Method for detecting and analyzing mutagenic impurities in key starting material 3-chlorobenzene propanol of atomoxetine hydrochloride

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a high-sensitivity liquid chromatography method for detecting mutagenic impurities in atomoxetine hydrochloride key starting material 3-chlorobenzene propanol, the method adopts an HPLC (High Performance Liquid Chromatography) analysis method, a chromatographic column adopts octadecylsilane chemically bonded silica as a filler, and the composition of a mobile phase A is a buffer solution; the mobile phase B is methanol or acetonitrile; carrying out gradient elution on the mobile phase; the flow velocity is 0.4 to 0.8 ml / min; the detection wavelength is 230 to 250 nm; by adopting an HPLC analysis method, the content of mutagenic impurities in the key starting material 3-chlorobenzene propanol of atomoxetine hydrochloride can be effectively separated and measured, and the method is high in sensitivity, strong in specificity and good in accuracy and repeatability, and has extremely important significance on quality control of the key starting material 3-chlorobenzene propanol of atomoxetine hydrochloride.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical detection, and in particular relates to a method for detecting and analyzing mutagenic impurities in 3-chlorophenylpropanol, a key starting material of atomoxetine hydrochloride. Background Art

[0002] 3-Chlorophenylpropanol is a key starting material for the API atomoxetine hydrochloride. Its quality control is of great significance to the quality of atomoxetine hydrochloride. In particular, in recent years, China has paid more and more attention to the study of impurity spectra in APIs and preparations, especially the evaluation of potential mutagenic impurities. 3-Chlorophenylpropanol may introduce 3-chlorophenylpropanone, a potential mutagenic impurity, during the synthesis process. Since there is no detailed toxicological data for this impurity, in order to strictly control the quality of the starting material-3-chlorophenylpropanol, according to ICH M7 and the "Guidelines for the Control of Genotoxic Impurities" of the 2020 edition of the Pharmacopoeia of the People's Republic of China, combined with the maximum daily dose (100 mg / day) of atomoxetine hydrochloride oral solution and atomoxetine hydrochloride capsules, the toxicological concern threshold (TTC) is used to calculate that the limit of 3-chlorophenylpropanone in 3-chlorophenylpropanol should not exceed 15 ppm.

[0003] At present, there are many studies on potential mutagenic impurities in the raw material atomoxetine hydrochloride, but there are few methods for detecting potential mutagenic impurities in the starting material 3-chlorophenylpropanol. In order to fully implement the concept of quality comes from control, the relevant factors that may affect the quality of the raw materials of atomoxetine hydrochloride should be controlled from the source. Due to the low control limit of 3-chlorophenylpropanol, higher requirements are placed on the detection sensitivity of the method. For this reason, we designed a high-sensitivity liquid chromatography method for detecting mutagenic impurities in 3-chlorophenylpropanol, a key starting material of atomoxetine hydrochloride. Summary of the invention

[0004] The purpose of the present invention is to provide a high-sensitivity liquid chromatography method for detecting mutagenic impurities in 3-chlorophenylpropanol, a key starting material of atomoxetine hydrochloride, which has the advantages of high sensitivity, economy and convenience for promotion, and avoids the disadvantages of high performance liquid chromatography-mass spectrometry, that is, expensive instruments and unfavorable for promotion.

[0005] In order to achieve the above technical objectives, the present invention is implemented through the following technical solutions.

[0006] A method for detecting and analyzing mutagenic impurities in 3-chlorophenylpropanol, a key starting material of atomoxetine hydrochloride, adopts an HPLC analysis method, a chromatographic column using octadecylsilane bonded silica gel as a filler, a mobile phase A comprising a buffer solution, a mobile phase B comprising methanol or acetonitrile, a mobile phase gradient elution, a flow rate of 0.4-0.8 ml / min, and a detection wavelength of 230-250 nm.

[0007] The present invention relates to a method for detecting and analyzing mutagenic impurities in 3-chlorophenylpropanol, a key starting material of atomoxetine hydrochloride, wherein the column temperature in the detection method is 20° C. to 30° C.

[0008] In the method for detecting and analyzing mutagenic impurities in 3-chlorophenylpropanol, a key starting material of atomoxetine hydrochloride, the mobile phase A is composed of a buffer solution; the mobile phase B is acetonitrile; wherein the ratio of the gradient elution time to the mobile phase volume is as follows: .

