Method for analyzing related substances of flomoxef sodium

Abstract

The invention relates to a method for analyzing related substances of flomoxef sodium, which is carried out by adopting a high performance liquid chromatograph, a chromatographic column taking octadecylsilane chemically bonded silica as a filler or a chromatographic column with equivalent efficiency is adopted, and a mixed solution of tetrabutylammonium bromide-phosphate buffer solution and methanol is adopted as a mobile phase. Elution is carried out by adopting a specific gradient, and the sample size is 3-20 [mu] g. The method provided by the invention can accurately and sensitively detect related substances with relatively low content and can more effectively separate impurities, so that the detection result is more accurate, and the effectiveness and safety of the flomoxef sodium medicine can be better ensured.

Description

Technical Field

[0001] The present invention belongs to the field of drug analysis and specifically relates to a method for analyzing related substances in fluoxetine sodium by high performance liquid chromatography. Technical Background

[0002] The chemical name of fluoxetine sodium is (6R,7R)-7-(2-((difluoromethyl)thio)acetylamino)-3-(((1-(2-hydroxyethyl)-1H-tetrazol-5-yl)thio)methyl)-7-methoxy-8-oxo-5-oxa-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate sodium, and has the following structural formula:

[0003]

[0004] Fluoxetine sodium is a white to light yellowish white powder or lyophilized product, odorless, highly soluble in water or methanol, soluble in ethanol or anhydrous ethanol, and almost insoluble in ether. It mainly has antibacterial effects on Staphylococci, Streptococci (except Enterococci), Streptococcus pneumoniae, Peptostreptococci, Catarrhal cocci, Neisseria gonorrhoeae, Escherichia coli, Klebsiella, Proteus, Haemophilus influenzae and Bacteroides.

[0005] Sodium fluoxetine for injection is a lyophilized product, which is suitable for the following diseases: moderate to severe infections caused by the above-mentioned bacteria: sepsis, infective endocarditis; secondary infections such as trauma and surgical wounds; pneumonia, peritonsillar abscess, empyema, bronchitis, bronchiectasis infection, acute exacerbation of chronic respiratory diseases; pyelitis, cystitis, prostatitis, gonococcal urethritis; cholecystitis, cholangitis; peritonitis, pelvic peritonitis, Douglas' abscess; uterine appendage inflammation, endometritis, pelvic inflammatory disease, parametritis, vestibular glanditis, etc.

[0006] Adopt high performance liquid chromatography to analyze the related substances of fluoxetine sodium, and the related method is recorded in the existing import registration standard. However, in the high performance liquid chromatography analysis method of the related substances of fluoxetine sodium for injection recorded in the existing import drug registration standard, there is interference peak interference at the position where the eluent gradient changes, for example, there are two interference peaks of fluoxetine sodium polymer 1 (its chemical structure see below) and fluoxetine sodium polymer 2 (its chemical structure see below) appear. In addition, the method can not effectively separate fluoxetine and fluoxetine sodium double bond displacement (specific structural formula see below). In addition, the injection volume specified in the method is 5 microliters, and the injection amount is low, which causes each impurity to be not easy to detect, and the impurity detection sensitivity is poor.

[0007] In pharmacy, impurities refer to substances in drugs that have no therapeutic effect or affect the stability and efficacy of drugs, or are even harmful to human health. In drug research, production, storage and clinical application, it is necessary to maintain the purity of drugs and reduce impurities in drugs to ensure the effectiveness and safety of drugs.

[0008] Therefore, it is necessary to develop a related substance analysis method for the fluoxetine sodium drug with better separation, higher detection sensitivity and no interference peaks at key positions, so as to better analyze and control the quality level of the fluoxetine sodium drug. Summary of the invention

[0009] In order to solve the above problems, the present invention provides an analytical method which can effectively separate the double bond displacement of fluoxetine and fluoxetine sodium and can sensitively and non-interferingly detect various related substances.

[0010] Specifically, the present invention provides an analysis method for related substances of fluoxetine sodium, which is carried out by high performance liquid chromatography, wherein a chromatographic column with octadecylsilane bonded silica gel as a filler or a chromatographic column with equivalent performance is used; a mobile phase A and a mobile phase B are used as eluents for gradient elution, wherein the mobile phase A is a mixture of tetrabutylammonium bromide-phosphate buffer and methanol in a volume ratio of (73-77): (23-27), and the mobile phase B is tetrabutylammonium bromide A mixed solution of ammonium salt-phosphate buffer and methanol in a ratio of (38-42):(58-62) by volume, wherein the tetrabutylammonium bromide salt-phosphate buffer is an aqueous solution composed of potassium dihydrogen phosphate, disodium hydrogen phosphate, and tetrabutylammonium bromide, the concentration of potassium dihydrogen phosphate is 6.90-6.98 g / l, the concentration of disodium hydrogen phosphate is 1.24-1.32 g / l, and the concentration of tetrabutylammonium bromide is 1.57-1.65 g / l; the elution gradient is shown in Table 1 below,

[0011] Table 1

[0012] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0-1 99-100 0-1 8-16 99-100 0-1 39-41 19-32 68-81 41-43 99-100 0-1 59-61 99-100 0-1

[0013] , using an ultraviolet detector with a detection wavelength of 245-247 nm, the test sample for measuring fluoxetine sodium is an aqueous solution with a concentration of 0.3-0.7 mg / ml.

