 0.81 29.46 100 1.01 38.81 120 1.21 44.27 160 1.61 59.98 200 2.02 77.13

[0105] With concentration (μg / ml) as the horizontal axis and the corresponding peak area as the vertical axis, linear regression was performed. The linear results and linear equation are shown in Figure 3-6 As shown, the results show that 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester, 2-amino-4-hydroxy-6-oxo-1,6-dihydropyridine-3-carboxylic acid methyl ester, 2-amino-4-chloro-6-oxopiperidine-3-carboxylic acid methyl ester and 2-amino-4-chloro-6-hydroxy-3-picolinic acid methyl ester are detected according to the chromatographic conditions described in Example 1. 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester has a good linear relationship in the concentration range of 0.50 μg / ml to 2.02 μg / ml, and the linear equation y=31.69x-1.3647, R 2 =0.9996; 2-amino-4-hydroxy-6-oxo-1,6-dihydropyridine-3-carboxylic acid methyl ester has a good linear relationship in the concentration range of 0.50μg / ml to 2.01μg / ml, and the linear equation is y=50.462x+1.2206, R 2 =0.9966; 2-amino-4-chloro-6-oxopiperidine-3-carboxylic acid methyl ester has a good linear relationship in the concentration range of 0.51μg / ml to 2.04μg / ml, and the linear equation is y=35.088x-2.9085, R 2 =0.9979; 2-amino-4-chloro-6-hydroxy-3-picolinic acid methyl ester has a good linear relationship in the concentration range of 0.50μg / ml to 2.01μg / ml, and the linear equation is 37.782x-0.0165, R 2 =0.9970.

[0106] The above content is a further detailed description of the technical solution provided in combination with the preferred implementation mode of the present invention. It cannot be determined that the specific implementation of the present invention is limited to the above descriptions. For ordinary technicians in the technical field to which the present invention belongs, without departing from the concept of the present invention, some simple deductions or substitutions can be made, which should be considered as falling within the protection scope of the present invention as needed.

Claims

1. A method for detecting specific impurities in an enastat S1 intermediate, characterized in that: The test sample was dissolved and tested by liquid chromatography; The liquid chromatography column is an octadecylsilane bonded silica gel column; Mobile phase A is an aqueous solution containing 0.1 wt % trifluoroacetic acid and mobile phase B is an acetonitrile solution containing 0.1 wt % trifluoroacetic acid; The column temperature of liquid chromatography was 20°C to 40°C and the detection wavelength was 270nm; The flow rate of the mobile phase was 0.8-1.2 ml / min, and the elution method was gradient elution; The injection volume for liquid chromatography is 5 to 15 μl; The specific impurities are 2-amino-4-hydroxy-6-oxo-1,6-dihydropyridine-3-carboxylic acid methyl ester, 2-amino-4-chloro-6-oxopiperidine-3-carboxylic acid methyl ester and 2-amino-4-chloro-6-hydroxy-3-picolinic acid methyl ester; The liquid chromatography method adopts an ultraviolet detector; The liquid chromatography method adopts a gradient elution mode; The detection steps of the liquid chromatography method are: Set the liquid chromatograph according to the chromatographic conditions until the baseline is stable and wait for injection; Preparation of impurity stock solution: Take 10 mg each of 2-amino-4-hydroxy-6-oxo-1,6-dihydropyridine-3-carboxylic acid methyl ester, 2-amino-4-chloro-6-oxopiperidine-3-carboxylic acid methyl ester and 2-amino-4-chloro-6-hydroxy-3-picolinic acid methyl ester, accurately weigh, place in a 100 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well; Test solution: Take about 10 mg of 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester, accurately weigh it, put it in a 10 ml volumetric flask, add dimethyl sulfoxide to dissolve it, dilute it to the scale, and shake it well; Separation solution: Take about 10 mg of 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester, accurately weigh it, put it in a 10 ml volumetric flask, add 1 ml of impurity stock solution, add dimethyl sulfoxide to dissolve it, dilute to the scale, and shake well; According to the above chromatographic conditions, the separation solution was sampled and the separation between the impurity 2-amino-4-chloro-6-oxopiperidine-3-carboxylic acid methyl ester, the impurity 2-amino-4-chloro-6-hydroxy-3-pyridinecarboxylic acid methyl ester, the impurity 2-amino-4-hydroxy-6-oxo-1,6-dihydropyridine-3-carboxylic acid methyl ester and the product 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester was examined; According to the above chromatographic conditions, the test solution was sampled and the peak area was recorded. The impurity content was calculated according to the area normalization method. The calculation formula is as follows: Where: A 杂 —The peak area of ​​impurities in the test solution; A 总 —The sum of the peak areas of all chromatographic peaks in the test solution.

2. The analysis method according to claim 1, characterized in that The detection conditions are as follows: the chromatographic column is a ZORBAX Eclipse XDB-C18 liquid chromatography column; the column temperature of the liquid chromatography is 30° C.; and the flow rate of the liquid chromatography mobile phase is 1.0 ml / min.

3. The method for detecting specific impurities in an enastat S1 intermediate according to claim 1, characterized in that: The liquid chromatography injection volume is 10 μl, the concentration of the test solution is 0.8-1.2 mg / ml, and the concentration of the impurity localization solution is 0.8-1.2 μg / ml.

4. The method for detecting specific impurities in an enastat S1 intermediate according to claim 1, characterized in that: The determination of the standard curves for methyl 2-amino-4,6-dichloropyridine-3-carboxylate, methyl 2-amino-4-hydroxy-6-oxo-1,6-dihydropyridine-3-carboxylate, methyl 2-amino-4-chloro-6-oxopiperidine-3-carboxylate and methyl 2-amino-4-chloro-6-hydroxy-3-picolinate includes: S01) accurately weigh 10 mg each of 2-amino-4,6-dichloropyridine-3-carboxylic acid methyl ester, 2-amino-4-hydroxy-6-oxo-1,6-dihydropyridine-3-carboxylic acid methyl ester, 2-amino-4-chloro-6-oxopiperidine-3-carboxylic acid methyl ester and 2-amino-4-chloro-6-hydroxy-3-picolinic acid methyl ester into a 10 ml volumetric flask, and dissolve to volume with dimethyl sulfoxide; then transfer 1 ml of the above solution to a 100 ml volumetric flask, dilute to volume with dimethyl sulfoxide, and prepare a single standard solution of 10 μg / ml; S02) using a pipette to accurately pipette 2.5 ml, 4.0 ml, 5.0 ml, 6.0 ml, 8.0 ml, and 10.0 ml into a 50 ml volumetric flask, and then dilute to scale with mobile phase to obtain standard curve solutions of 0.5 μg / ml, 0.8 μg / ml, 1.0 μg / ml, 1.2 μg / ml, 1.6 μg / ml, and 2.0 μg / ml; S03) taking the standard curve solution for high performance liquid chromatography analysis, the chromatographic conditions are: using a ZORBAX Eclipse XDB-C18 column as a HPLC column, with a specification of 4.6×150mm×5μm, the mobile phase is an aqueous solution of trifluoroacetic acid and an acetonitrile solution of trifluoroacetic acid, the flow rate is 1.0ml / min, and the column temperature is 30°C; the detector is a UV detector, and a standard curve is drawn according to the relationship between the concentration μg / ml and the chromatographic peak area.

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