Method for detecting related substances in tandospirone citrate

Abstract

The invention provides a method for detecting related substances in a tandospirone citrate product, which adopts high performance liquid chromatography and can effectively detect the related substances in the tandospirone citrate product. The detection method provided by the invention has the advantages of no baseline interference, high separation degree, low detection limit, strong specificity, high sensitivity, and good accuracy, precision and durability. According to the invention, new compounds, namely a compound IV, Tand-3, Tand-4, Tand-5, Tand-base-4, Tand-base-6 and Tand-base-8, are also found in the invention. The compound can be used as a reference substance for content analysis and / or quality control of a product containing tandospirone citrate or tandospirone.

Description

Technical Field

[0001] The invention relates to the technical field of high performance liquid chromatography, in particular to a method for detecting related substances in tandospirone citrate. Background Art

[0002] Tandospirone citrate is a new type of antianxiety drug developed by Sumitomo Pharma Co., Ltd. of Japan. It was approved for marketing in Japan in 1996 and entered the Chinese market in 2004. It has good therapeutic effects on anxiety caused by various neuroses, mild to moderate depression, and anxiety and / or depression associated with various physical diseases such as hypertension and peptic ulcer, and significantly improves patients' mental and physical symptoms. It is increasingly widely used in the field of anti-anxiety in China. Tandospirone citrate can selectively act on 5-HT1A receptors in the brain. The action sites are concentrated in the limbic system of the brain such as the hippocampus and amygdala of the emotional center and project to the raphe nucleus of the 5-HT nerve. By activating the presynaptic 5-HT1A receptor, it inhibits neuronal discharge and reduces the synthesis of 5-HT. At the same time, it has a partial agonist effect on the postsynaptic 5-HT1A receptor, thereby achieving a comprehensive regulation of the synaptic 5-HT function and exerting antianxiety effects. Long-term use can also exert an antidepressant effect. Compared with traditional sedatives and hypnotics, tandospirone citrate has the advantages of specific anti-anxiety effect, fewer side effects, weak sedative and hypnotic effect, no muscle relaxant effect, no dependence and withdrawal symptoms, no accumulation in the body after long-term use, etc., and has broad application prospects in the field of anti-anxiety. The structure of tandospirone citrate includes four parts: exomethylene hexahydrophthalic anhydride amide (referred to as spirone ring) (A), four carbon atom alkane chain bridge (B), piperazine pyrimidine (referred to as base) (C) and citric acid (D).

[0003]

[0004] At present, there have been many studies on the preparation method of tandospirone citrate, such as US4507303, US4818756, JP60087262, CN101362751A, CN101880274A, etc., which respectively disclose tandospirone and the preparation method of tandospirone citrate. In the process of preparing tandospirone citrate, a large number of impurities (i.e., related substances) will be produced, including the reaction raw materials introduced in the preparation process, reaction intermediates, reaction by-products generated, and degradation impurities generated by drug degradation. The research and control of impurities is an important link in risk control in drug development, a key element in drug safety assurance, and one of the core contents of drug quality control. It runs through the entire drug research. According to the definition of "Technical Guiding Principles for the Study of Impurities in Chemical Drugs", impurities contained in raw materials refer to any substance that affects the purity of the drug, is a substance that has no therapeutic effect in the drug, or affects the stability and efficacy of the drug, or is even harmful to human health. From the analysis of its chemical structure, it is a certain component that is different from the raw material. Whether the impurities in drugs can be reasonably and effectively controlled is directly related to the quality controllability and safety of the drugs.

[0005] Tandospirone citrate requires long-term use, and the risk of impurity intake should be fully considered. Therefore, it is urgent to study the impurities of tandospirone citrate. Summary of the invention

[0006] In order to improve the above technical problems, the present invention provides a method for detecting related substances in tandospirone citrate.

[0007] The present invention provides a method for detecting related substances in a product, wherein the product comprises tandospirone citrate; the detection method adopts high performance liquid chromatography, and the chromatographic conditions are:

[0008] Chromatographic column: octadecylsilane bonded silica gel is used as filler, such as C18; preferably, the size of the chromatographic column is 4.6 mm × 250 mm, 5 μm;

[0009] The mobile phase conditions are as follows: phase A is 0.001 mol / L to 0.1 mol / L (preferably 0.005 mol / L to 0.05 mol / L, for example 0.01 mol / L) potassium dihydrogen phosphate buffer solution-acetonitrile, and phase B is 0.001 mol / L to 0.1 mol / L (preferably 0.005 mol / L to 0.05 mol / L, for example 0.01 mol / L) potassium dihydrogen phosphate buffer solution-acetonitrile;

[0010] In phase A, the volume ratio of potassium dihydrogen phosphate buffer solution to acetonitrile is 70-90:10-30, preferably 85:15;

[0011] In phase B, the volume ratio of potassium dihydrogen phosphate buffer solution to acetonitrile is 30-70:30-70, preferably 50:50;

[0012] Gradient elution;

[0013] The detection wavelength is 220nm-250nm, for example 243nm;

[0014] The injection volume is 10 μL-50 μL, for example, 20 μL;

[0015] The column temperature is 20°C-40°C, for example 30°C;

[0016] The flow rate is 0.9 mL / min-1.5 mL / min, for example 1.0 mL / min.