[0014] The related substances detected by the method are as follows:

[0015] (0) Fluoxetine Sodium HTT

[0016] Chemical name: 1-(2-hydroxyethyl)-1H-tetrazolyl-5-sulfate sodium

[0017] Chemical formula: C3H5N4NaOS

[0018] Molecular weight: 168.15

[0019] Structural formula:

[0020]

[0021] (2) Fluoxetine Sodium DP-N

[0022] Chemical name: 4-hydroxy-3-((4-(2-hydroxyethyl)-5-thioxo-4,5-dihydro-1H-tetrazol-1-yl)methyl)-2-oxobutanoic acid

[0023] Chemical formula: C8H 12 N4O5S

[0024] Molecular weight: 276.27

[0025] Structural formula:

[0026]

[0027] (3) Fluoxetine Sodium DP-L

[0028] Chemical name: (2-difluoromethylthiazole)-N-((5aR,6R)-6-methoxy-1,7-dioxo-1,4,5a,6-tetrahydro-3H,7H-aza[2,1-b]furo[3,4-d][1,3]oxazin-6-yl)acetamide

[0029] Chemical formula: C 12 H 12 F2N2O6S

[0030] Molecular weight: 350.29

[0031] Structural formula:

[0032]

[0033] (4) Decarboxylation impurities of fluoxetine sodium DP-B

[0034] Chemical name: 2-((2-(difluoromethyl)thio)acetamido)(methoxy)methyl)-5-methylene-5,6-dihydro-2H-1,3-oxazine-4-carboxylic acid

[0035] Chemical formula: C 11 H 14 F2N2O5S

[0036] Molecular weight: 324.30

[0037] Structural formula:

[0038]

[0039] (5) Double bond displacement of fluoxetine sodium

[0040] Chemical name: (6R,7R)-7-(2-(difluoromethyl)thio)acetamido)-3-(((1-(2-hydroxyethyl)-1H-tetrazol-5-yl)thia)methyl)-7-methoxy-8-oxo-5-oxo-1-azabicyclo[4.2.0]oct-3-ene-2-carboxylic acid

[0041] Chemical formula: C 15 H 18 F2N6O7S2

[0042] Molecular weight: 496.46

[0043] Structural formula:

[0044]

[0045] (6) Fluoxetine sodium open ring lactone

[0046] Chemical Name: -2-(2-(difluoromethyl)thioacetylamino)-2-methoxy-2-( -7-Oxo-1,2,5,7-tetrahydro-4H-furan[3,4-d][1,3]oxazin-2-yl)acetic acid

[0047] Chemical formula: C 12 H 14 F2N2O7S

[0048] Molecular weight: 368.31

[0049] Structural formula:

[0050]

[0051] (7) Impurities in the ring opening of fluoxetine sodium

[0052] Chemical name: (2R)-2-[(3R)-3-carboxy-8,8-difluoro-5-oxo-4-aza-2-oxa-7-thiooct-3-yl]-5-({[1-(2-hydroxyethyl)-1,2,3,4-tetrazolyl-5-yl]thio}-m-ethyl}}-3H,6H,2H-1,3-oxazine-4-carboxylic acid

[0053] Chemical formula: C 15 H 20 F2N6O8S2

[0054] Molecular weight: 514.48

[0055] Structural formula:

[0056]

[0057] (8) Fluoxetine Sodium N-coupling

[0058] Chemical name: (6R,7R)-7-(2-(difluoromethyl)thia)acetamido)-3-((4-(2-hydroxyethyl)-5-thia-4,5-dihydro-1H-tetrazol-1-yl)methyl)-7-methoxy-8-oxo-5-oxa-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0059] Chemical formula: C 15 H 18 F2N6O7S2

[0060] Molecular weight: 496.46

[0061] Structural formula:

[0062]

[0063] (9) Fluoxetine Sodium Polymer Impurity 1

[0064] Chemical name: (6R,7R)-3-({[1-(2-{[(2R)-2-[(5R)-4-carboxy-5-{[4-(2-hydroxyethyl)-5-thiocarbonyl-4,5-dihydro-1H-tetrazol-1-yl)methyl)-5,6-dihydro-2H-1,3-oxazepin-2-yl)-2-([(difluoromethyl)thio]acetamido)-2-methoxyacetyl)oxy)ethyl)-1H-tetrazol-5-yl)thio)methyl)-7-([(difluoromethyl)thio]acetamido)-7-methoxy-8-oxo-5-oxa-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0065] Chemical formula: C 30 H 36 F4N 12 O 14 S4

[0066] Molecular weight: 992.92

[0067] Structural formula:

[0068]

[0069] (10) Fluoxetine Sodium Polymer Impurity 2

[0070] Chemical name: (6R,7R)-3-({[1-(2-{[(2R)-2-[(2R,5S)-4-carboxy-5-{[4-(2-hydroxyethyl)-5-thiocarbonyl-4,5-dihydro-1H-tetrazol-1-yl]methyl}-5,6-dihydro-2H-1,3-oxazepin-2-yl]-2-([(difluoromethyl)thio]acetylamino)-2-methoxyacetyl]oxy}ethyl)-1H-tetrazol-5-yl]thio}methyl)-7-([(difluoromethyl)thio]acetylamino)-7-methoxy-8-oxo-5-oxa-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0071] Chemical formula: C 30 H 36 F4N 12 O 14 S4

[0072] Molecular weight: 992.92

[0073] Structural formula:

[0074]

[0075] (11) Fluoxetine Sodium Polymer Impurity 3

[0076] Chemical name: (6S,7S)-3-((1-(2- -2-( -4-carboxy-5-methylene-5,6-dihydro-2H-1,3-oxazin-2-yl)-2-(2-(difluoromethyl)thioacetamido)-2-methoxyacetoxyethyl)-1H-tetrazol-5-yl)thia)methyl)-7-(2-(difluoromethyl)thia)acetamido)-7-methoxy-8-oxo-5-oxa-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

[0077] Chemical formula: C 27 H 30 F4N8O 13 S3

[0078] Molecular weight: 846.75

[0079] Structural formula:

[0080]

[0081] (12) Fluoxetine Sodium Polymer Impurity 4

[0082] Chemical name: (7aR,7R)-7-({2-[(difluoromethyl)thio]acetyl}amino)-3-[({1-[(5R)-5-(dihydroxymethyl)-10,10-difluoro-5-methoxy-4,7-dioxo-6-aza-3-oxa-9-thia-1-yl]-1,2,3,4-tetrazol-5-yl}thio)methyl]-7-methoxy-6-oxo-2,6,7,7a-tetrahydroaza[2,1-b][1,3]oxazine-4-carboxylic acid

[0083] Chemical formula: C 22 H 27 F4N7O 12 S3

[0084] Molecular weight: 753.67

[0085] Structural formula:

[0086]

[0087] On the other hand, the present invention provides a method for analyzing related substances of fluoxetine sodium, which is performed by high performance liquid chromatography, wherein the amount of fluoxetine sodium sample measured is 3-20 μg, preferably 5-15 μg, and more preferably 8-12 μg.

