Method for detecting imidazole in amperozine bulk drug

Abstract

The application discloses a kind of determination amiprilose raw drug in imidazole high performance liquid chromatography detection method, belong to the field of pharmaceutical analysis chemistry.The method uses chromatographic condition, including: high performance liquid chromatography system is Agilent 1260 high performance liquid chromatograph (with UV / DAD detector) or similar equipment;Chromatographic column is with amido bond ethylene bridge hybrid silica gel as filler chromatographic column;Mobile phase is composed of A, B, wherein A is acetate buffer solution, B is acetonitrile, the concentration of acetate in the acetate buffer solution is 0.01mol / L, pH is 7.5~8.5, the volume ratio of A and B is (9~11) :(89~91);Detection wavelength is 210nm;Injection amount is 10 μl;Column temperature is 30 DEG C;Flow rate is 0.5ml / min.The application method is simple in operation, strong in specificity, high in sensitivity, good in repeatability, high in accuracy, good in durability, and imidazole peak shape is excellent, can effectively overcome the problem that imidazole peak is seriously tailing in gas chromatography, ion pair chromatography column loss is big and method reproducibility is poor, can be used for amiprilose raw drug quality control, with good industrial practicability.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analytical chemistry, and specifically relates to a method for detecting imidazole in amisulpride raw material, particularly a method for determining the imidazole content in amisulpride raw material using high performance liquid chromatography. Background Technology

[0002] Amisulpride is a benzamide antipsychotic, a dopamine receptor antagonist, primarily used to treat schizophrenia, and also being investigated for depression. The chemical name of amisulpride is 4-amino- N -[(1-ethyl-2-pyrrolidinyl)methyl]-5-(ethylsulfonyl)-2-methoxybenzamide.

[0003] In the synthesis of amisulpride, the following are commonly used: N , N '-Carbonyldiimidazole (CDI) is used as a catalyst. During the condensation reaction, CDI forms the byproduct imidazole. Imidazole is a heterocyclic compound and has a certain degree of toxicity (oral LD50 in mice). 50 (The concentration is 18.80 mg / kg). Therefore, strict monitoring of imidazole is necessary in the quality control of amisulpride raw material.

[0004] Currently, the conventional methods for determining the imidazole content in amisulpride raw material are gas chromatography and ion-pair chromatography. However, gas chromatography has the following shortcomings in practical applications: (1) Imidazole is highly polar and easily adsorbed in the gas chromatography system, resulting in severe peak tailing and affecting quantitative accuracy; (2) Gas chromatography requires high injection port temperature and detector temperature, placing high demands on the equipment. Ion-pair chromatography has the following shortcomings in practical applications: (1) Ion-pairing reagents are irreversibly adsorbed onto the stationary phase of the chromatographic column, leading to stationary phase modification and reduced column efficiency; (2) System equilibration usually takes a long time, resulting in poor method stability and reproducibility.

[0005] Therefore, there is an urgent need to develop a highly sensitive, well-shaped, and accurate method for the detection of imidazole to meet the quality control requirements of amisulpride raw material. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for detecting imidazole in amisulpride raw material. This method uses high performance liquid chromatography, which can effectively separate and determine imidazole in amisulpride raw material. It has the advantages of strong specificity, high sensitivity, good repeatability, high accuracy, and excellent peak shape.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for detecting imidazole in amisulpride raw material, using high-performance liquid chromatography (HPLC), with the following chromatographic conditions:

[0009] The HPLC system is an Agilent 1260 high-performance liquid chromatograph (UV / DAD detector) or a similar device;

[0010] The chromatographic column was a Waters XBridge Amide column, 4.6 × 150 mm, 3.5 μm;

[0011] The mobile phase consists of A and B, where A is acetate buffer and B is acetonitrile. The concentration of the acetate buffer is 0.01 mol / L, the pH value is 7.5~8.5, and the volume ratio of mobile phase A to B is (9~11):(91~89).

