Method for determining tandospirone and its salt intermediates

Abstract

The present application relates to the field of pharmaceutical analysis chemistry, and specifically provides a determination method of tandospirone and salt intermediates, which adopts high performance liquid chromatography, and the chromatographic conditions include: adopting a chromatographic column with octadecylsilane bonded silica gel as a filler, and gradient elution is performed with mobile phase A and mobile phase B; wherein, the mobile phase A is composed of a first water phase and acetonitrile; the mobile phase B is composed of a second water phase and acetonitrile; the first water phase is an aqueous solution containing potassium dihydrogen phosphate and sodium hexanesulfonate, and the pH is 3.0-3.8; the second water phase is an aqueous solution containing potassium dihydrogen phosphate and sodium hexanesulfonate, and the pH is 1.8-2.6. The test method has good specificity, accuracy and repeatability, and can be used for quality monitoring of tandospirone bulk drug.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical analysis chemistry, and in particular, provides a method for determining tandospirone and its salt intermediates. BACKGROUND

[0002] Tandospirone is an important anxiolytic drug, which has a significant advantage in safety compared with traditional benzodiazepines, as it is less likely to cause significant sedation, muscle relaxation or drug dependence and other adverse reactions. With the rising prevalence of anxiety disorders worldwide, the drug is increasingly used in clinical practice and has become a common choice for anxiolytic therapy. To ensure the solubility and stability of the drug, its common drug form is tandospirone citrate. However, the current research on its impurity system is still not systematic, especially the quality control of 1-(2-pyrimidinyl)piperazine, a key intermediate used in the synthesis process, has not been fully valued. As a key starting material for constructing the nitrogen spiro ring skeleton in the molecule of tandospirone, if the reaction is incomplete or the purification is insufficient during the synthesis process, it may remain in the final product.

[0003] Currently, the quality control of tandospirone and its salts mainly focuses on the determination of the content of the main component and the routine related substance inspection. For example, CN101362751A and CN106963766A disclose analysis methods for tandospirone isomers and impurities produced by incomplete reduction reaction; CN115791985A and CN119224160A mainly involve the detection of bromine-containing impurities and potential genotoxicity impurities of nitrosamines. These methods mainly focus on the analysis of tandospirone isomers, bromine-containing impurities or nitrosamine substances, and lack of research on the key intermediate 1-(2-pyrimidinyl)piperazine of tandospirone and its salts, which is a necessary fragment of the side chain of tandospirone. This intermediate is likely to exist in the bulk drug, and its residual level directly affects the quality of the final product and is related to the safety and effectiveness of the drug.

[0004] Therefore, it is particularly important to develop a detection method for this intermediate. SUMMARY

[0005] In the process of establishing the detection method, it was found that 1-(2-pyrimidinyl)piperazine has weak retention ability in traditional chromatographic systems due to its strong polarity, and its chromatographic peak is easily overlapped with the solvent front, making it difficult to accurately quantify. In addition, this intermediate also has similar polarity with another key impurity, quaternary ammonium salt compound 8-(2-pyrimidinyl)-8-aza-5-azaspiro[4.5]decane bromide (referred to as "impurity B"), which is likely to exist in the synthesis process, and it is difficult to effectively separate them under conventional chromatographic conditions.

[0006] To this end, the present application provides a determination method of an intermediate (1-(2-pyrimidinyl)piperazine) of tandospirone and salts thereof, which adopts high performance liquid chromatography and specific chromatographic conditions to realize effective separation and accurate quantification of the target intermediate. Specifically, the chromatographic conditions include:

[0007] A chromatographic column with octadecylsilane-bonded silica gel as the filler is adopted, and gradient elution is performed with mobile phase A and mobile phase B; wherein,

[0008] The mobile phase A is composed of a first aqueous phase and acetonitrile in a volume ratio of (85-97):(15-3);

[0009] The mobile phase B is composed of a second aqueous phase and acetonitrile in a volume ratio of (20-30):(80-70);

[0010] The first aqueous phase is an aqueous solution containing potassium dihydrogen phosphate and sodium hexanesulfonate, and the pH is 3.0-3.8;

[0011] The second aqueous phase is an aqueous solution containing potassium dihydrogen phosphate and sodium hexanesulfonate, and the pH is 1.8-2.6.

[0012] The test method of the present application has good specificity, accuracy and repeatability: by introducing sodium hexanesulfonate as an ion pair reagent in the mobile phase, the retention of polar intermediates on the stationary phase is effectively enhanced, and the separation of 1-(2-pyrimidinyl)piperazine and the solvent peak is successfully realized. At the same time, by gradient elution with the optimized dual mobile phase system, the separation of the intermediate and the quaternary ammonium salt compound (impurity B) with similar properties is realized. The determination method of the present application can be used for quality control of tandospirone bulk drug.

[0013] In some embodiments of the present application, in the first aqueous phase, the concentration of potassium dihydrogen phosphate is 6.5-7.5 g / L, and the concentration of sodium hexanesulfonate is 0.8-1.0 g / L; in the second aqueous phase, the concentration of potassium dihydrogen phosphate is 3.2-3.8 g / L, and the concentration of sodium hexanesulfonate is 3.3-3.9 g / L.

[0014] In some embodiments of the present application, based on the total volume of the mobile phase A and the mobile phase B, the program of the gradient elution is as follows:

[0015] The volume percentage of the mobile phase B is kept at 14%-16% within 0-6 min;

[0016] The volume percentage of the mobile phase B is linearly increased to 54%-56% within 6-6.5 min;

[0017] The volume percentage of the mobile phase B is kept at 54%-56% within 6.5-20 min;

[0018] 20~20.5min, the volume percentage of mobile phase B linearly decreases to 14%~16%;

[0019] 20.5~30min, the volume percentage of mobile phase B remains at 14%~16%.

