Method for determining related substances in citalopram or its salts and formulations thereof

Abstract

The present application belongs to the field of analytical chemistry, and particularly relates to a method for determining related substances in citalopram or its salt and preparation thereof, which adopts HPLC to detect citalopram; wherein, mobile phase A is phosphate buffer with mass-volume ratio of 0.6-0.7% and pH of 6.0-7.0; mobile phase B is a mixed solution of methanol and acetonitrile with volume ratio of 30-50:70-50. The method can detect at least 12 known impurities in citalopram, and even 3 unknown impurities, with good peak shape, good separation degree, good reproducibility, and can realize effective separation and quantitative determination of citalopram.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry, and specifically relates to a method for determining related substances in citalopram or its salts and preparations. Background Technology

[0002] Citalopram hydrobromide, chemically named (±)-1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydro-5-isobenzofuran carboxylate hydrobromide, with the structural formula shown in Formula I, is a serotonin reuptake inhibitor used in the conventional treatment of depression and anxiety disorders.

[0003]

[0004] In 1989, citalopram hydrobromide tablets, an oral serotonin reuptake inhibitor (SSRI) jointly developed by Lundbeck and FOREST LABS of Denmark, were first approved for marketing in Denmark, and subsequently in Germany, the United Kingdom, the United States, and other countries. In June 2003, citalopram hydrobromide tablets manufactured by Lundbeck were approved in China, under the brand name Cipramil. Used to treat depression.

[0005] Impurity studies are an important approach to drug quality evaluation. According to the pharmacopoeia standards for citalopram hydrobromide / citalopram raw materials and preparations and related varieties, there may be 12 known organic impurities (Z2 to Z13) and other unknown impurities in citalopram or its salt preparations. Some of these impurities may have high levels, which may lead to changes in drug bioavailability and may also affect drug safety. Therefore, it is necessary to detect and monitor the above impurities in the related substances method of the product.

[0006] The known impurity codes, names, and structures of citalopram or its salts and their preparations are shown in Table 1.

[0007] Table 1

[0008]

[0009]

[0010]

[0011] Existing technologies also include studies on the determination of related substances in citalopram hydrobromide and its preparations, for example:

[0012] The paper "Determination of Citalopram Hydrobromide Raw Material Content and Related Substances by RP-HPLC" (Wei Jun and Xie Jianwei, Institute of Toxicology and Pharmacology, Academy of Military Medical Sciences) uses high performance liquid chromatography to determine the related substances and content in citalopram hydrobromide raw material. The chromatographic column is a Zobax SB-C18 250*4.6mm, 5μm, the mobile phase is methanol:0.1mol / L ammonium acetate = 60:40, the detection wavelength is 240nm, the flow rate is 1.0mL / min, and the column temperature is 25℃. However, this method can only detect 4 impurities.

[0013] The study, titled "Determination of Citalopram Hydrobromide and its Tablets and Related Substances by HPLC" (Zhao Zhongqiong, Liang Long, Zhu Qunbin, Chengdu Kelun Pharmaceutical Research Institute), used an ODS column (150*4.6mm, 5μm) with acetonitrile-0.05mol / L potassium dihydrogen phosphate solution as the mobile phase, a flow rate of 1.0mL / min, and a detection wavelength of 238nm. However, this method could only detect two impurities.

[0014] The aforementioned existing technologies all have drawbacks such as poor separation effect and small number of separated impurities. Summary of the Invention

[0015] To address the shortcomings of existing technologies in detecting related substances in citalopram hydrobromide and its preparations, such as poor separation efficiency and limited number of separated impurities, this invention provides a novel method for determining related substances in citalopram, its salts, and their preparations. This method can detect and separate 12 known impurities and 3 unknown impurities in citalopram, its salts, and their preparations.

[0016] The technical problem to be solved by this invention is to provide a method for determining related substances in citalopram or its salts and preparations. The method includes the following steps: detection by HPLC; wherein the chromatographic conditions are: mobile phase A is a phosphate buffer solution with a mass-to-volume ratio of 0.6-0.7% and a pH of 6.0-7.0; mobile phase B is a mixed solution of methanol and acetonitrile in a volume ratio of 30-50:70-50. The mass-to-volume ratio of 0.6-0.7% is expressed as 0.6-0.7 g of diammonium hydrogen phosphate dissolved in 100 mL of water.

[0017] Furthermore, in the above method for determining related substances in citalopram or its salts and preparations, the HPLC employs gradient elution, and the gradient elution conditions are as follows:

[0018] Time (min) Mobile phase A (%) Mobile phase B (%) 0 82-78 18-22 10 70 30 20 60 40 30 60 40 50 50 50 50.1 82-78 18-22 60 82-78 18-22

[0019] 0-10 min: (82-78%)-70% mobile phase A, (18-22%)-30% mobile phase B; 10-20 min: 70-60% mobile phase A, 30-40% mobile phase B; 20-30 min: 60% mobile phase A, 40% mobile phase B; 30-50 min: 60-50% mobile phase A, 40-50% mobile phase B; 50-50.1 min: 50-(82-78%)% mobile phase A, 50-(18-22%)% mobile phase B; 50.1-60 min: (82-78%)% mobile phase A, (18-22%)% mobile phase B; 60 min: End.