[0017] According to an embodiment of the present invention, in phase A and phase B, the potassium dihydrogen phosphate buffer solution is further adjusted to a pH value of 7.0-8.0, preferably 7.5±0.2, using a 10% sodium hydroxide solution.

[0018] According to an embodiment of the present invention, the elution gradient is:

[0019] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 10 100 0 25 0 100 50 0 100 51 100 0 58 100 0

[0020] According to an embodiment of the present invention, the product is selected from medicines, raw materials, preparations, etc. containing tandospirone citrate.

[0021] According to an embodiment of the present invention, the related substances include the following compounds:

[0022]

[0023] According to an embodiment of the present invention, the related substances further include the following compounds:

[0024]

[0025]

[0026] According to an embodiment of the present invention, the related substances further include the following compounds:

[0027]

[0028] According to an embodiment of the present invention, the related substances further include any one, two or more of the following compounds:

[0029]

[0030] According to an embodiment of the present invention, the related substances further include any one, two or more of the following compounds:

[0031]

[0032] According to an embodiment of the present invention, the related substances further include any one or two of the following compounds:

[0033]

[0034] According to an embodiment of the present invention, the related substances further include any one, two or more of the following compounds:

[0035]

[0036] According to an embodiment of the present invention, the related substances further include any one, two or more of the following compounds:

[0037]

[0038] According to an embodiment of the present invention, the related substances further include any one, two or more of the following compounds:

[0039]

[0040] According to an embodiment of the present invention, the related substances include the following compounds:

[0041]

[0042]

[0043] According to an embodiment of the present invention, the detection method comprises the following steps:

[0044] 1) Preparation of the test solution: take a product containing tandospirone citrate, accurately weigh it, put it in a volumetric bottle, add an appropriate amount of solvent to dissolve it, and make up to volume to obtain the test solution;

[0045] 2) Preparation of system suitability solution: Accurately weigh the above-mentioned related substances respectively, place them in a volumetric bottle, add an appropriate amount of solvent to dissolve, and make up to volume to prepare a system suitability solution;

[0046] 3) Detection: The relevant substances in the test solution and the system suitability solution were detected by high performance liquid chromatography. The chromatographic conditions were as described above.

[0047] According to the embodiment of the present invention, 2) the preparation of the system suitability solution is specifically as follows: accurately weigh the tandospirone citrate reference substance and the above-mentioned related substances, place them in a volumetric bottle, add an appropriate amount of solvent to dissolve, and make up to volume to prepare a system suitability solution.

[0048] According to an embodiment of the present invention, the concentration of the test solution in step 1) is 0.1 mg / mL-1.0 mg / mL, preferably 0.5 mg / mL.

[0049] According to an embodiment of the present invention, the concentration of the tandospirone citrate reference substance in step 2) is 0.1 mg / mL-1.0 mg / mL, preferably 0.5 mg / mL.

[0050] According to an embodiment of the present invention, the concentration of the relevant substance in step 2) is 0.1 μg / mL-1.0 μg / mL, preferably 0.5 μg / mL.

[0051] According to an embodiment of the present invention, the solvent used in step 1) and / or step 2) is a mixed solvent of acetonitrile-water, preferably a mixed solvent of acetonitrile-water in a volume ratio of 15:85.

[0052] The present invention also provides the following compounds or pharmaceutically acceptable salts thereof:

[0053]

[0054]

[0055] The present invention also provides the use of one, two or more of the above-mentioned compounds or pharmaceutically acceptable salts thereof in content analysis and / or quality control of products; preferably, the above-mentioned compounds are used as reference substances or standard substances for content analysis and / or quality control of products; preferably, the product contains tandospirone citrate or tandospirone.

[0056] According to an embodiment of the present invention, the product is selected from medicines, raw materials, preparations, etc.

[0057] The present invention also provides a method for content analysis and / or quality control of a product, the method comprising:

[0058] Using the above-mentioned compound or its pharmaceutically acceptable salt as a standard or reference substance, detecting the content of the compound or its pharmaceutically acceptable salt in the product, analyzing the content of the product and / or controlling the quality of the product;

[0059] Preferably, the product comprises tandospirone citrate or tandospirone.

