Reversed-phase high-performance liquid chromatography for quantitative analysis of multi-components by single marker of atomoxetine hydrochloride oral soluble impurities C, D and E

Abstract

The invention relates to the technical field of chemical medicine quality detection methods, in particular to a reversed-phase high-performance liquid chromatography method for quantitative analysis of impurities C, D and E of atomoxetine hydrochloride oral solution by single-marker multi-components. According to the determination method, a solid core particle chromatographic column bonded with a diphenyl stationary phase is adopted, a sodium octanesulfonate solution-methanol (88-92: 8-12) is taken as a mobile phase A, methanol is taken as a mobile phase B, and a gradient elution procedure is adopted for detection. The detection wavelength is 205-225 nm, the column temperature is 25-30 DEG C, and the flow velocity is 0.9-1.1 ml / min. The method is good in specificity, precision, repeatability and solution stability and strong in durability, and can solve the technical problem that various impurities with similar structures in atomoxetine hydrochloride are difficult to effectively separate and detect in the prior art. The method can be used for controlling the quality of the atomoxetine hydrochloride oral solution, improving the quality control level and ensuring the product quality, and has an ideal application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of chemical drug quality detection methods, and in particular to a reversed-phase high performance liquid chromatography method for one-test-multiple-evaluation detection of impurities C, D and E of oral solution of atomoxetine hydrochloride. Background Art

[0002] Atomoxetine hydrochloride is a selective norepinephrine reuptake inhibitor (SNRI) developed by Lilly Corporation of the United States. The drug was officially approved by the FDA in July 2002 and successfully launched in 2003. It is the first non-central stimulant approved by the US FDA for the treatment of attention deficit hyperactivity disorder (ADHD); it can prolong the release of norepinephrine in the synaptic cleft and is mainly used to treat ADHD in adults and children over 7 years old. Many studies at home and abroad have shown that atomoxetine hydrochloride has significant clinical efficacy, high patient compliance, and safe application.

[0003] The chemical name of atomoxetine hydrochloride is (-)-N-methyl-3-phenyl-3-(o-tolyloxy)-propan-1-amine hydrochloride [(R)-N-Methyl-3-phenyl-3-(o-tolyloxy)-propan-1-amine hydrochloride], and its molecular formula is C 17 H 21 NO·HCl, see formula I for the structural formula.

[0004]

[0005] During the synthesis of atomoxetine hydrochloride and the storage of preparations, relevant impurities may be introduced or degraded, such as atomoxetine hydrochloride impurities C, D, and E. The structures of atomoxetine impurities C, D, and E are as follows:

[0006] Formula I (impurity C): (3R)-N-methyl-3-(4-methylphenoxy)-3-phenylpropylamine, the structural formula is as follows:

[0007]

[0008] Formula II (impurity D): (3R)-N-methyl-3-(3-methylphenoxy)-3-phenylpropylamine, the structural formula is as follows:

[0009]

[0010] Formula III (impurity E): L-mandelic acid, the structural formula is as follows:

[0011]

[0012] If such impurities exceed the limit, serious adverse reactions will occur, and the structures of such impurities are very similar, so it is difficult to effectively separate them using existing methods. The Chinese invention patent with the patent publication number CN 115561358 A discloses a method for detecting impurities B and C in atomoxetine hydrochloride, which uses high performance liquid chromatography for detection, and the measurement conditions include: the chromatographic column uses cellulose-tris (3,5-dichlorophenylcarbamate) bonded silica gel as a filler; n-hexane-anhydrous ethanol-diethylamine is used as the mobile phase, and the detection wavelength is 278-280nm. This method effectively avoids large baseline fluctuations. However, the defect of this detection method is that impurity E cannot be detected at the same time, and the detection sensitivity needs to be further improved. Therefore, in order to better control the quality of this product, it is urgent to develop a one-test-multiple-evaluation method for rapid determination of the three impurities. Summary of the invention

[0013] The present invention aims to provide a reverse phase high performance liquid chromatography method for one-test multi-evaluation detection of impurities C, D and E in oral solution of atomoxetine hydrochloride, so as to solve the technical problem that multiple impurities with similar structures in atomoxetine hydrochloride are difficult to be effectively separated and detected in the prior art.

[0014] In order to achieve the above object, the present invention adopts the following technical scheme:

[0015] A reversed-phase high performance liquid chromatography method for one-test-multiple-evaluation detection of impurities C, D and E in atomoxetine hydrochloride oral solution, wherein the chromatographic conditions include: the chromatographic column filler uses solid core particles bonded with a diphenyl stationary phase; the mobile phase includes a mobile phase A and a mobile phase B; the mobile phase A is composed of a sodium octane sulfonate solution and methanol, and the mobile phase B is methanol.

[0016] Furthermore, the chromatographic column is a Shim-pack velox Biphenyl chromatographic column.

[0017] Furthermore, the specification of the chromatographic column is 4.6×250 mm, and the particle size of the chromatographic column filler is 5 μm.

[0018] Furthermore, the elution method adopts the gradient elution method; the procedure of the gradient elution is shown in the following table:

[0019] Time min Mobile phase A volume percentage (%) Mobile phase B volume percentage (%) 0 55 45 3 55 45 13 42 58 33 42 58 34 55 45 40 55 45

[0020] In mobile phase A, the volume ratio of sodium octane sulfonate solution to methanol is 88-92:8-12; the pH of the sodium octane sulfonate solution is 2.4-2.6.

[0021] Furthermore, the sodium octane sulfonate solution is prepared by the following method: anhydrous potassium dihydrogen phosphate is dissolved in water at a ratio of 2.72 g: 1000 ml, the pH is adjusted to 2.4-2.6 with phosphoric acid, and sodium octane sulfonate is added in a ratio of 4.32 g: 1000 ml of water to dissolve, and the mixture is mixed to obtain the solution.

[0022] Furthermore, the volume ratio of the sodium octane sulfonate solution to methanol is 90:10; and the pH value of the sodium octane sulfonate solution is 2.5.

[0023] Furthermore, an ultraviolet detector is used, and the detection wavelength is 200-225 nm; preferably, the detection wavelength is 205 nm.

[0024] Furthermore, the column temperature of the chromatographic column is 28°C to 32°C; preferably, the column temperature is 30°C.

[0025] Furthermore, the flow rate of the mobile phase is 0.9-1.1 ml / min; preferably, the flow rate is 1.0 ml / min.

[0026] Further, the separation degree between the chromatographic peaks of atomoxetine hydrochloride, impurity C, impurity D and impurity E and the adjacent chromatographic peaks is ≥1.2; preferably, the separation degree is ≥1.5;

[0027] The linear range of atomoxetine hydrochloride is 0.0658μg / ml~1.4040μg / ml, the linear range of impurity C is 0.0662μg / ml~0.4412μg / ml, the linear range of impurity D is 0.0764μg / ml~0.5096μg / ml, and the linear range of impurity E is 0.0801μg / ml~0.5341μg / ml.

[0028] The principle of this scheme is:

[0029] This technical solution uses a chromatographic column loaded with solid core particles bonded with a diphenyl stationary phase to detect impurities C, D, and E in atomoxetine hydrochloride oral solution. Among them, the mobile phase uses a mobile phase A composed of sodium octane sulfonate solution and methanol, and a mobile phase B composed of methanol, and the three impurities are effectively detected by gradient elution. Although there are some methods for detecting impurities in atomoxetine hydrochloride oral solution in the prior art, the methods of the prior art have problems such as unsatisfactory detection sensitivity and limited types of impurities detected, which cannot meet the needs of practical applications.

