A method for detecting impurity B and impurity C in atomoxetine hydrochloride

Abstract

The present invention provides a method for detecting impurities B and C in atomoxetine hydrochloride, and high performance liquid chromatography is used for detection, and the determination conditions include: chromatographic column cellulose-tris (3,5-dichlorophenyl carbamate) bonded silica gel as filler; n-hexane-anhydrous ethanol-diethylamine as mobile phase, and the detection wavelength is 278-280nm. The method of the present invention effectively avoids the large baseline fluctuation caused by the interference of impurity H during inspection, improves the sensitivity, and better meets the detection requirements of impurities B and impurity C in atomoxetine hydrochloride.

Description

Technical Field

[0001] The present invention relates to the field of drug detection, and in particular to a method for detecting impurities B and C in atomoxetine hydrochloride. Background Art

[0002] Atomoxetine Hydrochloride, English name: Atomoxetine Hydrochloride, chemical name: (-)-N-methyl-3-phenyl-3-(O-toluyloxy)-propylamine hydrochloride, molecular formula: C 17 H 21 NO.HCl, molecular weight: 291.82, CAS: 82248-59-7, chemical structure:

[0003]

[0004] Atomoxetine hydrochloride was developed by Eli Lilly and Company Limited. In 2002, atomoxetine hydrochloride capsules were launched on the market for the treatment of attention deficit hyperactivity disorder (ADHD).

[0005] The reaction starting material 2-fluorotoluene is a liquid, and the by-products of its preparation process (such as fluorobenzene, 3-fluorotoluene, 4-fluorotoluene, 2,6-difluorotoluene, etc.) are difficult to remove. The 3-fluorotoluene brought in participates in the synthesis reaction of atomoxetine to generate impurity B; the reaction starting material 2-fluorotoluene is a liquid, and the by-product 4-fluorotoluene of its preparation process participates in the subsequent reaction to form this impurity C.

[0006] (3R)-N-methyl-3-(3-methylphenoxy)-3-phenylpropylamine (impurity B) has the structural formula:

[0007]

[0008] (3R)-N-methyl-3-(4-methylphenoxy)-3-phenylpropylamine (impurity C) has the structural formula:

[0009]

[0010] Impurity B and impurity C are isomers of atomoxetine and cannot be effectively separated from atomoxetine under relevant material conditions. When impurities B and C are checked according to the standard method performed by the supplier (ZCL CHEMICALS LTD), they are interfered by impurity H, and the baseline fluctuates greatly, and the sensitivity cannot meet the detection requirements. Summary of the invention

[0011] In view of this, the present invention proposes a method for detecting impurities B and impurity C in atomoxetine hydrochloride, which can better meet the detection requirements of impurities B and impurity C in atomoxetine hydrochloride.

[0012] The technical solution of the present invention is achieved in this way:

[0013] A method for detecting impurities B and C in atomoxetine hydrochloride comprises the following steps: high performance liquid chromatography is used for detection, and the determination conditions include: chromatographic columns use cellulose-tris(3,5-dichlorophenylcarbamate) bonded silica gel as fillers; n-hexane-anhydrous ethanol-diethylamine (volume ratio 97.5-98.5:1-3:0.5) is used as mobile phase, preferably the volume ratio is 98:2:0.5, the detection wavelength is 278-280nm, and the preferred detection wavelength is 279nm.

[0014] Furthermore, the chromatographic column is a Chiralpak IC chromatographic column with specifications of 4.6 mm×250 mm, 5 μm; the column temperature is 25 to 35° C.; the injection volume is 15 to 25 μl; and the flow rate is 0.9 to 1.1 ml / min.

[0015] Furthermore, the test solution is prepared at a concentration of 0.8 to 1.2 mg / ml, the reference solution is prepared at a concentration of 8 to 12 μg / ml, and the diluent consists of anhydrous ethanol and a mobile phase.

[0016] Furthermore, the preparation of the test solution includes the following steps: taking a sample of atomoxetine hydrochloride, accurately weighing it, placing it in a volumetric flask, adding anhydrous ethanol to dissolve it, diluting it to the scale with a mobile phase, and shaking it well to prepare the test solution, wherein each 1 ml contains 0.8 to 1.2 mg of atomoxetine hydrochloride.