[0088] In another aspect, the present invention provides a method for analyzing related substances of fluoxetine sodium, which is performed by high performance liquid chromatography, wherein the column temperature of the chromatographic column is 36-45°C, preferably 38-42°C.

[0089] According to the present invention, the analysis method of related substances of fluoxetine sodium is determined in accordance with General Rules 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia.

[0090] The beneficial effects of the method of the present invention for determining the related substances of fluoxetine sodium are as follows:

[0091] (1) The method of the present invention can effectively separate and detect specific impurities such as fluoxetine sodium HTT body, fluoxetine sodium DP-N, fluoxetine sodium DP-L, fluoxetine sodium DP-B decarboxylation impurities, fluoxetine sodium open-ring lactone, fluoxetine sodium double bond displacement, fluoxetine sodium open-ring impurities, fluoxetine sodium-N-coupling, fluoxetine sodium polymer impurity 4, fluoxetine sodium polymer impurity 3, fluoxetine sodium polymer impurity 1, and fluoxetine sodium polymer impurity 2, and can help control the levels of these impurities, thereby better controlling the level of fluoxetine sodium active ingredients and the quality of fluoxetine sodium drugs, especially fluoxetine sodium for injection;

[0092] (2) Compared with the method included in the registration standard for imported drugs, the method of the present invention has no interference in the baseline during determination, and the separation degree between fluoxetine sodium and the double bond displacement of fluoxetine sodium is better, and the fluoxetine sodium polymer impurity 1 can be separated and accurately detected more effectively, thereby more accurately testing the quality of the fluoxetine sodium active ingredient or drug;

[0093] (3) The method of the present invention can detect a larger amount of samples, and the detection sensitivity of each impurity is better. Therefore, the impurity level obtained by detecting the active substance or drug of fluoxetine sodium by this method is higher, and the quality level of the active substance or drug of fluoxetine sodium controlled by this method is more accurate and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 It is a chromatogram of the sensitivity solution measured according to chromatographic condition 1 (the method of the present invention) in the system suitability test and the sensitivity test (Example D). In the figure, "floxacin" is "floxacin sodium" and 19.822 is the retention time.

[0095] Figure 2 It is a chromatogram of the sensitivity solution measured according to chromatographic condition 2 (the method included in the registration of imported drugs) in the system suitability test and the sensitivity test (Example D). In the figure, "floxacin" is "floxacin sodium" and 11.573 is the retention time.

[0096] Figure 3 This is a chromatogram of a mixed solution of related substances measured according to chromatographic condition 1 (the method of the present invention) in a separation test (Example E), in which the abbreviations of the names and retention times of the related substances are marked.

[0097] Figure 4 This is a chromatogram of a mixed solution of related substances measured according to chromatographic condition 2 (the method included in the registration of imported drugs) in the separation test (Example E), and the abbreviations and retention times of the related substances are marked in the figure.

[0098] Figure 5 The chromatogram is a chromatogram of a test solution of sodium fluoxetine for injection measured according to chromatographic condition 1 (the method of the present invention) in a related substance test experiment (Example G), in which the abbreviations of the names and retention times of the related substances are marked.

[0099] Figure 6 The chromatogram is a chromatogram of a reference solution of sodium fluoxetine for injection measured according to chromatographic condition 1 (the method of the present invention) in a related substance test experiment (Example G), in which the abbreviations of the names and retention times of the related substances are marked.

[0100] Figure 7The chromatogram is a chromatogram of a test solution of sodium fluoxetine for injection measured according to chromatographic condition 2 (the method included in the registration of imported drugs) in the related substance test experiment (Example G), and the abbreviation of the name and retention time of each related substance are marked in the figure.

[0101] Figure 8 The chromatogram is a chromatogram of a reference solution of sodium fluoxetine for injection measured according to chromatographic condition 2 (the method included in the registration of imported drugs) in the related substance test experiment (Example G), and the abbreviation of the name and retention time of each related substance are marked in the figure. DETAILED DESCRIPTION

[0102] The method conditions of the method for analyzing related substances of fluoxetine sodium according to the present invention are specifically described below.

[0103] According to the present invention, the method for analyzing related substances of fluoxetine sodium is carried out using a high performance liquid chromatograph.

[0104] In some preferred embodiments, in tetrabutylammonium bromide-phosphate buffer, the concentration of potassium dihydrogen phosphate is 6.92-6.96 g / l, more preferably 6.93-6.95 g / l; the concentration of sodium dihydrogen phosphate is 1.26-1.30 g / l, more preferably 1.27-1.29 g / l; the concentration of tetrabutylammonium bromide is 1.59-1.63 g / l, more preferably 1.60-1.62 g / l.

[0105] Tetrabutylammonium bromide-phosphate buffer can be prepared as follows: dissolve 6.93-6.95 g of potassium dihydrogen phosphate, 3.21-3.23 g of disodium hydrogen phosphate dodecahydrate, and 1.60-1.62 g of tetrabutylammonium bromide in water, and dilute with water to 1000 ml.

[0106] In some preferred embodiments, mobile phase A is a mixture of tetrabutylammonium bromide-phosphate buffer and methanol in a volume ratio of (74-76):(24-26), more preferably 75:25, and mobile phase B is a mixture of tetrabutylammonium bromide-phosphate buffer and methanol in a volume ratio of (39-41):(59-61), more preferably 40:60.

[0107] In some preferred embodiments, the chromatographic column filled with octadecylsilane bonded silica gel is preferably an Agilent 5HC-C18(2) chromatographic column purchased from Agilent, with a specification of 4.6 mm×250 mm 5 μm, or a chromatographic column with equivalent performance.

[0108] In some preferred embodiments, the detection wavelength is 246 nm.

[0109] In some preferred embodiments, the mobile phase flow rate is 0.5-1.5 ml / min, preferably 0.8-1.2 ml / min, more preferably 0.9-1.1 ml / min. In some embodiments, the mobile phase flow rate is 1.0 ml / min.

