Method for detecting nitrosamine impurity N-nitroso-atomoxetine in atomoxetine hydrochloride

Abstract

The invention discloses a method for detecting nitrosamine impurity N-nitroso-atomoxetine in atomoxetine hydrochloride, and belongs to the technical field of medicine detection. The method comprises the following steps: dissolving atomoxetine hydrochloride in a diluent to obtain a test solution; carrying out analysis and detection on the test solution by adopting an ultra-high performance liquid chromatography; chromatographic conditions are as follows: octadecylsilane chemically bonded silica is taken as a filling agent of a chromatographic column; a formic acid aqueous solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, and gradient elution is performed. When the method is adopted for detection, the atomoxetine peak can be effectively separated from the N-nitroso-atomoxetine peak, the detection time is 10 minutes, the sample pretreatment operation is simple, the detection cost is low, the quantitation limit is equivalent to 30% of the limit concentration (1ppm), the detection limit is equivalent to 15% of the limit concentration (1ppm), the sensitivity is high, and the content of the N-nitroso-atomoxetine in the sample can be accurately and quickly detected.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and particularly relates to a method for detecting nitrosamine impurity N-nitroso-tomoxetine in atomoxetine hydrochloride. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Atomoxetine Hydrochloride has the following chemical structure:

[0004]

[0005] Atomoxetine Hydrochloride is a selective norepinephrine reuptake inhibitor, suitable for the treatment of attention deficit hyperactivity disorder (ADHD). During the synthesis of atomoxetine hydrochloride, nitrosamine impurities N-nitrosoatomoxetine are generated. Nitrosamine impurities are high-risk carcinogens ("cohort of concern"). Their toxicity poses a great threat to the safety of medication, and their limits are low. Therefore, it is very important to accurately measure their content during the drug development process. According to the latest FDA regulations, the recommended acceptable intake of nitrosamine impurities in atomoxetine hydrochloride is 100 ng / day, and the maximum daily dose of atomoxetine hydrochloride is 100 mg / day. The calculated limit is not more than 1 ppm. At present, this API is included in the European Pharmacopoeia (EP11.6) and the United States Pharmacopoeia (USP2023), but neither includes a detection method for nitrosamine impurities. After literature search, only the detection method for atomoxetine hydrochloride and its isomers (impurity B, impurity C, chemical structure as follows) is found (patent CN115561358A), and no reports on the detection method for nitrosamine impurities in atomoxetine hydrochloride API and preparations have been found.

[0006]

[0007] Therefore, it is of great significance to establish a method for analyzing nitrosamine impurities in atomoxetine hydrochloride with strong specificity, high sensitivity, high accuracy, simplicity and speed, which can effectively control the quality of drugs and provide strong guarantee for the safety of drugs. Summary of the invention

[0008] In order to solve the deficiencies of the prior art, the object of the present invention is to provide a method for detecting nitrosamine impurities N-nitroso-tomoxetine in atomoxetine hydrochloride. The detection method provided by the present invention can quickly detect nitrosamine impurities in atomoxetine hydrochloride raw materials with high sensitivity and good accuracy.

[0009] In order to achieve the above object, the technical solution of the present invention is:

[0010] The first aspect of the present invention provides a method for detecting nitrosamine impurity N-nitroso-tomoxetine in atomoxetine hydrochloride, comprising the following steps:

[0011] Dissolving atomoxetine hydrochloride in a diluent to obtain a test solution;

[0012] The test solution is analyzed and detected by ultra-high performance liquid chromatography;

[0013] Chromatographic conditions included:

[0014] The chromatographic column is filled with octadecylsilane bonded silica gel;

[0015] The mobile phase A was a formic acid aqueous solution and the mobile phase B was acetonitrile, and the elution was performed in a gradient manner.

[0016] In some embodiments of the present invention, the structure of N-nitroso-atomoxetine is as follows:

[0017]

[0018] It should be noted that the molecular formula of N-nitroso-atomoxetine is C 17 H 20 N2O2, the reference substance was purchased from QUALITY CONTROL CHEMICALS INC, USA, QCC Cat No.: QA150923.