[0020] In some embodiments of the present application, the determination method comprises the following steps:

[0021] (1) providing a solution to be tested

[0022] The solution to be tested comprises a test solution and an intermediate control solution; wherein,

[0023] The test solution is a solution of citadopitant with a concentration of 0.25~2mg / mL, and the intermediate control solution is a solution of 1-(2-pyrimidinyl)piperazine hydrochloride;

[0024] (2) injecting each solution to be tested into a high performance liquid chromatograph for separation and detection;

[0025] Optionally, it further comprises:

[0026] (3) calculating the content of 1-(2-pyrimidinyl)piperazine in the test solution by using an external standard method.

[0027] Further, the content of 1-(2-pyrimidinyl)piperazine is not more than 0.1% based on the mass of citadopitant in the test solution.

[0028] Further, in step (1), the solution to be tested further comprises an impurity B control solution with a concentration of 10~100μg / mL, and the impurity B is 8-(2-pyrimidinyl)-8-aza-5-azaspiro[4.5]decane bromide.

[0029] Further, in step (1), each solution to be tested is prepared by using a diluent, and the diluent is prepared by mixing mobile phase A and mobile phase B at a volume ratio of (84~86):(16~14).

[0030] In some embodiments of the present application, the detection wavelength of the high performance liquid chromatograph is 235~245nm. Within this wavelength range, 1-(2-pyrimidinyl)piperazine has strong absorption and less solvent interference.

[0031] In some embodiments of the present application, the inner diameter of the chromatographic column is 4.6mm, the length is 100~250mm, and the particle size of the filler is 2.7~5μm.

[0032] In some embodiments of the present application, the sample injection volume of the high performance liquid chromatography is 15-25 μL, and the flow rate of the mobile phase is 0.9-1.1 mL / min. The sample injection volume can provide sufficient detection response and avoid overloading of the chromatographic column; in combination with the medium flow rate, the separation effect is ensured while the analysis efficiency is improved.

[0033] Other features and advantages of the present application will be described in detail in the following specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0034] The exemplary embodiments of the present application will be described in more detail by way of specific examples with reference to the accompanying drawings.

[0035] Figure 1 Chromatogram of the sample solution in Example 1;

[0036] Figure 2 Chromatogram of the sample solution in Example 1;

[0037] Figure 3 Chromatogram of the sample solution in Example 1;

[0038] Figure 4 Chromatogram of the sample solution in Example 1;

[0039] Figure 5 Chromatogram of the sample solution in Example 1;

[0040] Figure 6 Chromatogram of the sample solution in Example 1;

[0041] Figure 7 Chromatogram of the sample solution in Example 1;

[0042] Figure 8 Chromatogram of the sample solution in Example 1;

[0043] Figure 9 Chromatogram of the sample solution in Example 1. DETAILED DESCRIPTION

[0044] The specific embodiments of the present application will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely intended to illustrate and explain the present application, and are not intended to limit the present application.

[0045] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as the exact dimensions are not critical to the properties. The endpoints of the ranges and the individual points within the ranges are included in the ranges themselves. The exact numerical values include approximations within the range likely to result from measurement, precisions of the measurements, and variations of the manufacturer's process and equipment, typical variations and applications for the disclosure.

[0046] Tandospirone is a partial agonist of 5-HT1A receptor with high selectivity. To improve its solubility and bioavailability, it is usually formulated as a pharmaceutically acceptable salt, of which the citrate salt is the most common. One common synthetic method of tandospirone and its salts is to use norbornimide and the key intermediate 1-(2-pyrimidinyl)piperazine as raw materials, and to condense them with 1,4-dibromobutane under alkaline conditions (such as potassium carbonate) and polar aprotic solvents (such as N,N-dimethylformamide, DMF) through N-alkylation reaction to obtain tandospirone free base, which is then salted with citric acid to obtain the target product tandospirone citrate. The intermediate 1-(2-pyrimidinyl)piperazine can be prepared by reacting piperazine with 2-chloropyrimidine. Therefore, as one of the key raw materials, 1-(2-pyrimidinyl)piperazine, if it is not completely consumed in the final alkylation reaction or not effectively removed in the subsequent purification step, will remain as a process impurity in the final product. In addition, the linker precursor 1,4-dibromobutane used in the synthesis has bifunctional activity and is easy to have a competitive side reaction with 1-(2-pyrimidinyl)piperazine in the reaction system to generate 8-(2-pyrimidinyl)-8-aza-5-azaspiro[4.5]decane bromide (“Impurity B”). Currently, there is a lack of methods for qualitative and quantitative analysis of 1-(2-pyrimidinyl)piperazine in tandospirone and its salts, and Impurity B also has similar polarity with 1-(2-pyrimidinyl)piperazine, and it is difficult to effectively separate the two under conventional chromatographic conditions.

[0047] To solve the above problems, the present application provides a method for determining the intermediate of tandospirone and its salts. The salt of the tandospirone can be its citrate salt, i.e. tandospirone citrate, and the intermediate is 1-(2-pyrimidinyl)piperazine. The determination method uses high performance liquid chromatography (HPLC), and the chromatographic conditions include using a chromatographic column with octadecylsilane bonded silica gel (C18) as the filler, and gradient elution with mobile phase A and mobile phase B.

[0048] In the present application, the mobile phase A is composed of a first aqueous phase and acetonitrile in a volume ratio of (85-97):(15-3). As some examples, the volume ratio of the first aqueous phase and acetonitrile can be 85:15, 88:12, 90:10, 93:7, 93:7, 94:6, 95:5, 96:4, 97:3, preferably 95:5. The pH of the first aqueous phase is 3.0-3.8, for example 3.2, 3.3, 3.4, 3.5, 3.6, etc., preferably 3.4.