[0110] In some preferred embodiments, the amount of the fluoxetine sodium sample measured is 3-20 μg, preferably 5-15 μg, and more preferably 8-12 μg. Optionally, the amount of the fluoxetine sodium sample measured is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 μg. Compared with the existing method, the method of the present invention can detect a larger amount of fluoxetine sodium samples, and there is still a good separation between the impurity peaks.

[0111] In some preferred embodiments, the injection volume may be 15-30 μl, preferably 18-25 μl, more preferably 18-22 μl. In some embodiments, the injection volume may be 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 μl. In some preferred embodiments, the measured fluoxetine sodium sample is an aqueous solution with a concentration of 0.3-0.7 mg / ml, preferably 0.4-0.6 mg / ml, more preferably 0.5 mg / ml. During measurement, the fluoxetine sodium sample solution is injected into a high performance liquid chromatograph, and the chromatogram is recorded to complete the detection of fluoxetine sodium related substances.

[0112] In some preferred embodiments, the method uses a capture column. The capture column, also known as a capture column or a liquid ghost peak capture column, can be installed between the gradient mixer and the injector to adsorb and remove impurities with weaker polarity in the system, such as impurities in the mobile phase and impurities in the pipeline and mixer, thereby preventing the impurity peaks in the system from interfering with the target peak. The injected sample will not flow through the capture column. A capture column with a model number of 50 mm × 3.0 mm Ghost-Buster Column II of Yuexu Material Technology Co., Ltd. or a capture column with equivalent performance can be used.

[0113] In some embodiments, the column temperature may be 20-50° C., preferably 36-45° C., preferably 38-42° C. In some embodiments, the column temperature is 40° C. Compared with the existing method, the method of the present invention increases the column temperature of the chromatographic column, which is more beneficial for the separation of various impurities.

[0114] In some preferred embodiments, the elution gradient is shown in Table 2 below:

[0115] Table 2

[0116] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0-1 99-100 0-1 8-16 99-100 0-1 39-41 20-30 70-80 41-43 99-100 0-1 59-61 99-100 0-1

[0117] , more preferably the elution gradient in the following Table 3:

[0118] Table 3

[0119]

[0120]

[0121] , most preferably the elution gradient in Table 4 below:

[0122] Table 4

[0123] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 15 100 0 40 20 80 42 100 0 60 100 0 .

[0124] According to the present invention, by adopting the above elution gradient, each impurity peak is less affected by the baseline, and all impurity peaks can be accurately detected.

[0125] In some preferred embodiments of the method for analyzing the related substances of fluoxetine sodium, the column temperature is 20-50°C, preferably 36-45°C, preferably 38-42°C, the amount of the fluoxetine sodium sample measured is 3-20 μg, preferably 5-15 μg, more preferably 8-12 μg, and the elution gradient is as shown in Table 1 above, preferably the elution gradient is as shown in Table 2 above, and more preferably the elution gradient is as shown in Table 3 above. The above conditions are combined with other conditions such as the above-mentioned chromatographic column, mobile phase, detection wavelength, etc., and work synergistically to make the detection sensitivity higher, the baseline fluctuation smaller, the detection of impurity peaks more, the peak of fluoxetine sodium and the peak of fluoxetine sodium double bond displacement can be better separated, so that the content of fluoxetine sodium double bond displacement can be more accurately detected, and the result detection is more accurate at the same time. These effects were verified in the following experiments.

[0126] In some embodiments, the content of each related substance in fluoxetine sodium is determined by a self-control method. Specifically, a reference solution of fluoxetine sodium of 2-10 μg / ml can be prepared. During the determination, the fluoxetine sodium test sample and the reference solution of fluoxetine sodium are respectively injected into a high performance liquid chromatograph, and the determination is performed using the method of the present invention. The content of each related substance is calculated based on the peak area of ​​each related substance in the fluoxetine sodium test sample, the peak area of ​​the fluoxetine sodium reference solution, and the concentration of the fluoxetine sodium reference solution.

[0127] The sodium fluoxetine mentioned in the present application may be the active ingredient of sodium fluoxetine or a sodium fluoxetine preparation. The method of the present invention is particularly suitable for detecting and analyzing the content level of related substances in sodium fluoxetine for injection.

[0128] The present invention is further described below in conjunction with specific examples. In the following examples, various processes and methods not described in detail are conventional methods known in the art, and the reagents used without indicating the source and specifications are all commercially available analytical or chromatographically pure.

[0129] Example

[0130] In the examples, sodium fluoxetine for injection was used as the experimental subject.

[0131] A. The material information used in this example is as follows:

[0132] Potassium dihydrogen phosphate: analytical grade, source: Guangzhou Chemical Reagent Factory;

[0133] Disodium hydrogen phosphate dodecahydrate: analytical grade, source: Guangzhou Chemical Reagent Factory;

[0134] Tetrabutylammonium bromide: analytical grade, source: Bidex Pharmaceuticals / Aladdin;

[0135] Methanol: chromatographic grade, source: Tianjin Kangkede Technology Co., Ltd.;

[0136] Fluoxetine sodium for injection, source: Shandong Lukang Sanye Pharmaceutical Co., Ltd.

[0137] B. The test solution, control solution, sensitivity solution, and mobile phase solution used in this embodiment are prepared as follows:

[0138] Test solution: Take an appropriate amount of sodium fluoxetine for injection, weigh accurately, dissolve in water and dilute to a 0.50 mg / ml solution;

[0139] Control solution: Accurately pipette an appropriate amount of the test solution and dilute it with water to a 5.00 μg / ml solution;

[0140] Sensitivity solution: Accurately pipette an appropriate amount of control solution and dilute it with water to a 0.25 μg / ml solution;

[0141] Tetrabutylammonium bromide-phosphate buffer: accurately weigh 6.94g potassium dihydrogen phosphate, 3.22g disodium hydrogen phosphate dodecahydrate, and 1.61g tetrabutylammonium bromide, dissolve in water and dilute to 1000ml;

[0142] Mobile phase A: Use a measuring cylinder to measure 375 ml of tetrabutylammonium bromide-phosphate buffer and 125 ml of methanol and mix well;

[0143] Mobile phase B: Use a measuring cylinder to measure 200 ml of tetrabutylammonium bromide-phosphate buffer and 300 ml of methanol and mix them evenly.

