Method for detecting related substances in olanzapine or olanzapine fluoxetine hydrochloride preparation

Abstract

The invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for detecting related substances in olanzapine or olanzapine fluoxetine hydrochloride preparations, which comprises the following steps: taking a sample to be detected, and detecting by high performance liquid chromatography under the following chromatographic conditions: a chromatographic column takes octyl silane bonded silica gel as a filler; a mobile phase A is prepared from a solution A and acetonitrile in a volume ratio of (52-54): (46-48), and a mobile phase B is prepared from a solution A and acetonitrile in a volume ratio of 30: 70. The detection method is good in system applicability, high in specificity and high in accuracy, the detection limit and the quantification limit meet the requirements of Chinese pharmacopoeia, the quality of the olanzapine or olanzapine fluoxetine hydrochloride preparation can be effectively monitored, then the quality safety of the olanzapine or olanzapine fluoxetine hydrochloride preparation is guaranteed, and the application prospect is wide.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for detecting related substances in olanzapine or olanzapine hydrochloride fluoxetine preparation. Background Art

[0002] Bipolar depression is a difficult point in clinical treatment. During the onset, there are often suicidal thoughts, and in severe cases, there may even be thoughts or behaviors of expansive suicide, posing a serious threat to the life safety of patients and others. Moreover, usually, the duration of depressive episodes is longer than that of manic episodes. Therefore, there is an urgent need for a rapid and effective treatment plan in psychiatric clinics.

[0003] Olanzapine hydrochloride fluoxetine capsules (trade name: ) was developed by Eli Lilly and Company and is the first compound preparation composed of the atypical antipsychotic drug olanzapine and SSRI fluoxetine hydrochloride approved by the US FDA for marketing. Clinically, it can be used to treat depressive episodes of bipolar disorder and can also be used to treat major depressive disorder. The active components of olanzapine hydrochloride fluoxetine capsules are olanzapine (2-methyl-10-(4-methyl-1-piperazinyl)-4H-thieno[2,3-b][1,5]benzodiazepine) and fluoxetine hydrochloride (({3-phenyl-3-[4-(trifluoromethyl)-phenoxy]propyl})methylamine hydrochloride or (3RS)-N-methyl-3-phenyl-3-[4-(trifluoromethyl)-phenoxy]-1-propanamine hydrochloride). Olanzapine is a selective serotonin reuptake inhibitor with good antidepressant effects and is currently widely used as a first-line drug in clinical practice; fluoxetine hydrochloride is a selective serotonin reuptake inhibitor. Through dopamine and serotonin type 2 antagonism, it can enhance the function of serotonin neurotransmitters, block serotonin reuptake, and can also increase the transmission of norepinephrine and dopamine, thereby exerting antidepressant, anti-anxiety, and anti-obsessive effects, and then significantly improving and controlling the psychotic symptoms of patients.

[0004] The types and contents of related substances are directly related to the quality and safety of drugs. The simultaneous detection and analysis of various related substances under the same chromatographic conditions are of great significance for the quality control of drug raw materials, the production process of preparations, and product quality.

[0005] At present, the known impurities of olanzapine and fluoxetine hydrochloride capsules are shown in Table 1 below. The detection method for the related substances of olanzapine and fluoxetine hydrochloride capsules is not included in the Chinese Pharmacopoeia. USP2023 discloses an HPLC method for detecting the related substances of olanzapine and fluoxetine hydrochloride capsules, which controls the quality of olanzapine impurity I and fluoxetine impurities I / II / III. By reproducing this method, the peaks of fluoxetine impurity I and olanzapine impurity I overlap, and this method cannot effectively separate them. Moreover, the applicability of different chromatographs is poor, resulting in difficulty in controlling the product quality during the preparation process and affecting the safety of drug use. Olanzapine impurity A, olanzapine impurity C, and olanzapine impurity D are process impurities of olanzapine. In capsule products, it is still very important to investigate and monitor them. How to effectively separate the related substances in capsule products and ensure the quality and safety of drugs is of crucial importance.

[0006] N - Formylfluoxetine is an impurity generated by fluoxetine and the excipient pregelatinized starch under high - temperature conditions. For capsule products, the control of degradation impurities is also very important. How to effectively detect known impurities, active ingredients, and degradation impurities is one of the urgent problems to be solved in drug quality control.

[0007] Table 1 Related Substances of Olanzapine and Olanzapine and Fluoxetine Hydrochloride Capsules

[0008] Summary of the Invention

[0009] In order to effectively control the quality of olanzapine and olanzapine and fluoxetine hydrochloride preparations, the present invention provides a method for detecting related substances in olanzapine or olanzapine and fluoxetine hydrochloride preparations. This detection method is convenient and has a wide applicability, and can effectively detect the related substances in olanzapine and olanzapine and fluoxetine hydrochloride preparations.

[0010] The method for detecting related substances in olanzapine or olanzapine and fluoxetine hydrochloride preparations of the present invention includes the following steps: Take the test sample and detect it by high - performance liquid chromatography. The chromatographic conditions are as follows: Chromatographic column: Octadecylsilyl silica gel bonded silica is used as the filler; Mobile phase: Solution A and acetonitrile with a volume ratio of 52 - 54∶46 - 48 are used as mobile phase A, and solution A and acetonitrile with a volume ratio of 30∶70 are used as mobile phase B; The gradient elution program is:

[0011]

[0012] The solution A is an aqueous solution containing disodium edetate, phosphoric acid, and sodium dodecyl sulfate with a pH value of 3.3 - 3.7.

[0013] Further, it includes the following steps:

[0014] a. Take the test sample, dissolve it with mobile phase A to obtain the test solution.

[0015] b. Take olanzapine and fluoxetine hydrochloride reference substances, dissolve them in mobile phase A to obtain the reference substance solution.

[0016] c. Take olanzapine, fluoxetine hydrochloride and related substance reference substances, dissolve them in mobile phase A to obtain the system suitability solution.

[0017] d. Detect the test solution, reference substance solution and system suitability solution by high performance liquid chromatography. Among them, the chromatographic column in the chromatographic conditions is Agilent ZORBAX SB-C8, 4.6 mm×250 mm, 5 μm; mobile phase A is a solution A and acetonitrile with a volume ratio of 54:46.

[0018] Furthermore, every 1000 ml of the solution A contains 37 mg of disodium edetate, 3.3 ml of phosphoric acid, and 8.7 g of sodium dodecyl sulfate; adjust the pH value with NaOH solution, preferably adjust the pH value with 50% NaOH solution.