[0030] In order to develop a detection method that meets application requirements, the inventor has carried out a large number of experimental studies. The inventor first attempts to use the conventional polysaccharide bonded column of the prior art to detect impurities C, D, and E simultaneously. After a large number of parameter adjustments (for example: the composition of the mobile phase and the gradient elution conditions, etc.), it is found that although certain conditions can be used to ensure the detection of impurity E, it is very difficult to realize the separation of impurities C, D and the main component chromatographic peaks on this basis (see Example 2 Test 1). In order to develop and establish an effective method for multiple evaluations of three impurities, the inventor has then tried various types of chromatographic columns (including a series of C18 chromatographic columns and phenyl columns, etc.), and there will be impurities C and D and atomoxetine hydrochloride peak overlap or very low separation, impurities C and D peak peak type difference and other problems, which seriously affects the quantitative detection effect of impurities C, D, and E (see Example 2 Test 6). After a large number of experimental attempts, the inventor finds that the chromatographic column of the solid core particles loaded with the bonded diphenyl stationary phase of the present scheme can achieve the effective detection of three impurities. Since this is the first application of the above-mentioned chromatographic column (for example: Shim-packvelox Biphenyl) in the impurity detection of atomoxetine hydrochloride oral solution, the inventor needs to explore the appropriate detection conditions from the beginning. In the process of using a solid core particle chromatographic column bonded with a diphenyl stationary phase for detection, the inventor found that: the composition type of the mobile phase (see Example 2 Test 3), the detection wavelength (see Example 2 Test 4), and the program setting of the gradient elution (see Example 2 Test 2) are very critical to the realization of the detection effect. If the parameter setting of this scheme is not adopted, the detection results will show that impurities C and D overlap with the atomoxetine hydrochloride peak or the separation is very low, or the impurity response value is too low, or the chromatogram baseline drifts downward. Through the above research, a set of effective methods for detecting three impurities has been established, which not only realizes the effective quantitative detection of the three impurities, but also has a much higher sensitivity than similar methods in the prior art.

[0031] The beneficial effects of this solution are:

[0032] (1) The present invention realizes the effective separation and detection of atomoxetine hydrochloride and its impurities C, D, and E. The reverse phase chromatography system used has the advantages of stable chromatographic medium performance, strong separation ability, simple operation process, etc., which can more conveniently realize the quality control of the product production process and long-term storage, improve the quality of the final product, and ensure the safety and effectiveness of clinical medication.

[0033] (2) The liquid chromatography characteristic spectrum determination method provided by the present invention has the advantages of specificity, wide linear range, precision, repeatability, good solution stability, and wide durability. In particular, the detection method of this scheme has a significant improvement in detection sensitivity compared with the prior art, and has achieved unexpected technical effects.

[0034] (3) The liquid chromatography one-test-multiple-evaluation method provided by the present invention can control the intrinsic quality of the product, improve the quality control level, and better ensure the quality of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The HPLC spectrum of the blank solution of Example 3.

[0036] Figure 2 It is the HPLC spectrum of the blank auxiliary material solution of Example 3.

[0037] Figure 3 This is the HPLC spectrum of the atomoxetine hydrochloride localization solution of Example 3.

[0038] Figure 4 This is the HPLC spectrum of the impurity D localization solution of Example 3.

[0039] Figure 5 This is the HPLC spectrum of the impurity C localization solution of Example 3.

[0040] Figure 6 This is the HPLC spectrum of the solution for locating impurity E in Example 3.

[0041] Figure 7 The HPLC spectrum of the spiked test solution of Example 3.

[0042] Figure 8 The HPLC spectrum of the test solution of Example 3. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial channels. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art.

[0044] Example 1: Establishment of reverse phase high performance liquid chromatography

[0045] (1) The information of the instruments, equipment, reagents, test solutions, reference substances and test substances used in the test is detailed in Tables 1 to 5.

[0046] Table 1: Instrument information

[0047] Instrument Name model factory Electronic balance MS204T / 02 Mettler Electronic balance XPR105DR / A Mettler Electronic balance XPR2 / A Mettler pH Meter PHS-3E Shanghai Instrument and Electronics Science High performance liquid chromatography LC-2030Plus Shimadzu

[0048] Table 2: Reagents and test drug information

[0049] name batch number grade source Methanol 2023070105 HPLC Norsch Trifluoroacetic acid H2208018 HPLC Comeo Triethylamine 20200331 HPLC Comeo Anhydrous potassium dihydrogen phosphate 2024031201 HPLC Comeo Sodium octane sulfonate 20240305 —— Shandong Yucheng Sino-American Chromatography

[0050] Table 3: Reference Material Information

[0051] name batch number content source Atomoxetine hydrochloride reference substance 510143-201701 99.7％ China Inspection and Quarantine Agency Atomoxetine Hydrochloride Impurity C 29-FEB-24-03 99.64％ QCC Atomoxetine Hydrochloride Impurity D 11-APR-24-04 99.54％ QCC Atomoxetine Hydrochloride Impurity E 13-MAY-19-05 98.91％ QCC

[0052] Table 4: Test product information

[0053] Product Name batch number Specification Atomoxetine Hydrochloride Oral Solution 240101 <![CDATA[0.4% (100 mL: 0.4 g) (calculated as C 17 H 21 NO)]]> Blank Accessories XS240101 ——

[0054] Table 5: Column information

[0055]

[0056] (2) Chromatographic conditions

[0057] The optimal chromatographic conditions established by this technical solution are:

[0058] High performance liquid chromatograph: Agilent, Waters, Shimadzu and Thermo Fisher instruments can be used, preferably Agilent and Shimadzu. Shimadzu LC-2030Plus was specifically selected for subsequent experimental studies.

[0059] Chromatographic column: a solid core particle chromatographic column bonded with a diphenyl stationary phase; its model is preferably: Shim-packveloxBiphenyl (4.6×250mm, 5μm).

[0060] Mobile phase: Sodium octane sulfonate solution-methanol was used as mobile phase A, and methanol was used as mobile phase B.

[0061] The sodium octane sulfonate solution contains 2.72 g / L of anhydrous potassium dihydrogen phosphate and 4.32 g / L of an aqueous solution of sodium octane sulfonate, and the pH value is 2.4-2.6. It is prepared by the following method: 2.72 g of anhydrous potassium dihydrogen phosphate is added to 1000 ml of water to dissolve, mixed, then the pH is adjusted to 2.4-2.6 with phosphoric acid, 4.32 g of sodium octane sulfonate is added to dissolve, and mixed to prepare. The pH value of the sodium octane sulfonate solution is preferably 2.5.

[0062] The mobile phase A is composed of sodium octane sulfonate solution and methanol in a volume ratio of 88-92:8-12; the ratio of sodium octane sulfonate solution to methanol is preferably 90:10.

[0063] Detector: UV detector, detection wavelength is 200-225nm; detection wavelength is preferably 205nm.

[0064] Chromatographic column temperature: 28℃~32℃; the column temperature is preferably 30℃.

[0065] Mobile phase flow rate: 0.9-1.1 ml / min; preferably 1.0 ml / min.

[0066] Elution method: gradient elution. For details of the elution program, see Table 6.