[0144] C. Chromatographic Conditions

[0145] The high performance liquid chromatograph model used is: Ultimate 3000, manufacturer: Thermo Fisher.

[0146] The chromatographic conditions 1 used in the method of the present invention are as follows:

[0147] Detector: UV absorption detector;

[0148] Chromatographic column: A chromatographic column filled with octadecylsilane bonded silica gel, Agilent 5HC-C18 (2), specification 4.6 mm × 250 mm 5 μm, purchased from Agilent;

[0149] Impurity / ghost peak trapping column: purchased from Yuexu Technology (Shanghai) Co., Ltd., model: Ghost-Buster Column II, specification: 50mm×3mm;

[0150] Mobile phase A: a mixture of tetrabutylammonium bromide-phosphate buffer and methanol in a volume ratio of 75:25;

[0151] Mobile phase B: a mixture of tetrabutylammonium bromide-phosphate buffer and methanol in a volume ratio of 40:60;

[0152] Flow rate: 1.0ml / min;

[0153] Detection wavelength: 246nm;

[0154] Injection volume: 20 μl;

[0155] Column temperature: 40°C;

[0156] The gradient elution program is shown in Table 5:

[0157] Table 5

[0158] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 15 100 0 40 20 80 42 100 0 60 100 0 .

[0159] The chromatographic conditions of the related substance method included in the imported drug standards are as follows2.

[0160] The determination was carried out according to the high performance liquid chromatography method in accordance with the general rule 0512 of the fourth part of the 2015 edition of the Chinese Pharmacopoeia. The high performance liquid chromatograph model used was: Ultimate 3000, manufacturer: Thermo Fisher (same as described above).

[0161] The specific chromatographic conditions 2 are as follows:

[0162] Detector: Same as chromatographic condition 1;

[0163] Chromatographic column: octadecylsilane bonded silica gel filled chromatographic column, model: Develosil ODS-UG-5, specification: 4.6×150mm, purchased from Beijing Yuwei Technology Co., Ltd.;

[0164] Impurity / ghost peak trapping column: same as chromatographic conditions 1;

[0165] Mobile phase A: Same as chromatographic conditions 1;

[0166] Mobile phase B: Same as chromatographic conditions 1;

[0167] Flow rate: same as chromatographic condition 1;

[0168] Detection wavelength: same as chromatographic condition 1;

[0169] Injection volume: 5 μl;

[0170] Column temperature: 35°C;

[0171] The gradient elution program is shown in Table 6:

[0172] Table 6

[0173] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 15 100 0 40 40 60 42 100 0 60 100 0 .

[0174] D. System suitability test and sensitivity test

[0175] Test method:

[0176] Inject the prepared sensitivity solution into the liquid chromatograph for analysis. The chromatographic conditions are as described above under chromatographic condition 1. Record the chromatogram. Figure 1 ;

[0177] Inject the prepared sensitivity solution into the liquid chromatograph for analysis. The chromatographic conditions are as described above under chromatographic condition 2. Record the chromatogram. Figure 2 .

[0178] The analysis was performed using the software provided by the high performance liquid chromatograph, and the signal-to-noise ratio results are shown in Table 7 below:

[0179] Table 7

[0180] Method Name Chromatographic conditions Retention time Signal-to-Noise Ratio Method of the present invention Chromatographic conditions 1 19.822 25.8 Methods included in the standards for imported drugs Chromatographic conditions 2 11.573 12.2 .

[0181] According to General Rule 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, the signal-to-noise ratio of the fluoxetine sodium peak in the sensitivity solution chromatogram shall not be less than 10.

[0182] It can be seen from the above test results that both methods meet the system suitability requirements. The higher the signal-to-noise ratio, the better the sensitivity. It can be seen that compared with the method included in the imported drug standards, the detection sensitivity of the method of the present invention is significantly better.

[0183] E. Separation Test

[0184] This test is carried out using a mixed solution of the relevant substances.

[0185] The mixed solution of the relevant substances is prepared as follows:

[0186] Mixed solution: Take appropriate amount of fluoxetine sodium for injection, fluoxetine sodium polymer impurity 3, fluoxetine sodium polymer impurity 2, fluoxetine sodium-HTT body, fluoxetine sodium-N-coupling, fluoxetine sodium DP-L, fluoxetine sodium DP-N body, fluoxetine sodium open-ring lactone, fluoxetine sodium double bond displacement, fluoxetine sodium polymer impurity 4, fluoxetine DP-B decarboxylation impurity, fluoxetine sodium open-ring impurity, fluoxetine sodium polymer impurity 1 reference substance, dilute with water to make each 1 ml contain: fluoxetine sodium about 0.5mg, about 6μg of fluoxetine sodium polymer impurity 3, about 5μg of fluoxetine sodium polymer impurity 2, about 4μg of fluoxetine sodium polymer impurity 1, about 3μg of fluoxetine sodium-HTT body, about 2.5μg of fluoxetine sodium-N-coupling, fluoxetine sodium DP-L, and fluoxetine sodium DP-N body, and about 1μg of fluoxetine sodium open-ring lactone, fluoxetine sodium double bond displacement, fluoxetine sodium polymer impurity 4, fluoxetine DP-B decarboxylation impurity, and fluoxetine sodium open-ring impurity.