[0017] Furthermore, the preparation of the reference solution includes the following steps: taking the atomoxetine hydrochloride reference, accurately weighing it, placing it in a volumetric bottle, adding anhydrous ethanol to dissolve it, diluting it to the scale with the mobile phase, shaking it well, and using it as the reference stock solution;

[0018] Take the reference substance of atomoxetine related substance B (impurity B), accurately weigh it, put it in a volumetric bottle, add anhydrous ethanol to dissolve it, dilute it to the scale with the mobile phase, shake well, and use it as the reference substance stock solution of atomoxetine related substance B;

[0019] Take the reference substance of atomoxetine related substance C (impurity C), accurately weigh it, put it in a volumetric bottle, add anhydrous ethanol to dissolve it, dilute it to the scale with the mobile phase, shake it well, and use it as the reference substance stock solution of atomoxetine related substance C;

[0020] Accurately measure the reference substance stock solution, atomoxetine related substance B reference substance stock solution, and atomoxetine related substance C reference substance stock solution, respectively, add mobile phase for quantitative dilution to make a solution containing 8-12 μg of atomoxetine hydrochloride, atomoxetine related substance B, and atomoxetine related substance C per 1 ml as the reference substance solution.

[0021] Furthermore, the preparation of the system suitability solution includes the following steps: taking appropriate amounts of atomoxetine hydrochloride reference substance, atomoxetine related substance B reference substance and atomoxetine related substance C reference substance, adding anhydrous ethanol to dissolve them, and diluting them with a mobile phase to prepare a solution containing 0.8-1.2 mg of atomoxetine hydrochloride, 0.8-1.2 μg of atomoxetine related substance B and 0.8-1.2 μg of atomoxetine related substance C per 1 ml, as the system suitability solution.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The present invention provides a method for detecting impurities B and C in atomoxetine hydrochloride. The method has strong specificity and can better meet the requirements for detecting impurities B and C in atomoxetine hydrochloride through examinations such as linearity, limit of quantification, limit of detection, recovery rate, repeatability, intermediate precision, solution stability, and durability.

[0024] (2) The method of the present invention effectively avoids large baseline fluctuations caused by interference from impurity H during inspection, improves sensitivity, and better meets detection requirements.

[0025] (3) The present invention optimizes the detection method of impurities B and C in atomoxetine hydrochloride, effectively improves the quality control level of atomoxetine hydrochloride, and controls the safety of the drug from the raw material drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the HPLC spectrum of the system suitability test of the present invention, which, from top to bottom, are the chromatograms of blank solution, impurity mixed solution, system suitability test solution, and test sample solution.

[0027] Figure 2 This is the linear relationship diagram of impurity B in the present invention.

[0028] Figure 3 This is the linear relationship diagram of impurity C in the present invention.

[0029] Figure 4 It is the linear relationship diagram of atomoxetine hydrochloride of the present invention. DETAILED DESCRIPTION

[0030] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0031] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0032] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0033] Table 1 Reference material information

[0034] Reference factory batch number Purity (based on removal of hydrochloric acid) Atomoxetine Hydrochloride China Food and Drug Inspection Institute 510143-201701 99.7％ Impurity B EP 1.0 100％ Impurity C Huaxin Pharmaceutical HX-TMXT-C-201904 87.51％

[0035] Example

[0036] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0512).

[0037] (1) Chromatographic conditions:

[0038] Chromatographic column: Cellulose-tris(3,5-dichlorophenylcarbamate) bonded silica gel as filler (Chiralpak IC, 4.6 mm x 250 mm, 5 μm);

[0039] Detection wavelength: 279nm;

[0040] Flow rate: 1.0ml / min;

[0041] Column temperature: 30°C;

[0042] Injection volume: 20 μl

[0043] Mobile phase: n-hexane-anhydrous ethanol-diethylamine (v / v / v=98:2:0.5)

[0044] Diluent: anhydrous ethanol, mobile phase

[0045] (2) Solution preparation

[0046] Blank solution: Take 2.5 ml of anhydrous ethanol, place it in a 10 ml volumetric flask, dilute to the scale with the mobile phase, shake well, and you have it.