[0020] Furthermore, in the above method for determining related substances in citalopram or its salts and preparations, the gradient elution conditions are as follows:

[0021] Time (min) Mobile phase A (%) Mobile phase B (%) 0 80 20 10 70 30 20 60 40 30 60 40 50 50 50 50.1 80 20 60 80 20

[0022] 0-10 min: 80-70% mobile phase A, 20-30% mobile phase B; 10-20 min: 70-60% mobile phase A, 30-40% mobile phase B; 20-30 min: 60% mobile phase A, 40% mobile phase B; 30-50 min: 60-50% mobile phase A, 40-50% mobile phase B; 50-50.1 min: 50-80% mobile phase A, 50-20% mobile phase B; 50.1-60 min: 80% mobile phase A, 20% mobile phase B; 60 min: End.

[0023] In the above method for determining related substances in citalopram or its salts and preparations, triethylamine is added to the mobile phase A; preferably, 2 mL of triethylamine is added per 1000 mL of mobile phase A.

[0024] For example, the preparation method of a phosphate buffer solution with a mass-to-volume ratio of 0.66% and a pH of 6.1 is as follows: Dissolve 6.6g of diammonium hydrogen phosphate in 1000mL of water, add 2mL of triethylamine, shake well, and adjust the pH to 6.1 with phosphoric acid. The purpose of adding triethylamine is to reduce peak tailing.

[0025] In the above method for determining related substances in citalopram or its salts and preparations, the HPLC column is a reversed-phase column packed with octadecylsilane-bonded silica gel. Preferably, the column size is 4.6 × 150 mm. Preferably, the particle size of the column packing material is 3 μm or 3.5 μm.

[0026] In the above method for determining related substances in citalopram or its salts and preparations, the detection wavelength of the HPLC is UV220nm or 239nm.

[0027] In the above method for determining related substances in citalopram or its salts and preparations, the column temperature of the HPLC is 25–35°C. Preferably, it is 28–32°C. More preferably, it is 30°C.

[0028] In the above method for determining related substances in citalopram or its salts and preparations, the flow rate of the HPLC is 0.6–1.0 mL / min. Preferably, it is 0.7–0.9 mL / min. More preferably, it is 0.8 mL / min.

[0029] In the above method for determining related substances in citalopram or its salts and preparations, the HPLC injection volume is 5–20 μL, preferably 10 μL.

[0030] In the above method for determining related substances in citalopram or its salts and preparations, the sample to be tested is dissolved in a diluent to form a sample solution. The concentration of the sample solution is 0.2–2 mg / mL. Preferably, it is 0.5–2 mg / mL. More preferably, it is 1.0 mg / mL.

[0031] In the above method for determining related substances in citalopram or its salts and preparations, the diluent is a mixed solution of mobile phase A and mobile phase B. Preferably, the diluent is a mixed solution of mobile phase A and mobile phase B at a volume ratio of 70-85:30-15. More preferably, the diluent is a mixed solution of mobile phase A and mobile phase B at a volume ratio of 80:20.

[0032] Specifically, in the above method for determining related substances in citalopram or its salts and preparations, the citalopram salt is a pharmaceutically acceptable salt of citalopram. Preferably, the citalopram salt is citalopram hydrobromide or escitalopram oxalate.

[0033] In the above method for determining related substances in citalopram or its salts and preparations, citalopram hydrobromide or escitalopram oxalate, or other salts of citalopram, will dissociate into free citalopram in the mobile phase, separate from its related substances on the chromatographic column, and the active ingredient is represented by a citalopram chromatographic peak in the detection chromatogram.

[0034] Furthermore, in the above method for determining related substances in citalopram or its salts and their preparations, the preparations of citalopram or its salts are oral tablets, capsules, oral liquids, orally disintegrating films, orally disintegrating tablets, dispersible tablets, granules, or chewable tablets.