[0049] In some embodiments, the concentration of potassium dihydrogen phosphate in the first aqueous phase can be 6.5-7.5 g / L, for example 6.6 g / L, 6.7 g / L, 6.8 g / L, 6.9 g / L, 7.1 g / L, 7.3 g / L, etc., preferably 6.8 g / L; and the concentration of sodium hexanesulfonate can be 0.8-1.0 g / L, for example 0.85 g / L, 0.90 g / L, 0.93 g / L, 0.95 g / L, 0.97 g / L, 0.98 g / L, etc.

[0050] In some embodiments, the first aqueous phase is prepared by dissolving potassium dihydrogen phosphate and sodium hexanesulfonate in water to 1000 mL, and adjusting the pH to 3.0-3.8 with phosphoric acid. As a preferred example, the first aqueous phase is obtained by dissolving 6.8 g of potassium dihydrogen phosphate and 0.93 g of sodium hexanesulfonate in water to 1000 mL, and adjusting the pH to 3.4 with phosphoric acid.

[0051] In the present application, the mobile phase B is composed of a second aqueous phase and acetonitrile in a volume ratio of (20-30):(80-70). As some examples, the volume ratio of the second aqueous phase and acetonitrile can be 20:80, 23:77, 24:76, 25:75, 26:74, 27:73, 30:70, etc., preferably 25:75. The pH of the second aqueous phase is 1.8-2.6, for example 2.0, 2.1, 2.2, 2.3, 2.4, etc., preferably 2.2.

[0052] In some embodiments, the concentration of potassium dihydrogen phosphate in the second aqueous phase can be 3.2-3.8 g / L, for example 3.2 g / L, 3.3 g / L, 3.4 g / L, 3.7 g / L, 3.8 g / L, etc., preferably 3.4 g / L; and the concentration of sodium hexanesulfonate can be 3.2-3.8 g / L, for example 3.25 g / L, 3.30 g / L, 3.50 g / L, 3.52 g / L, 3.68 g / L, 3.72 g / L, 3.80 g / L, etc.

[0053] In some embodiments, the second aqueous phase is prepared by dissolving potassium dihydrogen phosphate and sodium hexanesulfonate in water to 1000 mL, and adjusting the pH to 1.8-2.6 with phosphoric acid. As a preferred example, the second aqueous phase is obtained by dissolving 3.4 g of potassium dihydrogen phosphate and 3.52 g of sodium hexanesulfonate in water to 1000 mL, and then adjusting the pH to 2.2 with phosphoric acid.

[0054] In this invention, pH adjustment of the first and second aqueous phases using phosphoric acid can typically be accomplished in two steps: first, chromatographic grade phosphoric acid (concentration can be 85%) is added to coarsely adjust the pH to near the target range, and then 30% dilute phosphoric acid is used for fine adjustment. As a specific example, when the target pH value of the first aqueous phase is 3.4, chromatographic grade phosphoric acid can be used to coarsely adjust the pH to the range of 3.5~3.7, and then dilute phosphoric acid can be used for fine adjustment to the target value; if the target pH value of the second aqueous phase is 2.2, chromatographic grade phosphoric acid is used to coarsely adjust the pH to the range of 2.5~3.0, and then dilute phosphoric acid can be used for fine adjustment to the target value.

[0055] In this invention, gradient elution is performed using mobile phase A and mobile phase B. In the initial stage of elution, 100% mobile phase A can be used, or a mixture of the two mobile phases can be used; the latter is preferred.

[0056] In some embodiments, the gradient elution procedure is based on the total volume of mobile phase A and mobile phase B:

[0057] Within 0 to 6 minutes, the volume percentage of mobile phase B remained between 14% and 16%.

[0058] Within 6 to 6.5 minutes, the volume percentage of mobile phase B increased linearly to 54% to 56%.

[0059] Within 6.5 to 20.0 min, the volume percentage of mobile phase B remained at 54% to 56%.

[0060] Within 20.0–20.5 min, the volume percentage of mobile phase B decreased linearly to 14%–16%.

[0061] The volume percentage of mobile phase B was maintained at 14% to 16% for 20.5 to 30.0 min.

[0062] This gradient program achieves efficient separation and system stability through multiple stages: In the initial stage (0–6 min), maintaining a low proportion of mobile phase B provides a stable chromatographic environment for polar intermediates, effectively avoiding solvent peak interference and achieving complete separation from adjacent quaternary ammonium salts, while significantly shortening retention time compared to using 100% mobile phase A, thus improving analytical efficiency; In the elution stage (6–20.5 min), rapidly increasing the proportion of mobile phase B and continuing elution enhances elution intensity, prompting the main component and strongly retained impurities to elute rapidly, shortening analysis time and avoiding the residue of the main component in the column, ensuring system stability under continuous injection conditions; In the equilibration stage (20.5–30 min), the mobile phase proportion is restored to the initial conditions and sufficient column equilibration is performed to ensure the reproducibility of the chromatographic system between multiple injections.

[0063] Furthermore, the gradient elution procedure is as follows:

[0064] Within 0 to 6 minutes, maintain the volume ratio of mobile phase A to mobile phase B at 85:15;

[0065] Within 6 to 6.5 minutes, the volume ratio of mobile phase A to mobile phase B is linearly and gradually changed to 45:55.

[0066] Maintain a volume ratio of mobile phase A to mobile phase B of 45:55 within 6.5 to 20 minutes.