[0047] Impurity B reference substance stock solution: Take about 2.5 mg of impurity B reference substance, accurately weigh it, put it in a 25 ml volumetric flask, add 5 ml of anhydrous ethanol to dissolve it, dilute it to the scale with mobile phase, shake well, and obtain it.

[0048] Impurity C reference substance stock solution: Take about 2.5 mg of impurity C reference substance, accurately weigh it, put it in a 25 ml volumetric flask, add 5 ml of anhydrous ethanol to dissolve it, dilute it to the scale with mobile phase, shake well, and obtain it.

[0049] Impurity reference solution: Take 1 ml of each impurity B and C reference stock solutions, accurately measure, place in a 10 ml volumetric flask, dilute to the scale with mobile phase, shake well, and obtain.

[0050] System applicable test solution: Take about 10 mg of this product, weigh it accurately, put it in a 10 ml volumetric flask, add 2.5 ml of anhydrous ethanol to dissolve it, then add 1 ml of impurity reference solution, accurately measure it, dilute it to the scale with mobile phase, shake well, and you are done.

[0051] Test solution: Take 10 mg of the product, weigh accurately, place in a 10 ml volumetric flask, add 2.5 ml of anhydrous ethanol to dissolve, dilute to the scale with mobile phase, shake well, and obtain.

[0052] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the scale with the mobile phase, shake well, then accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute it to the scale with the mobile phase, shake well, and you have it.

[0053] (3) System suitability test: Accurately measure 20 μl of the system suitability test solution and inject it into the liquid chromatograph. Record the chromatogram. The order of peaks is atomoxetine hydrochloride, impurity B and impurity C. The separation between the peaks should meet the requirements.

[0054] (4) Determination method: Accurately measure 20 μl of the test solution and the control solution, respectively, and inject them into the liquid chromatograph to record the chromatogram. If there are chromatographic peaks in the chromatogram of the test solution with the same retention time as impurities B and impurity C, they shall not be larger than the main peak area of ​​the control solution (0.1%).

[0055] (5) Calculation formula

[0056]

[0057] Where As: peak area of ​​impurity B (or impurity C) in the chromatogram of the test solution;

[0058] Ar: Main peak area in the chromatogram of the reference solution.

[0059] (6) Result determination

[0060] Impurity B and impurity C shall not exceed 0.1%.

[0061] The methodological investigation of the detection method of the present invention was carried out, and the results are as follows:

[0062] (1) Specificity (blank interference and separation test)

[0063] 1.1 Test methods

[0064] Blank solution: Take 2.5 ml of anhydrous ethanol, place it in a 10 ml volumetric flask, dilute to the scale with the mobile phase, shake well, and you have it.

[0065] Impurity B reference substance stock solution: Take about 2.5 mg of impurity B reference substance, place it in a 25 ml volumetric flask, add 5 ml of anhydrous ethanol to dissolve it, then dilute it to the scale with mobile phase, shake well, and you have it.

[0066] Impurity C reference substance stock solution: Take about 2.5 mg of impurity C reference substance, place it in a 25 ml volumetric flask, add 5 ml of anhydrous ethanol to dissolve it, then dilute it to the scale with mobile phase, shake well, and you have it.

[0067] Impurity reference solution: Take 2 ml of each impurity B and C reference stock solutions, accurately measure, place in a 20 ml volumetric flask, dilute to the scale with mobile phase, shake well, and obtain.

[0068] System applicable test solution: Take about 10 mg of this product, weigh it accurately, put it in a 10 ml volumetric flask, add 2.5 ml of anhydrous ethanol to dissolve it, then add 1 ml of impurity reference solution, accurately measure it, dilute it to the scale with mobile phase, shake well, and you are done.

[0069] Impurity B positioning solution: Accurately measure 1 ml of the impurity B reference stock solution and place it in a 100 ml volumetric flask, then dilute to the scale with the mobile phase and shake well.

[0070] Impurity C positioning solution: Accurately measure 1 ml of the impurity C reference stock solution, place it in a 100 ml volumetric flask, dilute to the scale with the mobile phase, shake well, and obtain.

[0071] Test solution: Take 10 mg of the product, weigh accurately, place in a 10 ml volumetric flask, add 2.5 ml of anhydrous ethanol to dissolve, dilute to the scale with mobile phase, shake well, and obtain.