[0019] In some embodiments of the present invention, the diluent is a methanol aqueous solution, preferably a 70% methanol aqueous solution.

[0020] It should be noted that 70% in the 70% methanol aqueous solution is a volume percentage, and the volume ratio of methanol to water in the 70% methanol aqueous solution is 7:3.

[0021] In some embodiments of the present invention, the concentration of the test solution is 0.09-0.11 g / mL, preferably 0.1 g / mL.

[0022] In some embodiments of the present invention, the mobile phase A is a 0.09-0.11% formic acid aqueous solution, preferably a 0.1% formic acid aqueous solution.

[0023] It should be noted that the 0.09-0.11% is a volume percentage, and a 0.09-0.11% formic acid aqueous solution is a solution containing 0.9-1.1 mL of formic acid per 1000 mL of solution.

[0024] In some embodiments of the present invention, the gradient elution is as follows, in volume percentage:

[0025] 0-1min, mobile phase A 80%, mobile phase B 20%;

[0026] From 1 to 8 min, mobile phase A was reduced from 80% to 10%, and mobile phase B was increased from 20% to 90%;

[0027] 8.1~10min, mobile phase A 80%, mobile phase B 20%.

[0028] In some embodiments of the present invention, the chromatographic conditions further include:

[0029] The flow rate is 0.35-0.45 mL / min, preferably 0.4 mL / min;

[0030] The column temperature is 35-45°C, preferably 40°C;

[0031] The chromatographic column was Hypersil GOLD, 100 mm × 2.1 mm, 1.9 μm;

[0032] The detection wavelength is 240-250nm, preferably 245nm;

[0033] The injector temperature was 4 °C.

[0034] In some embodiments of the present invention, the chromatographic conditions are:

[0035] The chromatographic column was filled with octadecylsilane bonded silica gel; the chromatographic column was Hypersil GOLD, 100 mm × 2.1 mm, 1.9 μm;

[0036] 0.1% formic acid aqueous solution was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed;

[0037] The flow rate was 0.4 mL / min, the column temperature was 40 °C, the injection volume was 10 μL, the detection wavelength was 245 nm, and the injector temperature was 4 °C.

[0038] The second aspect of the present invention provides a method for quantitatively detecting nitrosamine impurity N-nitroso-tomoxetine in atomoxetine hydrochloride, comprising:

[0039] Dissolving atomoxetine hydrochloride in a diluent to obtain a test solution; analyzing the test solution using the above-mentioned detection method to obtain a chromatogram of the test solution;

[0040] Dissolving N-nitroso-atomoxetine in a diluent to obtain a reference solution, and analyzing the reference solution using the above-mentioned detection method to obtain a chromatogram of the reference solution;

[0041] The content of N-nitroso-tomoxetine in atomoxetine hydrochloride was calculated by the peak area in the chromatogram.

[0042] It should be noted that the content of N-nitroso-tomoxetine in atomoxetine hydrochloride can be calculated by the external standard method, and the calculation formula is as follows:

[0043]

[0044] Where:

[0045] A U --N-nitroso-atomoxetine peak area in the test solution;

[0046] A S --N-nitroso-atomoxetine peak area in the reference solution;

[0047] C S --N-nitroso-atomoxetine concentration in the reference solution, μg / mL;

[0048] C U --Concentration of test solution, g / mL.

[0049] In some embodiments of the present invention, the diluent is a methanol aqueous solution, preferably a 70% methanol aqueous solution.

[0050] As above, 70% in the 70% methanol aqueous solution is a volume percentage, and the volume ratio of methanol to water in the 70% methanol aqueous solution is 7:3.

[0051] In some embodiments of the present invention, the concentration of the test solution is 0.09-0.11 g / mL, preferably 0.1 g / mL.

[0052] The third aspect of the present invention provides a method for evaluating the quality of atomoxetine hydrochloride, using the above-mentioned detection method or the above-mentioned quantitative detection method.