[0019] Furthermore, the purity of the sodium dodecyl sulfate is 97%-100.4%.

[0020] Furthermore, in the test solution in step a), the concentration of olanzapine is 0.2 mg / mL, and the concentration of fluoxetine is 0.83-1.7 mg / mL.

[0021] Further, every 1 mL of the reference substance solution in step b) contains 0.4 μg-2.4 μg of olanzapine, preferably 2 μg; 2.1 μg-8.3 μg of fluoxetine, preferably 7 μg.

[0022] Furthermore, every 1 mL of the system suitability solution in step c) contains 0.1-1 mg of olanzapine, 0.1-1 mg of fluoxetine hydrochloride, and 1-5 μg each of fluoxetine impurity I, fluoxetine impurity II, fluoxetine impurity III and olanzapine impurity I.

[0023] Further, the chromatographic conditions in step d) further include a flow rate of 1-2 mL per minute; a column temperature of 30-40 °C; a sample chamber temperature of 2-8 °C; a detection wavelength of 210-220 nm; an injection volume of 20-80 μL.

[0024] Furthermore, the flow rate is 1.5 mL per minute; the column temperature is 35 °C; the sample chamber temperature is 5 °C; the detection wavelength is 215 nm; the injection volume is 50 μL.

[0025] Further, the related substances include: olanzapine impurity I, fluoxetine impurity I, fluoxetine impurity II, fluoxetine impurity III, N-formylfluoxetine, olanzapine impurity A, olanzapine impurity C, olanzapine impurity D.

[0026] The detection method for related substances in olanzapine or olanzapine hydrochloride fluoxetine preparation of the present invention can effectively separate 8 related substances, namely olanzapine impurity A, olanzapine impurity I, olanzapine impurity C, olanzapine impurity D, fluoxetine impurity I, fluoxetine impurity II, fluoxetine impurity III, and N-formylfluoxetine in olanzapine and olanzapine hydrochloride fluoxetine preparation through specific solvents and chromatographic conditions. By using the detection method of the present invention to detect these 8 impurities in olanzapine or olanzapine hydrochloride fluoxetine preparation, the limits of impurities in olanzapine or olanzapine hydrochloride fluoxetine preparation can be accurately controlled, and the determination results are stable, reliable, and highly specific.

[0027] The methodological verification shows that the detection method of the present invention has good system suitability, strong specificity, high accuracy, and the detection limit and quantification limit meet the requirements of the Chinese Pharmacopoeia. It can effectively monitor the quality of olanzapine or olanzapine hydrochloride fluoxetine preparation, and thus ensure the quality and safety of the drug olanzapine or olanzapine hydrochloride fluoxetine preparation, with broad application prospects.

[0028] Obviously, based on the above content of the present invention, according to the common general knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modification, substitution, or variation can be made.

[0029] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Brief Description of the Drawings

[0030] Figure 1 It is the chromatogram of the system suitability solution for Example 1;

[0031] Figure 2 It is the chromatogram of the test solution for Example 1;

[0032] Figure 3 It is the chromatogram of the spiked test solution for Example 1;

[0033] Figure 4 It is the chromatogram of the test solution for Example 2;

[0034] Figure 5 It is the chromatogram of the spiked test solution for Example 2;

[0035] Figure 6 It is the chromatogram of the spiked test solution for Example 3;

[0036] Figure 7 It is the chromatogram of the spiked test solution for Example 4;

[0037] Figure 8 It is the chromatogram of the system suitability solution for Example 5;

[0038] Figure 9 Chromatogram of the spiked mixed solution for Example 6;

[0039] Figure 10 Chromatogram of the spiked test solution for Example 7;

[0040] Figure 11 Chromatogram of N-formylfluoxetine solution;

[0041] Figure 12 Chromatogram of the test solution for Example 8;

[0042] Figure 13 Chromatogram of the system suitability solution for Comparative Example 1;

[0043] Figure 14 Chromatogram of the system suitability solution for Comparative Example 2;

[0044] Figure 15 Chromatogram of the system suitability solution for Comparative Example 3;

[0045] Figure 16 Chromatogram of the system suitability solution for Comparative Example 4;

[0046] Figure 17 Chromatogram of the system suitability solution for Comparative Example 5;

[0047] Figure 18 Chromatogram of the system suitability solution in the methodological verification;

[0048] Figure 19 Chromatogram of the sensitivity solution in the methodological verification;

[0049] Figure 20 Chromatogram of the reference solution in the methodological verification. Detailed implementation manners

[0050] In the following examples, all reagents and materials not specifically described are commercially available products.

[0051] The implementation results of the present invention are judged according to the following description:

[0052] For the system suitability requirements, in the chromatogram of the system suitability solution, the peaks should elute in the order of fluoxetine impurity I, olanzapine impurity I, fluoxetine impurity II, fluoxetine impurity III, olanzapine, and fluoxetine. The resolution between olanzapine impurity I and fluoxetine impurity I should be not less than 1.9; in the sensitivity solution, the S / N of the fluoxetine impurity I peak should be not less than 3; the tailing factors of the olanzapine and fluoxetine peaks in the system suitability solution and the reference solution should not exceed 1.8; in the reference solution, the relative standard deviations of the peak areas of olanzapine and fluoxetine should not exceed 2.0%.

[0053] If there are chromatographic peaks in the chromatogram of the test solution with the same retention time as the olanzapine impurity I peak in the chromatogram of the system suitability solution, calculate according to the external standard method based on the peak area of olanzapine in the chromatogram of the reference solution, and then multiply by 0.6. It shall not exceed 0.20% of the labeled amount of olanzapine; if there are other impurity peaks before the olanzapine peak and after the fluoxetine peak, calculate according to the external standard method based on the peak area of olanzapine in the chromatogram of the reference solution, and it shall not exceed 0.20% of the labeled amount of olanzapine; the total amount of impurities of olanzapine shall not exceed 1.5% of the labeled amount of olanzapine, and impurity peaks with a content less than 0.1% of the labeled amount of olanzapine shall be ignored. If there are chromatographic peaks in the chromatogram of the test solution with the same retention time as the fluoxetine impurity I, II, and III peaks in the chromatogram of the system suitability solution, calculate according to the external standard method based on the peak area of fluoxetine in the chromatogram of the reference solution, and then multiply by 1.3, 1.3, and 1.2 respectively. They shall not exceed 0.25% of the labeled amount of fluoxetine; if there are other impurity peaks between the olanzapine peak and the fluoxetine peak, calculate according to the external standard method based on the peak area of fluoxetine in the chromatogram of the reference solution, and it shall not exceed 0.25% of the labeled amount of fluoxetine. The total amount of impurities of fluoxetine shall not exceed 0.40% of the labeled amount of fluoxetine, and impurity peaks with a content less than 0.1% of the labeled amount of fluoxetine shall be ignored. Olanzapine impurity A, olanzapine impurity C, and olanzapine impurity D are process impurities of olanzapine. Calculated as unknown impurities, they shall not exceed 0.20% of the labeled amount of olanzapine; N-formyl fluoxetine, calculated as an unknown impurity of fluoxetine, shall not exceed 0.25% of the labeled amount of fluoxetine.