[0072] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the scale with the mobile phase, shake well, then accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute it to the scale with the mobile phase, shake well, and you have it.

[0073] Impurity mixed solution: Weigh appropriate amounts of impurities A, B, C, D, E, F, G, H, I and J and dilute with mobile phase to make a solution containing 1 μg each of impurities A, B, C, E, F, G, H, I, J and 5 μg of impurity D per 1 ml.

[0074] Accurately measure 20 μl each of the blank solution, system suitability test solution, impurity B and C positioning solutions, test sample solution, control solution and impurity mixed solution, and inject them into the liquid chromatograph according to the above chromatographic conditions. Record the chromatogram to examine whether the blank solution and other impurities interfere with the detection of impurities B and C of this product; the order of peaks in the chromatogram of the system suitability test solution is atomoxetine hydrochloride, impurity B and impurity C, and the separation between each peak should meet the requirements.

[0075] 1.2 Acceptability criteria

[0076] The blank solvent and other impurities in the blank solution chromatogram do not interfere with the inspection of impurities B and C. The separation between the peaks in the system suitability test chromatogram should meet the requirements, with a separation of ≥3.

[0077] 1.3 Test results

[0078] 1) In the blank solution chromatogram, the blank solvent and other impurities do not interfere with the inspection of impurities B and C.

[0079] 2) In the chromatogram of the system suitability test solution, the order of peaks is atomoxetine hydrochloride, impurity B and impurity C, and the separation between each peak meets the requirements. The retention time of atomoxetine is 10.276 min; the retention time of impurity B is 12.220 min, and the separation is 4.240; the retention time of impurity C is 14.080 min, and the separation is 3.774.

[0080] (2) Linearity and range

[0081] 2.1 Test methods

[0082] Blank solution: Take 2.5 ml of anhydrous ethanol, place it in a 10 ml volumetric flask, dilute to the scale with the mobile phase, shake well, and you have it.

[0083] Impurity B reference substance stock solution: Take about 2.5 mg of impurity B reference substance, place it in a 25 ml volumetric flask, add 5 ml of anhydrous ethanol to dissolve it, then dilute it to the scale with mobile phase, shake well, and you have it.

[0084] Impurity C reference substance stock solution: Take about 2.5 mg of impurity C reference substance, place it in a 25 ml volumetric flask, add 5 ml of anhydrous ethanol to dissolve it, then dilute it to the scale with mobile phase, shake well, and you have it.

[0085] Impurity B, C linear mother solution: Accurately measure 2 ml of impurity B and C reference substance stock solutions, place them in a 20 ml volumetric flask, dilute to the scale with mobile phase, shake well, and obtain.

[0086] Tomoxetine hydrochloride reference substance stock solution: Take about 10 mg of Tomoxetine hydrochloride reference substance, accurately weigh it, put it in a 10 ml volumetric flask, add 2.5 ml of anhydrous ethanol to dissolve it, then dilute it to the scale with the mobile phase, shake well, and obtain it.

[0087] Atomoxetine hydrochloride linear stock solution: Accurately measure 1 ml of atomoxetine hydrochloride reference stock solution, place it in a 100 ml volumetric flask, dilute to the scale with mobile phase, shake well, and obtain.

[0088] Accurately measure an appropriate amount of linear mother solution, dilute it with mobile phase to prepare a quantitative limit solution, which is used as linear solution ①; then accurately measure 0.5ml, 0.8ml, 1ml, 1.5ml, and 2ml of the linear mother solution respectively, place them in a 10ml volumetric flask, dilute to the scale with mobile phase, and shake well to prepare linear test solutions ②③④⑤⑥.

[0089] According to the chromatographic conditions of this method, accurately measure 20 μl of each linear solution, inject it into the liquid chromatograph, and record the chromatogram. Perform linear regression with concentration (μg / ml) as the horizontal axis and peak area as the vertical axis to obtain the linear regression equation.

[0090] 2.2 Acceptability criteria

[0091] Range: limit of quantitation ~ 200% of limit; correlation coefficient r: ≥0.998.