[0053] The fourth aspect of the present invention provides the use of detecting N-nitroso-tomoxetine in the quality control of atomoxetine hydrochloride.

[0054] The beneficial effects of the present invention are:

[0055] The present invention provides a method for detecting nitrosamine impurity N-nitroso-tomoxetine in atomoxetine hydrochloride. Since there is no public report on the detection method of nitrosamine impurity N-nitroso-tomoxetine in atomoxetine hydrochloride at home and abroad, the present invention proposes for the first time to analyze and detect the nitrosamine impurity N-nitroso-tomoxetine in atomoxetine hydrochloride, which provides a strong guarantee for effectively controlling the quality of drugs and ensuring the safety of drugs.

[0056] The method of the present invention is used to detect the nitrosamine impurity N-nitroso-tomoxetine in the atomoxetine hydrochloride raw material, and the atomoxetine peak can be effectively separated from the N-nitroso-tomoxetine peak, and the method has good specificity; the instrument running time is 10 minutes, the sample pretreatment operation is simple, and the content of the nitrosamine impurity N-nitroso-tomoxetine in the sample can be quickly detected, effectively reducing the analysis cost, and accurately detecting the content of the nitrosamine impurity N-nitroso-tomoxetine in the atomoxetine hydrochloride raw material. And there is no need to use expensive instruments such as mass spectrometry, and detection can be achieved using only a UV detector, which greatly reduces the analysis cost.

[0057] The present invention also verifies the accuracy of nitrosamine impurities in the range of 50% to 150% of the limit concentration (1ppm), and the recovery rate of N-nitroso-tomoxetine is between 75% and 120%, with good accuracy; it also verifies the linearity of N-nitroso-tomoxetine in the range of 30% to 200% of the limit concentration, and N-nitroso-tomoxetine has r>0.999 within the above range, with good linearity. The quantitative limit of the method of the present invention is equivalent to 30% of the limit concentration, the detection limit is equivalent to 15% of the limit concentration, and the method has high sensitivity. The test solution and the reference solution used in the detection process of the present invention still have good stability after being placed at 4°C for 8 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0059] Figure 1 This is the blank solution spectrum of Example 1 of the present invention;

[0060] Figure 2 This is the N-nitroso-atomoxetine impurity reference solution spectrum of Example 1 of the present invention;

[0061] Figure 3 The sample solution spectrum of Example 1 of the present invention;

[0062] Figure 4 The system applicability solution spectrum of Example 1 of the present invention;

[0063] Figure 5 It is a specificity overlay diagram of Example 1 of the present invention. In the figure, from top to bottom are the general applicability solution spectrum (red line), the test solution spectrum (blue line), the N-nitroso-atomoxetine impurity reference solution spectrum (black line) and the blank solution spectrum (green line). DETAILED DESCRIPTION

[0064] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0065] Example 1: Specificity

[0066] 1-1 Solution Preparation

[0067] Dilution solution (blank solution): Measure 700 mL of methanol and 300 mL of water and mix well.

[0068] N-nitroso-atomoxetine impurity reference solution: Accurately measure an appropriate amount of N-nitroso-atomoxetine impurity reference solution and quantitatively dilute it with diluent to make a 0.1 μg / mL solution.

[0069] Test solution: Take about 1 g of atomoxetine hydrochloride raw material, place it in a 10 mL volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0070] System suitability solution: Take about 1 g of atomoxetine hydrochloride raw material, place it in a 10 mL volumetric flask, add an appropriate amount of N-nitroso-tomoxetine impurity, add diluent to dissolve by ultrasonic and dilute to the scale, shake well, and prepare a mixed solution containing about 100 mg of atomoxetine hydrochloride and 0.1 μg of N-nitroso-tomoxetine impurity per 1 mL.