[0009] The method for detecting and analyzing mutagenic impurities in 3-chlorophenylpropanol, a key starting material of atomoxetine hydrochloride, wherein the ratio of gradient elution time to mobile phase volume is as follows: .

[0010] The method for detecting and analyzing mutagenic impurities in 3-chlorophenylpropanol, a key starting material of atomoxetine hydrochloride, comprises a mobile phase A buffer solution of 0.02 mol / L potassium dihydrogen phosphate solution, and phosphoric acid is used to adjust the pH value to 3.0.

[0011] The detection and analysis method of mutagenic impurities in 3-chlorophenylpropanol, a key starting material of atomoxetine hydrochloride, comprises the following steps: (1) Prepare the reference stock solution: Take an appropriate amount of 3-chloropropiophenone reference substance, dissolve it in solvent and quantitatively dilute it to make a solution containing about 0.6 μg of 3-chloropropiophenone per 1 ml; (2) Prepare the reference solution: accurately measure an appropriate amount of the reference stock solution and quantitatively dilute it with a solvent to produce a solution containing approximately 0.03 μg of 3-chloropropiophenone per 1 ml; (3) Prepare the test solution: Take an appropriate amount of the product, weigh it accurately, dissolve it in solvent and quantitatively dilute it to make a solution containing about 2 mg per 1 ml; (4) Accurately measure the reference solution and the test solution and inject them into the liquid chromatograph respectively. The injection volume is 20 μl. Record the chromatogram and calculate the peak area according to the external standard method.

[0012] The solvent is acetonitrile-water (50:50).

[0013] The mutagenic impurity in 3-chlorophenylpropanol, the key starting material of atomoxetine hydrochloride, is 3-chlorophenylpropiophenone, and its specific structure is as follows:

[0014] 3-Chloropropiophenone The present invention can determine 3-chloropropiophenone in a high-concentration test solution, and is well separated from each impurity peak in the test solution. The method can effectively enrich 3-chloropropiophenone in 3-chlorophenylpropanol, so that its quantitative limit meets the quantitative analysis requirements of drugs, and has high precision and sensitivity, strong specificity, good reproducibility and high accuracy, and provides technical support for production enterprises and testing institutions to control the quality of 3-chlorophenylpropanol, a key starting material of the raw material drug atomoxetine hydrochloride. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the blank solvent chromatogram in Example 1 of the present invention.

[0016] Figure 2 It is a chromatogram of the reference substance solution in Example 1 of the present invention.

[0017] Figure 3 It is a chromatogram of the test solution in Example 1 of the present invention.

[0018] Figure 4 It is a chromatogram of the spiked test solution in Example 1 of the present invention.

[0019] Figure 5 It is the linear relationship diagram of 3-chloropropiophenone in Example 3 of the present invention.

[0020] Figure 6 This is the chromatogram of the pH 2.9 spiked test product in Example 6 of the present invention.

[0021] Figure 7 This is the chromatogram of the pH 3.1 spiked test product in Example 6 of the present invention.

[0022] Figure 8 This is the chromatogram of the spiked test sample at a column temperature of 23° C. in Example 6 of the present invention.

[0023] Fig. 9 This is the chromatogram of the spiked test sample at a column temperature of 27° C. in Example 6 of the present invention. DETAILED DESCRIPTION

[0024] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work are all within the protection scope of the present invention.

[0025] The experimental methods in the following examples are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0026] The research process of this application is Unless otherwise specified, the following measurement conditions are used in the examples of the present invention: chromatographic column: Waters XTerra RP18, 150 mm × 3 mm, 3.5 μm; detection wavelength: 242 nm; flow rate: 0.6 ml / min; .

[0027] Example 1 Specificity Solvent: methanol-water (18:82) Reference substance stock solution: Accurately weigh 6.482 mg of 3-chloropropiophenone reference substance, place in a 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well; Accurately measure 1 ml of reference substance stock solution, place in a 100 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0028] Reference substance solution: Accurately measure 1 ml of reference substance stock solution, place it in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0029] Test solution: Weigh 20.68 mg of the test sample into a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0030] Test sample solution (with spike): Accurately weigh 41.626 mg of the test sample, place it in a 20 ml volumetric flask, add solvent to dissolve, add 1 ml of reference sample stock solution, dilute to the scale with solvent, and shake well.