[0187] The sources of the substances involved in the mixed solution are shown in Table 8 below:

[0188] Table 8

[0189] Name of active ingredient and related substances source Fluoxetine Sodium for Injection (Active Ingredient) Shandong Lukang Sanye Pharmaceutical Co., Ltd. Fluoxetine Sodium Polymer Impurity 3 (DeHTT-dimer) Guangzhou Aqixi Pharmaceutical Technology Co., Ltd. Fluoxetine Sodium Polymer Impurity 2(5S-dimer) Guangzhou Aqixi Pharmaceutical Technology Co., Ltd. Fluoxetine Sodium Polymer Impurity 1 (5R-dimer and isomers) Guangzhou Aqixi Pharmaceutical Technology Co., Ltd. Fluoxetine sodium-HTT Guangzhou Aqixi Pharmaceutical Technology Co., Ltd. Fluoxetine Sodium-N-coupling Guangzhou Aqixi Pharmaceutical Technology Co., Ltd. Fluoxetine Sodium DP-L Guangzhou Aqixi Pharmaceutical Technology Co., Ltd. Fluoxetine Sodium DP-N Guangzhou Aqixi Pharmaceutical Technology Co., Ltd. Fluoxetine sodium octadecene Guangzhou Aqixi Pharmaceutical Technology Co., Ltd. Double bond displacement of fluoxetine sodium Guangzhou Aqixi Pharmaceutical Technology Co., Ltd. Fluoxetine Sodium Polymer Impurity 4 (Dimer-M753) Guangzhou Aqixi Pharmaceutical Technology Co., Ltd. Decarboxylation impurity of fluoxetine DP-B Guangzhou Aqixi Pharmaceutical Technology Co., Ltd. Impurities in the Open-ring of Fluoxetine Sodium Guangzhou Aqixi Pharmaceutical Technology Co., Ltd. .

[0190] The specific structural formula, chemical name and other information of each impurity can be found above.

[0191] 1. Comparison between the method of the present invention and the method included in the registration of imported drugs

[0192] Test method:

[0193] (1) Inject the prepared mixed solution into a liquid chromatograph for analysis. The chromatographic conditions are as described in chromatographic condition 1. Record the chromatogram. Figure 3 ;

[0194] (2) Inject the prepared mixed solution into a liquid chromatograph for analysis. The chromatographic conditions are as described in chromatographic condition 2. Record the chromatogram. Figure 4 .

[0195] The separation degree analysis of the obtained chromatogram data was performed using the software provided by the high performance liquid chromatograph, and the results are as follows.

[0196] The results obtained by the method of the present invention (chromatographic condition 1) are shown in Table 9 below:

[0197] Table 9

[0198] name Retention time Relative retention time Separation Fluoxetine sodium HTT 5.332 0.3 11.33 Fluoxetine Sodium DP-N 7.550 0.4 3.32 Fluoxetine Sodium DP-L 8.368 0.4 8.83 Decarboxylation impurity of fluoxetine sodium DP-B 11.387 0.6 1.67 Fluoxetine sodium octadecene 12.043 0.6 15.03 Fluoxetine Sodium 19.024 1.0 2.39 Double bond displacement of fluoxetine sodium 20.371 1.1 9.88 Impurities in the Open-ring of Fluoxetine Sodium 25.523 1.3 2.87 Fluoxetine Sodium N-coupling 26.808 1.4 16.42 Fluoxetine Sodium Polymer Impurity 4 34.766 1.8 4.45 Fluoxetine Sodium Polymer Impurity 3 36.518 1.9 10.95 Fluoxetine Sodium Polymer Impurity 1 38.949 2.0 2.36 Fluoxetine Sodium Polymer Impurity 1* 39.476 2.1 2.15 Fluoxetine Sodium Polymer Impurity 2 39.936 2.1 na

[0199] Note 1: Both Fluoxetine Sodium Polymer Impurity 1 and Fluoxetine Sodium Polymer Impurity 1* are 5R-dimers, which are two isomers of the same substance. In this article, they are both referred to as Fluoxetine Sodium Polymer Impurity 1. Since there are two elution peaks and two retention times, they are distinguished by asterisks "*" in Tables 9-10 and 14;

[0200] Note 2: Fluoxetine Sodium Polymer Impurity 2 is the last impurity peak to appear. There are no more impurity peaks after it, so there is no separation data.

[0201] The results obtained by using the method included in the imported drug standards (chromatographic condition 2) are shown in Table 10 below:

[0202] Table 10

[0203] name Retention time Relative retention time Separation Fluoxetine sodium HTT 3.173 0.3 8.16 Fluoxetine Sodium DP-N 4.493 0.4 2.14 Fluoxetine Sodium DP-L 4.977 0.4 5.54 Decarboxylation impurity of fluoxetine sodium DP-B 6.647 0.6 1.57 Fluoxetine sodium octadecene 7.043 0.6 10.68 Fluoxetine Sodium 11.130 1.0 na Double bond displacement of fluoxetine sodium 11.907 1.1 na Impurities in the Open-ring of Fluoxetine Sodium 15.957 1.4 1.82 Fluoxetine Sodium N-coupling 18.417 1.7 15.71 Fluoxetine Sodium Polymer Impurity 4 31.557 2.8 5.44 Fluoxetine Sodium Polymer Impurity 3 34.933 3.1 11.84 Fluoxetine Sodium Polymer Impurity 1 38.727 3.5 2.27 Fluoxetine Sodium Polymer Impurity 1* 39.447 3.5 2.11 Fluoxetine Sodium Polymer Impurity 2 40.117 3.6 na .

[0204] from Figure 3 It can be seen that in the results obtained by the method of the present invention (chromatographic condition 1), the peak of fluoxetine sodium and the peak of fluoxetine sodium double bond shift can be well separated, so this method can accurately detect the content of the substance of fluoxetine sodium double bond shift.

[0205] from Figure 4 It can be seen that in the results obtained by using the method included in the imported drug standards (chromatographic condition 2), the peak of the active ingredient fluoxetine sodium and the peak of the double bond shift of the impurity fluoxetine sodium are connected together and are not effectively separated. Therefore, the separation degree between the two peaks cannot be clearly determined. This method cannot accurately detect the content of substances related to the double bond shift of fluoxetine sodium.

[0206] Under the condition that the injection volume is increased (from 5 μl to 20 μl) and the sensitivity is improved, the double bond displacement of fluoxetine sodium can be better separated by the method of the present invention, which is unexpected.

[0207] 2. Gradient comparison test

[0208] In this experiment, the following gradients were tested:

[0209] Table 11 (Gradient 1: gradient of the present invention)

[0210] Table 11

[0211] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 15 100 0 40 20 80 42 100 0 60 100 0 ;

[0212] Table 12 (Gradient 2: Gradient in the method in the imported drug standard)

[0213] Table 12

[0214] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 15 100 0 40 40 60 42 100 0 60 100 0 ;

[0215] Table 13 (Gradient 3)

[0216] Table 13

[0217] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 100 10 100 100 40 30 70 42 100 0 60 100 0 .