[0092] 2.3 Test results

[0093] Table 2 Linearity test results

[0094] name Concentration (μg / ml) Linear equations r Correction Factor Impurity B 0.31～2.08 y=4002.2253x-104.0978 0.999 1.1 Impurity C 0.26～1.74 y=5011.4728x-198.7880 0.998 0.9 Atomoxetine 0.26～2.05 y=4271.1454x-168.5055 0.998 1.0

[0095] Conclusion: In the range of concentration of atomoxetine hydrochloride, impurity B and impurity C from the limit of quantification to 2 times the limit, the concentration and peak area show a good linear relationship, and the correlation coefficient is greater than or equal to 0.998, which meets the requirements and meets the verification requirements. The correction factors of impurities B and C are between 0.9 and 1.1, and the contents of impurities B and C can be calculated by the self-control method.

[0096] (3) Limit of quantification and detection limit

[0097] 3.1 Test methods

[0098] Take an appropriate amount of "linear mother solution under linear test item", accurately measure 20 μl and inject into liquid chromatograph, record the chromatogram, determine the quantitative limit with a signal-to-noise ratio of 10:1, and determine the detection limit with a signal-to-noise ratio of not less than 3:1. Take the quantitative limit solution and inject 6 injections continuously, and calculate the RSD value of the peak area of ​​6 injections.

[0099] 3.2 Acceptability criteria

[0100] Quantitative limit: ①Signal-to-noise ratio (S / N) ≥ 10; ②RSD ≤ 10%; ③The quantitative limit concentration shall not exceed 1 / 2 of the limit concentration.

[0101] Detection limit: ① Signal-to-noise ratio (S / N) S / N ≥ 3; ② Detection limit concentration is not greater than 1 / 3 of the limit concentration.

[0102] 3.3 Test results

[0103] Table 3 Quantitation limit test results

[0104]

[0105] Table 4 Detection limit test results

[0106] name Concentration (μg / ml) Mass(ng) Equivalent to test sample concentration (%) Impurity B 0.09 1.87 0.009 Impurity C 0.08 1.57 0.008 Atomoxetine 0.08 1.54 0.008

[0107] Conclusion: The quantitative limit concentrations of the main component, impurity B and impurity C are all less than 1 / 2 of the limit concentration, the detection limit concentrations are all less than 1 / 3 of the limit concentration, and the RSD values ​​of the quantitative limit peak areas of 6 consecutive needles are all less than 10%, which can meet the detection requirements.

[0108] (4) Recovery rate

[0109] 4.1 Test methods

[0110] Blank solution: Take 2.5 ml of anhydrous ethanol, place it in a 10 ml volumetric flask, dilute to the scale with the mobile phase, shake well, and you have it.

[0111] Impurity B reference substance stock solution: Take about 2.5 mg of impurity B reference substance, place it in a 25 ml volumetric flask, add 5 ml of anhydrous ethanol to dissolve it, then dilute it to the scale with mobile phase, shake well, and you have it.

[0112] Impurity C reference substance stock solution: Take about 2.5 mg of impurity C reference substance, place it in a 25 ml volumetric flask, add 5 ml of anhydrous ethanol to dissolve it, then dilute it to the scale with mobile phase, shake well, and you have it.

[0113] Impurity reference solution: Take 1 ml of each impurity B and C reference stock solutions, accurately measure, place in a 10 ml volumetric flask, dilute to the scale with mobile phase, shake well, and obtain.

[0114] Test solution: Take 10 mg of the product, weigh accurately, place in a 10 ml volumetric flask, add 2.5 ml of anhydrous ethanol to dissolve, dilute to the scale with mobile phase, shake well, and obtain.

[0115] Recovery rate 80% Test solution: Take about 10 mg of this product, weigh accurately, put it in a 10 ml volumetric bottle, add 2.5 ml of anhydrous ethanol to dissolve it, then add 0.8 ml of reference stock solution, dilute to the scale with mobile phase, shake well, and you have it. (Prepare 3 copies in parallel)

[0116] Recovery rate 100% Test solution: Take about 10 mg of this product, weigh accurately, put it in a 10 ml volumetric bottle, add 2.5 ml of anhydrous ethanol to dissolve, then add 1.0 ml of reference stock solution, dilute to the scale with mobile phase, shake well, and you have it. (Prepare 3 copies in parallel)

[0117] Recovery rate 120% Test solution: Take about 10 mg of this product, weigh accurately, put it in a 10 ml volumetric bottle, add 2.5 ml of anhydrous ethanol to dissolve, then add 1.2 ml of reference stock solution, dilute to the scale with mobile phase, shake well, and you have it. (Prepare 3 copies in parallel). Prepare other solutions according to the above method.

