A method for analyzing related substances of thiocarbamide bromide intermediates

Abstract

The present invention relates to the field of drug analysis, and in particular to a method for analyzing related substances of a thiophene bromide intermediate. (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophene-2-yl)acetate is an important intermediate compound for the preparation of thiophene bromide, and its related substances will have a direct impact on the safety and effectiveness of thiophene bromide. Currently, no analytical method has been reported to be applicable to the detection of related substances of this product. The method provided by the present invention can accurately determine the content of each impurity in (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophene-2-yl)acetate, and the separation degree between the main peak and the related substances is ≥1.5, the solvent does not interfere with the detection of impurities, the method has good specificity, and the detection method is simple, rapid, accurate, highly sensitive, repeatable and accurate, and can quickly and accurately perform qualitative and quantitative analysis of the content of each process impurity and degradation impurity in the test sample, thereby ensuring the controllability of the quality of this product.

Description

Technical Field

[0001] The invention relates to the field of drug analysis, and in particular to a method for analyzing related substances of a thiocarbamide bromide intermediate. Background Art

[0002] Thiogrenate bromide is a highly selective M3 cholinergic receptor inhibitor discovered in recent years. White crystal or powder, chemical name: 3R-1,1-dimethyl-3-(2-hydroxy-2,2-dithiophen-2-ylacetoxy)pyrrolidine bromide, molecular formula: C 16 H 20 O 3 NS 2 Br, the structural formula is shown in Formula Ⅰ.

[0003]

[0004] Thiotropium bromide is a new generation of innovative COPD drugs. Its strong drugability and low systemic exposure are expected to overcome the clinical defects of existing COPD drugs and achieve new COPD therapeutic drugs with better clinical effects. (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl)acetate is its important intermediate compound (Formula II). Its related substances will have a direct impact on the safety and effectiveness of thiotropium bromide. At present, no analytical method has been reported to be suitable for the detection of related substances of this product. Therefore, this patent adopts an independently developed analytical method to control the quality of related substances of (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl)acetate, which is of great significance. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a method for analyzing related substances of (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophene-2-yl)acetate.

[0006] The technical scheme of the present invention is as follows: a method for analyzing related substances of (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl)acetate, the method is a high performance liquid chromatography method, using a chromatographic column with octadecylsilane bonded silica gel as a filler, and using mobile phase A and mobile phase B as eluents, wherein mobile phase A is a sodium heptane sulfonate solution, and mobile phase B is acetonitrile.

[0007] In some embodiments, the mobile phase A is a 0.02 mol / L sodium heptane sulfonate solution.

[0008] In some embodiments, the volume ratio of mobile phase A to mobile phase B is 69:31.

[0009] In some embodiments, the 0.02 mol / L sodium heptane sulfonate solution contains 0.5% triethylamine, and the pH value is adjusted to 3.5 with phosphoric acid.

[0010] In some embodiments, the flow rate of the mobile phase is 0.75-1 ml / min; preferably, the flow rate of the eluent is 1 ml / min.

[0011] In some embodiments, the chromatographic column is Agilent ZORBAX SB-C18, with specifications of 250 mm×4.6 mm and 5 μm.

[0012] In some embodiments, the analysis method is performed on a high performance liquid chromatograph using a diode array detector or a UV detector; in some more typical embodiments, the analysis method is performed on a high performance liquid chromatograph using a UV detector, and its detection wavelength is 200nm to 300nm; preferably 237nm.

[0013] In some embodiments, the column temperature of the chromatographic column is 20-30°C; in some more typical embodiments, the column temperature of the reverse phase chromatographic column is 25°C.