[0035] This invention provides a high-performance liquid chromatography (HPLC) method for determining related substances in citalopram or its salts and preparations. The method can detect at least 12 known substances in citalopram, and even 3 unknown substances. It also features good peak shape, good separation, high specificity, and good reproducibility, achieving effective separation and quantitative determination of citalopram related substances. The chromatographic column has good durability. Attached Figure Description

[0036] Figure 1 Liquid chromatogram of Example 1 of this invention;

[0037] Figure 2 Liquid chromatogram of Example 2 of this invention;

[0038] Figure 3 Liquid chromatogram of Example 3 of this invention;

[0039] Figure 4 Liquid chromatogram of Example 4 of this invention;

[0040] Figure 5 Liquid chromatogram of Example 5 of this invention;

[0041] Figure 6 Liquid chromatogram of Example 6 of this invention;

[0042] Figure 7 Liquid chromatogram of Example 7 of this invention;

[0043] Figure 8 Liquid chromatogram of Example 8 of this invention;

[0044] Figure 9 Liquid chromatogram of Example 9 of this invention;

[0045] Figure 10 Liquid chromatogram of Example 10 of the present invention;

[0046] Figure 11 Liquid chromatogram of Example 11 of this invention;

[0047] Figure 12 Liquid chromatogram of blank solution in Example 12 of this invention;

[0048] Figure 13 Liquid chromatogram of blank excipient solution in Example 12 of this invention;

[0049] Figure 14 Liquid chromatogram of the test solution in Example 12 of this invention;

[0050] Figure 15 The liquid chromatogram of Comparative Example 1 of this invention;

[0051] Figure 16 The liquid chromatogram of Comparative Example 2 of this invention;

[0052] Figure 17 The liquid chromatogram of Comparative Example 3 of this invention;

[0053] Figure 18 The liquid chromatogram of Comparative Example 5 of this invention has a detection wavelength of 239 nm.

[0054] Figure 19 The liquid chromatogram of Comparative Example 5 of this invention has a detection wavelength of 220 nm.

[0055] Figure 20 The liquid chromatogram of Comparative Example 6 of this invention. Detailed Implementation

[0056] Example 1

[0057] Diluent: Phosphate buffer and organic mixed solution are mixed at a ratio of 80:20 (v / v); wherein, phosphate buffer: take 6.6g of diammonium hydrogen phosphate, add 1000mL of water to dissolve, add 2mL of triethylamine, shake well, and adjust the pH value to 6.1 with phosphoric acid; organic mixed solution: methanol-acetonitrile = 40:60 (v / v).

[0058] Mixed impurity stock solution: Accurately weigh approximately 2.5 mg of each of the 12 impurity reference standards Z2-Z13, place them in 10 mL volumetric flasks, dissolve them with diluent and make up to volume, shake well, and use them as stock solutions for each impurity; accurately measure 0.5 mL of each impurity stock solution, add it to a 10 mL volumetric flask, make up to volume with diluent, shake well, and use it as mixed impurity stock solution.

[0059] First system suitability solution: Accurately weigh 25 mg of citalopram hydrobromide raw material (source: Qionglai Branch of Sichuan Kelun Pharmaceutical Co., Ltd., batch 1), place it in a 20 mL volumetric flask, then accurately measure 2 mL of mixed impurity stock solution, dissolve and dilute with diluent, shake well to obtain the first system suitability solution.

[0060] Accurately measure 10 μL of the first system suitability solution described above and inject it into the liquid chromatograph. Analyze the solution under the chromatographic conditions described below, record the chromatogram, and the experimental data and results are shown in Table 3. (The chromatogram is shown in the image below.) Figure 1 .

[0061] The chromatographic conditions are as follows:

[0062] Chromatographic column: Reversed-phase column packed with octadecylsilane-bonded silica gel (Shiseido C18MGⅡ, 4.6×150mm, 3μm);

[0063] Mobile phase A: 0.66% phosphate buffer (dissolve 6.6g of diammonium hydrogen phosphate in 1000mL of water, add 2mL of triethylamine, shake well, and adjust the pH to 6.0 with phosphoric acid); Mobile phase B: methanol-acetonitrile (40:60, v:v);

[0064] Flow rate: 0.8 mL / min, detection wavelength: 220 nm, column temperature: 30 °C;

[0065] Gradient elution conditions are shown in Table 2:

[0066] Table 2

[0067]

[0068]

[0069] Table 3

[0070] impurities Retention time (min) Theoretical number of plates Resolution Unknown impurity 1 8.43 35523 - Impurity Z13 9.53 52012 6.32 impurity Z2 11.64 71228 12.34 Unknown impurity 2 12.16 73456 2.91 Impurity Z3 13.08 83320 5.11 Impurity Z4 16.09 104152 15.78 Impurity Z5 18.76 109687 12.53 Unknown impurity 3 21.05 163108 10.49 Impurity Z12 22.38 169968 6.25 Impurity Z6 22.88 180778 2.30 Impurity Z7 23.85 207133 4.56 Citalopram 24.40 44537 1.63 Impurity Z8 25.69 137031 3.48 Impurity Z9 30.12 82610 12.71 Impurity Z10 41.45 121434 25.28 Impurity Z11 44.64 137527 6.64

[0071] Combined with Table 3 and Figure 1 As can be seen, the method of the present invention detected 12 known impurities and 3 unknown impurities in the first system suitability solution of citalopram hydrobromide. The peak shapes were good, and baseline separation was achieved between all impurities and between the impurities and citalopram, with a resolution of more than 1.5, indicating good separation effect.