[0067] Within 20 to 20.5 minutes, the volume ratio of mobile phase A to mobile phase B is linearly and gradually changed to 85:15.

[0068] Maintain a volume ratio of mobile phase A to mobile phase B of 85:15 for 20.5 to 30 minutes.

[0069] In some embodiments, the detection wavelength of the high-performance liquid chromatography is 235-245 nm, such as 235 nm, 238 nm, 240 nm, 245 nm, etc. 1-(2-pyrimidinyl)piperazine has maximum absorption in this wavelength range, thus achieving high sensitivity.

[0070] In some embodiments, the chromatographic column has an inner diameter of 4.6 mm and a length of 100-250 mm, such as 100 mm, 150 mm, 200 mm, 250 mm, etc., and the packing material particle size is 2.7-5 μm, such as 2.7 μm, 3.5 μm, 5 μm, etc. Preferably, the chromatographic column has an inner diameter of 4.6 mm, a length of 250 mm, and a packing material particle size of 5 μm. The chromatographic column can be an Agilent ZORBAX SB-C18 purchased from Agilent Technologies, or a RIGOL Compass(2)C18 purchased from RPG Technologies.

[0071] In some embodiments, the injection volume of the high-performance liquid chromatography (HPLC) is 15–25 μL, such as 15 μL, 20 μL, or 25 μL, preferably 20 μL; the total flow rate of the mobile phase is 0.9–1.1 mL / min, such as 0.9 mL / min, 1.0 mL / min, or 1.1 mL / min, preferably 1.0 mL / min. This injection volume provides sufficient detection response while avoiding column overload; combined with moderate flow rates, it improves analytical efficiency while ensuring separation effectiveness.

[0072] According to some specific embodiments, the determination method of the present invention includes the following steps:

[0073] (1) Provide the solution to be tested

[0074] The test solution includes the test solution and the intermediate reference solution;

[0075] (2) Inject the solutions prepared in step (1) into the high performance liquid chromatograph and perform separation and detection according to the above chromatographic conditions.

[0076] In step (1), the test solution can be a tandospirone citrate solution, and the concentration of tandospirone citrate can be 0.25~2 mg / mL, for example, 0.25 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, etc. Based on the high sensitivity of the method of the present invention, it is preferable to use a lower concentration for testing, that is, the concentration of tandospirone citrate in the test solution is ≤1 mg / mL. This can help save sample consumption while ensuring accurate determination.

[0077] In step (1), the intermediate reference solution can be a 1-(2-pyrimidinyl)piperazine hydrochloride solution, with a concentration of 0.25~1 μg / mL based on its free base (1-(2-pyrimidinyl)piperazine), for example 0.25 μg / mL, 0.45 μg / mL, 0.5 μg / mL, 0.75 μg / mL, 1 μg / mL, etc. The reference standard is used in the form of the intermediate hydrochloride to ensure stability and accurate weighing. The hydrochloride will dissociate in the mobile phase, and its free base portion is the target of the UV detector. Therefore, in the quantitative calculation process, the mass of the hydrochloride obtained by precise weighing needs to be converted into an equivalent mass of free base based on its molecular weight ratio to the free base. Unless otherwise stated, the concentrations and injection volumes of the intermediate reference solutions in the following examples and comparative examples are based on their free base (1-(2-pyrimidinyl)piperazine).

[0078] In some embodiments, the content limit of 1-(2-pyrimidinyl)piperazine in the test sample is set to no more than 0.1% based on the mass of tandospirone citrate in the test sample, for example, 0.1%. Thus, the qualification of the test sample can also be directly assessed based on the chromatographic detection results of step (2).

[0079] The present invention does not impose any particular restrictions on the source of the test sample; it may be a self-made product or a commercially purchased product. As a specific example, the product can be obtained by the following method: First, piperazine hexahydrate and 2-chloropyrimidine are refluxed in isopropanol at a molar ratio of (1.8~2.2):1 for 8~10 h. After post-treatment purification, the intermediate 1-(2-pyrimidinyl)piperazine is obtained. Then, the intermediate is dissolved in DMF with norbornene and 1,4-dibromobutane at a molar ratio of 1:(0.8~1.2):(0.8~1.2) and reacted at 85~90℃ for 10~12 h. The crude product is decolorized and recrystallized from ethanol to obtain the free base of tandospirone. Finally, the free base is reacted with citric acid in methanol at a molar ratio of 1:(1.1~1.3) for 2~3 h to form a salt. After crystallization purification, the final product of tandospirone citrate is obtained.

[0080] In step (1), the test solution may further include a reference solution of impurity B, wherein impurity B is 8-(2-pyrimidinyl)-8-aza-5-azaspirocyclic [4.5]decane bromide (CAS: 81461-73-6), and its concentration may be 10~100 μg / mL. By injecting this solution for chromatographic analysis, the retention time of impurity B in the chromatogram can be determined, which is used to accurately locate the peak of impurity B in the test sample and to assist in evaluating its separation from adjacent chromatographic peaks (intermediates, main peak of the test sample).

[0081] In some embodiments, the test solution further includes a blank solvent and / or a system suitability solution. In step (2), by injecting a blank solvent (with the same diluent composition as the test solution), it can be verified that the chromatographic system is free from interference at the target peak position. The system suitability solution is a solution containing the test sample, impurity B, and intermediate reference standard. In step (2), by injecting this solution, it can be verified whether the resolution of the chromatographic system meets the determination requirements, ensuring the effectiveness of the analytical method.