[0060] According to an embodiment of the present invention, the product is selected from medicines, raw materials, preparations, etc.

[0061] The present invention also provides a method for preparing the above-mentioned compound IV or a pharmaceutically acceptable salt thereof, the preparation method comprising the following steps:

[0062] Compound IV can be obtained by reacting tandospirone with an oxidant in a solvent;

[0063] The reaction formula is as follows:

[0064]

[0065] According to an embodiment of the present invention, the oxidizing agent includes hydrogen peroxide (eg, 30% hydrogen peroxide), perchloric acid, peracetic acid, or meta-chloroperbenzoic acid.

[0066] According to an embodiment of the present invention, the solvent is selected from methanol, ethanol, dichloromethane, toluene, isopropyl acetate or acetonitrile.

[0067] According to an embodiment of the present invention, the mass volume ratio of tandospirone to the solvent is 1g:5mL-1g:20mL, preferably 1g:5mL-1g:10mL, for example 1g:8mL.

[0068] According to an embodiment of the present invention, the preparation method comprises the following steps:

[0069] Tandospirone and a solvent are mixed and stirred, and after the system becomes clear, an oxidant is added and the mixture is refluxed to obtain compound IV.

[0070] According to an embodiment of the present invention, the preparation method optionally further comprises the following steps:

[0071] After the reaction is completed, the reactants are stirred with anhydrous sodium sulfite for a period of time (to remove excess oxidant), filtered, washed, and concentrated;

[0072] The concentrate is dissolved, washed with water, and the organic phase is dried, filtered, and concentrated;

[0073] The concentrate is then separated and purified to obtain compound IV; preferably, the concentrate is separated and purified using silica gel column chromatography; more preferably, the eluent used in the silica gel column chromatography is: dichloromethane / methanol = 10:1.

[0074] Beneficial Effects

[0075] (1) The present invention provides a method for detecting related substances in a product, wherein the product contains tandospirone citrate; the method adopts high performance liquid chromatography. The method can effectively detect related substances in tandospirone citrate, and the method has no interference in the baseline, high separation, low detection limit, strong specificity, high sensitivity, good accuracy, precision and durability.

[0076] (2) The detection method is particularly effective in separating compounds IV and Tand-6 in products containing tandospirone citrate, and can further separate a variety of related substances, such as Tand-3, Tand-4, Tand-5, Tand-base-4, Tand-base-6, Tand-base-8 and other unknown compounds and impurities II, Tand-base-10, Tamd-qas, impurity I, Tand-base-3 and other known compounds.

[0077] (3) The present invention also discovered new compounds - Compound IV, Tand-3, Tand-4, Tand-5, Tand-base-4, Tand-base-6, Tand-base-8. The compounds can be used as reference substances for content analysis and / or quality control of products containing tandospirone citrate or tandospirone. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 is the liquid chromatogram of blank solvent;

[0079] Figure 2 is the liquid chromatogram of the system suitability solution;

[0080] Figure 3 is the liquid chromatogram of the test solution;

[0081] Figure 4 is the liquid chromatogram of compound IV in Comparative Example 1;

[0082] Figure 5 This is the liquid chromatogram of Tand-6 in Comparative Example 1. DETAILED DESCRIPTION

[0083] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0084] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0085] Tandospirone citrate reference substance, impurity I, impurity II, Tand-qas, Tand-base-3, and Tand-base-10 are all commercially available products.

[0086] Example 1 Preparation of Compounds

[0087] 1. Preparation of Compound IV

[0088]

[0089] Add 50.00g of tandospirone to 400mL of methanol, heat to 50°C, add 130ml of 30% hydrogen peroxide, heat to reflux for 3 hours, and monitor the reaction to end by TLC. Add 120.00g of anhydrous sodium sulfite and stir for 0.5 hours, filter, wash with 50mL of methanol, and concentrate the filtrate under reduced pressure at 50±5°C to obtain 35.00g of concentrate. Add 100mL of dichloromethane to the concentrate, wash once with 30ml of water, dry the organic phase with 50.00g of anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure at 50±5°C. The concentrate is separated and purified by 100-200 mesh silica gel column chromatography, and the eluent is: dichloromethane / methanol = 10:1. The eluent is concentrated under reduced pressure to obtain 28.10g of compound IV.

[0090] Compound IV 1 H spectrum, 13 C spectrum and LC-MS information are as follows:

[0091] 1 H NMR(400MHz,DMSO-d6)δ8.403-8.391(d,2H),6.692-6.668(t,1H),4.463-4.4 29(d,2H),3.673-3.356(t,2H),3.268-3.203(t,2H),3.166-3.124(m,4H),2. 953-2.926(d,2H),2.664(s,2H),2.492(s,2H),1.823-1.737(m,2H),1.573-1 .439(m,4H),1.323-1.304(d,2H),1.178-1.136(d,1H),1.001-0.974(d,1H).