[0067] Table 6: HPLC elution program

[0068]

[0069]

[0070] Example 2: Detection method screening

[0071] (1) Test 1

[0072] The specific chromatographic conditions are:

[0073] Chromatographic column: polysaccharide bonded column (CHIRALPAK IB N-3, 4.6×250 mm, 3 μm), and a guard column (IB N-3, 4.0 mmI.D×10 mmL, 3 μm) was used.

[0074] Mobile phase: A mixture of 0.1% n-propylamine solution and acetonitrile in a volume ratio of 90:10 was used as mobile phase A; acetonitrile was used as mobile phase B. 0.1% n-propylamine solution was prepared by the following method: 1 ml of n-propylamine was measured, 1000 ml of water was added, mixed, pH was adjusted to 8.6 with phosphoric acid, and filtered through a 0.45 μm filter membrane. Gradient elution was performed according to Table 7.

[0075] Table 7: Gradient elution program for Test 1

[0076] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 55 45 30 45 55 35 55 45 45 55 45

[0077] Detection wavelength: 225nm; column temperature: 23℃; flow rate: 0.4ml / min.

[0078] 10 μl of impurities C, D, E, atomoxetine control solution (all with a concentration of 10 μg / ml) and the mixed solution were injected respectively.

[0079] Test results: Impurity E is the first to elute, and can be well separated from atomoxetine and the other two impurities. Impurities C and D elute at similar times and basically overlap with the peak of atomoxetine hydrochloride, so the conditions of this method cannot meet the detection requirements of impurities C and D. Using a polysaccharide bonded column as an HPLC column cannot achieve effective detection of the three impurities. Even with the gradient elution method, it is difficult to achieve effective separation of impurities, especially the two impurities with similar structures (impurity C and impurity D). The structural formulas of impurities C, impurity D and atomoxetine hydrochloride differ only in one methyl position, which makes it difficult to separate the several substances.

[0080] In addition, it should be noted that in HPLC, the resolution of characteristic peaks is a very important parameter, which measures the degree of separation between two adjacent peaks. Good resolution is critical to ensure accurate qualitative identification and quantitative analysis. The resolution (RS) is calculated by the following formula:

[0081] RS = 2(Ta-Tb) / (Wa+Wb); Ta and Tb are the retention times of two adjacent peaks A and B, respectively; Wa and Wb are the baseline widths of the two peaks, respectively.

[0082] It is generally believed that when the resolution is ≥1.5, the two peaks can be considered to have achieved "complete separation" and baseline separation. This means that there is enough space between the two peaks to clearly distinguish each other without causing measurement errors due to overlap. In this test, the retention times of the impurity C peak, impurity D peak, and atomoxetine hydrochloride peak are very similar (basically overlapping), resulting in a resolution between the two that is far less than 1.5, and accurate detection of the target substance cannot be guaranteed.

[0083] (2) Test 2

[0084] The specific chromatographic conditions are:

[0085] Chromatographic column: Shim-pack Velox Biphenyl, 4.6×250 mm, 5 μm.

[0086] Detection wavelength: 225nm; column temperature: 30℃; flow rate: 1.0ml / min.

[0087] Mobile phase: Mobile phase A: phosphate buffer salt (weigh 2.72 g of anhydrous potassium dihydrogen phosphate, add 1000 ml of water to dissolve, and adjust the pH of the solution to 2.5 with trifluoroacetic acid); Mobile phase B: methanol; the volume ratio of mobile phase A and B is 30:70.

[0088] Elution mode: isocratic elution

[0089] 10 μl of impurities C, D, E (all with a concentration of 10 μg / ml), atomoxetine reference solution and mixed solution were injected respectively.

[0090] Test results: E peaks quickly with good separation, impurities C, D and atomoxetine peaks are almost the same (the peaks of the three peaks are similar to test 1), and the separation effect is very poor. If the appropriate mobile phase and elution procedure are not used, it is difficult to separate and detect impurities and main components.

[0091] (3) Test 3

[0092] The specific chromatographic conditions are:

[0093] Chromatographic column: Shim-pack Velox Biphenyl, 4.6×250 mm, 5 μm.

[0094] Detection wavelength: 225nm; column temperature: 30℃; flow rate: 1.0ml / min.

[0095] Mobile phase A: Mobile phase A: phosphate buffer salt (weigh 2.72 g of anhydrous potassium dihydrogen phosphate, add 1000 ml of water to dissolve, and adjust the pH of the solution to 2.5 with trifluoroacetic acid); Mobile phase B: methanol.

[0096] Elution mode: gradient elution. For details of the elution program, see Table 8.

[0097] Table 8: Gradient elution program for test 3

[0098] Time (min) 0 20 30 Mobile phase A percentage (%) 60 45 45 Mobile phase B ratio (%) 40 55 55

[0099] 10 μl of impurities C, D, E (all with a concentration of 10 μg / ml), atomoxetine reference solution and mixed solution were injected respectively.

[0100] Test results: Impurity E peaks quickly with good separation, impurities C and D are basically separated from the atomoxetine hydrochloride peak, but the baseline has been drifting downward seriously under the conditions of this method. It is planned to adjust the mobile phase system to examine the separation of the baseline and each known impurity. Among them, "baseline drift downward" refers to the phenomenon that the baseline on the chromatogram gradually moves in the direction of decreasing response value over time. Under normal circumstances, when no sample components pass through the detector, the baseline should remain at a relatively stable horizontal position, indicating the background signal or noise. When the "baseline drifts downward" phenomenon occurs, you will see that the baseline is not a straight line, but a gradually decreasing curve, and the detected signal intensity is gradually decreasing. This will affect the accurate measurement of the sample peak, because peak height and peak area are key parameters for quantitative analysis, and changes in the baseline will directly affect the accuracy of these measurements. Baseline drift downward is different from normal baseline noise or occasional small fluctuations. It is a continuous trend, indicating that there are links in the entire HPLC system that need further improvement. There may be many reasons for the phenomenon of "baseline drift downward", including but not limited to mobile phase, detector settings, chromatographic column status, etc. In order to ensure the reliability of the analysis results, the inventors conducted research and exploration on the causes of the above phenomenon.

[0101] (4) Test 4

[0102] According to the spectrum obtained from the previous injection, the response values ​​of atomoxetine hydrochloride, impurity C and impurity D at a wavelength of 225nm are all small. According to the absorption spectrum of each known impurity in the obtained spectrum, it is found that the absorption of each known impurity in the low wavelength region is stronger, which is conducive to improving the response value of each known impurity. Therefore, the detection wavelength was changed from 225nm to 205nm for subsequent experimental research.

[0103] The specific chromatographic conditions are:

[0104] Chromatographic column: Shim-pack Velox Biphenyl, 4.6×250 mm, 5 μm.

[0105] Detection wavelength: 205nm; column temperature: 30℃; flow rate: 1.0ml / min.

[0106] Mobile phase A: Mobile phase A: phosphate buffer salt (weigh 2.72 g of anhydrous potassium dihydrogen phosphate, add 1000 ml of water to dissolve, and mix well); Mobile phase B: methanol.

[0107] Elution mode: gradient elution, elution program is the same as Table 8 above.

[0108] 10 μl of impurities C, D, E (all with a concentration of 10 μg / ml), atomoxetine reference solution and mixed solution were injected respectively.

[0109] Test results: Under the conditions of this method, impurity E peaks rapidly with good separation, while the separation of impurity C peak and atomoxetine hydrochloride peak is poor (the separation between the two peaks is less than 1.5), but impurity C and impurity D peaks are both at the smooth baseline. This test did not adopt the method of "adjusting the pH of the solution to 2.5 with trifluoroacetic acid" in test 3, and the problem of downward drift of the baseline was solved, but the impurity peak and the atomoxetine hydrochloride peak were difficult to separate, and quantitative detection of impurities could not be achieved, and the detection conditions needed to be further improved.