[0082] In some embodiments, each test solution is prepared using a diluent, which is a mixture of mobile phase A and mobile phase B at a volume ratio of (84~86):(16~14). In this case, the diluent used for all test solutions is the same as the mobile phase ratio in the initial stage of gradient elution, which can eliminate the solvent effect caused by solvent strength mismatch, avoid chromatographic peak distortion and retention time drift, and easily obtain sharp, symmetrical chromatographic peaks. As some specific examples, the volume ratio of mobile phase A to mobile phase B in the diluent can be 84:16, 85:15, 86:14, etc., preferably 85:15.

[0083] In step (2), the test solution and the intermediate reference solution are injected into a high-performance liquid chromatograph (HPLC) to obtain their respective chromatograms. Based on the chromatograms and using the chromatographic response of the reference solution as a benchmark, the following objectives are achieved: firstly, it can be determined whether the content of 1-(2-pyrimidinyl)piperazine in the test sample exceeds the limit (i.e., whether it is greater than the specified limit); secondly, it can be quantitatively determined the content of the intermediate in the test sample. The chromatographic conditions in step (2) are as described above and will not be repeated here.

[0084] To achieve quantitative analysis of 1-(2-pyrimidinyl)piperazine in the test sample, preferably, the test method further includes: (3) calculating the content of 1-(2-pyrimidinyl)piperazine in the test sample using the external standard method. This quantitative method relies only on the comparison of the peak area of ​​the impurity itself and the reference standard, and is not affected by the main peak. Specifically, the content of the intermediate in the test sample is calculated by the following formula:

[0085] Formula 1

[0086] Among them, X 杂 : The percentage content of 1-(2-pyrimidinyl)piperazine in the test sample;

[0087] A 杂 Peak area of ​​1-(2-pyrimidinyl)piperazine in the test solution;

[0088] A 对 Peak area of ​​1-(2-pyrimidinyl)piperazine in intermediate reference solution;

[0089] C 对 : Concentration of 1-(2-pyrimidinyl)piperazine in the intermediate reference solution, g / mL;

[0090] b 对 : Content (purity) of 1-(2-pyrimidinyl)piperazine hydrochloride reference standard;

[0091] m 样 : Sample weight, in g;

[0092] V : The dilution volume of the test solution, in mL.

[0093] The assay method established in this invention is highly specific and accurate, effectively supporting the quality control of tandospirone. This method is applicable in the following ways: as a quality control method for active pharmaceutical ingredients (APIs) to ensure that the product meets preset impurity limits; for monitoring the residues of key intermediates in the synthesis process, providing data support for process optimization and control; and also suitable for long-term and accelerated stability studies of APIs to track changes in their impurity profiles.

[0094] The following describes embodiments of the present invention. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0095] Example 1

[0096] 1. Preparation of solution

[0097] 1) Preparation of mobile phase

[0098] Dissolve 6.8 g of potassium dihydrogen phosphate and 0.93 g of sodium hexanesulfonate in water and bring the volume to 1000 mL. Adjust the pH to 3.4 with phosphoric acid to obtain the first aqueous phase. Mix the first aqueous phase with acetonitrile at a volume ratio of 95:5 to obtain the mobile phase A.

[0099] Dissolve 3.4 g of potassium dihydrogen phosphate and 3.52 g of sodium hexanesulfonate in water and bring the volume to 1000 mL. Adjust the pH to 2.2 with phosphoric acid to obtain the second aqueous phase. Mix the second aqueous phase with acetonitrile at a volume ratio of 25:75 to obtain mobile phase B.

[0100] 2) Preparation of the test solution

[0101] Blank solvent: composed of mobile phase A and mobile phase B in a volume ratio of 85:15.

[0102] Test solution: Weigh approximately 25 mg of tandospirone citrate (provided by Shenyang Huatai Pharmaceutical Research Co., Ltd., batch number S200803) accurately, place it in a 50 mL volumetric flask, dissolve and dilute to the mark with blank solvent, shake well to obtain the test solution (approximately 0.5 mg / mL).

[0103] Intermediate reference solution: Weigh approximately 12.2 mg of 1-(2-pyrimidinyl)piperazine hydrochloride (purity 99.9%, the same below) accurately, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with blank solvent, and shake well to obtain the reference stock solution; accurately measure 1 mL of the stock solution and place it in a 100 mL volumetric flask, dilute to the mark with blank solvent, and shake well; then accurately measure 1 mL of the stock solution and place it in a 20 mL volumetric flask, dilute to the mark with blank solvent, and shake well to obtain the intermediate reference solution (concentration approximately 0.5 μg / mL based on free base).

[0104] Impurity B reference solution: Weigh approximately 10 mg of impurity B accurately and place it in a 10 mL volumetric flask. Dissolve and dilute to the mark with a blank solvent, and shake well to obtain the impurity B stock solution (approximately 1 mg / mL). Then, accurately measure 1 mL of the solution and place it in a 10 mL volumetric flask. Dissolve and dilute to the mark with a blank solvent, and shake well to obtain the impurity B reference solution (approximately 100 μg / mL).

[0105] System suitability solution: Accurately weigh approximately 10 mg of the test sample and place it in a 10 mL volumetric flask. Add 1 mL of intermediate reference stock solution and 1 mL of impurity B stock solution, dissolve and dilute to the mark with blank solvent, and shake well.

[0106] 2. Chromatographic analysis

[0107] Column: Agilent ZORBAX SB-C18, octadecylsilane bonded silica (C18) column (250 mm × 4.6 mm, 5 μm) from Agilent Technologies.

[0108] The detection wavelength is 240nm;

[0109] The injection volume was 20 μL;

[0110] The flow rate was 1.0 mL / min;

[0111] The column temperature is 40℃.

[0112] Blank solvent, test solution, intermediate reference solution, impurity B reference solution, and system suitability solution were injected into the high-performance liquid chromatograph (equipped with a UV detector, the same below), and eluted according to the gradient program shown in Table 1. Chromatograms were recorded until the main component peak was completely eluted, and the test results are shown below. Figures 1-4 As shown.