[0092] 13 C NMR (100MHz, DMSO-d6) δ179.1,161.2,158.5,111.0,69.9,63.2,48.4,40.0,38.5,38.2,33.2,27.9,25.3,19.4.

[0093] LC-MS: m / z = 400.12 [M+H] + .

[0094] 2. Preparation of Tand-6

[0095]

[0096] 100.00g of quaternary ammonium salt, 49.20g of phthalamide, and 138.60g of anhydrous potassium carbonate were added to 600mL of DMF, and the temperature was raised to 145±5℃ for reaction. The reaction was monitored by TLC until it was complete. Filtered, the filtrate was allowed to stand at 5±5℃ for crystallization for 8 hours. Filtered, a small amount of DMF was used to wash the filter cake, and the filter cake was dried under reduced pressure at 55±5℃ to obtain 70.99g of intermediate 1; 2600mL of water was added to the filtrate, stirred at 5±5℃ for crystallization for 3h, filtered, washed, and the filter cake was dried under reduced pressure at 55±5℃ to obtain 24.80g of intermediate 1.

[0097] 94.20g of intermediate 1 and 25.60g of hydrazine hydrate were added to 600mL of anhydrous ethanol, and the reaction was refluxed and monitored by TLC until the reaction was complete. Filter, adjust the pH of the filtrate to less than 3 with concentrated hydrochloric acid, stir and crystallize at 20±5℃ for 3 hours, filter, dissolve the filter cake with 600mL of purified water, filter, adjust the pH of the filtrate to more than 14 with sodium hydroxide solution, extract with 150mL*3 dichloromethane, and dry the organic phase with 25.00g of anhydrous sodium sulfate. Filter, concentrate the filtrate under reduced pressure at 45±5℃ to obtain 26.00g of Tand-6.

[0098] Tand-6 1 H spectrum, 13 C spectrum and LC-MS information are as follows:

[0099] 1 H NMR(400MHz, CDCl3-d)δ8.302–8.290(d,2H),6.480-6.456(t,1H),3.843-3.818(t,4H) ,2.737-2.703(t,2H),2.507-2.481(t,4H),2.400-2.363(m,2H),1.591-1.460(m,6H).

[0100] 13 C NMR (100MHz, CDCl3-d) δ161.5, 157.8-157.3, 109.6-109.4, 58.5, 53.2-52.8, 43.7-43.3, 42.00, 31.8-31.4, 24.63-24.0.

[0101] LC-MS: m / z=236.09 [M+H] + .

[0102] 3. Preparation of Tand-3

[0103]

[0104] Weigh 1.00 g of tandospirone citrate, add it to 20 mL of 30% hydrogen peroxide, adjust the pH to about 7 with saturated K2HPO4 solution, add an equal volume of water to dilute, heat to 70°C and react for 4 hours. The reaction solution is separated by multiple preparative chromatography to obtain 0.31 g of Tand-3.

[0105] 1 H NMR(400MHz,DMSO-d6)δ8.37(s,2H),6.66(s,1H),4.43(m,2H),3.99(m,1H),3.60(m,2H),3.35(m,2H),3.23(m,2H),3.16(m,2H),3.00(m ,2H),2.76(m,1H),2.60(m,1H),2.58(m,1H),2.44(m,1H),1.75(m,2H),1.66(m,1H),1.45(m,2H),1.29(m,1H),1.15(m,1H),0.88(m,1H).

[0106] 13 C NMR (100MHz, DMSO-d6) δ178.6, 178.5, 161.2, 158.5, 111.1, 85.0, 69.5, 63.0, 48.0, 44.1, 42.8, 38.5, 38.3, 38.2, 35.2, 29.6, 25.2, 19.3.

[0107] LC-MS: m / z=432.22 [M+H] + .

[0108] 4. Preparation of Tand-4

[0109]

[0110] Weigh 2.00 g of tandospirone citrate, add it to 40 mL of 30% hydrogen peroxide, adjust the pH to about 7 with saturated K2HPO4 solution, add an equal volume of water to dilute, heat to 70°C and react for 5 hours. The reaction solution is separated by multiple preparative chromatography to obtain 0.65 g of Tand-4.

[0111] 1H NMR(400MHz,DMSO-d6)δ8.37(d,2H),6.66(t,1H),4.43(d,2H),4.36(m,1H),3.60(m,2H),3.36(m,2H),3.24(m,2H),3.16(m,2H),3.10(d ,1H),3.00(m,2H),2.72(m,1H),2.65(m,1H),2.39(m,1H),1.87(m,1H),1.75(m,2H),1.45(m,2H),1.28(m,1H),1.00(m,1H),0.87(m,1H).