[0110] (5) Test 5

[0111] The specific chromatographic conditions are:

[0112] Chromatographic column: Shim-pack Velox Biphenyl, 4.6×250 mm, 5 μm.

[0113] Detection wavelength: 205nm; column temperature: 30℃; flow rate: 1.0ml / min; injection volume: 10μL.

[0114] Mobile phase: Mobile phase A: Phosphate buffer and methanol are mixed in a volume ratio of 90:10; Phosphate buffer is prepared as follows: Weigh 2.72 g of anhydrous potassium dihydrogen phosphate, add 1000 ml of water to dissolve, adjust the pH of the solution to 2.5 with phosphoric acid, add 4.32 g of sodium octane sulfonate to dissolve, and mix well. Mobile phase B: Methanol.

[0115] Elution mode: gradient elution. For details of the elution program, see Table 9.

[0116] Table 9: Gradient elution program for Test 5

[0117]

[0118] Take 10 μl of blank solvent, blank excipient solution, impurities C, D, E (all with a concentration of 10 μg / ml), tomoxicillin test solution and test solution with impurities C, D, E respectively for injection.

[0119] Test results: Under the conditions of this method, the blank solution and blank excipients did not interfere with the detection of impurities C, D and E. In the spiked test solution, the known impurity peaks were well separated from the adjacent impurity peaks (the separation degree was greater than 1.5). It was preliminarily determined that the conditions of this method were feasible for the detection of impurities C, D and E in atomoxetine hydrochloride oral solution. For more detailed experimental results, please refer to the specificity study results below.

[0120] (6) Test 6

[0121] In addition to the above research, this technical solution also screened a large number of different chromatographic columns, including:

[0122] (a) C18 column: Waters Xbridge, Thermo Hypersil GOLD, Kromasil 100-5-C18, Welch Ultimate XB-C18, FLM Superlu C18, Agilent ZORBAX SB Aq;

[0123] (b) Phenyl column: XBridge BEH Phenyl Column, HyperSep TM Phenyl Column, (Agilent) Zorbax XDB-Phenyl.

[0124] The technical parameters of the above chromatographic columns are as follows: the stationary phase particle size is 5 μm, and the chromatographic column specifications are 4.6×250 mm. Except for the different chromatographic column selection, the other technical parameters are set the same as test 5.

[0125] The performance of the chromatographic column is judged according to the number and peak shape of the impurity chromatographic peaks. The results show that all the above C18 chromatographic columns can only detect impurity E, but impurities C and D overlap with the peak of atomoxetine hydrochloride, so this type of chromatographic column is not suitable for one-time testing of multiple batches of this type of impurities. The three impurities can be detected under the detection conditions of the three phenyl columns. Impurity E peaks normally, but the peak shape of impurities C and D is very poor (the peak shape is not sharp, it is in the shape of a steamed bun, and the tailing is serious). The separation degree between the impurities C, D and atomoxetine hydrochloride peaks is less than 1.0, so it is not suitable for one-time testing of multiple batches of these three impurities. The study found that the Shimadzu phenyl column Shim-pack Velox Biphenyl (4.6×250mm, 5μm) has the best performance, so the Shimadzu phenyl column was finally selected.

[0126] Example 3: Specificity study

[0127] (1) Solution preparation

[0128] Blank solution: diluent (methanol-water 1:1).

[0129] Blank excipient solution: Accurately measure 5 ml of blank excipient (a solution without main ingredients composed of a certain proportion of sodium benzoate, sorbitol, xylitol, sucralose, raspberry essence, sodium hydroxide, sodium dihydrogen phosphate, phosphoric acid and purified water), place it in a 50 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0130] Atomoxetine hydrochloride localizing solution: Take an appropriate amount of atomoxetine hydrochloride reference substance, weigh it accurately, and add diluent to dissolve it into a 10μg / ml atomoxetine hydrochloride reference substance localizing solution.

[0131] Atomoxetine hydrochloride impurity C localization solution: Take an appropriate amount of Atomoxetine hydrochloride EP impurity C reference substance, accurately weigh it, and add diluent to dissolve it into 10μg / ml.

[0132] Atomoxetine hydrochloride impurity D localization solution: Take an appropriate amount of Atomoxetine hydrochloride EP impurity D reference substance, accurately weigh it, and add diluent to dissolve it to 10μg / ml.

[0133] Atomoxetine hydrochloride impurity E localization solution: Take an appropriate amount of Atomoxetine hydrochloride EP impurity E reference substance, accurately weigh it, and add diluent to dissolve it to 10μg / ml.

[0134] Mixed impurity reference substance stock solution: Weigh appropriate amounts of impurity C, D and E reference substances, add diluent to dissolve and quantitatively dilute to make 1 ml containing approximately 10 μg of each impurity, thereby obtaining impurity C, D and E reference substance stock solutions.

[0135] Accurately measure 5 ml of each impurity C, D, and E reference substance stock solution, place in a 50 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0136] System suitability solution: Accurately measure 5 ml of the test sample and place it in a 50 ml volumetric flask. Accurately add 2 ml of the mixed impurity reference stock solution, then dilute to the scale with diluent, shake well, and obtain.

[0137] Test solution: Accurately measure 5 ml of the test sample, place it in a 50 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0138] Control solution: Accurately measure 3 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with diluent, shake well, accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0139] Sensitivity solution: Accurately measure 3 ml of the control solution, place it in a 20 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0140] (2) Determination

[0141] Under the determined chromatographic conditions (see the optimal chromatographic conditions of Example 1 or Test 5 of Example 2), 10 μl of each of blank solvent, system suitability solution, control solution, blank excipient solution, sensitivity solution, test solution, atomoxetine hydrochloride localization solution, atomoxetine hydrochloride impurity C localization solution, atomoxetine hydrochloride impurity D localization solution, and atomoxetine hydrochloride impurity E localization solution were injected into the HPLC. For details, see the chromatographic Figures 1 to 7 The results are shown in Table 10.

[0142] Table 10: Results of specificity tests for impurities C, D and E

[0143]

[0144] (3) Specificity conclusion

[0145] The peaks of blank solution, blank excipient solution and test solution did not interfere with the detection of known impurities. The separation degree between each known impurity peak and adjacent peak in the system suitability solution spectrum was ≥1.5. The results showed that the method had good specificity for detecting impurities C, D and E in atomoxetine hydrochloride oral solution.

[0146] Example 4: Degradation test

[0147] (1) Solution preparation

[0148] Undegraded solution: Atomoxetine hydrochloride oral solution

[0149] Blank solution: diluent

[0150] Blank excipient solution: Accurately measure 5 ml of blank excipient, place in a 50 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0151] Test solution: Accurately measure 5 ml of the test sample, place it in a 50 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0152] Acid Degradation Solution

[0153] Blank solution: Accurately measure 5 ml of diluent, place in a 50 ml volumetric flask, add 5 ml of 1 mol / L hydrochloric acid solution and 5 ml of 1 mol / L sodium hydroxide solution, shake well, then dilute to the scale with diluent, shake well, and obtain.