[0072] Example 2

[0073] Except for mobile phase A, which was 0.66% phosphate buffer at pH 7.0, the other chromatographic conditions were the same as in Example 1. The suitability solution of the first system was analyzed, and the results are shown in Table 4. Figure 2 .

[0074] Table 4

[0075]

[0076]

[0077] Combined with Table 4 and Figure 2 As can be seen, the method of the present invention detected 12 known impurities and 3 unknown impurities in the first system suitability solution of citalopram hydrobromide. The peak shapes were good, and baseline separation was achieved between all impurities and between the impurities and citalopram, with a resolution of more than 1.5, indicating good separation effect.

[0078] Example 3

[0079] Second system suitability solution: Accurately weigh 25 mg of citalopram hydrobromide raw material (source: Qionglai Branch of Sichuan Kelun Pharmaceutical Co., Ltd., batch 2), place it in a 20 mL volumetric flask, then accurately measure 2 mL of the mixed impurity stock solution in Example 1, dissolve and dilute with the diluent in Example 1, shake well, and the second system suitability solution is obtained.

[0080] Except for the column temperature of 28℃, all other chromatographic conditions were the same as in Example 1. The suitability solution for the second system was analyzed, and the results are shown in Table 5. Figure 3 .

[0081] Example 4

[0082] Except for the column temperature of 32℃, all other chromatographic conditions were the same as in Example 1. The suitability solution for the second system was analyzed, and the results are shown in Table 5. Figure 4 .

[0083] Table 5

[0084]

[0085]

[0086] Combined with Table 5, Figure 3 and Figure 4 As can be seen, the detection method provided by this invention can effectively separate 12 known impurities within a column temperature range of 28-32℃, with a separation degree of over 1.5, demonstrating good separation effect and peak shape, thus meeting the detection requirements.

[0087] Example 5

[0088] Except for the detection wavelength of 239 nm, the other chromatographic conditions were the same as in Example 1. The suitability solution of the first system was analyzed, and the results are shown in Table 6. Figure 5 .

[0089] Table 6

[0090] impurities Retention time (min) Theoretical number of plates Resolution Unknown impurity 1 8.48 35762 - Impurity Z13 9.54 53230 6.14 impurity Z2 11.89 70639 13.62 Unknown impurity 2 12.60 65372 3.77 Impurity Z3 13.18 84594 3.06 Impurity Z4 16.29 104367 16.18 Impurity Z5 18.89 114081 12.24 Unknown impurity 3 21.58 156663 12.15 Impurity Z12 22.58 172413 4.59 Impurity Z6 23.14 181175 2.58 Impurity Z7 23.94 197908 3.69 Citalopram 24.67 45694 2.13 Impurity Z8 25.85 128137 3.16 Impurity Z9 31.19 77707 14.46 Impurity Z10 42.11 123324 23.53 Impurity Z11 45.34 141871 6.71

[0091] In this embodiment, the number of impurities detected was consistent with the 220nm wavelength. A total of 12 known impurities and 3 unknown impurities were detected. The peak shapes were good, and baseline separation was achieved between all impurities and between the impurities and citalopram. The resolution was greater than 1.5, and the separation effect was good.

[0092] Example 6

[0093] The gradient elution conditions are shown in Table 7, and other chromatographic conditions are the same as in Example 1. The suitability solution for the second system was analyzed, and the results are shown in Table 9. Figure 6 .

[0094] Table 7

[0095]

[0096]

[0097] Example 7

[0098] The gradient elution conditions are shown in Table 8, and other chromatographic conditions are the same as in Example 1. The suitability solution for the second system was analyzed, and the results are shown in Table 9. Figure 7 .

[0099] Table 8

[0100] Time (min) Mobile phase A (%) Mobile phase B (%) 0 78 22 10 70 30 20 60 40 30 60 40 50 50 50 50.1 78 22 60 78 22

[0101] Table 9

[0102]

[0103] Combined with Table 9, Figure 6 and Figure 7 As can be seen, the detection method provided by the present invention can effectively separate 12 known impurities within the initial mobile phase ratio range of (82-78):(18-22), with a separation degree of more than 1.5, good separation effect, good peak shape, and meets the detection requirements.

[0104] Example 8

[0105] Except for the mobile phase flow rate of 0.7 mL / min, all other conditions were the same as in Example 1. The suitability solution for the second system was tested and analyzed; the results are shown in Table 10 and... Figure 8 .

[0106] Example 9

[0107] Except for the mobile phase flow rate of 0.9 mL / min, all other conditions were the same as in Example 1. The suitability solution for the second system was tested and analyzed; the results are shown in Table 10. Figure 9 .