[0218] Test method: Inject the mixed solution of related substances into a high performance liquid chromatograph, wherein the chromatographic condition 1 of the method of the present invention is adopted, but the gradients are respectively the above three gradients, and the separation degree is analyzed.

[0219] The respective separation results are shown in Table 14:

[0220] Table 14

[0221] name Gradient 1 resolution Gradient 3 resolution Gradient 2 resolution Fluoxetine Sodium 2.39 2.50 2.20 Fluoxetine sodium HTT 11.33 1.75 10.39 Fluoxetine Sodium DP-N 3.32 3.04 2.34 Fluoxetine Sodium DP-L 8.83 9.06 8.78 Decarboxylation impurity of fluoxetine sodium DP-B 1.67 1.88 1.94 Double bond displacement of fluoxetine sodium 9.88 9.33 7.80 Fluoxetine sodium octadecene 15.03 14.59 13.35 Impurities in the Open-ring of Fluoxetine Sodium 2.87 1.49 3.22 Fluoxetine Sodium N-coupling 16.42 16.22 15.40 Fluoxetine Sodium Polymer Impurity 4 4.45 3.84 5.91 Fluoxetine Sodium Polymer Impurity 3 10.95 1.49 10.21 Fluoxetine Sodium Polymer Impurity 1 2.36 2.57 1.46 Fluoxetine Sodium Polymer Impurity 1* 2.15 2.27 Affected by baseline, not detected Fluoxetine Sodium Polymer Impurity 2 na na na .

[0222] In the above three gradient tests, the same chromatographic column and the same chromatographic conditions were used, and only the elution gradients were different. From the above content, it can be seen that in gradient 2, the elution gradient affected the baseline fluctuation, resulting in one of the impurity peaks not being detected. It can be seen that the gradient effect of the method of the present invention is better.

[0223] F. Detection limit, quantification limit, and accuracy test

[0224] Compared with the method included in the imported drug standards, the method of the present invention can detect more impurities with lower content, and the detection method is accurate. The detection limit, quantitative limit and accuracy of the method of the present invention are verified as follows.

[0225] 1. Detection limit and quantification limit

[0226] Preparation of detection limit solution: Take appropriate amount of each impurity reference substance and dilute with water to prepare the quantitative limit concentration;

[0227] Preparation of quantitative limit solution: Take an appropriate amount of each impurity reference substance and dilute it with water to prepare the detection limit concentration.

[0228] Test method: Inject the detection limit solution and the quantitative limit solution into the high performance liquid chromatograph respectively, and use the chromatographic condition 1. The detection limit concentration, quantitative limit concentration and the obtained results are shown in Table 15 below:

[0229] Table 15

[0230]

[0231]

[0232] It can be seen that the method of the present invention can detect impurities at very low concentrations.

[0233] 2. Accuracy

[0234] The accuracy test was carried out using the sample recovery method.

[0235] The solutions involved in this experiment were prepared as follows.

[0236] (1) Test solution: Take 10.44 mg of fluoxetine sodium for injection, weigh accurately, place in a 20 ml volumetric flask, dissolve with water and dilute to scale, shake well. The concentration of fluoxetine sodium test solution is 0.522 mg / ml. Prepare 2 portions in parallel.

[0237] (2) Reference solution: Accurately measure 1 ml of the test solution and place it in a 100.0 ml volumetric flask. Add water to dilute to the mark and shake well. The concentration of the reference solution of fluoxetine sodium is 5.22 μg / ml.

[0238] (3) Impurity stock solutions: Accurately weigh appropriate amounts of fluoxetine sodium polymer impurity 3 (DeHTT body-dimer), fluoxetine sodium polymer impurity 2 (5S-dimer), fluoxetine sodium polymer impurity 1 (5R-dimer and isomers), fluoxetine sodium-HTT body, fluoxetine sodium-N-coupling, fluoxetine sodium DP-L, fluoxetine sodium DP-N, fluoxetine sodium open-ring lactone, fluoxetine sodium double bond displacement, fluoxetine sodium polymer impurity 4 (Dimer-M753), fluoxetine DP-B decarboxylation impurity, and fluoxetine sodium open-ring impurity, respectively, dissolve in water and dilute to scale. Prepare impurity stock solutions with concentrations as shown in Table 16 below.

[0239] (4) Accuracy solution 1 (add 50% level, prepare 3 solutions in parallel)

[0240] Take 5.22 mg of the test sample of fluoxetine sodium for injection, accurately weigh it, put it in a 10 ml volumetric bottle, add 0.2 ml of fluoxetine sodium polymer impurity 3, fluoxetine sodium polymer impurity 2, fluoxetine sodium-HTT body, fluoxetine sodium N-coupling, fluoxetine sodium DP-L, fluoxetine sodium DP-N stock solution, 0.1 ml of fluoxetine sodium open-ring lactone, fluoxetine sodium double bond displacement, fluoxetine sodium polymer impurity 4, fluoxetine DP-B decarboxylation impurity, fluoxetine sodium open-ring impurity stock solution, 0.5 ml of fluoxetine sodium polymer impurity 1 stock solution, dilute to the scale with water, and shake well. The amount of each impurity added in the accuracy solution 1 is shown in Table 16 below. Accuracy solution 1 is freshly prepared before use.

[0241] (5) Accuracy solution 2 (add 100% level, prepare 3 solutions in parallel)

[0242] Take 5.22 mg of the test sample of fluoxetine sodium for injection, accurately weigh it, put it in a 10 ml volumetric bottle, add 0.4 ml of each of the stock solutions of fluoxetine sodium polymer impurity 3, fluoxetine sodium polymer impurity 2, fluoxetine sodium-HTT body, fluoxetine sodium-N-coupling, fluoxetine sodium DP-L, and fluoxetine sodium DP-N body, 0.2 ml of each of the stock solutions of fluoxetine sodium open-ring lactone, fluoxetine sodium double bond displacement, fluoxetine sodium polymer impurity 4, fluoxetine DP-B decarboxylation impurity, and fluoxetine sodium open-ring impurity reference substance, and 1 ml of the stock solution of fluoxetine sodium polymer impurity 1 reference substance, dilute to the scale with water, and shake well. The amount of each impurity added in the accuracy solution 2 is shown in Table 16 below. Accuracy solution 2 is freshly prepared before use.