[0012] The detection wavelength is 210nm;

[0013] The column temperature is 28~32℃;

[0014] The flow rate is 0.4~0.6 ml / min;

[0015] The injection volume was 10 μl.

[0016] Preferably, the acetate buffer is an ammonium acetate buffer, and the pH is adjusted to 8.0 with ammonia.

[0017] Preferably, the volume ratio of the mobile phase A to B is 10:90.

[0018] Preferably, the column temperature is 30°C.

[0019] Preferably, the flow rate is 0.5 ml / min.

[0020] Furthermore, the method includes the following steps:

[0021] (1) Preparation of reference solution: Take an appropriate amount of imidazole reference standard, dissolve and dilute it with solvent to prepare a reference solution containing 1 μg of imidazole per 1 ml.

[0022] (2) Preparation of test solution: Take an appropriate amount of ammonia sulfadiazine test sample, dissolve and dilute it with solvent to prepare a test solution containing 1 mg of ammonia sulfadiazine per 1 ml.

[0023] (3) Take 10 μl of the reference solution and the test solution respectively, inject them into the high performance liquid chromatograph, and perform the determination under the above chromatographic conditions, and record the chromatogram.

[0024] (4) Calculate the content of imidazole in the test solution using the external standard method.

[0025] In summary, the chromatographic conditions for the high-performance liquid chromatography (HPLC) method for the determination of imidazole in amisulpride of the present invention are as follows:

[0026] HPLC system Agilent 1260 high performance liquid chromatograph (UV / DAD detector) or similar equipment chromatographic column Waters XBridge Amide, 4.6×150mm, 3.5um mobile phase 0.01 mol / L ammonium acetate (pH 8.0) - acetonitrile (10:90) Flow rate 0.5 ml / min Column temperature 30℃ Detection wavelength 210nm Injection volume 10μl solvent 0.01 mol / L ammonium acetate (pH 8.0) - acetonitrile (10:90) Reference solution Take an appropriate amount of imidazole reference standard, dissolve and dilute it with a solvent to prepare a reference standard solution containing 1 μg of imidazole per 1 ml. Test solution Take an appropriate amount of amisulpride test sample, dissolve and dilute it with a solvent to prepare a test solution containing 1 mg of amisulpride per 1 ml.

[0027] This invention utilizes a Waters XBridge Amide column (amide-bonded phase), which is based on ethylene-bridged hybrid particle (BEH) technology and features a triple-bonded amide phase, making it less prone to Schiff base formation and thus offering higher quantitative accuracy. Unlike traditional C18 reversed-phase columns, the Amide column exhibits excellent retention and peak shape for polar compounds such as imidazoles. Furthermore, this column demonstrates excellent chemical stability, tolerating mobile phase conditions ranging from pH 2 to 11.

[0028] This invention selects 0.01 mol / L ammonium acetate (pH 8.0)-acetonitrile (10:90) as the mobile phase. The high proportion of acetonitrile (90%) provides a favorable hydrophilic interaction chromatography (HILIC) environment for the amide column, enabling the effective retention and separation of the polar compound imidazole. The ammonium acetate buffer (pH 8.0) in the mobile phase not only adjusts the pH of the system to improve peak shape but also forms appropriate ionic interactions with imidazole molecules, further enhancing the separation selectivity.

[0029] This invention selects 210nm as the detection wavelength, as imidazole has strong ultraviolet absorption at this wavelength, which can ensure that the detection sensitivity meets the detection requirements for imidazole impurity limits in amisulpride raw material.

[0030] In the method of this invention, the concentration of the test solution is 1 mg / ml and the concentration of the reference solution is 1 μg / ml. Under these concentration conditions, the imidazole peak has a good response value and peak shape, which can meet the requirements of quantitative analysis.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The present invention adopts the hydrophilic interaction chromatography (HILIC) mode, which effectively solves the problem that imidazole polar compounds are poorly retained in reversed phase chromatography and severely adsorbed in gas chromatography. The imidazole peaks are symmetrical and there is no tailing phenomenon.