[0054] In the examples,

[0055] The preparation methods of the olanzapine impurity I stock solution, fluoxetine impurity I / II / III stock solution, and mixed impurity stock solution involved in olanzapine hydrochloride and fluoxetine capsules (6 / 25 mg specification) are as follows:

[0056] A. Olanzapine impurity I stock solution: Take about 20 mg of olanzapine impurity I, accurately weigh it, place it in a 100 ml volumetric flask, dissolve it with acetonitrile and dilute to the mark, and shake well. (0.2 mg / ml)

[0057] B. Fluoxetine impurity I / II / III stock solution: Weigh 20.75 mg each of fluoxetine impurity I, II, and III, place them in 20 ml volumetric flasks respectively, dissolve them with acetonitrile and dilute to the mark, and shake well. (1.0375 mg / ml)

[0058] C. Mixed impurity stock solution: Transfer 5 ml each of the olanzapine impurity I stock solution, fluoxetine impurity I, II, and III stock solutions to the same 50 ml volumetric flask, dilute to the mark with mobile phase A, and shake well. (Olanzapine impurity I: 20 μg / ml; Fluoxetine impurity I / II / III: 103.75 μg / ml each)

[0059] The preparation methods of olanzapine impurity I stock solution and fluoxetine impurities I / II / III stock solution involved in olanzapine hydrochloride fluoxetine capsules (3 / 25 mg specification) are as follows:

[0060] A. Olanzapine impurity I stock solution: Take about 20 mg of olanzapine impurity I, accurately weigh it, place it in a 100 ml volumetric flask, dissolve it with acetonitrile and dilute to the mark, shake well; then measure 1 ml and place it in a 10 ml volumetric flask, dilute it to the mark with mobile phase A, and shake well. (0.02 mg / ml)

[0061] B. Fluoxetine impurities I / II / III stock solution: Weigh 20.9 mg of fluoxetine impurity I, II, and III respectively, place them in 20 ml volumetric flasks respectively, dissolve them with acetonitrile and dilute to the mark, shake well; then measure 1 ml of each and place them in 10 ml volumetric flasks respectively, dilute them to the mark with mobile phase A, and shake well. (0.1045 mg / ml)

[0062] Olanzapine impurities A / C / D stock solution: Take about 2 mg of olanzapine impurity A, impurity C, and impurity D respectively, place them in 10 ml volumetric flasks respectively, dissolve them with acetonitrile and dilute to the mark, shake well; measure 1 ml of the olanzapine impurities A / C / D solutions respectively and place them in 10 ml volumetric flasks respectively, dilute them to the mark with mobile phase A, and shake well. (Olanzapine A / C / D: 0.02 mg / ml each)

[0063] Example 1 Determination of related substances in olanzapine hydrochloride fluoxetine capsules

[0064] Accurately prepare the system suitability solution, test solution and reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0065] The test solution is freshly prepared before use. Take an appropriate amount of the content of olanzapine hydrochloride fluoxetine capsules (6 / 25 mg specification), place it in a 50 ml volumetric flask, add about 35 ml of mobile phase A, shake for 5 minutes, then dilute to the mark with mobile phase A, shake well, filter or centrifuge to prepare a solution containing 0.2 mg of olanzapine and 0.83 mg of fluoxetine per 1 ml.

[0066] Reference solution Take appropriate amounts of olanzapine reference substance and fluoxetine hydrochloride reference substance, accurately weigh them, dissolve and dilute with mobile phase A to prepare a solution containing 2 μg of olanzapine and 7 μg of fluoxetine per 1 ml.

[0067] System suitability solution Take appropriate amounts of olanzapine, fluoxetine hydrochloride, fluoxetine impurity I, fluoxetine impurity II, fluoxetine impurity III and olanzapine impurity I reference substances, dissolve and dilute with mobile phase A to prepare a solution containing 0.1 mg of olanzapine, 0.11 mg of fluoxetine hydrochloride, and 2 μg of each of fluoxetine impurity I, fluoxetine impurity II, fluoxetine impurity III and olanzapine impurity I per 1 ml.

[0068] Sensitivity solution: Accurately measure 3 ml of the system suitability solution, transfer it to a 100-ml volumetric flask, dilute it to the mark with mobile phase A to prepare a solution containing 3 μg of olanzapine, 3 μg of fluoxetine hydrochloride, 0.06 μg each of fluoxetine impurity I, fluoxetine impurity II, fluoxetine impurity III, and olanzapine impurity I per 1 ml.

[0069] Spiked test solution: Take the contents of olanzapine and fluoxetine hydrochloride capsules (6 / 25 mg specification: 383.3 mg), accurately weigh, transfer to a 50-ml volumetric flask, add about 35 ml of mobile phase A, shake for 5 minutes, accurately transfer 1.0 ml of the mixed impurity stock solution, then dilute to the mark with mobile phase A, shake well, filter through a 0.45-μm filter membrane, and take the subsequent filtrate. (Olanzapine 0.2 mg, fluoxetine 0.83 mg, olanzapine impurity I 0.4 μg / ml, fluoxetine impurities I / II / III 2.075 μg / ml each)

[0070] Chromatographic conditions: Use octadecylsilane-bonded silica gel as the filler (Agilent ZORBAX SB-C8, 4.6 mm × 250 mm, 5 μm); prepare solution A with sodium dodecyl sulfate with a purity of 100.4%; use solution A - acetonitrile (54:46) as mobile phase A and solution A - acetonitrile (30:70) as mobile phase B, and perform gradient elution as follows: flow rate is 1.5 ml per minute; column temperature is 35 °C; sample chamber temperature is ica 5 °C; detection wavelength is 215 nm; injection volume is 50 μl.

[0071]

[0072] The purity of the sodium dodecyl sulfate is determined by ion exchange titration.