[0014] On the other hand, the present invention provides a method for analyzing related substances of (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl)acetate, characterized in that:

[0015] The analysis method is carried out on a high performance liquid chromatograph, which uses octadecylsilane bonded silica gel as a chromatographic column with a stationary phase;

[0016] The analytical method uses an ultraviolet detector with a detection wavelength of 237 nm;

[0017] The analysis method column temperature is 25°C; sample plate temperature is 4°C; injection volume is 10 μl;

[0018] The analysis method uses mobile phase A and mobile phase B as eluents, wherein mobile phase A is a sodium heptane sulfonate solution, mobile phase B is acetonitrile, and the sodium heptane sulfonate solution is a 0.02 mol / L sodium heptane sulfonate aqueous solution;

[0019] The analytical method uses isocratic elution:

[0020] And in the isocratic elution process, the sum of the proportion of mobile phase A and the proportion of mobile phase B is 100%; wherein the proportion of mobile phase A refers to the percentage of the volume of mobile phase A in the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of mobile phase B in the total volume of the eluent;

[0021] The eluent flow rate was 1 ml / min.

[0022] In some specific embodiments, the present invention provides a method for analyzing related substances of (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl)acetate, comprising the following steps:

[0023] Preparation of test sample solution: Take an appropriate amount of test sample, add mobile phase to dissolve and dilute to make the test sample solution.

[0024] Preparation of control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with the mobile phase to prepare the control solution.

[0025] Determination: Agilent ZORBAX SB-C18 was used as the chromatographic column, 0.02 mol / L sodium heptane sulfonate solution-acetonitrile (69:31) was used as the mobile phase, the flow rate was 1.0 ml per minute; the detection wavelength was 237 nm; the column temperature was 25°C; the sample plate temperature was 4°C; the injection volume was 10 μl; the test solution and the control solution were taken and injected into the liquid chromatograph respectively, and the chromatogram was recorded;

[0026] Calculation of impurity content:

[0027]

[0028] Where: Ru is the peak area of ​​impurities in the test solution;

[0029] Rs is the peak area of ​​the main peak in the control solution;

[0030] L is the dilution factor of the control solution;

[0031] F is the correction factor for impurities.

[0032] In the present invention, unless otherwise specified, the compound of formula II used for "test preparation" includes but is not limited to a newly prepared or stored compound of formula II or a pharmaceutical composition comprising a compound of formula II, preferably a newly prepared or stored compound of formula II.

[0033] In the present invention, an appropriate amount means that the amount of each compound is within the detection limit or quantitative limit of its high performance liquid chromatography according to the experimental purpose.

[0034] The beneficial effects of the present invention are as follows: the method provided by the present invention can accurately determine the contents of various impurities in (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl)acetate, and the separation between the main peak and its known impurity peak and the adjacent impurity peak is ≥1.5, the solvent does not interfere with the impurity detection, the method has good specificity, and the detection method is simple, rapid, highly sensitive, repeatable and accurate, and can quickly and accurately perform qualitative and quantitative analysis of the contents of various process impurities and degradation impurities in the test sample, thereby ensuring the controllability of the quality of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the system suitability solution spectrum;

[0036] Figure 2 is the sample solution spectrum;

[0037] Figure 3 This is the reference solution spectrum. DETAILED DESCRIPTION

[0038] The following embodiments can enable those skilled in the art to more fully understand the present invention, but the present invention is not limited to the scope of the embodiments.

[0039] (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl)acetate is the main research object of the present invention. The reference substance is derived from self-developed calibration, with a content of 98.49%. The preparation process is as follows:

[0040] Under the condition of toluene reflux and water separation, methyl 2,2-dithienyl glycolate (starting material 1) and (R)-1-methyl-3-hydroxypyrrolidine (starting material 2) undergo ester exchange reaction under high temperature conditions and the action of cesium carbonate, and then undergo post-treatment steps such as filtration, water washing, drying, concentration and pulping to obtain (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl)acetate.

[0041]

[0042] Impurity A is the starting material in the synthesis of (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl)acetate and is also a degradation impurity. The reference substance comes from Sinco and has a content of 99.80%. The impurity structure is as follows:

[0043]

[0044] Impurity B is a process impurity in the synthesis of (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl)acetate. The reference substance comes from Sinco, with a content of 98.76%. The impurity structural formula is as follows:

[0045]

[0046] Impurity C is a process impurity in the synthesis of (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl)acetate. The reference substance is from Enamine, with a content of 95%. The impurity structure is as follows:

[0047]

[0048] Impurity J is a degradation impurity in the process of synthesizing (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl)acetate. The reference substance is from Sinco, with a content of 93.97%. The impurity structural formula is as follows:

[0049]

[0050] Example 1 Sample detection

[0051] Preparation of test sample solution: Take an appropriate amount of the test sample, dissolve it in the mobile phase and dilute it to make a solution containing about 0.5 mg per 1 ml.