[0033] (2) The present invention adopts isocratic elution, which is simple to operate, has a short system equilibration time, and has good reproducibility and stability.

[0034] (3) The present invention has high detection sensitivity and a quantitation limit of up to 0.5 μg / ml, which can meet the detection requirements of imidazole impurity limit (usually not higher than 0.1%) in amisulpride raw material.

[0035] (4) The mobile phase of the present invention does not use ion-pairing reagents, thus avoiding irreversible damage to the chromatographic column caused by ion-pairing reagents, extending the service life of the chromatographic column and reducing the detection cost. Attached Figure Description

[0037] Figure 1 This is the chromatogram of the blank solution in Example 1 of the present invention.

[0038] Figure 2 This is a typical chromatogram of the imidazole reference solution in Example 1 of the present invention.

[0039] Figure 3 This is a typical chromatogram of the amisulpride test solution in Example 6 of the present invention.

[0040] Figure 4 This is a typical chromatogram of the 100% spiked test solution of amisulpride in Example 6 of the present invention.

[0041] Figure 5 This is a graph showing the linear relationship between the peak area and concentration of imidazole in Example 4 of the present invention.

[0042] Figure 6 This is a typical chromatogram for the determination of imidazole by gas chromatography in Comparative Example 1 of the present invention, showing the imidazole peak tailing phenomenon. Detailed Implementation

[0044] The present invention will be further described below through specific embodiments and comparative examples, but the present invention is not limited to the following embodiments.

[0045] Example 1 Chromatographic conditions and system suitability test

[0046] 1. Instruments and reagents

[0047] High performance liquid chromatograph: Agilent 1260 high performance liquid chromatograph, equipped with ultraviolet detector;

[0048] Column: Waters XBridge Amide, 4.6 × 150 mm, 3.5 μm;

[0049] Imidazole reference standard: TCI, purity 99.3%;

[0050] Amisulpride test sample: TCI, purity 99.9%;

[0051] Acetonitrile: ThermoFisher, chromatographic grade;

[0052] Ammonium acetate: Aladdin, chromatographic grade;

[0053] Water: Ultrapure water.

[0054] 2. Solution preparation

[0055] Mobile phase: Dissolve 0.77 g of ammonium acetate in water and dilute to 1000 ml. Adjust the pH to 8.0 with ammonia to obtain 0.01 mol / L ammonium acetate buffer (pH 8.0). Mix 0.01 mol / L ammonium acetate buffer (pH 8.0) and acetonitrile at a volume ratio of 10:90 to obtain the mobile phase.

[0056] Solvent: Same as mobile phase.

[0057] Reference stock solution: Weigh approximately 10 mg of imidazole reference standard accurately, place it in a 100 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well to obtain the reference stock solution (concentration approximately 100 μg / ml).

[0058] Reference solution: Accurately measure 1 ml of the reference stock solution, place it in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain a reference solution with a concentration of 1 μg / ml.

[0059] Test solution: Weigh approximately 20 mg of amisulpride test sample accurately, place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well to obtain a test solution with a concentration of 1 mg / ml.

[0060] 3. Chromatographic conditions

[0061] Column: Waters XBridge Amide, 4.6 × 150 mm, 3.5 μm;

[0062] Mobile phase: 0.01 mol / L ammonium acetate (pH 8.0) - acetonitrile (10:90);

[0063] Detection wavelength: 210nm;

[0064] Column temperature: 30℃;

[0065] Flow rate: 0.5 ml / min;

[0066] Injection volume: 10 μl.

[0067] 4. System suitability test

[0068] Take the reference solution and inject it six times consecutively under the chromatographic conditions described above. Record the peak area of ​​imidazole and calculate the theoretical plate number, tailing factor, and peak area RSD. Take another reference solution, inject it, and calculate the recovery rate. The system suitability acceptance criteria are: theoretical plate number of the main peak not less than 5000, tailing factor not greater than 2.0, peak area RSD not greater than 2.0%, and recovery rate between 98.0% and 102.0%.