[0112] 13 C NMR (100MHz, DMSO-d6) δ180.0, 178.9, 161.2, 158.5, 111.1, 82.9, 69.5, 62.9, 48.2, 43.3, 40.8, 39.2, 38.4, 38.2, 33.7, 31.8, 25.2, 19.3.

[0113] LC-MS: m / z=432.22 [M+H] + .

[0114] 5. Preparation of Tand-5

[0115]

[0116] Weigh 1.50g of norbornane dianhydride (exo form) and 1.72g of 4-(4-(2-pyrimidinyl)-1-piperazinyl)-1-butylamine, add them to 20mL of methanol, and react at 20±5°C for 3 hours. Filter the reaction liquid, collect the filter cake, add the filter cake to 50mL of methanol, then add 30% sodium hydroxide aqueous solution and stir, concentrate the reaction liquid under reduced pressure, add 30mL of ethyl acetate to the concentrate, stir for 0.5 hours, filter, collect the filter cake, and dry the filter cake to obtain 2.50g of Tand-5.

[0117] 1 H NMR(400MHz,MeOD-d4)δ8.338-8.326(d,2H),6.621-6.597(t,1H),3.838-3.812(t,4H),3.329-3.226(m,1H),3.112-3. 096(m,1H),2.579-2.488(m,6H),2.454-2.415(m,3H),2.287-2.266(m,2H),1.606-1.522(m,6H),1.522-1.161(m,3H).

[0118] 13C NMR (100MHz, MeOD-d4) δ182.0, 177.0, 162.9, 159.1, 111.3, 59.5, 56.4, 54.1, 44.5, 42.3, 40.7, 40.2, 37.2, 30.5, 30.0, 28.4, 25.0, 24.3.

[0119] LC-MS: m / z = 399.81 [M-Na] - .

[0120] 6. Preparation of Tand-base-4

[0121] The reaction formula is:

[0122]

[0123] 30.00 g of norbornane dicarboximide (external form), 117.64 g of 1,4-dibromobutane, and 37.65 g of anhydrous potassium carbonate were weighed and added to 300 mL of acetone. The mixture was refluxed for 3 hours. The reaction solution was filtered and the filtrate was concentrated under reduced pressure. The concentrate was separated and purified by column chromatography to obtain 54.50 g of intermediate 1.

[0124] 20.00 g of 2-piperazinone, 22.88 g of 2-chloropyrimidine and 21.17 g of anhydrous sodium carbonate were weighed and added into 150 mL of water for reflux reaction for 2 hours. The reaction solution was cooled to 20±5°C, filtered and the filter cake was dried to obtain 30.00 g of intermediate 2.

[0125] Weigh 18.00g of intermediate 1, 10.00g of intermediate 2, and 6.73g of sodium hydride (content 60%), add to 300mL of DMF, and react at -5±5°C for 2 hours. Add 300mL of dichloromethane, extract with water, dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The concentrate is separated and purified by column chromatography to obtain 2.50g of Tand-base-4.

[0126] 1 H NMR(400MHz,MeOD-d4)δ8.396-8.384(d,2H),6.707-6.683(t,1H),4.354(s,2H),4.072 -4.045(t,2H),3.519-3.467(m,6H),2.671(s,2H),2.598(s,2H),1.706-1.264(m,10H).

[0127] 13C NMR (100MHz, CDCl3-d) δ183.6, 171.3, 164.5, 161.8, 114.7, 59.4, 51.5, 49.9, 49.8, 44.3, 43.5, 41.6, 36.4, 31.4, 28.5, 27.8.

[0128] LC-MS: m / z = 420.12 [M+Na] + .

[0129] 7. Preparation of Tand-base-6

[0130] The reaction formula is:

[0131]

[0132] Weigh 15.00 g of norbornane dianhydride (external form) and 0.45 g of triethylamine, add to 150 mL of dichloromethane, cool to 0°C, add 12.20 g of dimethylamine, and react at 0±5°C for 3 hours. Concentrate the reaction solution under reduced pressure to obtain 19.00 g of the intermediate.

[0133] 5.20 g of the intermediate, 6.40 g of 4-(4-(2-pyrimidinyl)-1-piperazinyl)-1-butylamine, 9.30 g of DIEPA, and 20.90 g of HATU were added to 50 mL of dichloromethane and reacted at 20±5°C for 2 hours. The reaction solution was concentrated under reduced pressure, and the concentrate was separated and purified by column chromatography to obtain 3.60 g of Tand-base-6.

[0134] LC-MS: m / z = 451.18 [M+Na] + .