[0113] Table 1

[0114]

[0115] pass Figures 1-4 Analysis shows that the test method in this embodiment has strong specificity and good system applicability. First, in a blank solvent ( Figure 2No interference was observed at the target analyte peak position, indicating that the solvent does not affect the detection. The test solution ( Figure 1 Retention time (t) in ) R The chromatographic peak at 5.230 min was compared with the superimposed chromatogram. Figure 3 The comparison confirmed it to be 1-(2-pyrimidinyl)piperazine. Figure 3 Peak 1 is a characteristic peak of 1-(2-pyrimidinyl)piperazine. Additionally, the superimposed chromatogram also shows that peak 2 is impurity B, and peak 3 is the main peak of tandospirone citrate; peaks 1 through 3 are clearly separated. System suitability solution ( Figure 4 The results further validated the separation of the key chromatographic peaks, including 1-(2-pyrimidinyl)piperazine (t). R =5.156min), impurity B (t R =5.997min) and tandospirone citrate main peak (t R (11.487 min) all achieved baseline separation, meeting the system applicability requirements.

[0116] 3. Quantitative analysis

[0117] The content of 1-(2-pyrimidinyl)piperazine in the test sample was calculated to be 0.0036% by substituting the peak area of ​​1-(2-pyrimidinyl)piperazine in the test sample and the peak area of ​​the reference standard into the external standard method formula in Equation 1.

[0118] Example 2: Methodological Validation

[0119] 1. Limit of Quantification (LOQ) and Precision of LOQ

[0120] Accurately weigh an appropriate amount of 1-(2-pyrimidinyl)piperazine hydrochloride reference standard, dissolve it in a blank solvent (as in Example 1, the same below), and gradually dilute it to obtain reference standard solutions of different concentrations. Analyze the reference standard solutions according to the chromatographic analysis method in Example 1.

[0121] When the concentration of 1-(2-pyrimidinyl)piperazine in the solution was 0.0052 µg / mL, the target chromatographic peak (t) was measured. R The signal-to-noise ratio (S / N = 13.158) of this method was 5.302 min, therefore the limit of quantitation was determined to be 0.0052 µg / mL, which is equivalent to 0.104 ng based on the injection volume.

[0122] To verify the precision of the limit of quantitation level, six solutions of 1-(2-pyrimidinyl)piperazine hydrochloride at the limit of quantitation concentration were prepared independently according to the above method. Chromatographic analysis was performed on each solution under the chromatographic conditions of Example 1, and the peak areas were recorded. The results are shown in Table 2.

[0123] Table 2

[0124]

[0125] As shown in Table 2, the relative standard deviation (RSD) of the peak area measured in the six measurements was 9.98% (<10%), indicating that the method in Example 1 has good precision at the limit of quantitation concentration and can perform reliable and accurate quantitative analysis.

[0126] 2. Limit of Detection (LOD)

[0127] Accurately weigh an appropriate amount of 1-(2-pyrimidinyl)piperazine hydrochloride reference standard, dissolve it in a blank solvent, and gradually dilute it to obtain reference standard solutions of different concentrations. Analyze the reference standard solutions according to the chromatographic analysis method described in Example 1.

[0128] When the concentration of 1-(2-pyrimidinyl)piperazine in the solution was 0.00156 µg / mL, the target chromatographic peak (t) was measured. R The signal-to-noise ratio (S / N) of the sample (5.296 min) was 3.710, therefore the limit of detection (LOD) of this method was determined to be 0.00156 µg / mL, which is equivalent to 0.0312 ng based on the injection volume.

[0129] 3. Linearity and Range Examination

[0130] Weigh an appropriate amount of 1-(2-pyrimidinyl)piperazine hydrochloride reference standard, dissolve and dilute it with blank solvent to prepare stock solutions with concentrations of 5.07 µg / mL and 0.507 µg / mL, respectively. A series of standard solutions of different concentrations were prepared by accurately measuring different volumes of the above stock solutions and quantitatively diluting them with blank solvent.

[0131] Under the chromatographic conditions described in Example 1, accurately measure 20 µL of each standard solution and inject them into the liquid chromatograph, recording the peak area.

[0132] Linear regression analysis was performed with the injection volume W (ng) of 1-(2-pyrimidinyl)piperazine as the x-axis and the corresponding peak area A as the y-axis. The results are shown in Table 3, and the linear regression relationship is shown in [Table 3]. Figure 7 .

[0133] Table 3

[0134]

[0135] Combination Figure 7 The regression equation is A = 5.6522W + 1.4745, and the correlation coefficient r = 0.9992.

[0136] The results showed that the injection amount of 1-(2-pyrimidinyl)piperazine exhibited a good linear relationship with the chromatographic peak area in the range of 0.10~20.28 ng.

[0137] 4. Solution stability

[0138] Accurately weigh an appropriate amount of 1-(2-pyrimidinyl)piperazine hydrochloride reference standard, dissolve and dilute it with blank solvent to prepare a solution with a 1-(2-pyrimidinyl)piperazine concentration of approximately 0.5 μg / mL. Place the solution at room temperature and take samples at 0 hours after preparation and at time points of 2, 4, 6, and 24 hours.

[0139] Under the chromatographic conditions described in Example 1, 20 μL of each solution at different storage times was precisely measured and injected into the liquid chromatograph for analysis. The peak area was recorded and the relative standard deviation (RSD) was calculated. The results are shown in Table 4.

[0140] Table 4

[0141]

[0142] As shown in Table 4, the peak area RSD at each time point was 2.63%, indicating that the 1-(2-pyrimidinyl)piperazine hydrochloride reference solution had good stability within 24 hours at room temperature.