[0052] Preparation of control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with the mobile phase to make a solution containing 5 μg per 1 ml.

[0053] Determination: Agilent ZORBAX SB-C18 was used as the chromatographic column, 0.02 mol / L sodium heptane sulfonate solution-acetonitrile (69:31) was used as the mobile phase, the flow rate was 1.0 ml per minute; the detection wavelength was 237 nm; the column temperature was 25°C; the sample plate temperature was 4°C; the injection volume was 10 μl; the test solution and the control solution were injected into the liquid chromatograph respectively, and the chromatogram was recorded. The sample detection graph is shown in Figure 2 As shown, the control solution spectrum is as Figure 3 As shown;

[0054] Calculation of impurity content:

[0055]

[0056] Where: Ru is the peak area of ​​impurities in the test solution;

[0057] Rs is the peak area of ​​the main peak in the control solution;

[0058] L is the dilution factor of the control solution;

[0059] F is the correction factor for impurities.

[0060] Example 2 Specificity Test

[0061] Preparation of system suitability solution: Take appropriate amounts of (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl) acetate and impurity A, impurity B, impurity C, and impurity J reference substances, add mobile phase to dissolve and dilute to make a mixed solution containing approximately 0.5 mg of (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl) acetate, 50 μg of impurity A, 10 μg of impurity J, and 5 μg each of impurity B and impurity C per 1 ml.

[0062] Impurity A positioning solution: Take an appropriate amount of impurity A reference substance, add mobile phase to dissolve and dilute to make a solution containing approximately 50 μg of impurity A per 1 ml.

[0063] Impurity B localization solution: Take an appropriate amount of impurity B reference substance, add mobile phase to dissolve and dilute to make a solution containing approximately 5 μg of impurity B per 1 ml.

[0064] Impurity C localization solution: Take an appropriate amount of impurity C reference substance, add mobile phase to dissolve and dilute to make a solution containing approximately 5 μg of impurity C per 1 ml.

[0065] Impurity J localization solution: Take an appropriate amount of impurity J reference substance, add mobile phase to dissolve and dilute to make a solution containing approximately 10 μg of impurity C per 1 ml.

[0066] Test sample solution: Take an appropriate amount of the test sample, dissolve it in the mobile phase and dilute it to make a solution containing about 0.5 mg per 1 ml.

[0067] Control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with the mobile phase to make a solution containing 5 μg per 1 ml.

[0068] Test process: blank solvent, system suitability solution, each impurity location solution, test solution and control solution were injected respectively, measured according to the chromatographic conditions of Example 1, and the chromatogram was recorded. The specific test data are shown in the table below.

[0069] Table 1 Specificity test results

[0070]

[0071] Conclusion: The blank solvent has no interference, and the system suitability solution chromatogram is as follows Figure 1 As shown, the order of peaks is impurity J, (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl)acetate, impurity C, impurity A and impurity B. The separation between the peaks of each component meets the requirements. The theoretical plate number of (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl)acetate is greater than 5000, and the specificity is good.

[0072] Example 3 Correction Factor Test

[0073] Take appropriate amounts of (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl)acetate and impurity A, impurity B, impurity C, and impurity J reference substances, add mobile phase to dissolve and dilute to prepare a linear series solution of a certain concentration, the concentration range is shown in Table 2, and measure according to the chromatographic conditions of Example 1, record the peak area, perform linear regression with concentration as the abscissa and peak area as the ordinate, obtain the regression equation and draw a standard curve.