[0069] The results are shown in Table 1. The retention time of the imidazole peak was approximately 5.5 min, the RSD of the peak area after 6 injections was 0.16%, the recovery rate of the standard solution was 100.6%, the tailing factor was no higher than 1.33, and the theoretical plate number was no lower than 12275, indicating that the system has good suitability.

[0070] Table 1 System Suitability Test Results

[0071]

[0072] Example 2 Specificity Test

[0073] Take 10 μl each of blank solution (0.01 mol / L ammonium acetate (pH 8.0) - acetonitrile (10:90)), reference solution and test solution, and inject them according to the chromatographic conditions of Example 1, and record the chromatograms.

[0074] The results are shown in Table 2. No interfering peaks appeared at the retention time of the imidazole peak in the blank solution, indicating that the method of this invention has good specificity.

[0075] Table 2 Specificity test results

[0076]

[0077] Example 3: Limit of Quantitation and Limit of Detection Test

[0078] Take the imidazole reference solution, gradually dilute it with solvent, and inject it according to the chromatographic conditions of Example 1. Record the chromatogram. The limit of quantitation (LOQ) is the concentration with a signal-to-noise ratio (S / N) ≥ 10, and the limit of detection (LOD) is the concentration with a signal-to-noise ratio (S / N) ≥ 3.

[0079] The results are shown in Tables 3 and 4. The limit of detection (LOD) for imidazole was 0.1019 μg / ml (equivalent to 0.01% of the concentration of the test solution), and the limit of quantitation (LOQ) was 0.5094 μg / ml (equivalent to 0.05% of the concentration of the test solution). The results indicate that the sensitivity of the method of this invention meets the detection requirements for imidazole impurities in amisulpride raw material.

[0080] Table 3 Results of the detection limit test

[0081]

[0082] Table 4 Results of Limit of Quantitation Test

[0083]

[0084] Example 4 Linearity Test

[0085] Accurately measure an appropriate amount of imidazole reference standard stock solution and dilute it with solvent to prepare a series of linear solutions of different concentrations: 0.5 μg / ml, 0.8 μg / ml, 1.0 μg / ml, 1.5 μg / ml, and 2.0 μg / ml (covering the limit of quantitation to 200% of the limit concentration). Inject and determine the concentrations according to the chromatographic conditions of Example 1, and record the peak areas. Perform linear regression with imidazole concentration (μg / ml) as the x-axis (X) and peak area as the y-axis (Y).

[0086] The results are shown in Table 5. Imidazole showed good linearity in the concentration range of 0.5094–2.0376 μg / ml, with a regression equation of Y = 96.0493X - 0.6912 and a correlation coefficient r of 1.0000.

[0087] Table 5 Results of Linear Experiments

[0088]

[0089] Example 5 Repeatability Test

[0090] Take the same batch of amisulpride raw material, add imidazole reference standard equivalent to 100% of the imidazole limit, prepare 6 parallel test solutions, and inject them into the test solutions according to the chromatographic conditions of Example 1. Calculate the recovery rate and RSD value of imidazole in each test solution.

[0091] The results are shown in Table 6. The recoveries of imidazole in the six test solutions ranged from 98.53% to 100.40%, with an average recovery rate of 99.4% and an RSD of 0.62%, indicating that the method of the present invention has good repeatability.

[0092] Table 6 Repeatability Test Results

[0093]

[0094] Example 6 Accuracy Test

[0095] Accuracy tests were conducted using the spiked recovery method. Ammoniasulfamethoxazole raw material with a known imidazole content was taken, and three concentrations of imidazole reference standard (equivalent to 50%, 100%, and 200% of the imidazole limit in the test sample, respectively) were added. Three replicates of each concentration were prepared, and the samples were injected and analyzed under the chromatographic conditions described in Example 1. The recovery rate and RSD were calculated.