[0143] 5. Instrument precision

[0144] Weigh an appropriate amount of 1-(2-pyrimidinyl)piperazine hydrochloride reference standard, dissolve and dilute it with blank solvent to prepare a solution containing approximately 0.5 μg of 1-(2-pyrimidinyl)piperazine per 1 mL.

[0145] Accurately measure 20 μL of the solution and inject it 6 times consecutively under the chromatographic conditions described in Example 1. Record the peak area of ​​each chromatographic peak. The results are shown in Table 5.

[0146] Table 5

[0147]

[0148] As shown in Table 5, the relative standard deviation (RSD) of the peak area measured by 6 consecutive injections was 0.25%, which proves that the instrument has good precision.

[0149] 6. Repeatability

[0150] Following the method for determining 1-(2-pyrimidinyl)piperazine in Example 1, six independent parallel solutions of the test sample, intermediate reference solution, and impurity B reference solution were prepared and analyzed sequentially. The content (%) of 1-(2-pyrimidinyl)piperazine in the test sample solution was calculated, and the results are shown in Table 6.

[0151] Table 6

[0152]

[0153] The results showed that the 1-(2-pyrimidinyl)piperazine content results from the six independent determinations were highly consistent, with extremely low relative standard deviations (RSD), demonstrating that the test method in Example 1 has excellent repeatability and reliable test results.

[0154] 7. Accuracy

[0155] To verify the accuracy of the quantitative analysis, a recovery test was conducted.

[0156] (1) Provide blank sample solution and reference solution before sample addition.

[0157] Accurately weigh 25 mg of tandospirone citrate, dissolve it in a blank solvent and dilute to 50 mL to prepare a blank sample solution before adding the sample.

[0158] Accurately weigh an appropriate amount of 1-(2-pyrimidinyl)piperazine hydrochloride reference standard, dissolve it in blank solvent and dilute it quantitatively to prepare a reference solution with a concentration of approximately 0.5 µg / mL of 1-(2-pyrimidinyl)piperazine.

[0159] (2) Preparation of test solution

[0160] Accurately weigh 9 portions of tandospirone citrate, each approximately 25 mg, and place them in separate 50 mL volumetric flasks for later use;

[0161] Accurately weigh three portions of 1-(2-pyrimidinyl)piperazine hydrochloride at three different levels (weighing amounts equivalent to 80%, 100%, and 120% of the reference sample weight), dissolve and dilute them with blank solvent to prepare solutions containing approximately 20 µg, 25 µg, and 30 µg of 1-(2-pyrimidinyl)piperazine per mL, respectively; then accurately measure 1 mL of each solution and place them in the prepared volumetric flasks, dilute to the mark with blank solvent, and obtain a total of nine test solutions.

[0162] (3) Chromatographic analysis

[0163] Under the chromatographic conditions described in Example 1, 20 µL of each of the blank sample solution, 9 test solutions, and reference solution were precisely measured and injected into the liquid chromatograph. The chromatograms were recorded and the recovery rates were calculated. The results are shown in Table 7.

[0164] Table 7

[0165]

[0166] Note: The amount added is calculated based on the free alkali of the intermediate reference standard.

[0167] The results showed that 1-(2-pyrimidinyl)piperazine had an average recovery rate of 100.9% in the concentration range of 80%–120%, and the precision of the recovery rate at each concentration level was good (RSD = 1.07%). These results meet the accuracy validation requirements of the analytical method (RSD ≤ 2%).

[0168] 8. Durability

[0169] The determination of 1-(2-pyrimidinyl)piperazine was carried out according to the method of Example 1, except that the chromatographic conditions were adjusted. Specifically, the key parameters such as the chromatographic wavelength, the initial mobile phase ratio, the flow rate, the pH value of the aqueous phase, and the different brands of chromatographic columns were adjusted. The test solution and the intermediate reference solution were chromatographically detected and quantitatively analyzed. The test results are shown in Tables 8 to 12. The "standard" in the table corresponds to the corresponding chromatographic conditions of Example 1.

[0170] Table 8

[0171]

[0172] Table 9

[0173]

[0174] Table 10

[0175]

[0176] Table 11

[0177]

[0178] Table 12

[0179]

[0180] The results showed that 1-(2-pyrimidinyl)piperazine could be stably detected under varying chromatographic parameters. Although the measured values ​​fluctuated, the absolute content changes were minimal, and all results were well below the conventional quality control limit (0.1%). This indicates that the error introduced by the variation of these parameters is acceptable in rigorous pharmaceutical quality control practices, fully demonstrating the robustness of the method.

[0181] Example 3

[0182] The analysis was performed according to the method for determining 1-(2-pyrimidinyl)piperazine established in Example 1, except that the test solution was replaced with other tandospirone citrate samples (Sample I and Sample II). The results are shown in Table 13.

[0183] Table 13

[0184]

[0185] Note: Sample I was provided by Guangzhou Jiatu Technology Co., Ltd. (CATO); Sample II was provided by the China National Institutes for Food and Drug Control.

[0186] As can be seen, the method for determining 1-(2-pyrimidinyl)piperazine in the examples can effectively detect impurities, and the test results meet the standard requirements, thus ensuring product quality.

[0187] Comparative Example 1

[0188] Column: Agilent ZORBAX SB-C18, octadecylsilane bonded silica (C18) column (250 mm × 4.6 mm, 5 μm) from Agilent Technologies.

[0189] The detection wavelength is 239nm;

[0190] The injection volume was 20 μL;

[0191] The flow rate was 1.0 mL / min;

[0192] The column temperature is 40℃.