[0154] Blank auxiliary material solution: accurately measure 5 ml of blank auxiliary material, add 5 ml of 1 mol / L hydrochloric acid solution, shake well, seal, place in a boiling water bath for 5.5 min, take out and cool to room temperature, add 5 ml of 1 mol / L sodium hydroxide solution to neutralize, shake well, dilute to the scale with diluent, shake well, and obtain.

[0155] Test sample solution: Accurately measure 5 ml of the test sample and prepare it in the same way as the blank excipient solution.

[0156] Alkaline degradation solution

[0157] Blank solution: Accurately measure 5 ml of diluent, place in a 50 ml volumetric flask, add 5 ml of 1 mol / L sodium hydroxide solution and 5 ml of 1 mol / L hydrochloric acid solution, then dilute to the scale with diluent, shake well, and obtain.

[0158] Blank auxiliary material solution: accurately measure 5 ml of blank auxiliary material, place in a 50 ml volumetric flask, add 5 ml of 1 mol / L sodium hydroxide solution, shake well, seal, and place in a boiling water bath for 1 hour. Take out and cool to room temperature, add 5 ml of 1 mol / L hydrochloric acid solution to neutralize, and then dilute to the scale with a diluent, shake well, and the solution is ready.

[0159] Test sample solution: Accurately measure 5 ml of the test sample and prepare it in the same way as the blank excipient solution.

[0160] Oxidative degradation solution

[0161] Blank solution: Accurately measure 5 ml of diluent, place in a 50 ml volumetric flask, add 2 ml of 30% hydrogen peroxide solution, dilute to the scale with diluent, shake well, and the solution is ready.

[0162] Blank auxiliary material solution: accurately measure 5 ml of blank auxiliary material, place it in a 50 ml volumetric flask, add 2 ml of 30% hydrogen peroxide solution, leave it at room temperature for 1 hour, add diluent to dilute to the scale, shake well, and obtain.

[0163] Test sample solution: Accurately measure 5 ml of the test sample and prepare it in the same way as the blank excipient solution.

[0164] High temperature degradation solution

[0165] Blank solution: diluent

[0166] Blank auxiliary material solution: accurately measure 5 ml of blank auxiliary material, put it into a 50 ml volumetric flask, seal it tightly, and place it in a boiling water bath for 2 hours. Take it out and cool it to room temperature, then dilute it to the scale with a diluent, shake it well, and you have it.

[0167] Test sample solution: Accurately measure 5 ml of the test sample and prepare it in the same way as the blank excipient solution.

[0168] Photodegradation solution

[0169] Blank solution: diluent

[0170] Blank auxiliary material solution: Take about 20 ml of blank auxiliary material, place it in a 25 ml colorless transparent volumetric bottle, seal it tightly, and irradiate it under sunlight and ultraviolet light for 15 days (illuminance is 5000 Lux, 90 μw / cm 2 ), take it out, accurately measure 5 ml of the blank auxiliary material after illumination, put it in a 50 ml volumetric flask, dilute it to the scale with diluent, shake well, and you have it.

[0171] Test sample solution: Accurately measure 20 ml of the test sample and prepare it in the same way as the blank excipient solution.

[0172] (2) Determination

[0173] According to the determined chromatographic conditions (see the optimal chromatographic conditions of Example 1 or Test 5 of Example 2), 10 μl of each of the above solutions was injected into the chromatograph, injected into the liquid chromatograph, and the chromatogram was recorded. The results are shown in Table 11.

[0174] Table 11: Degradation test results

[0175]

[0176]

[0177] (3) Conclusion of degradation experiment

[0178] Under alkaline, oxidative, light, and high temperature conditions, the test solution did not produce obvious degradation products; under strong acid conditions, the test solution was significantly degraded, but the degradation product peaks did not interfere with the detection of known impurities. The results show that the method has good specificity for the detection of impurities C, D, and E in atomoxetine hydrochloride oral solution.

[0179] Example 5: System Applicability

[0180] (1) Solution preparation

[0181] System suitability solution: Take the system suitability solution in Example 3.

[0182] (2) Determination

[0183] According to the determined chromatographic conditions (see the optimal chromatographic conditions of Example 1 or Test 5 of Example 2), 10 μl of the system suitability solution was injected into the chromatograph, and the injection was repeated 5 times. The chromatogram was recorded. The results are shown in Tables 12 to 14.

[0184] Table 12: Peak area statistics results

[0185] name Atomoxetine Hydrochloride EP Impurity C EP Impurity D EP Impurity E System Suitability Solution 1 20723556 15333 18095 13930 System Suitability Solution 2 20720997 15669 18558 13829 System Suitability Solution 3 20697982 15364 18428 14038 System Suitability Solution 4 20706457 15512 18853 13971 System Suitability Solution 5 20720667 15425 18635 14091 Peak area RSD 0.05％ 0.87％ 1.51％ 0.72％

[0186] Table 13: Retention time statistics

[0187]

[0188] Table 14: Statistical results of the separation of EP impurities C, D and E from adjacent peaks

[0189] name EP Impurity C EP Impurity D EP Impurity E System Suitability Solution 1 3.0 2.3 1.6 System Suitability Solution 2 3.0 2.3 1.6 System Suitability Solution 3 3.0 2.3 1.5 System Suitability Solution 4 3.0 2.3 1.5 System Suitability Solution 5 3.0 2.3 1.5

[0190] (3) Conclusion on system applicability

[0191] In the spectrum obtained by continuous injection of 5 injections of the system suitability solution, the RSD of the peak area and retention time of each target component was less than 2.0%, and the separation degree between each known impurity peak and the adjacent peak was greater than 1.5. This shows that the system suitability of this method for detecting impurities C, D, and E in atomoxetine hydrochloride oral solution is good.

[0192] Example 6: Sensitivity

[0193] (1) Limit of quantification

[0194] The limit of quantitation refers to the lowest concentration or amount of the target analyte that can be quantitatively determined with acceptable accuracy and precision. The lowest concentration or amount corresponding to the signal-to-noise ratio of 10:1 is the limit of quantitation. When the target analyte is greater than or equal to this concentration or amount, the target analyte can be quantitatively determined. The determination method is: prepare a series of standard solutions of different concentrations to cover the expected limit of quantitation range, then inject the samples for HPLC analysis, and calculate and determine the specific value of the limit of quantitation based on the test results.

[0195] (1.1) Solution preparation

[0196] Blank solution: diluent

[0197] Quantitative limit stock solution: Accurately measure 1 ml of the atomoxetine hydrochloride reference stock solution of Example 3 and each known impurity reference stock solution, place in a 200 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0198] Quantitation limit solution: Accurately measure 3 ml of the quantitation limit stock solution, place it in a 20 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0199] (1.2) Determination

[0200] According to the determined chromatographic conditions (see the optimal chromatographic conditions of Example 1 or Test 5 of Example 2), 10 μl of the quantitative limit solution and the detection limit solution were accurately measured and injected into the liquid chromatograph respectively to record the chromatogram. The results are shown in Tables 15 to 18.

[0201] Table 15: Impurity C Quantitation Limit Results

[0202]

[0203]

[0204] Table 16: Impurity D Quantitation Limit Results

[0205]

[0206] Table 17: Impurity E Quantitation Limit Results

[0207]

[0208] Table 18: Limit of quantitation results of atomoxetine hydrochloride

[0209]

[0210] (2) Detection limit

[0211] The detection limit is the lowest concentration or amount that can reliably distinguish between the signal and the background noise. In other words, it is the minimum amount of the target analyte that can be reported in the sample. The lowest concentration corresponding to the signal-to-noise ratio of 3:1 is the detection limit, which refers to the minimum amount that can reliably distinguish between the signal and the background noise, that is, the minimum concentration of the target analyte that can be reported in the sample. The determination method is: prepare a series of standard solutions of different concentrations to cover the expected quantitative limit range, then inject the samples for HPLC analysis, and calculate and determine the specific value of the detection limit based on the test results.