[0243] (6) Accuracy solution 3 (add 3 solutions at 150% level)

[0244] Take 5.22 mg of the test sample of fluoxetine sodium for injection, accurately weigh it, put it in a 10 ml volumetric bottle, add 0.6 ml of fluoxetine sodium polymer impurity 3, fluoxetine sodium polymer impurity 2, fluoxetine sodium HTT body, fluoxetine sodium N-coupling, fluoxetine sodium DP-L, fluoxetine sodium DP-N body stock solution, 0.3 ml of fluoxetine sodium open-ring lactone, fluoxetine sodium double bond displacement, fluoxetine sodium polymer impurity 4, fluoxetine DP-B decarboxylation impurity, fluoxetine sodium open-ring impurity stock solution, 1.5 ml of fluoxetine sodium polymer impurity 1 stock solution, dilute to the scale with water, and shake well. The amount of each impurity added in the accuracy solution 3 is shown in Table 16 below. Accuracy solution 3 is freshly prepared before use.

[0245] Table 16

[0246]

[0247] Note: The data in the above table are approximate values, which are the concentrations prepared and the values ​​of the impurities to be added, and are not the actual values ​​in actual operation. The actual accurate values ​​in actual operation are shown in Table 17 below.

[0248] Test method: Inject the control solution, the test solution, the solution before adding each impurity, and the solution after adding different amounts of each impurity into the high performance liquid chromatograph. Calculate the sample recovery rate based on the obtained chromatogram data and the self-control method. The results are shown in Table 17:

[0249] Table 17

[0250]

[0251]

[0252]

[0253] From the verification results, it can be seen that the method of the present invention can accurately determine the concentration of each impurity content.

[0254] G. Related substances determination test

[0255] Test method:

[0256] (1) Inject the prepared reference solution and test solution into a liquid chromatograph for analysis. The chromatographic conditions are as described in chromatographic condition 1. Record the chromatogram. Figure 5 and 6 ;

[0257] (2) Inject the prepared reference solution and test solution into the liquid chromatograph for analysis respectively. The chromatographic conditions are as described in chromatographic condition 2. Record the chromatogram. Figure 7 and 8 .

[0258] The chromatogram data obtained were analyzed using the software provided by the HPLC, and the results are as follows. The impurity standards are the standards in the method included in the registration of imported drugs.

[0259] The results obtained using chromatographic condition 1 are shown in Table 18 below:

[0260] Table 18

[0261]

[0262] The results obtained using chromatographic condition 2 are shown in Table 19 below:

[0263] Table 19

[0264]

[0265] The impurity content is calculated according to the following formula:

[0266]

[0267] A u : The peak area of ​​each impurity in the test solution;

[0268] A s : Peak area of ​​control solution.

[0269] From the results in the above table, it can be seen that compared with the detection results using chromatographic condition 1 (the method of the present invention), the results of the related substances detected using chromatographic condition 2 are lower, and impurities with a smaller content cannot be detected. Obviously, the detection performed using the method of the present invention can detect more related substances, and the results are more sensitive and accurate.

[0270] Therefore, the method developed by the present invention can detect the impurities in the active ingredient or drug of fluoxetine sodium, especially fluoxetine sodium for injection, more sensitively and accurately, so that technicians can more effectively control the impurities of the active ingredient or drug, and can better ensure the effectiveness and safety of the drug.

Claims

1. A method for analyzing related substances of fluoxetine sodium, which is carried out by high performance liquid chromatography, wherein Gradient elution was performed using mobile phase A and mobile phase B as eluents, wherein mobile phase A was a mixture of tetrabutylammonium bromide-phosphate buffer and methanol in a volume ratio of (73-77):(23-27), and mobile phase B was a mixture of tetrabutylammonium bromide-phosphate buffer and methanol in a volume ratio of (38-42):(58-62), The elution gradient is as follows: 。 2. The method for analyzing the related substances of fluoxetine sodium according to claim 1, wherein the elution gradient is as follows:

3. The method for analyzing related substances of sodium fluoxetine according to claim 1 or 2, wherein the amount of the sodium fluoxetine sample measured is 3-20 μg, preferably 5-15 μg.

4. The method for analyzing related substances of fluoxetine sodium according to claim 1 or 2, wherein the injection volume is 15-25 μl.

5. The method for analyzing the related substances of sodium fluoxetine according to claim 1 or 2, wherein the sodium fluoxetine sample to be measured is an aqueous solution with a concentration of 0.3-0.7 mg / ml, preferably 0.4-0.6 mg / ml.

6. The method for analyzing related substances of fluoxetine sodium according to claim 1 or 2, wherein the column temperature of the chromatographic column is 36-45°C.

7. The method for analyzing the related substances of fluoxetine sodium according to claim 1 or 2, wherein a chromatographic column using octadecylsilane bonded silica gel as a filler or a chromatographic column with equivalent performance is used.

8. The method for analyzing the related substances of fluoxetine sodium according to claim 1 or 2, wherein an ultraviolet detector is used and the detection wavelength is 245-247nm.

9. The method for analyzing related substances of fluoxetine sodium according to claim 1 or 2, wherein the mobile phase flow rate is 0.8-1.2 ml / min.

10. The method for analyzing related substances of fluoxetine sodium according to claim 1 or 2, wherein the tetrabutylammonium bromide-phosphate buffer is an aqueous solution composed of potassium dihydrogen phosphate, disodium hydrogen phosphate, and tetrabutylammonium bromide, wherein the concentration of potassium dihydrogen phosphate is 6.90-6.98 g / l, the concentration of disodium hydrogen phosphate is 1.24-1.32 g / l, and the concentration of tetrabutylammonium bromide is 1.57-1.65 g / l.

Images