[0108] Table 10

[0109]

[0110] Combined with Table 10, Figure 8 and Figure 9 As can be seen, the detection method provided by this invention can effectively separate 12 known impurities within a flow rate range of 0.7-0.9 mL / min, with a separation degree of over 1.5, demonstrating good separation effect and peak shape, thus meeting the detection requirements.

[0111] Example 10

[0112] Except for mobile phase A: the concentration of diammonium hydrogen phosphate in phosphate buffer was 0.63%, other conditions were the same as in Example 1. The suitability solution for the second system was tested and analyzed; the results are shown in Table 11 and... Figure 10 .

[0113] Example 11

[0114] Except for mobile phase A: the concentration of diammonium hydrogen phosphate in phosphate buffer was 0.69%, other conditions were the same as in Example 1. The suitability solution for the second system was tested and analyzed; the results are shown in Table 11 and... Figure 11 .

[0115] Table 11

[0116]

[0117]

[0118] Combined with Table 11, Figure 10 and Figure 11 As can be seen, the detection method provided by this invention can effectively separate 12 known impurities when the concentration of diammonium hydrogen phosphate in the mobile phase A (phosphate buffer) is in the range of 0.63-0.69%, with a separation degree of more than 1.5, good separation effect, good peak shape, and meets the detection requirements.

[0119] As can be seen from Examples 1 to 11 above, the detection method provided by the present invention can effectively separate various impurities and meet the detection requirements when the pH value of the mobile phase, column temperature, detection wavelength, gradient elution conditions, mobile phase flow rate, and the concentration of diammonium hydrogen phosphate in the phosphate buffer of mobile phase A vary within a certain range. The method has good durability.

[0120] Example 12

[0121] Blank solution: Take the diluent from Example 1 as a blank solution, and test the blank solution according to the chromatographic conditions of Example 1. The test results are shown in [Figure 1]. Figure 12 .

[0122] Blank excipient solution: Weigh approximately 104 mg of the relevant excipients for citalopram hydrobromide tablets (source: Anyue Branch of Sichuan Kelun Pharmaceutical Co., Ltd.), place in a 20 mL volumetric flask, add an appropriate amount of diluent, shake to disperse evenly, sonicate for 10 min, cool to room temperature, add diluent to make up to volume, shake well, filter through a 0.45 μm filter membrane, and take the filtrate as the blank excipient solution; detect using the chromatographic conditions of Example 1, and the test results are shown in [Figure 1]. Figure 13 .

[0123] Test solution: Take citalopram hydrobromide tablets (source: Anyue Branch of Sichuan Kelun Pharmaceutical Co., Ltd.), place them in a 200mL volumetric flask, add an appropriate amount of diluent, shake to disperse evenly, sonicate for 10min, cool to room temperature, add diluent to make up to volume, shake well, filter through a 0.45μm filter membrane, and take the filtrate as the test solution (citalopram concentration approximately 1.0mg / mL). Detect the solution using the chromatographic conditions of Example 1. The test results are shown in [Figure 1]. Figure 14 .

[0124] from Figure 12-14 It can be seen that the blank solvent and blank excipient do not interfere with the determination of related substances in the test solution, and the detection method provided by the present invention has good specificity.

[0125] Example 13

[0126] Reference solution: Accurately weigh approximately 12.5 mg of citalopram hydrobromide reference standard, place it in a 100 mL volumetric flask, dissolve and dilute to volume with diluent, and shake well. Accurately measure 1 mL of the above solution, place it in a 100 mL volumetric flask, dilute to volume with diluent, and shake well. The citalopram concentration is approximately 1.0 μg / mL. Prepare two parallel aliquots for calculating the impurity content of the test sample.

[0127] Test solution: Accurately weigh approximately 25 mg of citalopram hydrobromide raw material (source: Qionglai Branch of Sichuan Kelun Pharmaceutical Co., Ltd.), place it in a 20 mL volumetric flask, add an appropriate amount of diluent, shake to dissolve, and dilute to the mark. The citalopram concentration is approximately 1.0 mg / mL. Prepare 6 parallel solutions.

[0128] Accurately measure 10 μL of each of the above solutions and inject them into the liquid chromatograph. Detection is performed using the chromatographic conditions described in Example 1. The content of each impurity in the six test samples is calculated using the principal component self-comparison method with correction factors. The results are shown in Table 12.

[0129] Table 12

[0130]

[0131] Note: "ND" indicates that the impurity was not detected in the test sample.

[0132] Test results: Impurity Z3 was detected at 0.04%, impurity Z12 at 0.02%, and impurity Z7 at 0.01% in the test samples. An unknown impurity was also detected, with a total impurity content of 0.13%. The types of impurities detected in the six test samples were consistent, and the contents of each impurity and the total impurity content were basically consistent. The resolution between all impurities and between impurities and citalopram was greater than 1.5. This method can accurately detect related substances in citalopram hydrobromide raw material and has good repeatability.