[0071] 1-2 Chromatographic conditions

[0072] Column: Hypersil GOLD, 100 mm × 2.1 mm, 1.9 μm;

[0073] Mobile Phase:

[0074] Mobile phase A was 0.1% formic acid in water;

[0075] Mobile phase B was acetonitrile;

[0076] Elution mode: gradient elution;

[0077] Time (min) Mobile phase A (%) Mobile phase B (%) 0～1 80 20 1～8 80～10 20～90 8.1～10 80 20

[0078] The detection wavelength is 245nm;

[0079] Flow rate: 0.4 mL / min;

[0080] The column temperature was 40°C;

[0081] The injection volume was 10 μL;

[0082] The injector temperature was 4 °C.

[0083] 1-3 Experimental conclusion:

[0084] Accurately measure the above blank solution, N-nitroso-atomoxetine impurity reference solution, test solution and system suitability solution, inject them into the ultra-high performance liquid chromatograph respectively, and record the chromatogram. The results are shown in Table 1, and the blank solution spectrum is shown in Figure 1 , N-nitroso-atomoxetine impurity reference solution see Figure 2 , test solution see Figure 3 , System Suitability Solutions See Figure 4 See the specific overlay Figure 5 .

[0085] Table 1 System suitability and impurity localization results

[0086]

[0087] Conclusion: The blank solvent and the test solution do not interfere with the detection of N-nitroso-atomoxetine impurities, and the specificity of the present invention is good.

[0088] Example 2: Linearity and range experiments

[0089] 2-1 Solution preparation

[0090] Accurately weigh an appropriate amount of N-nitroso-atomoxetine impurity reference substance and prepare a series of concentration linearity test solutions equivalent to about 0.3ppm (quantification limit), 0.5ppm, 1ppm, 1.5ppm, and 2ppm of the test sample concentration.

[0091] 2-2 Chromatographic conditions are the same as those in Example 1.

[0092] 2-3 Conclusion:

[0093] The above series of concentration linear investigation solutions were accurately measured and injected into the ultra-high performance liquid chromatograph to record the chromatogram. The results are shown in Table 2. A linear regression equation was made with concentration as the abscissa and peak area as the ordinate.

[0094] Table 2 Results of linearity test of N-nitroso-atomoxetine impurities

[0095] Element Linear equations r Nitrosamine impurities y=20880.9485x-107.0041 0.999

[0096] Conclusion: In the range of 0.3-2 ppm, which is equivalent to the concentration of the test sample, the linear relationship of N-nitroso-atomoxetine impurity is good and meets the methodological requirements.

[0097] Example 3: Limit of Quantitation and Limit of Detection

[0098] 3-1 Solution preparation

[0099] The concentration series linear investigation solution in Example 2 was taken and diluted step by step, and the concentration when the signal-to-noise ratio S / N≈3 was taken as the detection limit, and the concentration when S / N≈10 was taken as the quantification limit.

[0100] 3-2 Chromatographic conditions are the same as those in Example 1.

[0101] 3-3 Conclusion:

[0102] Accurately measure the above linear solution, inject it into the ultra-high performance liquid chromatograph, record the chromatogram, and the results are shown in Table 3.

[0103] Table 3 Quantitation limit and detection limit test results

[0104]

[0105] Conclusion: The quantitative limit of N-nitroso-tomoxetine impurity (0.3ppm) is lower than the impurity reporting limit of this product (1ppm). Both the detection limit and the quantitative limit of the present invention meet the requirements for quantitative detection of N-nitroso-tomoxetine impurities in the test sample, and the method has high sensitivity.

[0106] Example 4: Accuracy Experiment

[0107] The accuracy was investigated through recovery test, and the study covered the limit concentration range of 50% to 150%.

[0108] 4-1 Solution preparation

[0109] N-nitroso-atomoxetine impurity stock solution (100 μg / mL): Take an appropriate amount of N-nitroso-atomoxetine impurity reference substance, accurately weigh it, add 70% methanol to dissolve it and dilute it to make a 100 μg / mL solution.

[0110] N-nitroso-atomoxetine impurity stock solution (1 μg / mL): Accurately measure 1 mL of N-nitroso-atomoxetine impurity stock solution (100 μg / mL), place in a 100 mL volumetric flask, dilute to the scale with 70% methanol, and shake well.