[0073] The preparation method of solution A is as follows: Take 37 mg of disodium edetate (EDTA-2Na·2H2O), dissolve it in 1000 ml of water, add 3.3 ml of phosphoric acid, then adjust the pH to 3.5 with 50% NaOH solution, add 8.7 g of sodium dodecyl sulfate with a purity of 100.4%, and stir to dissolve to obtain solution A.

[0074] Figure 1 It is the chromatogram of the system suitability solution in Example 1. It can be seen from the figure that the detection method of the present invention can well separate and locate olanzapine, fluoxetine, olanzapine impurity I, fluoxetine impurity I, fluoxetine impurity II, and fluoxetine impurity III; Figure 2 It is the liquid chromatogram of the test solution. It can be known from the figure that fluoxetine impurities I, II, and III are not detected in the olanzapine and fluoxetine hydrochloride capsules; Figure 3It is the liquid chromatogram of the spiked test solution. As can be seen from the figure, this detection method can well separate and locate olanzapine, fluoxetine, olanzapine impurity I, fluoxetine impurity I, fluoxetine impurity II, and fluoxetine impurity III in olanzapine hydrochloride and fluoxetine capsules.

[0075] Example 2: Determination of related substances in olanzapine hydrochloride and fluoxetine capsules

[0076] The difference from Example 1 is as follows:

[0077] Test solution: freshly prepared before use. Take the contents of olanzapine hydrochloride and fluoxetine capsules (3 / 25 mg specification: about 766.7 mg), place them in a 50 ml volumetric flask, add about 35 ml of mobile phase A, shake for 5 minutes, then dilute to the mark with mobile phase A, shake well, filter or centrifuge to obtain a solution containing 0.2 mg of olanzapine and 1.67 mg of fluoxetine per 1 ml.

[0078] Spiked test solution: Take the contents of olanzapine hydrochloride and fluoxetine capsules (3 / 25 mg specification: about 766.7 mg), accurately weigh, place them in a 50 ml volumetric flask, add about 35 ml of mobile phase A, shake for 5 minutes, accurately transfer 1.0 ml of olanzapine impurity I stock solution and 2.0 ml of fluoxetine impurity I / II / III stock solution, then dilute to the mark with mobile phase A, shake well, filter through a 0.45 μm filter membrane, and take the subsequent filtrate. (0.2 mg / ml of olanzapine, 1.67 mg / ml of fluoxetine, 0.4 μg / ml of olanzapine impurity I; 4.18 μg / ml each of fluoxetine impurity I, fluoxetine impurity II, and fluoxetine impurity III)

[0079] Figure 4 It is the chromatogram of the test solution in Example 2. As can be seen from the figure, the detection method of the present invention can effectively separate olanzapine and fluoxetine in the preparation, and is applicable to the detection of related substances in olanzapine hydrochloride and fluoxetine capsules of different specifications; Figure 5 It is the chromatogram of the spiked test solution. As can be seen from the figure, the detection method of the present invention can effectively separate olanzapine, fluoxetine, olanzapine impurity I, fluoxetine impurity I, fluoxetine impurity II, and fluoxetine impurity III in the spiked test solution.

[0080] Example 3: Determination of related substances in olanzapine hydrochloride and fluoxetine capsules

[0081] The difference from Example 1 is as follows:

[0082] The preparation method of solution A is as follows: Take 37 mg of disodium edetate (EDTA-2Na·2H2O), dissolve it in 1000 ml of water, add 3.3 ml of phosphoric acid, then adjust the pH to 3.3 with 50% NaOH solution, add 8.7 g of sodium dodecyl sulfate with a purity of 100.4%, and stir to dissolve to obtain solution A.

[0083] Figure 6 Chromatogram of the spiked test sample solution for Example 3. As can be seen from the figure, the detection method of the present invention can effectively separate olanzapine, fluoxetine, olanzapine impurity I, fluoxetine impurity I, fluoxetine impurity II, and fluoxetine impurity III in the test sample solution.

[0084] Determination of related substances in olanzapine hydrochloride and fluoxetine capsules in Example 4

[0085] The difference from Example 1 is as follows:

[0086] The preparation method of solution A is as follows: Take 37 mg of disodium edetate (EDTA-2Na·2H2O), dissolve it in 1000 ml of water, add 3.3 ml of phosphoric acid, then adjust the pH to 3.7 with 50% NaOH solution, add 8.7 g of sodium dodecyl sulfate with a purity of 100.4%, and stir to dissolve to obtain solution A.

[0087] Figure 7 Chromatogram of the spiked test sample solution for Example 4. As can be seen from the figure, the detection method of the present invention can effectively separate olanzapine, fluoxetine, olanzapine impurity I, fluoxetine impurity I, fluoxetine impurity II, and fluoxetine impurity III in the test sample solution.

[0088] Determination of related substances in olanzapine hydrochloride and fluoxetine capsules in Example 5

[0089] The difference from Example 1 is as follows:

[0090] The preparation method of solution A is as follows: Take 37 mg of disodium edetate (EDTA-2Na·2H2O), dissolve it in 1000 ml of water, add 3.3 ml of phosphoric acid, then adjust the pH to 3.5 with 50% NaOH solution, add 8.7 g of sodium dodecyl sulfate with a purity of 100.0%, and stir to dissolve to obtain solution A.

[0091] Figure 8 Chromatogram of the system suitability solution for Example 5. As can be seen from the figure, the detection method of the present invention can effectively separate olanzapine, olanzapine impurity I, fluoxetine, fluoxetine impurity I, fluoxetine impurity II, and fluoxetine impurity III.

[0092] Determination of related substances in olanzapine hydrochloride and fluoxetine capsules in Example 6

[0093] The difference from Example 1 is that a spiked mixed solution is used instead of the spiked test sample solution for detection. The specific preparation method of the spiked mixed solution is as follows:

[0094] Spiked mixed solution: Take about 10 mg of olanzapine raw material and about 46.5 mg of fluoxetine hydrochloride raw material, accurately weigh, place in the same 50 ml volumetric flask, add about 35 ml of mobile phase A, shake for 5 minutes, then accurately add 1 ml of mixed impurity stock solution, 1 ml each of olanzapine impurity A / C / D stock solutions, dilute to the mark with mobile phase A, and shake well. (Olanzapine 0.2 mg / ml, fluoxetine hydrochloride calculated as fluoxetine 0.83 mg / ml, olanzapine impurity A, olanzapine impurity I, olanzapine impurity C, olanzapine impurity D each 0.4 μg / ml, fluoxetine impurity I / II / III each 2.075 μg / ml)

[0095] Figure 9 For the chromatogram of the spiked mixed solution in Example 6, from Figure 9 it can be seen that the detection method of the present invention can simultaneously detect multiple substances in the analyte, and olanzapine, olanzapine impurity I, olanzapine impurity A, olanzapine impurity C, olanzapine impurity D, fluoxetine, fluoxetine impurity I, fluoxetine impurity II, and fluoxetine impurity III in the analyte can be effectively separated.