[0133] Example 14

[0134] Reference solution: Accurately weigh approximately 25 mg of citalopram hydrobromide reference standard, place it in a 100 mL volumetric flask, dissolve and dilute to volume with diluent, and shake well; accurately measure 1 mL of the above solution, place it in a 100 mL volumetric flask, dilute to volume with diluent, and shake well. The citalopram concentration is approximately 2.0 μg / mL. Prepare two parallel aliquots for calculating the impurity content of the test sample.

[0135] Test solution: Take citalopram hydrobromide tablets (source: Anyue Branch of Sichuan Kelun Pharmaceutical Co., Ltd.) and place them in a 200mL volumetric flask. Add an appropriate amount of diluent, shake to disperse evenly, sonicate for 10 minutes, cool to room temperature, add diluent to make up to volume, shake well, filter through a 0.45μm PTFE filter membrane, and take the filtrate to obtain a citalopram concentration of approximately 1.0mg / mL. Prepare 6 parallel solutions.

[0136] Accurately measure 10 μL of each of the above solutions and inject them into the liquid chromatograph. Detect the solutions under the chromatographic conditions of Example 1. Calculate the content of each impurity in the six test samples using the principal component self-comparison method with correction factors.

[0137] Experimental results: Impurity Z3 was detected at 0.04%, impurity Z6 at 0.01%, and impurity Z7 at 0.01% in the test samples. An unknown impurity was also detected, with a total impurity content of 0.14%. The types of impurities detected in the six test samples were consistent, and the content of each impurity and the total impurity content were basically consistent. The resolution between all impurities and between impurities and citalopram was greater than 1.5. This method can accurately detect related substances in citalopram hydrobromide tablets and has good repeatability.

[0138] Example 15

[0139] Blank solution: Take the diluent from Example 1 as a blank solution;

[0140] Blank excipient solution: Weigh approximately 100 mg of the relevant excipients for citalopram hydrobromide capsules (source: Anyue Branch of Sichuan Kelun Pharmaceutical Co., Ltd.), place them in a 20 mL volumetric flask, add an appropriate amount of diluent, shake to disperse evenly, sonicate for 10 min, cool to room temperature, add diluent to make up to volume, shake well, filter through a 0.45 μm filter membrane, and take the filtrate as the blank excipient solution;

[0141] Test solution: Take citalopram hydrobromide capsules (source: Anyue Branch of Sichuan Kelun Pharmaceutical Co., Ltd.), accurately weigh the contents (approximately equivalent to 20 mg of citalopram), place in a 20 mL volumetric flask, add an appropriate amount of diluent, shake to disperse evenly, sonicate for 10 min, cool to room temperature, add diluent to make up to volume, shake well, filter through a 0.45 μm PTFE filter membrane, and take the filtrate. The concentration of citalopram is approximately 1.0 mg / mL.

[0142] Reference solution: Same as the reference solution in Example 14.

[0143] High humidity destructive sample solution: Take citalopram hydrobromide capsules that have been placed under high humidity conditions (RH 92.5%) for 10 days and prepare a high humidity destructive sample solution according to the test solution preparation method. The citalopram concentration is approximately 1.0 mg / mL.

[0144] 10 μL of each of the above solutions was precisely measured and injected into the liquid chromatograph, and the solutions were detected under the chromatographic conditions of Example 1.

[0145] Experimental Results: The blank solvent and blank excipients did not interfere with the determination of related substances in the test solution, indicating that the detection method provided by this invention has good specificity. Eleven related substances were detected in the high-humidity-degraded sample solution. Calculated using the principal component self-comparison method with correction factors, the contents of impurity Z2 were 0.01%, Z3 0.04%, Z5 0.01%, Z6 0.05%, Z7 0.01%, Z8 0.01%, Z12 0.02%, and the unknown impurity was 0.04%. The resolution between the main peak and each impurity was greater than 1.5, demonstrating that this method can accurately detect related substances in citalopram hydrobromide capsules.

[0146] Comparative Example 1

[0147] Mobile phase A: 0.66% phosphate buffer (dissolve 6.6 g of diammonium hydrogen phosphate in 1000 mL of water, add 2 mL of triethylamine, shake well, and adjust the pH to 5.0 with phosphoric acid). Other chromatographic conditions were the same as in Example 1. The suitability solution of the first system was analyzed, and the results are shown in Table 13. Figure 15 .