[0212] (2.1) Solution preparation

[0213] Detection limit solution: Accurately measure 2 ml of the aforementioned quantitative limit stock solution, place it in a 20 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0214] (2.2) Determination

[0215] According to the determined chromatographic conditions, 10 μl of the quantitative limit solution and the detection limit solution were accurately measured and injected into the liquid chromatograph respectively, and the chromatograms were recorded. The results are shown in Tables 19 to 22.

[0216] Table 19: Impurity C detection limit results statistics

[0217]

[0218] Table 20: Impurity D detection limit results statistics

[0219]

[0220] Table 21: Statistics of detection limit results of impurity E

[0221]

[0222] Table 22: Detection limit statistics of atomoxetine hydrochloride

[0223]

[0224] (2.3) Sensitivity conclusion

[0225] The quantification limit of impurity C is 0.662ng, the quantification limit of impurity D is 0.764ng, the quantification limit of impurity E is 0.801ng, and the quantification limit of atomoxetine hydrochloride is 0.658ng; the quantification limit of each known impurity is less than 0.2% of the concentration of the main component of the test sample (the limit shall not exceed 0.10%), and the RSD of the peak area of ​​the target component in the spectrum obtained by continuous injection of 5 injections is less than 5%, and the peak height signal-to-noise ratio is greater than 10.

[0226] The detection limit of impurity C is 0.441ng, the detection limit of impurity D is 0.510ng, the detection limit of impurity E is 0.534ng, and the detection limit of atomoxetine hydrochloride is 0.439ng; the detection limits of each known impurity are less than 0.015% of the concentration of the main component of the test sample (the limit shall not exceed 0.10%), and in the spectrum obtained by three consecutive injections, the signal-to-noise ratio of the peak height of each target component is greater than 3.

[0227] Example 7: Linearity and Range

[0228] (1) Solution preparation

[0229] Blank solution: diluent.

[0230] Atomoxetine hydrochloride linear stock solution: Accurately measure 10 ml of the atomoxetine hydrochloride reference substance stock solution of Example 3, place it in a 50 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0231] Mixed impurity linear stock solution: Accurately measure 2 ml of each of the EP impurity C, EP impurity D, and EP impurity E reference stock solutions under the specificity item, place them in the same 100 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0232] Mixed impurity linear sample solution: Accurately measure 2ml, 3ml, 3.5ml, 4ml and 5ml of mixed impurity linear stock solution respectively and place them in 20ml volumetric flasks, add diluent to dilute to the scale, shake well, and obtain.

[0233] Atomoxetine hydrochloride linear 1 solution: Accurately measure 1 ml of atomoxetine hydrochloride linear stock solution, place it in a 100 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0234] Atomoxetine hydrochloride linear 2 solution: Accurately measure 1 ml of the atomoxetine hydrochloride linear stock solution, place it in a 50 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0235] Atomoxetine hydrochloride linear 3 solution: Accurately measure 2 ml of the atomoxetine hydrochloride linear stock solution, place it in a 50 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0236] Atomoxetine hydrochloride linear 4 solution: Accurately measure 3 ml of atomoxetine hydrochloride linear stock solution, place it in a 50 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0237] Atomoxetine hydrochloride linear 5 solution: Accurately measure 4 ml of atomoxetine hydrochloride linear stock solution, place it in a 50 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0238] (2) Determination

[0239] According to the determined chromatographic conditions (see the optimal chromatographic conditions of Example 1 or Test 5 of Example 2), 10 μl of each of the above linear solutions was accurately measured and injected into the liquid chromatograph respectively, and the chromatogram was recorded. The linear regression curve was drawn with the concentration as the abscissa and the peak area as the ordinate, and the regression equation and correlation coefficient were calculated.

[0240] (3) Linearity and range conclusions

[0241] The concentration of the main component and the peak area of ​​atomoxetine hydrochloride in the concentration range of 0.0658 μg / ml to 1.4040 μg / ml showed a good linear relationship. The linear equation was: y=53503x+103.74, the correlation coefficient r was 0.999 (greater than 0.995), and the ratio of the Y-axis intercept to the 100% concentration point response value was 0.16% (less than 10%).

[0242] The concentration of impurity C in the range of 0.0662 μg / ml to 0.4412 μg / ml has a good linear relationship with the peak area. The linear equation is: y=44797x-302.33, the correlation coefficient r is 0.997 (greater than 0.995), and the ratio of the Y-axis intercept to the 100% concentration point response value is 1.72% (less than 10%).

[0243] The concentration of impurity D is in the range of 0.0764 μg / ml to 0.5096 μg / ml, and the main component concentration and peak area show a good linear relationship. The linear equation is: y=59111x+15.459, the correlation coefficient r is 0.999 (greater than 0.995), and the ratio of the Y-axis intercept to the 100% concentration point response value is 0.07% (less than 10%).

[0244] The concentration of impurity E in the range of 0.0801 μg / ml to 0.5341 μg / ml has a good linear relationship with the peak area. The linear equation is: y=31812x-620.27, the correlation coefficient r is 1.000 (greater than 0.995), and the ratio of the Y-axis intercept to the 100% concentration point response value is 5.12% (less than 10%).

[0245] (4) Correction factor

[0246] The correction factors of each impurity calculated according to the linear test results are: the correction factor of impurity C is 1.19, and the correction factor of impurity E is 1.68, which are within the range of 0.20 to 5.00, but not between 0.90 and 1.10. Therefore, when calculating the content of impurities C and impurity E, the corrected peak area multiplied by the correction factor must be used; the correction factor of impurity D is 0.91, which is within the range of 0.90 to 1.10. Therefore, when calculating the content of impurity D, the peak area does not need to be corrected.

[0247] Example 8: Accuracy

[0248] (1) Solution preparation

[0249] Blank solution: diluent.

[0250] Mixed impurity reference substance stock solution: Accurately measure 5 ml of each of the atomoxetine hydrochloride EP impurity C, D, and E reference substance stock solutions of Example 3, place them in the same 100 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0251] System suitability solution: Accurately measure 5 ml of the test sample and place it in a 50 ml volumetric flask. Add 2 ml of the mixed impurity reference stock solution, dilute to the scale with diluent, shake well, and obtain.

[0252] Test solution: Accurately measure 5 ml of the test sample, place it in a 50 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0253] Control solution: Place 3 ml of the precise test solution in a 100 ml volumetric flask, add diluent to dilute to the mark, shake well, accurately measure 1 ml, place in a 10 ml volumetric flask, add diluent to dilute to the mark, shake well, and the solution is ready.

[0254] Sensitivity solution: Accurately measure 3 ml of the control solution, place it in a 20 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0255] Accuracy solution: Accurately measure 5 ml of the test sample, place it in a 50 ml volumetric flask, add 2 ml of the mixed impurity reference stock solution, dilute to the scale with diluent, shake well, and obtain (prepare 6 copies in parallel).

[0256] Accuracy control solution: Place 3 ml of precise accuracy solution in a 100 ml volumetric flask, add diluent to dilute to the mark, shake well, accurately measure 1 ml, place in a 10 ml volumetric flask, add diluent to dilute to the mark, shake well, and you have the solution (corresponding to the 6 accuracy solutions mentioned above).