[0096] Determination of related substances in olanzapine and fluoxetine hydrochloride capsules in Example 7

[0097] The difference from Example 1 is that:

[0098] The mobile phase A is: Solution A - acetonitrile (52:48).

[0099] Figure 10 For the chromatogram of the spiked test solution in Example 7, it can be seen from the figure that the detection method of the present invention can effectively separate olanzapine, fluoxetine, olanzapine impurity I, fluoxetine impurity I, fluoxetine impurity II, and fluoxetine impurity III in the spiked test solution.

[0100] Determination of related substances in olanzapine and fluoxetine hydrochloride capsules in Example 8

[0101] The difference from Example 1 is that: The olanzapine and fluoxetine hydrochloride capsules are placed at 60 °C for degradation for 30 days, and the degraded content is taken as the test sample. The test sample solution of this example is prepared according to the preparation method of the test sample solution in Example 1 and detected.

[0102] Preparation of N - formylfluoxetine solution: Take about 1.5 mg of N - formylfluoxetine reference substance, place in a 10 ml volumetric flask, dissolve with acetonitrile and dilute to the mark, and shake well to obtain the stock solution. Accurately pipette 1 ml of the stock solution, place in a 10 ml volumetric flask, and then dilute to the mark with mobile phase A, and shake well. (15 μg / ml)

[0103] Figure 11 For the chromatogram of N - formylfluoxetine solution, Figure 12This is the chromatogram of the test solution. As can be seen from the figure, new impurities have been generated in the degraded content. Through process tests and comparison with the reference of N-formylfluoxetine solution, it is determined that during the high-temperature degradation process of olanzapine and fluoxetine hydrochloride capsules, fluoxetine hydrochloride reacts with pregelatinized starch to generate the degradation impurity N-formylfluoxetine. The detection method of the present invention can effectively detect the degradation impurity N-formylfluoxetine. From Figure 9 , Figure 11 and Figure 12 it can be seen that the related substances method of the present invention can effectively separate olanzapine, olanzapine impurity I, olanzapine impurity A, olanzapine impurity C, olanzapine impurity D, fluoxetine, fluoxetine impurity I, fluoxetine impurity II, fluoxetine impurity III and N-formylfluoxetine.

[0104] Comparative Example 1

[0105] The difference from Example 1 is that: the mobile phase and chromatographic parameters of the USP2023 method are adopted, specifically:

[0106] Mobile phase: Buffer solution (weigh 37 mg of disodium edetate (EDTA-2Na·2H2O), dissolve it in 1000 ml of water, add 3.3 ml of phosphoric acid, and then adjust the pH to 2.5 with 50% NaOH solution. Weigh 8.7 g of sodium dodecyl sulfate, dissolve it in the above solution, shake well, and filter)-acetonitrile = 50:50

[0107] Chromatographic conditions

[0108] Chromatographic column: Agilent ZORBAX SB-C8, 4.6 mm × 250 mm, 5 μm;

[0109] Column temperature: 35 °C; Flow rate: 1.5 ml / min; Detection wavelength: 215 nm; Injection volume: 50 μl; Collection time: 45 minutes.

[0110] Figure 13 This is the chromatogram of the system suitability solution of Comparative Example 1. As can be seen from the figure, the resolution of fluoxetine impurity II and fluoxetine impurity III is 1.2 < 1.5, and baseline separation is not achieved, not meeting the requirements.

[0111] Comparative Example 2

[0112] The difference from Example 1 is that:

[0113] Chromatographic conditions: Octadecylsilyl silica gel bonded silica was used as the filler (Agilent ZORBAX SB-C8, 4.6 mm × 250 mm, 5 μm); Solution A was prepared with sodium dodecyl sulfate with a purity of 100.4%; The pH of Solution A was 2.5. Solution A was used as mobile phase A, and acetonitrile was used as mobile phase B. Gradient elution was carried out as follows: The flow rate was 1.5 ml per minute; The column temperature was 35 °C; The sample chamber temperature was 5 °C; The detection wavelength was 215 nm; The injection volume was 50 μl.

[0114]

[0115] System suitability solution: Appropriate amounts of olanzapine, fluoxetine hydrochloride, fluoxetine impurity I, fluoxetine impurity II, fluoxetine impurity III, olanzapine impurity I, and olanzapine A / C / D reference substances were taken, dissolved and diluted with mobile phase A to prepare a solution containing 0.1 mg of olanzapine, 0.11 mg of fluoxetine hydrochloride, and 2 μg each of fluoxetine impurity I, fluoxetine impurity II, fluoxetine impurity III, and each impurity of olanzapine per 1 ml.

[0116] Figure 14 It is the chromatogram of the system suitability solution for Comparative Example 2. As can be seen from the figure, the peaks of fluoxetine impurity I and olanzapine impurity I overlap, not meeting the requirements; At the same time, olanzapine impurity C was not detected.

[0117] Comparative Example 3

[0118] The difference from Example 1 is that:

[0119] Chromatographic conditions: Octadecylsilyl silica gel bonded silica was used as the filler (Agilent ZORBAX SB-C8, 4.6 mm × 250 mm, 5 μm); Solution A was prepared with sodium dodecyl sulfate with a purity of 100.4%; The pH of Solution A was 3.5. Solution A:acetonitrile = 50:50 was used as the mobile phase; The flow rate was 1.5 ml per minute; The column temperature was 35 °C; The sample chamber temperature was 5 °C; The detection wavelength was 215 nm; The injection volume was 50 μl.

[0120] The preparation method of the system suitability solution was the same as that of Comparative Example 2.

[0121] Figure 15 It is the chromatogram of the system suitability solution for Comparative Example 3. As can be seen from the figure, the peaks of fluoxetine impurity II and fluoxetine impurity III overlap, not meeting the requirements.

[0122] Comparative Example 4

[0123] The difference from Example 1 is that: The purity of sodium dodecyl sulfate was 73.10%.