[0148] Table 13

[0149] impurities Retention time (min) Theoretical number of plates Resolution Unknown impurity 1 9.03 32548 - Impurity Z13 11.00 55037 10.14 impurity Z2 11.14 52357 0.74 Impurity Z3 13.01 84482 9.98 Impurity Z4 15.82 105462 15.02 Impurity Z5 18.72 37060 9.92 Unknown impurity 3 20.01 154004 4.33 Impurity Z12 22.18 175895 10.41 Impurity Z6 22.53 184940 1.69 Citalopram + Impurity Z7 24.09 42316 4.60 Impurity Z8 25.87 136231 4.75 Impurity Z9 28.15 106163 7.25 Impurity Z10 40.58 119190 30.44 Impurity Z11 43.68 148646 6.70

[0150] Experimental results: combined with Table 13 and Figure 15 As can be seen, under these conditions, unknown impurity 2 could not be detected, impurity Z7 overlapped with the citalopram chromatographic peak and could not be separated, and the resolution between impurities Z2 and Z13 was only 0.74, which does not meet the requirements. This method cannot effectively separate the impurities, cannot achieve the detection purpose, and is not suitable for the detection of citalopram related substances.

[0151] Comparative Example 2

[0152] Mobile phase A: 0.66% phosphate buffer (dissolve 6.6g of diammonium hydrogen phosphate in 1000ml of water, add 2ml of triethylamine, shake well, and adjust the pH to 3.0 with phosphoric acid). Other chromatographic conditions were the same as in Example 1. The suitability solution of the first system was analyzed, and the results are shown in Table 14. Figure 16 .

[0153] Table 14

[0154]

[0155]

[0156] Experimental results: combined with Table 14 and Figure 16 As can be seen, impurity Z7 coincides with citalopram, and impurity Z2 coincides with unknown impurity 1; the peak of impurity Z5 is broadened and cannot be detected. This method has poor detection performance and cannot achieve the detection objective.

[0157] Comparative Example 3

[0158] The column temperature was 45℃, and the remaining chromatographic conditions were the same as in Example 1. The suitability solution for the first system was analyzed, and the results are shown in Table 15. Figure 17 .

[0159] Table 15

[0160] impurities Retention time (min) Theoretical number of plates Resolution Impurity Z13 8.58 33670 - impurity Z2 11.41 50105 14.49 Impurity Z3 12.08 54573 3.22 Impurity Z4 15.06 68963 13.67 Impurity Z5 17.18 83447 9.06 Impurity Z12 21.07 107662 15.68 Impurity Z6 21.41 111234 1.34 Impurity Z7 21.91 132499 2.00 Citalopram 23.09 54232 3.68 Impurity Z8 23.89 116945 2.38 Impurity Z9 30.67 54223 16.78 Impurity Z10 39.28 72510 15.49 Impurity Z11 42.47 83156 5.44

[0161] Experimental results: combined with Table 15 and Figure 17 It is evident that impurity Z8 and the citalopram peak did not achieve baseline separation, and the separation degree between impurity Z12 and impurity Z6 was only 1.34. The separation effect of this method is poor and does not meet the requirements.

[0162] Comparative Example 4

[0163] The mobile phase flow rate was 1.5 mL / min, and the remaining chromatographic conditions were the same as in Example 1. The suitability solution of the first system was analyzed.

[0164] Test results: The column pressure reached 320 bar, the column was severely worn, and the durability of the column was affected.

[0165] Comparative Example 5

[0166] Solution preparation:

[0167] Diluent: Methanol-water (1:1, v:v).

[0168] Mixed impurity stock solution: Accurately weigh approximately 2.5 mg of each of the 12 impurity reference standards Z2-Z13, place them in 10 mL volumetric flasks, dissolve them with diluent and make up to volume, shake well, and use them as stock solutions for each impurity; accurately measure 0.5 mL of each impurity stock solution, add it to a 10 mL volumetric flask, make up to volume with diluent, shake well, and use it as mixed impurity stock solution.

[0169] Third System Suitability Solution: Accurately weigh 25 mg of citalopram hydrobromide raw material (source: Qionglai Branch of Sichuan Kelun Pharmaceutical Co., Ltd., batch 1), place it in a 20 mL volumetric flask, accurately measure 2 mL of mixed impurity stock solution, dissolve and dilute with diluent, shake well to obtain the third system suitability solution.

[0170] Chromatographic conditions:

[0171] Chromatographic column: octylsilane-bonded silica gel as the packing material (Shiseido Capcell pak C8 4.6×150mm, 3μm);

[0172] Mobile phase: 0.1% acetate buffer (dissolve 1g of sodium acetate in 800mL of water, add 6mL of triethylamine, adjust pH to 4.6 with glacial acetic acid, and add water to 1000mL) - acetonitrile = 80:20;

[0173] Isocratic elution, flow rate: 0.8 mL / min

[0174] Detection wavelengths: 220nm, 239nm;

[0175] Column temperature: 45℃.

[0176] Sample testing: Accurately measure 20 μL of the third system suitability solution and inject it into the liquid chromatograph. Record the chromatograms under different wavelength conditions. Experimental data and results are shown in Table 16, and the chromatograms are shown below. Figure 18 , Figure 19 .