[0118] Accurately measure 20 μl of blank solution, control solution, test solution and test solution with recovery rates of 80%, 100% and 120%, respectively, and inject them into liquid chromatograph according to the chromatographic conditions of the detection method of impurity B and impurity C, record the chromatogram, calculate the measured amount of each impurity in the added test solution by peak area according to the external standard method, and calculate the recovery rate of each impurity by the ratio of the added amount after deducting the background value.

[0119] 4.2 Acceptability criteria

[0120] 1. Recovery rate range: 80-120%;

[0121] 2. Recovery rate RSD: ≤6%.

[0122] 4.3 Test results

[0123] The average recovery rate of impurity B was 95.02%, and the RSD value was 3.76% (n=9);

[0124] The average recovery rate of impurity C was 95.72%, and the RSD value was 5.39% (n=9).

[0125] Conclusion: Among the 9 samples, the average recoveries of impurity B and impurity C were 95.02% and 95.72%, respectively. The RSDs of the 9 recovery results were all less than 6%, indicating that the method had good accuracy.

[0126] (5) Repeatability

[0127] 5.1 Test methods

[0128] Blank solution: Take 2.5 ml of anhydrous ethanol, place it in a 10 ml volumetric flask, dilute to the scale with the mobile phase, shake well, and you have it.

[0129] Impurity B reference substance stock solution: Take about 2.5 mg of impurity B reference substance, place it in a 25 ml volumetric flask, add 5 ml of anhydrous ethanol to dissolve it, then dilute it to the scale with mobile phase, shake well, and you have it.

[0130] Impurity C reference substance stock solution: Take about 2.5 mg of impurity C reference substance, place it in a 25 ml volumetric flask, add 5 ml of anhydrous ethanol to dissolve it, then dilute it to the scale with mobile phase, shake well, and you have it.

[0131] Impurity reference solution: Take 1 ml of each impurity B and C reference stock solutions, accurately measure, place in a 10 ml volumetric flask, dilute to the scale with mobile phase, shake well, and obtain.

[0132] Add test solution: Take about 10 mg of the product, weigh accurately, place in a 10 ml volumetric flask, add 2.5 ml of anhydrous ethanol to dissolve, then add 1 ml of impurity reference solution, place in a 10 ml volumetric flask, dilute to the scale with mobile phase, shake well, and obtain.

[0133] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the scale with the mobile phase, shake well, then accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute it to the scale with the mobile phase, shake well, and you have it.

[0134] Accurately measure 20 μl of blank solution, test solution and control solution respectively, and inject them into liquid chromatograph according to the chromatographic conditions of the impurity B and impurity C detection methods, and record the chromatograms. If there are impurity B and C peaks in the chromatogram of the test solution, calculate the impurity content by the self-control method.

[0135] 5.2 Acceptability criteria

[0136] The RSD value (n=6) of the contents of impurity B and impurity C is ≤20%.

[0137] 5.3 Test results

[0138] The average content of impurity B was 0.088%, and the RSD value was 4.95% (n=6);

[0139] The average content of impurity C was 0.091%, and the RSD value was 6.75% (n=6).

[0140] Conclusion: The RSD values ​​of the impurity B and impurity C contents measured in 6 samples were all less than 10%, and the repeatability of the method was good.

[0141] (6) Intermediate precision

[0142] 6.1 Test methods

[0143] The "reproducibility" test was repeated by another analyst at a different time and using a different instrument. The average content and RSD value of the 12 sample results obtained by the two analysts were calculated.

[0144] 6.2 Acceptability criteria

[0145] The RSD value (n=12) of the contents of impurity B and impurity C is ≤30%.

[0146] 6.3 Test results

[0147] The average content of impurity B was 0.086%, and the RSD value was 5.19% (n=12);

[0148] The average content of impurity C was 0.092%, and the RSD value was 5.79% (n=12).