[0257] (2) Determination

[0258] According to the determined chromatographic conditions (see the optimal chromatographic conditions of Example 1 or Test 5 of Example 2), 10 μl of each of the above solutions was accurately measured and injected into the liquid chromatograph respectively, and the chromatogram was recorded.

[0259] (3) Accuracy conclusion: In the test results of the six accuracy solutions spiked with limit concentrations, the recovery rates of each known impurity were between 80% and 105%, which met the requirements of the relevant guidelines (75% to 120%), and the RSD of the recovery rates of each known impurity was less than 10%. This shows that the accuracy of this method for detecting impurities C, D, and E in atomoxetine hydrochloride oral solution is good.

[0260] Example 9: Precision

[0261] (1) Repeatability

[0262] (1.1) Solution preparation

[0263] Blank solution: diluent.

[0264] Impurity reference substance stock solution: the same as the known impurity reference substance stock solution in Example 3.

[0265] Mixed impurity reference stock solution: Accurately measure 5 ml of EP impurity C, D, and E reference stock solutions, place them in the same 50 ml volumetric flask, add diluent to dilute to the scale, shake well, and it is ready.

[0266] System suitability solution: Accurately measure 5 ml of the test sample and place it in a 50 ml volumetric flask. Accurately add 2 ml of the mixed impurity reference stock solution, then dilute to the scale with diluent and shake well.

[0267] Test solution: Accurately measure 5 ml of blank auxiliary material, place it in a 50 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain (prepare 6 portions in parallel).

[0268] Control solution: Accurately measure 3 ml of the above test solution, place it in a 100 ml volumetric flask, dilute to the scale with diluent, shake well, accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0269] Sensitivity solution: Accurately measure 3 ml of the control solution, place it in a 20 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0270] (1.2) Determination

[0271] According to the determined chromatographic conditions (see the optimal chromatographic conditions of Example 1 or Test 5 of Example 2), 10 μl of each of the above solutions was accurately measured and injected into the liquid chromatograph respectively, and the chromatogram was recorded.

[0272] (1.3) Repeatability conclusion: None of the known impurities were detected in the 6 test solutions, and the repeatability was good.

[0273] (2) Intermediate precision

[0274] To investigate the impact of random variation on precision, another analyst (operator B) independently established the system, re-prepared 6 samples for testing, used different instruments, and performed the tests on different dates. The precision results and repeatability results were required to calculate the intermediate precision of the 12 samples.

[0275] (2.1) Solution preparation

[0276] Blank solution: diluent.

[0277] Impurity reference substance stock solution: the same as the known impurity reference substance stock solution in Example 3.

[0278] Mixed impurity reference stock solution: Accurately measure 5 ml of EP impurity C, D, and E reference stock solutions, place them in the same 50 ml volumetric flask, add diluent to dilute to the scale, shake well, and it is ready.

[0279] System suitability solution: Accurately measure 5 ml of the test sample and place it in a 50 ml volumetric flask. Accurately add 2 ml of the mixed impurity reference stock solution, then dilute to the scale with diluent and shake well.

[0280] Test solution: Accurately measure 5 ml of blank auxiliary material, place it in a 50 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain (prepare 6 portions in parallel).

[0281] Control solution: Accurately measure 3 ml of the above test solution, place it in a 100 ml volumetric flask, dilute to the scale with diluent, shake well, accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0282] Sensitivity solution: Accurately measure 3 ml of the control solution, place it in a 20 ml volumetric flask, add diluent to dilute to the scale, shake well, and the solution is ready.

[0283] (2.2) Determination

[0284] According to the determined chromatographic conditions, accurately measure 10 μl of each of the above solutions, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0285] (2.3) Precision conclusion: Two experimenters selected different instruments at different times and prepared 6 test solutions each, totaling 12 test solutions. None of the known impurities were detected. The results showed that the precision of this method for detecting impurities C, D, and E in atomoxetine hydrochloride oral solution was good.

[0286] Example 10: Solution Stability

[0287] (1) Solution preparation

[0288] Test solution: the same as the test solution spiked with that in Example 3.

[0289] Control solution: same as the control solution in Example 3.

[0290] (2) Determination

[0291] According to the determined chromatographic conditions (see the optimal chromatographic conditions of Example 1 or Test 5 of Example 2), the control solution and the spiked test solution were taken and placed at room temperature for 0 hours, 4 hours, 8 hours, 12 hours, and 24 hours, respectively, and the samples were analyzed and the spectra were recorded.

[0292] (3) Conclusion of solution stability experiment: In the spectra obtained by injecting the control solution and the spiked test solution at various time points after standing at room temperature for 24 hours, the RSD of the target peak area was less than 4%. The results showed that the control solution and the test solution were basically stable for at least 24 hours under room temperature conditions, which can meet the testing time requirements for multiple batches of test products.

[0293] Example 11: Durability

[0294] (1) Solution preparation

[0295] Blank solution: diluent.

[0296] Impurity reference substance stock solution: the same as the known impurity reference substance stock solution in Example 3.

[0297] Mixed impurity reference stock solution: Accurately measure 1 ml of each EP impurity C, D, and E reference stock solution, place them in the same 10 ml volumetric flask, add diluent to dilute to the scale, shake well, and it is ready.

[0298] System suitability solution: Accurately measure 5 ml of the test sample and place it in a 50 ml volumetric flask. Accurately add 2 ml of the mixed impurity reference stock solution, then dilute to the scale with diluent and shake well.

[0299] Blank excipient solution: Accurately measure 5 ml of blank excipient, place in a 50 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0300] Test solution: Accurately measure 5 ml of the sample solution, place it in a 50 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0301] Control solution: Accurately measure 3 ml of the spiked test solution, place it in a 100 ml volumetric flask, dilute to the mark with diluent, shake well, accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute to the mark with diluent, shake well, and obtain.

[0302] Sensitivity solution: Accurately measure 3 ml of the control solution, place it in a 20 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0303] (2) Durable chromatographic conditions

[0304] See Table 23 for details.

[0305] Table 23: Rugged Chromatographic Conditions

[0306]

[0307] (3) Determination

[0308] According to the above chromatographic conditions, 10 μl of each of the above solutions was accurately measured and injected into the liquid chromatograph to record the chromatogram. The results are shown in Tables 24 to 26.

[0309] Table 24: Test results statistics

[0310]

[0311]

[0312] Table 25: System suitability results statistics

[0313]

[0314] Table 26: Statistics of interference between blank solution and blank excipients

[0315]

[0316]

[0317] (4) Durability conclusion:

[0318] During the durability investigation, the column temperature, flow rate, mobile phase ratio, mobile phase pH fine-tuning and different filler batches of chromatographic columns in the method conditions were tested for the test solution. Under each durability condition, no known impurities were detected in the test solution. The peak shape of each known impurity in the system suitability solution was good and the separation degree with the adjacent peak was not less than 1.2. The blank solution and blank excipient peaks did not interfere with the detection of each known impurity. The results show that the method conditions have good durability for detecting impurities C, D, and E in atomoxetine hydrochloride oral solution.

[0319] Example 12: Detection results of impurities C, D and E in homemade products

[0320] According to the above experimental results, the methodological validation results of the impurity C, D, and E detection method showed that the specificity, system applicability, sensitivity, linearity and range, accuracy, precision, durability, and solution stability all met acceptable standards, indicating that the conditions of this method can be used for the detection of impurities C, D, and E in atomoxetine hydrochloride oral solution.