[0074] In order to accurately determine the correction factor of the impurity relative to the main component, the correction factor was determined by the standard curve method to control various error factors in the process of calculating the correction factor; considering the durability of the chromatographic system, the correction factor was determined by different brands of instrument systems. The correction factor f of each known impurity of (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl) acetate relative to (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl) acetate was calculated based on the slope of the linear equation between (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl) acetate and impurity J, impurity C, impurity A, and impurity B, f=k (R)-1-甲基吡咯烷-3-基-2-羟基-2，2-二(噻吩-2-基)乙酸酯 / k 杂质 , where k (R)-1-甲基吡咯烷-3-基-2-羟基-2，2-二(噻吩-2-基)乙酸酯 is the slope of the linear equation for (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl)acetate, k 杂质 is the slope of the impurity linear equation. See the table below for specific test data.

[0075] Table 2 Correction factor test results

[0076]

[0077] Conclusion: The correlation coefficients between (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl)acetate and impurity A, impurity B, impurity C and impurity J within the selected concentration range are all greater than 0.990, showing a good linear relationship. After calculation, the average correction factors of impurity A, impurity B, impurity C and impurity J are 0.74, 5.0, 0.68 and 0.69, respectively. When the above impurities are detected in the test solution, the self-control method with correction factors added should be used to calculate the content of each impurity.

[0078] Example 4 Accuracy Investigation

[0079] By using the standard addition method, a quantitative amount of impurity A, impurity B, impurity C, and impurity J were added to a sample of (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophene-2-yl)acetate with a known content, and the content of each impurity was calculated by the self-control method with a correction factor added. The specific test data are shown in the table below.

[0080] Table 3 Impurity A recovery accuracy test results

[0081]

[0082]

[0083] Table 4 Impurity B recovery accuracy test results

[0084]

[0085] Table 5 Impurity C recovery accuracy test results

[0086]

[0087]

[0088] Table 6 Impurity J recovery accuracy test results

[0089]

[0090] Conclusion: The recoveries of impurities A, B, C and J within the selected concentration range were all between 90% and 110%, and the RSD was less than 5%, indicating that the determination method and the proposed impurity content calculation method had good accuracy.

Claims

1. A method for analyzing related substances of (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl)acetate, the method comprising high performance liquid chromatography, using a chromatographic column with octadecylsilane bonded silica gel as filler, and using mobile phase A and mobile phase B as eluents, wherein mobile phase A is sodium heptane sulfonate solution, and mobile phase B is acetonitrile; The mobile phase A is 0.02 mol / L sodium heptane sulfonate solution; The 0.02 mol / L sodium heptane sulfonate solution contains 0.5% triethylamine, and the pH value is adjusted to 3.5 with phosphoric acid; The volume ratio of the mobile phase A to the mobile phase B is 69:31; The flow rate of the mobile phase is 1 ml / min; The chromatographic column is Agilent ZORBAX SB-C18, with specifications of 250 mm × 4.6 mm, 5 μm; The analysis method is carried out on a high performance liquid chromatograph, using an ultraviolet detector, and its detection wavelength is 237nm; The column temperature of the chromatographic column is 25°C; The related substances are 2. A method for analyzing related substances of (R)-1-methylpyrrolidin-3-yl-2-hydroxy-2,2-di(thiophen-2-yl)acetate, comprising the following steps: Preparation of test solution: Take an appropriate amount of test sample, add mobile phase to dissolve and dilute to make test solution; Preparation of control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with the mobile phase to prepare the control solution; Determination: Agilent ZORBAX SB-C18 was used as the chromatographic column, the mobile phase was 0.02 mol / L sodium heptane sulfonate solution-acetonitrile, the volume ratio was 69:31, and the flow rate was 1.0 ml per minute; the detection wavelength was 237 nm; the column temperature was 25°C; the sample plate temperature was 4°C; the injection volume was 10 μl; the test solution and the control solution were taken and injected into the liquid chromatograph respectively, and the chromatogram was recorded; The related substances are Calculation of impurity content: Where: Ru is the peak area of ​​impurities in the test solution; Rs is the peak area of ​​the main peak in the control solution; L is the dilution factor of the control solution; F is the correction factor for impurities.

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