[0124] Figure 16It is the chromatogram of the system suitability solution for Comparative Example 4. As can be seen from the figure, when the purity of sodium dodecyl sulfate used is 73.10%, the peaks of Ola-Imp I and Flu-Imp I in the system suitability solution overlap, not meeting the requirements.

[0125] Comparative Example 5

[0126] The difference from Example 1 is that:

[0127] Isocratic elution is adopted; the mobile phase ratio is 52:48. The system suitability solution is the same as that in Comparative Example 2.

[0128] Figure 17 It is the chromatogram of the system suitability solution. It can be seen that when isocratic elution is adopted and the mobile phase ratio is solution A - acetonitrile (52:48) 52:48, the elution times of the main peaks of olanzapine and fluoxetine hydrochloride are within 25 minutes, but the resolution of Ola-Imp I and Flu-Imp I is 1.18, not meeting the requirements; at the same time, olanzapine impurity C is not detected.

[0129] Experimental Example 1 Methodological Verification

[0130] The method shown in Example 1 above was subjected to methodological verification:

[0131] 1. System Suitability

[0132] Olanzapine reference stock solution: Take about 20 mg of olanzapine reference substance, accurately weigh it, place it in a 100 ml volumetric flask, dissolve it with acetonitrile and dilute it to the mark, and shake well. (Olanzapine: 0.2 mg / ml)

[0133] Fluoxetine hydrochloride reference stock solution: Take about 20 mg of fluoxetine hydrochloride reference substance, accurately weigh it, place it in a 25 ml volumetric flask, dissolve it with acetonitrile and dilute it to the mark, and shake well. (Fluoxetine hydrochloride: 0.8 mg / ml)

[0134] Reference solution: Accurately measure 1 ml each of the olanzapine reference stock solution and the fluoxetine hydrochloride reference stock solution, place them in the same 100 ml volumetric flask, dilute it to the mark with mobile phase A, and shake well. (Olanzapine: 2 μg / ml, Fluoxetine hydrochloride: 8 μg / ml)

[0135] Olanzapine impurity I stock solution: Take about 20 mg of olanzapine impurity I, accurately weigh it, place it in a 100 ml volumetric flask, dissolve it with acetonitrile and dilute it to the mark, and shake well. (0.2 mg / ml)

[0136] Fluoxetine impurity I / II / III stock solutions: Weigh 20.75 mg each of fluoxetine impurity I, fluoxetine impurity II, and fluoxetine impurity III, place them in 20 ml volumetric flasks respectively, dissolve them with acetonitrile and dilute them to the mark, and shake well.

[0137] (1.0375 mg / ml)

[0138] Fluoxetine impurity I / II / III positioning solution: Measure 1 ml of each of the stock solutions of fluoxetine impurity I / II / III, transfer them into 10-ml volumetric flasks respectively, dilute to the mark with mobile phase A, and shake well. (103.75 μg / ml, 50-fold limit concentration)

[0139] System suitability solution: Weigh accurately 5 mg of olanzapine reference substance and 5.5 mg of fluoxetine hydrochloride reference substance, transfer them into the same 50-ml volumetric flask. Then accurately measure 0.5 ml of the stock solution of olanzapine impurity I and 1 ml of each of the fluoxetine impurity I / II / III positioning solutions, dissolve with mobile phase A and dilute to the mark, and shake well. (Olanzapine: 0.1 mg / ml, fluoxetine hydrochloride: 0.11 mg / ml, olanzapine impurity I and fluoxetine impurity I / II / III: 0.002 mg / ml each)

[0140] Sensitivity solution: Accurately measure 1.5 ml of the system suitability solution, transfer it into a 50-ml volumetric flask, dilute to the mark with mobile phase A, and shake well. (Olanzapine: 3 μg / ml, fluoxetine hydrochloride: 3 μg / ml, olanzapine impurity I and fluoxetine impurity I / II / III: 0.06 μg / ml each)

[0141] Assay method: Respectively take 50 μl of the reference solution, the system suitability solution and the sensitivity solution, inject them into the liquid chromatograph, and record the chromatogram.

[0142] System suitability requirements: In the chromatogram of the system suitability solution, the resolution between fluoxetine impurity I and olanzapine impurity I should be not less than 1.9; in the chromatogram of the sensitivity solution, the S / N of the fluoxetine impurity I peak should be not less than 3; the tailing factors of the olanzapine and fluoxetine peaks in the system suitability solution and the reference solution should not exceed 1.8; when the reference solution is injected continuously for 6 times, the RSD of the retention times of the olanzapine and fluoxetine peaks should not exceed 1.0%, and the RSD of the peak areas of the olanzapine and fluoxetine peaks should not exceed 2.0%.

[0143] Figure 18 For the chromatogram of the system suitability solution, the resolution between fluoxetine impurity I and olanzapine impurity I is 2.67, which is greater than 1.9; Figure 19 For the chromatogram of the sensitivity solution, the S / N of the fluoxetine impurity I peak is 26.28, which is greater than 3; the maximum tailing factors of the olanzapine and fluoxetine peaks in the system suitability solution and the reference solution ( Figure 20 ) are 1.73, both of which do not exceed 1.8; when the reference solution is injected continuously for 6 times, the RSD of the retention times of the olanzapine and fluoxetine peaks are 0.1% and 0.0% respectively, both of which are less than 1.0%, and the RSD of the peak areas of the olanzapine and fluoxetine peaks are 0.5% and 0.1% respectively, both of which are less than 2.0%; the system suitability meets the requirements.

[0144] 2. Specificity

[0145] Blank solvent: Mobile phase A

[0146] Blank excipient solution: Weigh about 332 mg of blank excipient, place it in a 50-ml volumetric flask, add about 35 ml of mobile phase A, shake for 5 minutes, then dilute to the mark with mobile phase A, shake well, filter through a 0.45-μm filter membrane, and take the subsequent filtrate.