[0135] 8. Preparation of Tand-base-8

[0136] The reaction formula is:

[0137]

[0138] Weigh 6.10 g of norbornane dianhydride (external form) and 6.70 g of 4-(4-(2-pyrimidinyl)-1-piperazinyl)-1-butanol, add them to 70 mL of methyl tert-butyl ether, and react at 20±5°C for 4 hours. Filter the reaction liquid, collect the filter cake, and dry the filter cake to obtain 7.75 g of the intermediate.

[0139] 3.00 g of the intermediate, 1.76 g of 4-(4-(2-pyrimidinyl)-1-piperazinyl)-1-butanol, 4.72 g of DCC, and 0.06 g of DMAP were weighed and added to 30 mL of dichloromethane and refluxed for 4 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The obtained concentrate was separated and purified by column chromatography to obtain 1.30 g of Tand-base-8.

[0140] LC-MS: m / z = 643.26 [M+Na] + .

[0141] Example 2 Growth and accumulation of compound IV in the formulation

[0142] The inventors found in the process of studying tandospirone citrate and its preparations that as the storage time of tandospirone citrate and its preparations increases, the content of compound IV shows an increasing trend. It has been detected in multiple batches of tandospirone citrate samples. It is preliminarily judged that compound IV is a degradation impurity caused by oxidation of tandospirone citrate during storage.

[0143] Test conditions: 30±2℃, RH65%±5%

[0144] Table 1 Changes in the content of compound IV in samples during storage

[0145]

[0146] Composition of tandospirone citrate tablets: tandospirone citrate, lactose, corn starch, carboxymethylcellulose calcium, polyvinyl alcohol (partially saponified), magnesium stearate, hypromellose, glycerin, and titanium dioxide.

[0147] The method for content determination is as in Example 3.

[0148] As shown in Table 1, during the storage of tandospirone citrate tablets at 30±2°C and RH65%±5%, the content of compound IV in the preparation increased significantly with time; at 6 months, the content of compound IV in the preparation reached 0.02%. Moreover, according to this trend, it can be expected that the content of compound IV will also increase sharply as the storage time continues to increase. Since tandospirone citrate and its preparations are long-term medications, in order to ensure the safety of medication, it is of great significance to conduct quality control and content detection of compound IV in tandospirone citrate.

[0149] Example 3 Determination of related substances

[0150] Determined by high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia).

[0151] 1. Measurement conditions

[0152] Chromatographic column: Octadecylsilane bonded silica gel (Phenomenex Gemini C18, 250×4.6 mm, 5 μm) was used as filler.

[0153] Mobile phase A: 0.01 mol / L potassium dihydrogen phosphate buffer (pH adjusted to 7.5 with 10% sodium hydroxide solution)-acetonitrile (85:15, V / V).

[0154] Mobile phase B: 0.01 mol / L potassium dihydrogen phosphate buffer (adjust pH to 7.5 with 10% sodium hydroxide solution)-acetonitrile (50:50, V / V)

[0155] Detection wavelength: 243nm.

[0156] Injection volume: 20 μL.

[0157] Flow rate: 1.0mL / min.

[0158] Column temperature: 30℃.

[0159] Elution gradient:

[0160] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 10 100 0 25 0 100 50 0 100 51 100 0 58 100 0

[0161] 2. Preparation of solution:

[0162] Test solution: Take 50 mg of the product containing tandospirone citrate, accurately weigh it, place it in a 100 mL volumetric flask, add solvent to dissolve it, dilute it to the scale, shake well, and obtain it.

[0163] System suitability solution: Weigh appropriate amounts of tandospirone citrate reference substance, Tand-qas, Tand-base-3, Tand-base-4, Tand-base-6, Tand-base-8, Tand-base-10, impurity I, impurity II, compound IV, Tand-3, Tand-4, Tand-5, and Tand-6 reference substances, add solvent to make a mixed solution containing approximately 0.5 mg of tandospirone citrate working reference substance and approximately 0.5 μg of each impurity reference substance per 1 mL.

[0164] Solvent: acetonitrile-water (15:85).

[0165] 3. Determination:

[0166] Blank: Take 20 μL of blank (solvent), inject it into the liquid chromatograph, and record the chromatogram. The baseline of the blank spectrum should be stable and free of abnormal peaks.

[0167] System suitability solution: Take 20 μL, inject into liquid chromatograph, and record the chromatogram.

[0168] Sample determination: Accurately measure 20 μL of the test solution and inject it into the liquid chromatograph respectively, with one injection for each solution, and record the chromatogram.