[0111] Reference solution: Accurately measure 1 mL of N-nitroso-atomoxetine impurity stock solution (1 μg / mL), place in a 10 mL volumetric flask, dilute to the mark with 70% methanol, and shake well.

[0112] Accuracy solution: Weigh approximately 1 g of each atomoxetine hydrochloride bulk drug into 10 mL volumetric flasks, accurately measure 0.5 mL, 1.0 mL and 1.5 mL of N-nitroso-atomoxetine impurity stock solution (1 μg / mL), respectively, dissolve in diluent and dilute to the scale, shake well to obtain 50%, 100%, and 150% accurate solutions, and prepare 3 replicates in parallel for each spiked concentration level.

[0113] 4-2 Chromatographic conditions are the same as those in Example 1.

[0114] 4-3 Conclusion:

[0115] Accurately measure the above solution, inject it into ultra-high performance liquid chromatography, record the chromatogram, and calculate the accuracy by peak area according to the external standard method. The results are shown in Table 4.

[0116] Table 4 Accuracy test results

[0117]

[0118]

[0119] Conclusion: Under 50%, 100%, and 150% accuracy solutions, the recovery rate of N-nitroso-atomoxetine impurities is between 75% and 120%, and the RSD is 3.1%. The test results show that the accuracy of the present invention is good.

[0120] Example 5: Precision Experiment

[0121] 5-1 Solution preparation

[0122] Weigh about 1 g of atomoxetine hydrochloride raw material, place it in a 10 mL volumetric flask, add 1.0 mL of N-nitroso-atomoxetine impurity stock solution (1 μg / mL), add diluent to dissolve and dilute to the scale, shake well, and prepare 6 copies in parallel.

[0123] Control solution: same as Example 4.

[0124] 5-2 Chromatographic conditions are the same as those in Example 1.

[0125] 5-3 Conclusion:

[0126] Accurately measure the above solution, inject it into the ultra-high performance liquid chromatograph, record the chromatogram, calculate the impurity content, and the precision results are shown in Table 5.

[0127] Table 5 Precision test results

[0128] sample N-Nitroso-atomoxetine (ppm) 1 0.80 2 0.78 3 0.78 4 0.82 5 0.76 6 0.85 average(%) 0.80 RSD(%) 4.1

[0129] Conclusion: The test results of 6 replicate sample solutions prepared in parallel showed that the RSD value of N-nitroso-atomoxetine impurity was less than 10%, which proved that the present invention had good repeatability.

[0130] Example 6: Solution stability experiment

[0131] 6-1 Solution preparation

[0132] Take the test solution and the reference solution in Example 1, place them in a sample injector at 4°C, and inject and test them at different time points.

[0133] 6-2 Chromatographic conditions are the same as those in Example 1.

[0134] 6-3 Conclusion:

[0135] The above solution was accurately measured and injected into the ultra-high performance liquid chromatograph to record the chromatogram. The solution stability results are shown in Table 6.

[0136] Table 6 Solution stability test results

[0137]

[0138] Conclusion: After the test solution and the reference solution were placed at 4°C for 8 hours, the peak area of ​​N-nitroso-atomoxetine impurity did not change significantly, indicating that the test solution and the reference solution were stable within 8 hours.

[0139] Example 7: Durability Test

[0140] Durability refers to the degree to which the test results are not affected by slight changes in the chromatographic conditions. The preparation of the system suitability solution, the test solution and the reference solution is the same as in Example 1. On the basis of the test conditions of Example 1, the durability of the test method is changed under the conditions of temperature, flow rate, formic acid content and initial ratio of mobile phase B. The test results are shown in Table 7.

[0141] Table 7 Durability test results

[0142]

[0143]

[0144] Note: The test sample content in Table 7 refers to the content of N-nitroso-atomoxetine impurities in the test sample solution.