[0147] Olanzapine positioning solution: Take about 20 mg of olanzapine raw material, accurately weigh it, place it in a 100-ml volumetric flask, dissolve and dilute to the mark with mobile phase A, and shake well. (0.2 mg / ml)

[0148] Fluoxetine hydrochloride positioning solution: Take about 18.6 mg of fluoxetine hydrochloride raw material, place it in a 20-ml volumetric flask, dissolve and dilute to the mark with mobile phase A, and shake well. (Calculated as fluoxetine, 0.83 mg / ml)

[0149] Olanzapine impurity I stock solution: Take about 20 mg of olanzapine impurity I, accurately weigh it, place it in a 100-ml volumetric flask, dissolve and dilute to the mark with acetonitrile, and shake well. (0.2 mg / ml)

[0150] Olanzapine impurities A, C, D stock solution: Take about 2 mg of each of olanzapine impurity A, impurity C, and impurity D, place them in separate 10-ml volumetric flasks, dissolve and dilute to the mark with acetonitrile, and shake well. (0.2 mg / ml)

[0151] Olanzapine impurity positioning solutions: Measure 1 ml of each olanzapine impurity stock solution respectively, place them in separate 10-ml volumetric flasks, dilute to the mark with mobile phase A, and shake well. (20 μg / ml, 50 times the limit concentration)

[0152] Fluoxetine impurities I / II / III positioning solution: Weigh 20.75 mg of each of fluoxetine impurities I, II, and III respectively, place them in separate 20-ml volumetric flasks, dissolve and dilute to the mark with acetonitrile, and shake well; measure 1 ml of each of the fluoxetine impurities I / II / III stock solutions respectively, place them in separate 10-ml volumetric flasks, dilute to the mark with mobile phase A, and shake well. (103.75 μg / ml, 50 times the limit concentration)

[0153] Test solution: Take about 383.3 mg of the content of this product (6 / 25 mg specification), accurately weigh it, place it in a 50-ml volumetric flask, add about 35 ml of mobile phase A, shake for 5 minutes, then dilute to the mark with mobile phase A, shake well, filter through a 0.45-μm filter membrane, and take the subsequent filtrate. (Ola: 0.2 mg / ml, Flu: 0.83 mg / ml)

[0154] Mixed solution: Weigh accurately about 10 mg of olanzapine raw material and about 46.5 mg of fluoxetine hydrochloride raw material, place them in the same 50-ml volumetric flask, add about 35 ml of mobile phase A, shake for 5 minutes, then accurately add 1 ml of olanzapine impurity A, impurity I, impurity C, and impurity D positioning solutions respectively, and 1 ml of fluoxetine impurity I, impurity II, and impurity III positioning solutions respectively, dilute to the mark with mobile phase A, and shake well. (Olanzapine 0.2 mg / ml, fluoxetine hydrochloride calculated as fluoxetine 0.83 mg / ml, olanzapine impurity A / I / C / D each 0.4 μg / ml, fluoxetine impurity I / II / III each 2.075 μg / ml)

[0155] Precisely measure 50 μl each of blank solvent, blank excipient solution, olanzapine and fluoxetine hydrochloride positioning solutions, olanzapine and fluoxetine impurity positioning solutions, test solution, and mixed solution, inject them into the liquid chromatograph respectively, and record the chromatograms; the results of interference and positioning tests are shown in Table 2 below.

[0156] The blank solvent and blank excipient should not interfere with the impurity determination; the resolution between the main peak and each known impurity and between each known impurity in the mixed solution should be not less than 1.5, and the resolution between the main peak and each impurity and between each impurity in the test solution should be not less than 1.5.

[0157] Table 2 Results of interference and positioning tests

[0158]

[0159]

[0160] The blank solvent and blank excipient do not interfere with the impurity determination; the minimum resolution between the main peak and each known impurity and between each known impurity in the mixed solution is 2.08, all greater than 1.5; no unknown impurities are detected in the test solution; the interference and impurity positioning tests all meet the requirements.

[0161] 3. Detection limit and quantitation limit

[0162] Olanzapine reference stock solution: As described above. (Olanzapine: 0.2 mg / ml)

[0163] Fluoxetine hydrochloride reference stock solution: Weigh accurately about 23.19 mg of fluoxetine hydrochloride reference substance, place it in a 20-ml volumetric flask, dissolve it with acetonitrile and dilute to the mark, and shake well. (Calculated as fluoxetine 1.0375 mg / ml)

[0164] Olanzapine impurity I stock solution: As described above. (0.2 mg / ml)

[0165] Fluoxetine impurity I, II, and III stock solutions: As described above. (1.0375 mg / ml)

[0166] Accurately measure an appropriate amount of the above stock solution, dilute it step by step, inject samples for detection in sequence, and use the injection concentration when the signal-to-noise ratio (S / N) is not less than 10 as the quantitation limit, and the injection concentration when the signal-to-noise ratio (S / N) is not less than 3 as the detection limit; Table 3 shows the test results of the detection limit and quantitation limit.

[0167] Inject the quantitation limit solution continuously for 6 times. The RSD of the retention time shall not exceed 1.0%, and the RSD of the peak area shall not be greater than 10%. The S / N of each substance peak meets the requirements.

[0168] Table 3 Test Results of Detection Limit and Quantitation Limit

[0169]

[0170]

[0171] The detection limit concentration of olanzapine is 0.0071 μg / ml, and the quantitation limit concentration is 0.0238 μg / ml. The quantitation limit solution is injected continuously for 6 times. The RSD of the retention time is 0.2%, and the RSD of the peak area is 6.2%; the detection limit concentration of olanzapine impurity I is 0.0035 μg / ml, and the quantitation limit concentration is 0.0118 μg / ml. The quantitation limit solution is injected continuously for 6 times. The RSD of the retention time is 0.1%, and the RSD of the peak area is 0.7%; the detection limit concentration of fluoxetine is 0.0093 μg / ml, and the quantitation limit concentration is 0.031 μg / ml. The quantitation limit solution is injected continuously for 6 times. The RSD of the retention time is 0.1%, and the RSD of the peak area is 8.6%; the detection limit concentration of fluoxetine impurity I is 0.0094 μg / ml, and the quantitation limit concentration is 0.0314 μg / ml. The quantitation limit solution is injected continuously for 6 times. The RSD of the retention time is 0.2%, and the RSD of the peak area is 1.2%; the detection limit concentration of fluoxetine impurity II is 0.0094 μg / ml, and the quantitation limit concentration is 0.0312 μg / ml. The quantitation limit solution is injected continuously for 6 times. The RSD of the retention time is 0.2%, and the RSD of the peak area is 1.5%; the detection limit concentration of fluoxetine impurity III is 0.0182 μg / ml, and the quantitation limit concentration is 0.0606 μg / ml. The quantitation limit solution is injected continuously for 6 times. The RSD of the retention time is 0.1%, and the RSD of the peak area is 2.6%; the S / N of each substance peak meets the requirements.

[0172] 4. Accuracy

[0173] Reference solution, system suitability solution, sensitivity solution: as described above.