[0177] Table 16

[0178]

[0179] Note: "ND" indicates that the impurity was not detected.

[0180] Experimental results: (Refer to Table 16) Figure 18 and Figure 19As can be seen, at a detection wavelength of 239 nm, impurities Z10 and Z11 showed low responses and could not be detected. The chromatographic peaks of impurities Z6 and Z12 overlapped and could not be separated. The resolution between citalopram and impurity Z7 was 1.20, indicating poor resolution. At a detection wavelength of 220 nm, the baseline fluctuated significantly, and only five impurities could be detected, indicating a low number of impurities and poor peak shape. This method cannot effectively separate and detect the impurities, thus failing to achieve the detection objective and is not suitable for the detection of citalopram-related substances.

[0181] Comparative Example 6

[0182] Chromatographic conditions:

[0183] Stationary phase: Octadecylsilane-bonded silica gel or inorganic oxide microparticle stationary phase as filler (Shiseido C18MGⅡ, 4.6×150mm, 3μm);

[0184] Mobile phase: phosphate buffer-methanol-acetonitrile (55:38:7, v:v:v), pH adjusted to 6.5 with phosphate;

[0185] Isocratic elution, flow rate: 0.8 mL / min;

[0186] Detection wavelength: 239nm;

[0187] Column temperature: 45℃.

[0188] Sample testing:

[0189] Accurately measure 20 μl of the third system suitability solution and inject it into the liquid chromatograph, recording the chromatogram. Experimental data and results are shown in Table 17, and the chromatograms are shown in [reference needed]. Figure 20 .

[0190] Table 17

[0191]

[0192]

[0193] Experimental results: combined with Table 17 and Figure 20 It is evident that impurities Z10 and Z11 have low responses, impurity Z6 overlaps with impurity Z12, and the resolutions of impurity Z13 with impurity Z2, impurity Z4 with unknown impurity 2, unknown impurity 3 with its adjacent impurity peak, and impurity Z7 with citalopram are 1.10, 0.98, 1.31, and 0.55, respectively. This method has poor resolution and cannot effectively separate the impurities, thus failing to achieve the detection objective and is not suitable for the detection of citalopram-related substances.

Claims

1. A method for determining related substances in citalopram salt and its preparations, characterized in that: Includes the following steps: HPLC was used for detection; the chromatographic conditions were as follows: mobile phase A was phosphate buffer solution with a mass-to-volume ratio of 0.6-0.7% and a pH of 6.0-7.0; mobile phase B was a mixed solution of methanol and acetonitrile in a volume ratio of 30-50:70-50. The HPLC employed gradient elution, and the gradient elution conditions were as follows: The column temperature of the HPLC is 28–32 °C; The flow rate of the HPLC was 0.7–0.9 mL / min; The citalopram salt is citalopram hydrobromide; The HPLC column is a reversed-phase column packed with octadecylsilane-bonded silica gel, with a column size of 4.6 × 150 mm and a packing particle size of 3 μm or 3.5 μm.

2. The method for determining related substances in citalopram salt and its preparations according to claim 1, characterized in that: The HPLC employed gradient elution, and the gradient elution conditions were as follows: 。 3. The method for determining related substances in citalopram salt and its preparations according to claim 1 or 2, characterized in that: Triethylamine was added to the mobile phase A.

4. The method for determining related substances in citalopram salt and its preparations according to claim 3, characterized in that: In the mobile phase A, 2 mL of triethylamine is added for every 1000 mL of mobile phase A.

5. The method for determining related substances in citalopram salt and its preparations according to claim 1, characterized in that: The detection wavelength of the HPLC is UV220nm or 239nm.

6. The method for determining related substances in citalopram salt and its preparations according to claim 1, characterized in that: The column temperature of the HPLC was 30°C.

7. The method for determining related substances in citalopram salt and its preparations according to claim 1, characterized in that: The flow rate of the HPLC was 0.8 mL / min.

8. The method for determining related substances in citalopram salt and its preparations according to claim 1, characterized in that: The sample to be tested is dissolved in a diluent to form a sample solution; the diluent is a mixed solution of mobile phase A and mobile phase B.

9. The method for determining related substances in citalopram salt and its preparations according to claim 8, characterized in that: The diluent is a mixed solution of mobile phase A and mobile phase B in a volume ratio of 70-85:30-15.

10. The method for determining related substances in citalopram salt and its preparations according to claim 9, characterized in that: The diluent is a mixed solution of mobile phase A and mobile phase B in a volume ratio of 80:

20.

11. The method for determining related substances in citalopram salt and its preparations according to claim 1, characterized in that: The citalopram salt is prepared in the form of oral tablets, capsules, oral liquids, orally disintegrating films, orally disintegrating tablets, dispersible tablets, granules, or chewable tablets.

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