[0096] The results are shown in Table 7. The average recovery rates at each concentration level ranged from 99.18% to 101.51%, with an overall average recovery rate of 100.0% and an RSD of 0.65%, indicating that the method of the present invention has good accuracy.

[0097] Table 7 Accuracy Test Results

[0098]

[0099] Example 7 Durability Test

[0100] The robustness of the method of the present invention under slight changes in chromatographic conditions was investigated. The mobile phase ratio (0.01 mol / L ammonium acetate (pH 8.0) - acetonitrile (10:90) within ±1%), pH value (pH 8.0 ± 0.5), column temperature (30℃ ± 2℃), and flow rate (0.5 ml / min ± 0.1 ml / min) were adjusted respectively, and the sample was injected and determined according to the chromatographic conditions of Example 1 to investigate the consistency of the system suitability parameters and the results of imidazole content determination.

[0101] The results are shown in Table 8. Under various minor variations, the theoretical plate number of the reference solution was not less than 11682, the tailing factor was not higher than 1.35, the peak area RSD was not greater than 1.4%, the recovery rate was between 99.4% and 101.2%, and the RSD of the imidazole content determination result in the test sample was 0.01%, indicating that the method of the present invention has good robustness under minor changes in chromatographic conditions.

[0102] Table 8 Durability Test Results

[0103]

[0104] Comparative Example 1: Determination of imidazole by Gas Chromatography

[0105] The imidazole in amisulpride raw material was determined by gas chromatography under the following chromatographic conditions:

[0106] Column: Agilent DB-5, 30m × 0.53mm × 5μm;

[0107] Detector: Flame Ionization Detector (FID);

[0108] Inlet temperature: 260℃;

[0109] Detector temperature: 270℃;

[0110] Carrier gas: nitrogen, column inlet pressure 4.0 psi;

[0111] Flow split ratio: 20:1;

[0112] Programmed temperature rise: Initial temperature 40℃, hold temperature for 5 min, then rise to 230℃ at a rate of 10℃ / min, hold temperature for 10 min;

[0113] Injection volume: 1 μl.

[0114] Results: When determining imidazole using gas chromatography, imidazole, being a polar heterocyclic compound, is easily adsorbed in the gas chromatography system, resulting in severe peak tailing. The tailing factor of the imidazole peak was 5.42 (much greater than 2.0), seriously affecting the accuracy of quantitative analysis. Furthermore, gas chromatography requires high injection and detector temperatures, placing high demands on the instrument.

[0115] In contrast, this invention employs high-performance liquid chromatography (HPLC) with a Waters XBridge Amide column and a specific mobile phase system for separation and detection. The imidazole peak tailing factor is between 1.30 and 1.33, the peak shape is symmetrical, the quantitative accuracy is high, and the operating conditions are mild, making it more suitable for routine quality control analysis of imidazole in amisulpride API.

Claims

1. A method for detecting imidazole in amisulpride by high performance liquid chromatography, wherein the chromatographic conditions are as follows: the HPLC system is an Agilent 1260 high performance liquid chromatograph (UV / DAD detector) or a similar device; the mobile phase consists of A and B, wherein A is acetate buffer and B is acetonitrile.

2. The detection method according to claim 1, characterized in that, The chromatographic column was a Waters XBridgeAmide, 4.6 × 150 mm, 3.5 μm.

3. The detection method according to claim 1, characterized in that, The mobile phase A is ammonium acetate with a pH of 7.5-8.5 and a concentration of 0.01 mol / L.

4. The detection method according to claim 3, characterized in that, The mobile phase A has a pH of 8.

0.

5. The detection method according to claim 4, characterized in that, The volume ratio of the mobile phases A and B is (9~11):(89~91).

6. The detection method according to claim 5, characterized in that, The volume ratio of the mobile phases A and B is 10:

90.

7. The detection method according to claim 1, characterized in that, Under the specified chromatographic conditions, the detection wavelength is 210 nm.

8. The detection method according to claim 1, characterized in that, The chromatographic conditions are as follows:

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