[0193] Mobile phase: Weigh 1.0 g of sodium heptanesulfonate and place it in a 1000 mL volumetric flask. Add water to dissolve and dilute to the mark, shake well, and then add hydrochloric acid dropwise to adjust the pH to 3.0 to obtain the stock solution. Mix the stock solution with acetonitrile at a volume ratio of 70:30 to obtain the mobile phase.

[0194] Test solution: Weigh approximately 25 mg of tandospirone citrate (batch number S200803) accurately, place it in a 50 mL volumetric flask, dissolve and dilute to the mark with the mobile phase, and shake well to obtain the test solution (approximately 0.5 mg / mL).

[0195] Intermediate reference solution: Accurately weigh approximately 12.2 mg of 1-(2-pyrimidinyl)piperazine hydrochloride, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with the mobile phase, and shake well to obtain the reference stock solution; accurately measure 1 mL of the stock solution and place it in a 100 mL volumetric flask, dilute to the mark with the mobile phase, and shake well; then accurately measure 1 mL of the stock solution and place it in a 20 mL volumetric flask, dilute to the mark with the mobile phase, and shake well to obtain the intermediate reference solution (concentration approximately 0.5 μg / mL based on free base).

[0196] The test solution and intermediate reference solution were injected into a high-performance liquid chromatograph for analysis, and the chromatograms were recorded.

[0197] Test results are as follows Figures 8-9 As shown, Figure 9 The characteristic peak of 1-(2-pyrimidinyl)piperazine (t)R =3.542min) and solvent peak (t R =3.312min) Not separated, Figure 8 As can be seen from the chromatogram of the test sample (enlarged view), the solvent peak (t) R =3.872 min) and 1-(2-pyrimidinyl)piperazine (t R =3.563min) overlaps, indicating that the solvent peak interferes with the characteristic peak of the intermediate, and the comparative method is not suitable for detecting intermediates.

[0198] The various embodiments of the present invention have been described above. These embodiments are merely illustrative of the technical solutions of the present invention and are not intended to limit it, nor are they exhaustive, nor are they limited to the disclosed embodiments. Those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention. In particular, as long as there is no structural conflict, the various technical features mentioned in the various embodiments can be combined in any way. The present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A method for determining tandospirone and its salt intermediates, characterized by, The intermediate is 1-(2-pyrimidinyl)piperazine, and is determined by high performance liquid chromatography, and the chromatographic conditions include: A chromatographic column with octadecylsilane-bonded silica gel as the filler is used, and gradient elution is performed with mobile phase A and mobile phase B; wherein, The mobile phase A is composed of a first aqueous phase and acetonitrile in a volume ratio of (85-97):(15-3); The mobile phase B is composed of a second aqueous phase and acetonitrile in a volume ratio of (20-30):(80-70); The first aqueous phase is an aqueous solution containing potassium dihydrogen phosphate and sodium hexanesulfonate, and the pH is 3.0-3.8; The second aqueous phase is an aqueous solution containing potassium dihydrogen phosphate and sodium hexanesulfonate, and the pH is 1.8-2.6; In the first aqueous phase, the concentration of potassium dihydrogen phosphate is 6.5-7.5 g / L, and the concentration of sodium hexanesulfonate is 0.8-1.0 g / L; in the second aqueous phase, the concentration of potassium dihydrogen phosphate is 3.2-3.8 g / L, and the concentration of sodium hexanesulfonate is 3.3-3.9 g / L; The program of the gradient elution is as follows based on the total volume of the mobile phase A and the mobile phase B: The volume percentage of the mobile phase B is kept at 14%-16% within 0-6 min; The volume percentage of the mobile phase B is linearly increased to 54%-56% within 6-6.5 min; The volume percentage of the mobile phase B is kept at 54%-56% within 6.5-20 min; The volume percentage of the mobile phase B is linearly decreased to 14%-16% within 20-20.5 min; The volume percentage of the mobile phase B is kept at 14%-16% within 20.5-30 min.

2. The assay method according to claim 1, characterized by The method comprises the following steps: (1) providing a solution to be tested The solution to be tested includes a test sample solution and an intermediate control solution; wherein, The test sample solution is a citandosperone solution with a concentration of 0.25-2 mg / mL, and the intermediate control solution is a 1-(2-pyrimidinyl)piperazine hydrochloride solution; (2) injecting each solution to be tested into a high performance liquid chromatograph for separation and detection; Optionally, the method further comprises: (3) calculating the content of 1-(2-pyrimidinyl)piperazine in the test sample by an external standard method.

3. The assay method according to claim 2, characterized in that, The content of 1-(2-pyrimidinyl)piperazine in the test sample, based on the mass of citandosperone, is not more than 0.1%.

4. The assay method according to claim 2, characterized by, In step (1), the solution to be tested further includes an impurity B control solution with a concentration of 10-100 μg / mL, and the impurity B is 8-(2-pyrimidinyl)-8-aza-5-azaspiro[4.5]decane bromide.

5. The assay method according to claim 2, characterized by, In step (1), each solution to be tested is prepared by diluting with a diluent, and the diluent is obtained by mixing the mobile phase A and the mobile phase B in a volume ratio of (84-86):(16-14).

6. The assay method according to claim 1, characterized by, The detection wavelength of the high performance liquid chromatograph is 235-245 nm.

7. The assay method according to claim 1, characterized by, The inner diameter of the chromatographic column is 4.6 mm, the length is 100-250 mm, and the particle size of the filler is 2.7-5 μm.

8. The assay method of claim 1, wherein The injection volume of the high performance liquid chromatograph is 15-25 μL, and the flow rate of the mobile phase is 0.9-1.1 mL / min.

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