[0169] Injection sequence:

[0170] Table 2 Injection sequence table

[0171] Serial number Sample name Number of injection needles standard 1 blank 1 The baseline is stable and has no abnormal peaks. 2 System Suitability Solutions 1 —— 3 Test solution 1 ——

[0172] Calculation formula:

[0173]

[0174] Total impurities = ∑ single impurity

[0175] Result determination: Single impurity shall not exceed 0.10%, and total impurities shall not exceed 0.2%.

[0176] Test results: Figure 1 (blank), Figure 2 and Table 4 (System Suitability Solutions), Figure 3 and Table 5 (test solution).

[0177] Table 3 Impurity names, impurity structures and impurity sources

[0178]

[0179]

[0180]

[0181] Table 4 Summary of signal information of each peak in the chromatogram of system suitability solution

[0182]

[0183] Table 5 Information summary of peak signals in the chromatogram of the test solution

[0184]

[0185] Example 4 Analytical Method Verification

[0186] According to the purpose of impurity research and detection requirements, the present invention carried out verification items such as specificity, quantitative limit, detection limit, accuracy, etc. of the analytical method.

[0187] Preparation of sample interference solution:

[0188] 1) Impurity stock solution: Take appropriate amount of Tand-qas, impurity I, impurity II, compound IV, Tand-3, Tand-4, Tand-5, Tand-6, Tand-base-3, Tand-base-4, Tand-base-6, Tand-base-8, and Tand-base-10 reference substances, accurately weigh them, dissolve them in solvents and dilute them to prepare stock solutions containing 0.025 mg of impurities per 1 mL;

[0189] 2) Sample interference solution: Take 25 mg of tandospirone citrate reference substance, accurately weigh it, and place it in a 50 mL volumetric flask. Then accurately measure 1.0 mL of each impurity stock solution, place it in a 50 mL volumetric flask, dissolve it with solvent and dilute it to the scale, mix well, and obtain.

[0190] The analytical method validation data and results are shown in Table 6.

[0191] Table 6 Summary of test method validation

[0192]

[0193]

[0194]

[0195]

[0196] The detection method of the present invention realizes the simultaneous separation and quantitative detection of at least 13 process impurities and degradation impurities in tandospirone citrate for the first time, thereby effectively controlling the quality of tandospirone citrate. The detection method provided by the present invention has no interference in the baseline, high separation, low detection limit, strong specificity, high sensitivity, good accuracy, precision and durability.

[0197] Comparative Example 1: Chromatographic conditions in reference patent CN101362751B

[0198] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0199] Mobile phase: 0.01 mol / L potassium dihydrogen phosphate solution (adjusted to pH 7.5 with 10% sodium hydroxide solution)-acetonitrile (60:40);

[0200] Wavelength: 243nm;

[0201] Injection volume: 20 μL;

[0202] Flow rate: 1.0 mL / min;

[0203] The column temperature was 30°C.

[0204] Solution preparation:

[0205] Impurity localization solution: Weigh appropriate amounts of compound IV and Tand-6 impurity reference substance respectively, add solvent to prepare impurity localization solutions containing 0.5 μg of impurity reference substance per 1 mL.

[0206] Take 20 μL of blank (solvent) and each impurity location solution, inject them into the liquid chromatograph, and record the chromatogram.

[0207] Test results: The retention time of compound IV is 2.817min ( Figure 4 ), Tand-6 retention time is 2.860min( Figure 5 ). Under the chromatographic conditions, the retention time of compound IV and Tand-6 is very close, that is, compound IV and Tand-6 cannot be effectively separated.

[0208] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting related substances in a product, wherein the product contains tandospirone citrate; the detection method adopts high performance liquid chromatography, and the chromatographic conditions are: Chromatographic column: octadecylsilane bonded silica gel is used as filler, such as C18; preferably, the size of the chromatographic column is 4.6 mm × 250 mm, 5 μm; The mobile phase conditions are as follows: phase A is 0.001 mol / L to 0.1 mol / L (preferably 0.005 mol / L to 0.05 mol / L, for example 0.01 mol / L) potassium dihydrogen phosphate buffer solution-acetonitrile, and phase B is 0.001 mol / L to 0.1 mol / L (preferably 0.005 mol / L to 0.05 mol / L, for example 0.01 mol / L) potassium dihydrogen phosphate buffer solution-acetonitrile; In phase A, the volume ratio of potassium dihydrogen phosphate buffer solution to acetonitrile is 70-90:10-30, preferably 85:15; In phase B, the volume ratio of potassium dihydrogen phosphate buffer solution to acetonitrile is 30-70:30-70, preferably 50:50; Gradient elution; The detection wavelength is 220nm-250nm, for example 243nm; The injection volume is 10 μL-50 μL, for example, 20 μL; The column temperature is 20°C-40°C, for example 30°C; The flow rate is 0.9 mL / min-1.5 mL / min, for example 1.0 mL / min.