[0174] Stock solution of olanzapine impurity I: as described above. (0.2 mg / ml)

[0175] Stock solutions of Fluoxetine Impurity I, II, and III: As described above. (1.0375 mg / ml)

[0176] Accuracy stock solution: Pipette 5 ml each of the stock solutions of Olanzapine Impurity I, Fluoxetine Impurity I, II, and III into the same 50-ml volumetric flask, dilute to the mark with Mobile Phase A, and mix well. (Olanzapine Impurity I: 20 μg / ml; Fluoxetine Impurity I / II / III: 103.75 μg / ml each)

[0177] Test solution: Weigh accurately 383.3 mg of the content of this product (6 / 25 mg specification), place it in a 50-ml volumetric flask, add about 35 ml of Mobile Phase A, shake for 5 minutes, then dilute to the mark with Mobile Phase A, mix well, filter through a 0.45-μm filter membrane, and take the subsequent filtrate.

[0178] Preparation of test solution for recovery test: Weigh accurately 383.3 mg of the content of this product (6 / 25 mg specification), place it in a 50-ml volumetric flask, and prepare 9 portions in the same way. Prepare the test solution for recovery test according to the following table, dissolve with Mobile Phase A and dilute to the mark, mix well, filter, and take the subsequent filtrate. Table 4 shows the accuracy test results.

[0179] Table 4 Accuracy Test Results

[0180]

[0181] Precisely measure 50 μl of each of the above solutions, inject them into the liquid chromatograph respectively, and record the chromatograms. For Olanzapine Impurity I at 50% limit concentration, 100% limit concentration, and 150% limit concentration, the recoveries in each test sample should be within the range of 80% - 125%, and the RSD values of each recovery should not exceed 6%; for Fluoxetine Impurity I, Impurity II, and Impurity III at 50% limit concentration, 100% limit concentration, and 150% limit concentration, the recoveries in each test sample should be within the range of 80% - 125%, and the RSD values of each recovery should not exceed 4%. Calculate the recovery using the following formula; Table 5 shows the recovery test results.

[0182]

[0183] Table 5 Recovery Test Results

[0184]

[0185]

[0186] At the 50% limit concentration, 100% limit concentration, and 150% limit concentration of olanzapine impurity I, the recoveries in each test sample were between 112.87% and 120.46%, and the RSD values of each recovery were 2.3%, less than 6%; for fluoxetine impurity I, impurity II, and impurity III at the 50% limit concentration, 100% limit concentration, and 150% limit concentration, the recovery of fluoxetine impurity I was between 95.66% and 98.03%, and the RSD value of each recovery was 1.0%, the recovery of fluoxetine impurity II was between 110.25% and 113.07%, and the RSD value of each recovery was 0.8%, the recovery of fluoxetine impurity III was between 92.35 and 94.33%, and the RSD value of each recovery was 0.7%. The RSD values of each recovery were all less than 4%; the accuracy test met the requirements.

[0187] As can be seen from the above, the detection method for related substances of the olanzapine and fluoxetine hydrochloride preparation of the present invention can effectively separate various impurities from the active ingredient substances, and the test is simple and convenient; the system suitability of the determination method is good, the specificity is strong, the accuracy is high, and the detection limit and quantification limit meet the requirements.

Claims

1. A method for detecting related substances in olanzapine or an olanzapine hydrochloride-fluoxetine preparation, characterized in that: It includes the following steps: Take the sample to be tested and detect it by high performance liquid chromatography. The chromatographic conditions are as follows: Chromatographic column: Octadecylsilyl silica gel bonded silica as the filler; Mobile phase: Solution A and acetonitrile with a volume ratio of 52 - 54∶46 - 48 as mobile phase A, and solution A and acetonitrile with a volume ratio of 30∶70 as mobile phase B; The gradient elution program is: The solution A is an aqueous solution containing disodium edetate, phosphoric acid and sodium dodecyl sulfate with a pH value of 3.3 - 3.

7.

2. The detection method according to claim 1, wherein: It includes the following steps: a. Take the sample to be tested and dissolve it with mobile phase A to obtain the test solution. b. Take olanzapine and fluoxetine hydrochloride reference substances and dissolve them with mobile phase A to obtain the reference solution. c. Take olanzapine, fluoxetine hydrochloride and related substance reference substances and dissolve them with mobile phase A to obtain the system suitability solution. d. Detect the test solution, reference solution and system suitability solution by high performance liquid chromatography. Among them, in the chromatographic conditions, the chromatographic column is Agilent ZORBAX SB - C8, 4.6mm×250mm, 5μm; Mobile phase A is solution A and acetonitrile with a volume ratio of 54∶46.

3. The detection method according to claim 1 or 2, characterized in that: Each 1000 ml of the solution A contains 37 mg of disodium edetate, 3.3 ml of phosphoric acid and 8.7 g of sodium dodecyl sulfate; Adjust the pH value with NaOH solution, preferably adjust the pH value with 50% NaOH solution.

4. The detection method according to claim 3, characterized in that: The purity of the sodium dodecyl sulfate is 97% - 100.4%.

5. The detection method according to claim 2, characterized in that: The concentration of olanzapine in the test solution in step a) is 0.2 mg / mL, and the concentration of fluoxetine is 0.83 - 1.7 mg / mL.

6. The detection method according to claim 2, wherein: Each 1 mL of the reference solution in step b) contains 0.4 μg - 2.4 μg of olanzapine, preferably 2 μg; 2.1 μg - 8.3 μg of fluoxetine, preferably 7 μg.

7. The detection method according to claim 2, characterized in that: Each 1 mL of the system suitability solution in step c) contains 0.1 - 1 mg of olanzapine, 0.1 - 1 mg of fluoxetine hydrochloride, and 1 - 5 μg each of fluoxetine impurity I, fluoxetine impurity II, fluoxetine impurity III and olanzapine impurity I.

8. The detection method according to claim 2, characterized in that: The chromatographic conditions in step d) also include a flow rate of 1 - 2 mL per minute; Column temperature 30 - 40°C; Sample chamber temperature 2 - 8°C; Detection wavelength 210 - 220 nm; Injection volume 20 - 80 μL.

9. The detection method according to claim 8, characterized in that: The flow rate is 1.5 mL per minute; Column temperature 35°C; Sample chamber temperature 5°C; Detection wavelength 215 nm; Injection volume 50 μL.

10. The detection method according to any one of claims 1-9, characterized in that: The related substances include: olanzapine impurity I, fluoxetine impurity I, fluoxetine impurity II, fluoxetine impurity III, N - formylfluoxetine, olanzapine impurity A, olanzapine impurity C, olanzapine impurity D.

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