[0031] Accurately measure the solvent, reference solution, test solution and test solution (with spike), inject them into the liquid chromatograph respectively, and record the chromatogram. The chromatogram of the solvent is as follows: Figure 1 As shown, the chromatogram of the reference solution is as follows Figure 2 As shown, the chromatogram of the test solution is as follows Figure 3 The chromatogram of the test solution (spiked) is shown in Figure 4 The specific spectrum data can be seen in Table 1.

[0032] Table 1

[0033] Note: “—” indicates not applicable From the data in Table 1, it can be seen that the solvent does not interfere with the detection and has good specificity.

[0034] Example 2 Detection Limit Reference substance stock solution: Accurately weigh 6.866 mg of 3-chloropropiophenone reference substance, place in a 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0035] Reference solution: Accurately measure 1 ml of reference stock solution, place it in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well; accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0036] Quantitation limit solution: Accurately measure 5 ml of the reference solution, place it in a 25 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0037] Accurately measure 3 ml of the reference solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, shake well, and use this as the quantitative limit solution.

[0038] Accurately measure 3 ml of the quantitative limit solution and place it in a 10 ml volumetric flask. Dilute to the mark with solvent and shake well to use as the detection limit solution.

[0039] Accurately measure the quantitative limit solution and the detection limit solution, inject them into the liquid chromatograph respectively, and record the chromatograms.

[0040] Table 2

[0041] From the data in Table 2, it can be seen that the method can ensure the detection of 3-chloropropiophenone.

[0042] Example 3 Linear range Take 1 ml of the reference substance stock solution in Example 2, place it in a 200 ml volumetric flask, dilute to scale with solvent, shake well, and use it as the linear stock solution. Take the linear stock solution and dilute it according to Table 3 to obtain each linear solution.

[0043] Table 3

[0044] Take each linear solution and inject it into the liquid chromatograph respectively, and record the chromatogram. Figure 5 The standard curve was drawn with concentration as the horizontal axis and peak area as the vertical axis. The results are shown in Table 4.

[0045] Table 4

[0046] From the data in Table 4, it can be seen that 3-chloropropiophenone has good linearity.

[0047] Example 4 Accuracy (1) Reference substance stock solution Reference substance stock solution (1): Accurately weigh 6.835 mg of 3-chloropropiophenone reference substance, place in a 100 ml volumetric flask, add solvent to dissolve and dilute to scale, shake well; Accurately weigh 1 ml of reference substance stock solution, place in a 100 ml volumetric flask, dilute to scale with solvent, shake well.

[0048] Reference substance stock solution (2): Accurately weigh 6.671 mg of 3-chloropropiophenone reference substance, place in a 100 ml volumetric flask, add solvent to dissolve and dilute to scale, shake well; Accurately weigh 1 ml of reference substance stock solution, place in a 100 ml volumetric flask, dilute to scale with solvent, shake well.

[0049] (2) Reference solution Reference substance solution (1): Accurately measure 1 ml of reference substance stock solution (1) and place it in a 20 ml volumetric flask. Dilute to the mark with solvent and shake well.

[0050] Reference substance solution (2): Accurately measure 1 ml of reference substance stock solution (2) and place it in a 20 ml volumetric flask. Dilute to the mark with solvent and shake well.

[0051] (3) Quantitative limit stock solution Accurately measure 10 ml of the reference stock solution (1) and place it in a 50 ml volumetric flask. Dilute to the mark with solvent and shake well.

[0052] (4) Preparation of test solution Take about 20 mg of this product, grind it into fine powder, weigh it accurately, put it into a 10 ml volumetric bottle, label it and set aside.

[0053] Test solution: Take No. 1 volumetric flask, add solvent to dissolve and dilute to the scale, shake well.

[0054] (4) Quantitation limit spiked test sample solution: Take volumetric flasks No. 2 to 4, add an appropriate amount of solvent, shake to dissolve, add 1 ml of quantitation limit stock solution to each, dilute to the mark with solvent, and shake well.

[0055] 50% spiked test sample solution: Take a No. 5 to 7 volumetric flask, add an appropriate amount of solvent, shake to dissolve, add 0.5 ml of reference sample stock solution (1), dilute to the mark with solvent, and shake well.

[0056] 100% spiked test sample solution: Take a No. 8-10 volumetric flask, add an appropriate amount of solvent, shake to dissolve, add 1 ml of reference sample stock solution (1), dilute to the mark with solvent, and shake well.

[0057] 150% spiked test sample solution: Take a No. 11-13 volumetric flask, add an appropriate amount of solvent, shake to dissolve, and add 1.5 ml of the reference sample stock solution (1), dilute to the mark with solvent, and shake well.