[0149] Conclusion: The RSD values ​​of impurity B and C measured in 12 samples were less than 10%, and the intermediate precision of the method was good.

[0150] (7) Solution stability

[0151] 7.1 Stability of test solution

[0152] 7.1.1 Test method: Take about 10 mg of this product, weigh it accurately, put it in a 10 ml volumetric flask, add 2.5 ml of anhydrous ethanol to dissolve it, dilute it to the scale with mobile phase, shake it well, and the test solution is obtained. Place the test solution at room temperature, accurately measure 20 μl at 0, 4, 8, 12, and 24 hours after preparation, and inject it into the liquid chromatograph according to the chromatographic conditions of impurity B and impurity C, record the chromatogram, and examine the stability within 24 hours.

[0153] 7.1.2 Acceptability criteria: The maximum change value is ≤20%, and no new chromatographic peaks appear, indicating that the solution is stable.

[0154] 7.1.3 Test results: The test solution contains no new impurities within 24 hours at room temperature and the solution is stable.

[0155] 7.2 Control solution stability

[0156] 7.2.1 Test methods

[0157] Control solution (room temperature): Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the mark with the mobile phase, shake well, then accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute it to the mark with the mobile phase, shake well, and you have it. Accurately measure 20 μl of the control solution, inject it into the liquid chromatograph according to the chromatographic conditions of the detection method of impurities B and C, record the chromatogram, and examine the stability of the control solution at room temperature for 0 and 2 hours.

[0158] Control solution (4°C): Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the mark with the mobile phase, shake well, then accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute it to the mark with the mobile phase, shake well, and you have it. Accurately measure 20 μl of the control solution, inject it into the liquid chromatograph according to the chromatographic conditions of the detection method of impurities B and C, record the chromatogram, and examine the stability of the control solution at 4°C for 0, 2, 4, 8, and 10 hours.

[0159] 7.2.2 Acceptable standard: RSD value of main peak area ≤ 6% within 10 hours at 4℃.

[0160] 7.2.3 Test results: When the control solution was placed at room temperature for 2 hours, the RSD value of the peak area of ​​the main component in the chromatogram obtained at each time point was 11.37%, indicating that the solution was unstable; when it was placed at 4°C for 10 hours, the RSD value of the peak area of ​​the main component in the chromatogram obtained at each time point was 3.14%, indicating that the solution was stable at 4°C.

[0161] (8) Durability

[0162] 8.1 Test methods

[0163] Blank solution: Take 2.5 ml of anhydrous ethanol, place it in a 10 ml volumetric flask, dilute to the scale with the mobile phase, shake well, and you have it.

[0164] Impurity B reference substance stock solution: Take about 2.5 mg of impurity B reference substance, place it in a 25 ml volumetric flask, add 5 ml of anhydrous ethanol to dissolve it, then dilute it to the scale with mobile phase, shake well, and you have it.

[0165] Impurity C reference substance stock solution: Take about 2.5 mg of impurity C reference substance, place it in a 25 ml volumetric flask, add 5 ml of anhydrous ethanol to dissolve it, then dilute it to the scale with mobile phase, shake well, and you have it.

[0166] Impurity reference solution: Take 2 ml of each impurity B and C reference stock solutions, accurately measure, place in a 20 ml volumetric flask, dilute to the scale with mobile phase, shake well, and obtain.

[0167] Add test solution: Take about 10 mg of the product, weigh accurately, place in a 10 ml volumetric flask, add 2.5 ml of anhydrous ethanol to dissolve, then add 1 ml of impurity reference solution, place in a 10 ml volumetric flask, dilute to the scale with mobile phase, shake well, and obtain.

[0168] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the scale with the mobile phase, shake well, then accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute it to the scale with the mobile phase, shake well, and you have it.

[0169] The durability of the method was investigated by changing the flow rate, column temperature, wavelength, injection volume, mobile phase ratio, etc. According to the chromatographic condition change parameter settings in Table 5 below, 20 μl of blank solution, sample solution and control solution were accurately measured, and injected into the liquid chromatograph according to the chromatographic conditions of the impurity B and impurity C detection methods, and the chromatograms were recorded. The peak order in the sample solution was investigated to be atomoxetine hydrochloride, impurity B and impurity C, and the separation between the peaks should meet the requirements. The content of each impurity in the sample solution was changed.