[0321] The above-mentioned method for detecting impurities C, D and E in atomoxetine hydrochloride oral solution was used to detect 6 batches of samples, namely 220501, 220502, 220503, 240101, 240103 and 240104. The test results are shown in Table 27.

[0322] Table 27: Test results of impurities C, D and E in homemade products

[0323] batch number Impurity C content Impurity D content Impurity E content 220501 Not detected Not detected Not detected 220502 Not detected Not detected Not detected 220503 Not detected Not detected Not detected 240101 Not detected Not detected Not detected 240103 Not detected Not detected Not detected 240104 Not detected Not detected Not detected

[0324] Comparative Example 1

[0325] Chinese patent CN115561358A (a method for detecting impurities B and C in atomoxetine hydrochloride) detects impurities C and D involved in this scheme. The detection method is:

[0326] The HPLC column uses cellulose-tris(3,5-dichlorophenylcarbamate) bonded silica gel as filler (such as Chiralpak IC column), n-hexane-anhydrous ethanol-diethylamine as mobile phase, and the detection wavelength is 278-280 nm.

[0327] According to the above method, the quantitative limits of impurity C (named as impurity C in CN115561358A) and impurity D (named as impurity B in CN115561358A) are 5.23ng and 6.24ng, respectively, and the detection limits are 1.57ng and 1.87ng, respectively. Both the quantitative limit and the detection limit are relatively high, which does not meet the detection requirements of trace impurities in practical applications, and the detection method needs to be improved as a whole. In addition, the method of this comparative example cannot simultaneously detect impurity E. If a one-test-multiple-evaluation method for three impurities can be developed, it will be more in line with the needs of practical applications.

[0328] The inventor made improvements with reference to the chromatographic column conditions of the comparative patent, in order to further reduce the quantitative limit and detection limit, and simultaneously realize the detection of impurity E. After adjusting the elution procedure of the mobile phase (test 1 of visible embodiment 2), it was found that after adjusting the parameter conditions, impurity C and impurity D were difficult to effectively separate from the atomoxetine hydrochloride peak. The target substance is difficult to effectively separate, let alone how to reduce the effective quantitative limit and detection limit. Therefore, it is more difficult to further improve the detection effect on the Chinese patent CN115561358A method, and other detection method improvements must be considered, and a detection method completely different from this comparative example method is adopted.

[0329] In order to further reduce the limit of quantitation and the limit of detection, the inventors used a new type of chromatographic column filler (solid core particles bonded to a diphenyl stationary phase), but under the new detection conditions, it is difficult to accurately detect impurities C and D, mainly manifested in the difficulty in effectively separating impurities C and D from the atomoxetine hydrochloride peak, and the instability of the chromatogram baseline. Through a large number of experimental adjustments, the optimal detection conditions described in Example 1 were determined. Under the above conditions, not only can the separation between each target chromatographic peak and the adjacent peak be guaranteed to be greater than or equal to 1.5, but the limit of quantitation and the limit of detection can also be further reduced, and the detection sensitivity can be improved. Compared with Chinese patent CN115561358A, the quantitative limit of impurity C in this scheme is 0.662ng, the quantitative limit of impurity D is 0.764ng, the quantitative limit of impurity E is 0.801ng, and the quantitative limit of atomoxetine hydrochloride is 0.658ng (the quantitative limit is almost one order of magnitude lower than the prior art); the detection limit of impurity C is 0.441ng, the detection limit of impurity D is 0.510ng, the detection limit of impurity E is 0.534ng, and the detection limit of atomoxetine hydrochloride is 0.439ng. The sensitivity is far superior to that of Chinese patent CN115561358A, and an ideal effect is achieved.

[0330] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A reverse phase high performance liquid chromatography method for one-test-multiple-evaluation detection of impurities C, D and E in atomoxetine hydrochloride oral solution, characterized in that: The chromatographic conditions include: the chromatographic column filler uses solid core particles bonded with a diphenyl stationary phase; the mobile phase includes a mobile phase A and a mobile phase B; the mobile phase A is composed of a sodium octane sulfonate solution and methanol, and the mobile phase B is methanol.

2. The method for detecting impurities C, D and E of atomoxetine hydrochloride oral solution by reverse phase high performance liquid chromatography according to claim 1, characterized in that: The chromatographic column is a Shim-pack velox Biphenyl chromatographic column.

3. The method for detecting impurities C, D and E of atomoxetine hydrochloride oral solution by reverse phase high performance liquid chromatography according to claim 2, characterized in that: The specification of the chromatographic column is 4.6×250 mm, and the particle size of the chromatographic column filler is 5 μm.

4. The method for detecting impurities C, D and E of atomoxetine hydrochloride oral solution by reverse phase high performance liquid chromatography according to claim 1, characterized in that: The elution method adopts the gradient elution method; the procedure of gradient elution is shown in the following table: In mobile phase A, the volume ratio of sodium octane sulfonate solution to methanol is 88-92:8-12; the pH of the sodium octane sulfonate solution is 2.4-2.

6.

5. The method for detecting impurities C, D and E of atomoxetine hydrochloride oral solution by reverse phase high performance liquid chromatography according to claim 4, characterized in that: The sodium octane sulfonate solution is prepared by the following method: dissolve anhydrous potassium dihydrogen phosphate in water at a ratio of 2.72 g: 1000 ml, adjust the pH to 2.4-2.6 with phosphoric acid, add sodium octane sulfonate in a ratio of 4.32 g: 1000 ml of water to dissolve, mix well and prepare.

6. The method for detecting impurities C, D and E of atomoxetine hydrochloride oral solution by reverse phase high performance liquid chromatography according to claim 5, characterized in that: The volume ratio of the sodium octane sulfonate solution to methanol is 90:10; the pH value of the sodium octane sulfonate solution is 2.

5.

7. The method for detecting impurities C, D and E of atomoxetine hydrochloride oral solution by reverse phase high performance liquid chromatography according to claim 1, characterized in that: An ultraviolet detector is used, and the detection wavelength is 200-225 nm; preferably, the detection wavelength is 205 nm.

8. The method for detecting impurities C, D and E of atomoxetine hydrochloride oral solution by reverse phase high performance liquid chromatography according to claim 1, characterized in that: The column temperature of the chromatographic column is 28°C to 32°C; preferably, the column temperature is 30°C.

9. The method for detecting impurities C, D and E of atomoxetine hydrochloride oral solution by reverse phase high performance liquid chromatography according to claim 1, characterized in that: The flow rate of the mobile phase is 0.9-1.1 ml / min; preferably, the flow rate is 1.0 ml / min.

10. A reverse phase high performance liquid chromatography method for one-detection-multiple-evaluation detection of impurities C, D, and E in atomoxetine hydrochloride oral solution according to any one of claims 1 to 9, characterized in that: The separation degree between the chromatographic peaks of atomoxetine hydrochloride, impurity C, impurity D and impurity E and the adjacent chromatographic peaks is ≥1.2; preferably, the separation degree is ≥1.5; The linear range of atomoxetine hydrochloride is 0.0658μg / ml~1.4040μg / ml, the linear range of impurity C is 0.0662μg / ml~0.4412μg / ml, the linear range of impurity D is 0.0764μg / ml~0.5096μg / ml, and the linear range of impurity E is 0.0801μg / ml~0.5341μg / ml.

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