[0058] Take the reference solution and the test solution, inject them into the liquid chromatograph respectively, and record the chromatogram; calculate the recovery rate and RSD value by peak area according to the external standard method.

[0059] Table 5 Accuracy test results

[0060] From the data in Table 5, it can be seen that the recovery rates of 3-chloropropiophenone are between 80% and 115%, with good accuracy.

[0061] Example 5 Precision Take the 100% spiked test solution in Example 4 and prepare 3 more portions accordingly, to obtain 6 test solutions in total.

[0062] Take the test solution and the reference solution under Example 4, inject them into the liquid chromatograph respectively, and record the chromatogram. Calculate the content and RSD value by peak area according to the external standard method, and the results are shown in the table below.

[0063] Table 6 Repeatability test results

[0064] From the data in Table 6, it can be seen that the RSD value of the 3-chloropropiophenone detection result is less than 4%, which meets the requirements and the method has good repeatability.

[0065] Example 6 Durability Inspection conditions: Column temperature: 23℃, 25℃, 27℃ pH: 2.9, 3.0, 3.1 Take the reference solution and 100% spiked test solution under Example 4, inject them into the liquid chromatograph according to the above conditions, and record the chromatogram.

[0066] Table 7 Durability test results

[0067] From the data in Table 7, it can be seen that when the chromatographic conditions are slightly changed, there is no significant difference in the chromatographic behavior of 3-chlorophenylacetone, and there is no significant difference in the content of the detection results, and the method has good durability.

[0068] Obviously, the above embodiments are merely examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. The purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and it cannot be used to limit the protection scope of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for detecting and analyzing mutagenic impurities in 3-chlorophenylpropanol, a key starting material of atomoxetine hydrochloride, characterized in that: The method adopts HPLC analysis method, and the chromatographic column uses octadecylsilane bonded silica gel as a filler. The composition of mobile phase A is a buffer solution; mobile phase B is methanol or acetonitrile; mobile phase gradient elution; flow rate is 0.4~0.8ml / min; detection wavelength is 230~250nm.

2. A method for detecting and analyzing mutagenic impurities in 3-chlorophenylpropanol, a key starting material of atomoxetine hydrochloride according to claim 1, wherein the mutagenic impurity is 3-chlorophenylpropiophenone.

3. The method for detecting and analyzing mutagenic impurities in 3-chlorophenylpropanol, a key starting material of atomoxetine hydrochloride according to claim 1, wherein the column temperature in the detection method is 20°C to 30°C.

4. According to the method for detecting and analyzing mutagenic impurities in 3-chlorophenylpropanol, a key starting material of atomoxetine hydrochloride, according to claim 1, the composition of mobile phase A is a buffer solution; mobile phase B is acetonitrile; wherein the ratio of gradient elution time to mobile phase volume is as follows: 。 5. The method for detecting and analyzing mutagenic impurities in 3-chlorophenylpropanol, a key starting material of atomoxetine hydrochloride according to claim 1, wherein the ratio of gradient elution time to mobile phase volume is as follows: 。 6. A method for detecting and analyzing mutagenic impurities in 3-chlorophenylpropanol, a key starting material of atomoxetine hydrochloride according to claim 1, wherein the mobile phase A buffer is a 0.02 mol / L potassium dihydrogen phosphate solution, and the pH value is adjusted to 3.0 with phosphoric acid.

7. The method for detecting and analyzing mutagenic impurities in 3-chlorophenylpropanol, a key starting material of atomoxetine hydrochloride according to claim 1, wherein the detection method is: (1) Prepare the reference stock solution: Take an appropriate amount of 3-chloropropiophenone reference substance, dissolve it in solvent and quantitatively dilute it to make a solution containing about 0.6 μg of 3-chloropropiophenone per 1 ml; (2) Prepare the reference solution: accurately measure an appropriate amount of the reference stock solution and quantitatively dilute it with a solvent to produce a solution containing approximately 0.03 μg of 3-chloropropiophenone per 1 ml; (3) Prepare the test solution: Take an appropriate amount of the product, weigh it accurately, dissolve it in solvent and quantitatively dilute it to make a solution containing about 2 mg per 1 ml; (4) Accurately measure the reference solution and the test solution and inject them into the liquid chromatograph respectively. The injection volume is 20 μl. Record the chromatogram and calculate the peak area according to the external standard method.

8. The solvent according to claim 7 is acetonitrile-water (50:50).

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