[0170] Table 5 Chromatographic conditions range

[0171]

[0172]

[0173] 8.2 Acceptability criteria

[0174] 1. The system applicability meets the requirements;

[0175] 2. The RSD of each impurity content within the variable condition group is ≤30%.

[0176] 8.3 Test results

[0177] Under various changing conditions, the separation of each impurity peak in the sample solution met the requirements, and the RSD values ​​of impurities B and C were less than 10%, indicating that this method has good durability.

[0178] In summary, the method of the present invention has strong specificity and has passed the tests of linearity, limit of quantification, limit of detection, recovery rate, repeatability, intermediate precision, solution stability, durability, etc., and meets the detection requirements of impurity B and impurity C in atomoxetine hydrochloride.

[0179] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting impurities B and C in atomoxetine hydrochloride, characterized in that: The following steps are involved: The detection was carried out by high performance liquid chromatography, and the determination conditions included: the chromatographic column used cellulose-tris (3, 5-dichlorophenyl carbamate) bonded silica gel as a filler; n-hexane-anhydrous ethanol-diethylamine is used as the mobile phase, wherein the volume ratio of n-hexane, anhydrous ethanol and diethylamine in the mobile phase is 97.5-98.5:1-3:0.5; the detection wavelength is 278-280 nm; the chromatographic column is a ChiralpakIC chromatographic column; The structural formula of impurity B is: ； The structural formula of impurity C is: 。 2. The detection method according to claim 1, characterized in that: The detection wavelength is 279 nm.

3. The detection method according to claim 1, characterized in that: The column temperature is 25~35℃; the injection volume is 15~25μl; the flow rate is 0.9~1.1ml / min.

4. The detection method according to claim 1, characterized in that: The specifications of the chromatographic column are 4.6 mm×250 mm, 5 μm.

5. The detection method according to claim 1, characterized in that: The concentration of the test solution is 0.8~1.2mg / ml, the concentration of the reference solution is 8~12μg / ml, and the diluent consists of anhydrous ethanol and mobile phase.

6. The detection method according to claim 5, characterized in that: The test solution preparation comprises the following steps: Take a sample of atomoxetine hydrochloride, accurately weigh it, place it in a volumetric flask, add anhydrous ethanol to dissolve it, dilute it to the scale with mobile phase, shake well, and use it as the test solution. Each 1 ml contains 0.8-1.2 mg of atomoxetine hydrochloride.

7. The detection method according to claim 5, characterized in that: The preparation of the reference substance solution comprises the following steps: Take the atomoxetine hydrochloride reference substance, weigh it accurately, put it in a volumetric bottle, add anhydrous ethanol to dissolve it, dilute it to the scale with the mobile phase, shake it well, and use it as the reference substance stock solution; Take the reference substance B of atomoxetine, accurately weigh it, put it in a volumetric bottle, add anhydrous ethanol to dissolve it, dilute it to the scale with the mobile phase, shake well, and use it as the reference substance stock solution of atomoxetine related substance B; Take the reference substance C of atomoxetine, accurately weigh it, put it in a volumetric bottle, add anhydrous ethanol to dissolve it, dilute it to the scale with the mobile phase, shake well, and use it as the reference substance stock solution of atomoxetine related substance C; Accurately measure the reference substance stock solution, atomoxetine related substance B reference substance stock solution, and atomoxetine related substance C reference substance stock solution, respectively, add mobile phase for quantitative dilution to make a solution containing 8-12 μg of atomoxetine hydrochloride, atomoxetine related substance B, and atomoxetine related substance C per 1 ml as the reference substance solution.

8. The detection method according to any one of claims 5 to 7, characterized in that: The preparation of the system suitability solution includes the following steps: taking atomoxetine hydrochloride reference substance, atomoxetine related substance B reference substance and atomoxetine related substance C reference substance, adding anhydrous ethanol to dissolve them, and diluting them with a mobile phase to prepare a solution containing 0.8-1.2 mg of atomoxetine hydrochloride, 0.8-1.2 μg of atomoxetine related substance B and 0.8-1.2 μg of atomoxetine related substance C per 1 ml, as the system suitability solution.

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