2. The detection method according to claim 1, characterized in that: In phase A and phase B, the potassium dihydrogen phosphate buffer solution is further adjusted to pH 7.0-8.0, preferably 7.5±0.2, with 10% sodium hydroxide solution; and / or, the elution gradient is: And / or, the product is selected from medicines, raw materials, preparations, etc. containing tandospirone citrate.

3. The detection method according to claim 1 or 2, characterized in that: The related substances include the following compounds: Preferably, the related substances further include the following compounds: Preferably, the related substances further include the following compounds: Preferably, the related substances further include any one, two or more of the following compounds: Preferably, the related substances further include any one, two or more of the following compounds: Preferably, the related substances further include any one or two of the following compounds: Preferably, the related substances further include any one, two or more of the following compounds: Preferably, the related substances further include any one, two or more of the following compounds: Preferably, the related substances further include any one, two or more of the following compounds: Preferably, the related substances include the following compounds:

4. The detection method according to any one of claims 1 to 3, characterized in that: The detection method comprises the following steps: 1) Preparation of the test solution: take a product containing tandospirone citrate, accurately weigh it, put it in a volumetric bottle, add an appropriate amount of solvent to dissolve it, and make up to volume to obtain the test solution; 2) Preparation of system suitability solution: accurately weigh the relevant substances respectively, place them in a volumetric bottle, add an appropriate amount of solvent to dissolve, and make up to volume to prepare a system suitability solution; 3) Detection: Detect the related substances in the test solution and the system suitability solution by high performance liquid chromatography, and the chromatographic conditions are as described in any one of claims 1-3.

5. The detection method according to claim 4, characterized in that: 2) The preparation of the system suitability solution is specifically as follows: accurately weigh the tandospirone citrate reference substance and related substances, respectively, place them in a volumetric bottle, add an appropriate amount of solvent to dissolve, and make up to volume to prepare a system suitability solution; and / or, the concentration of the test solution in step 1) is 0.1 mg / mL-1.0 mg / mL, preferably 0.5 mg / mL; and / or, in step 2), the concentration of the tandospirone citrate reference substance is 0.1 mg / mL-1.0 mg / mL, preferably 0.5 mg / mL; and / or, in step 2), the concentration of the related substance is 0.1 μg / mL-1.0 μg / mL, preferably 0.5 μg / mL; And / or, the solvent used in step 1) and / or step 2) is a mixed solvent of acetonitrile-water, preferably a mixed solvent of acetonitrile-water in a volume ratio of 15:

85.

6. The following compound or a pharmaceutically acceptable salt thereof:

7. Use of the compound according to claim 6 or a pharmaceutically acceptable salt thereof in content analysis and / or quality control of a product; Preferably, the product contains tandospirone citrate or tandospirone; Preferably, the product is selected from medicines, raw materials, preparations, etc.

8. A method for content analysis and / or quality control of a product, the method comprising: Using the compound or pharmaceutically acceptable salt thereof as claimed in claim 6 as a standard or reference substance, detecting the content of the compound or pharmaceutically acceptable salt thereof in the product, analyzing the content of the product and / or controlling the quality of the product; Preferably, the product contains tandospirone citrate or tandospirone; Preferably, the product is selected from medicines, raw materials, preparations, etc.

9. A method for preparing compound IV or a pharmaceutically acceptable salt thereof, the method comprising the following steps: Compound IV can be obtained by reacting tandospirone with an oxidant in a solvent; The reaction formula is as follows:

10. The preparation method according to claim 9, characterized in that: Oxidants include hydrogen peroxide (e.g., 30% hydrogen peroxide), perchloric acid, peracetic acid, or meta-chloroperbenzoic acid; and / or, the solvent is selected from methanol, ethanol, dichloromethane, toluene, isopropyl acetate or acetonitrile; and / or, the mass volume ratio of tandospirone to the solvent is 1 g:5 mL-1 g:20 mL, preferably 1 g:5 mL-1 g:10 mL, for example 1 g:8 mL; And / or, the preparation method comprises the following steps: mixing and stirring tandospirone and a solvent, adding an oxidant after the system is dissolved, and performing a reflux reaction to obtain compound IV; And / or, the preparation method may further optionally include the following steps: after the reaction is completed, stirring the reactants with anhydrous sodium sulfite for a period of time (to remove excess oxidant), filtering, washing, and concentrating; dissolving the concentrate, washing with water, drying the organic phase, filtering, and concentrating; then separating and purifying the concentrate to obtain compound IV; preferably, using silica gel column chromatography to separate and purify the concentrate; more preferably, the eluent used in the silica gel column chromatography is: dichloromethane / methanol = 10:1.

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