[0145] Conclusion: When changing (column temperature ±5°C, flow rate ±0.05mL / min, formic acid ±10%, initial proportion of mobile phase B ±5%), the blank solution and the test solution did not interfere with the determination of N-nitroso-atomoxetine impurities, the system suitability parameters all met the requirements, and no impurities were detected in the test samples. The durability of the method of the present invention is good.

[0146] Based on the above experimental results, the detection method of the present invention is used to detect the nitrosamine impurity N-nitroso-tomoxetine in the atomoxetine hydrochloride raw material. The blank solvent and the main component do not interfere with the detection of nitrosamine impurities. The method has high sensitivity and can provide a basis for the quality control of the atomoxetine hydrochloride raw material, thereby ensuring its drug safety.

[0147] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting nitrosamine impurity N-nitroso-tomoxetine in atomoxetine hydrochloride, characterized in that: The steps include: Dissolving atomoxetine hydrochloride in a diluent to obtain a test solution; The test solution is analyzed and detected by ultra-high performance liquid chromatography; Chromatographic conditions included: The chromatographic column is filled with octadecylsilane bonded silica gel; The mobile phase A was a formic acid aqueous solution and the mobile phase B was acetonitrile, and the elution was performed in a gradient manner.

2. The detection method according to claim 1, characterized in that The structure of the N-nitroso-atomoxetine is as follows:

3. The detection method according to claim 1, characterized in that The diluent is a methanol aqueous solution, preferably a 70% methanol aqueous solution; Preferably, the concentration of the test solution is 0.09-0.11 g / mL, preferably 0.1 g / mL.

4. The detection method according to claim 1, characterized in that The mobile phase A is a 0.09-0.11% formic acid aqueous solution, preferably a 0.1% formic acid aqueous solution.

5. The detection method according to claim 1, characterized in that In volume percentage, the gradient elution is as follows: 0-1min, mobile phase A 80%, mobile phase B 20%; From 1 to 8 min, mobile phase A was reduced from 80% to 10%, and mobile phase B was increased from 20% to 90%; 8.1~10min, mobile phase A 80%, mobile phase B 20%.

6. The detection method according to claim 1, characterized in that The chromatographic conditions also include: The flow rate is 0.35-0.45 mL / min, preferably 0.4 mL / min; The column temperature is 35-45°C, preferably 40°C; The chromatographic column was Hypersil GOLD, 100 mm × 2.1 mm, 1.9 μm; The detection wavelength is 240-250nm, preferably 245nm; The injector temperature was 4 °C.

7. The detection method according to claim 1, characterized in that The chromatographic conditions are: The chromatographic column was filled with octadecylsilane bonded silica gel; the chromatographic column was Hypersil GOLD, 100 mm × 2.1 mm, 1.9 μm; 0.1% formic acid aqueous solution was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed; The flow rate was 0.4 mL / min, the column temperature was 40 °C, the injection volume was 10 μL, the detection wavelength was 245 nm, and the injector temperature was 4 °C.

8. A method for quantitatively detecting nitrosamine impurity N-nitroso-tomoxetine in atomoxetine hydrochloride, characterized in that: include: Dissolving atomoxetine hydrochloride in a diluent to obtain a test solution; Analyze the test solution using the detection method described in any one of claims 1 to 7 to obtain a chromatogram of the test solution; Dissolving N-nitroso-atomoxetine in a diluent to obtain a reference solution, and analyzing the reference solution using the detection method described in any one of claims 1 to 7 to obtain a chromatogram of the reference solution; The content of N-nitroso-tomoxetine in atomoxetine hydrochloride was calculated by the peak area in the chromatogram; Preferably, the diluent is a methanol aqueous solution, preferably a 70% methanol aqueous solution; Preferably, the concentration of the test solution is 0.09-0.11 g / mL, preferably 0.1 g / mL.

9. A quality assessment method for atomoxetine hydrochloride, characterized in that: The detection method according to any one of claims 1 to 7 or the quantitative detection method according to claim 8 is used.

10. Application of detection of N-nitroso-atomoxetine in quality control of atomoxetine hydrochloride.

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