Method for detecting content of 1-aminocyclopentyl formonitrile in 2-butyl-1, 3-diazaspiro [4, 4] nonyl-1-ene-4-one hydrochloride

Abstract

The invention provides a method for detecting the content of 1-aminocyclopentyl formonitrile in 2-butyl-1, 3-diazaspiro [4, 4] nonyl-1-ene-4-one hydrochloride by adopting a high performance liquid chromatography, and the chromatographic conditions are as follows: a chromatographic column is adopted: YMC-Pack ODS-AQ, 4.6 mm * 150 mm, 5 [mu] m; an ultraviolet detector is adopted, the detection wavelength is 254 nm, the column temperature is 25 DEG C, the flow rate is 0.8 ml / min, and the sample injection volume is 50 microliters; gradient elution is adopted, and a mobile phase A is a phosphoric acid aqueous solution with the concentration of 0.1%; the mobile phase B is methanol.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis, specifically to a method for detecting the content of 1-aminocyclopentylformonitrile in 2-butyl-1,3-diazaspirocyclic[4,4]non-1-en-4-one hydrochloride. Background Technology

[0002] 2-Butyl-1,3-diazaspirocyclic[4,4]non-1-en-4-one is a key synthetic intermediate used in the preparation of irbesartan, a drug for treating hypertension. Related studies have found that 1-aminocyclopentylcarboxylate, an impurity, may be present in 2-butyl-1,3-diazaspirocyclic[4,4]non-1-en-4-one and may remain in the final product, irbesartan.

[0003]

[0004] According to ICH (The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) guidelines, many molecular structures currently lack detailed toxicological experimental data. Therefore, the risk is generally estimated based on whether the target molecule contains a warning structure fragment to assess the risk posed by the impurity to drug safety. 1-Aminocyclopentylformonitrile is an impurity containing a genotoxic warning structure, requiring content monitoring and control to meet product safety requirements.

[0005] Regarding the detection methods for impurities in irbesartan and its intermediates, Zhang Wenhui et al. reported a method using LC-MS / MS to detect the content of impurities in irbesartan in an article published in Volume 59, Issue 7 of the Chinese Pharmaceutical Journal. The impurities involved were a series of molecules containing the irbesartan core skeleton. The chromatographic conditions used were: a C-18 column, with 0.1% formic acid solution-acetonitrile (volume ratio 62:38) as the mobile phase, and isocratic elution. Liang Yanfen et al. reported a method using HPLC to detect the content of impurities in irbesartan in an article published in Volume 17, Issue 4 of Food and Drug. The impurity molecule was related substance A. The separation conditions used were: a C-18 column, with acetonitrile-0.025 mol / L KH2PO4 solution (35:65, adjusted to pH 6.0 with triethylamine) as the mobile phase, and isocratic elution.

[0006] It is evident that, in the current state of technology, there are no reports on the detection of 1-aminocyclopentylformonitrile in irbesartan or its intermediates, and no relevant methods are available. Therefore, there is a need to establish a method with sufficient precision and sensitivity for the detection of 1-aminocyclopentylformonitrile in irbesartan or its intermediates to meet the quality control requirements of pharmaceutical production. Summary of the Invention

[0007] Based on the above technical background, the present invention provides a method for detecting the content of 1-aminocyclopentylformonitrile in 2-butyl-1,3-diazaspirocyclic[4,4]non-1-en-4-one hydrochloride. The method has high detection sensitivity, and its system applicability and specificity meet the requirements, which can meet the needs of drug production quality monitoring.

[0008] This invention provides a method for detecting the content of 1-aminocyclopentylformonitrile in 2-butyl-1,3-diazaspirocyclic [4,4]non-1-en-4-one hydrochloride, a synthetic intermediate of irbesartan, comprising the following steps: (1) Solution preparation: Take 1-aminocyclopentylformitrile hydrochloride reference standard and prepare a reference standard solution with 50% methanol-water solution; take 2-butyl-1,3-diazaspirocyclo[4,4]non-1-en-4-one hydrochloride sample to be tested and prepare a test solution with 50% methanol-water solution. (2) Inject the test solution and the reference solution into the high performance liquid chromatograph. The chromatographic conditions are as follows: column: YMC-Pack ODS-AQ, 4.6 mm × 150 mm, 5 μm; UV detector, detection wavelength: 254 nm, column temperature: 25 ℃, flow rate: 0.8 ml / min, injection volume: 50 μl; gradient elution is used, mobile phase A is 0.1% phosphoric acid aqueous solution; mobile phase B is methanol.

[0009] Furthermore, the elution gradient of the gradient elution is: .

[0010] A more specific operating procedure is as follows: Solution preparation: Diluent: 50% methanol aqueous solution; Blank solution: diluent; 1-Aminocyclopentylformitrile stock solution: Accurately weigh 1-aminocyclopentylformitrile hydrochloride reference standard, place it in a volumetric flask, dissolve and dilute to the mark with diluent, and shake well. (Concentration of 1-aminocyclopentylformitrile hydrochloride: 666 μg / ml, equivalent to a concentration of 500 μg / ml for 1-aminocyclopentylformitrile) Reference solution: Accurately measure 1.0 ml of 1-aminocyclopentylformitrile stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, shake well, and incubate in a constant temperature water bath at 80°C for 2 hours. Allow to cool to room temperature. (Concentration of 1-aminocyclopentylformitrile hydrochloride: 66.6 μg / ml, equivalent to a 1-aminocyclopentylformitrile concentration of 50.0 μg / ml) Sensitivity solution: Accurately measure 1.0 ml of 1-aminocyclopentylformitrile stock solution into a 20 ml volumetric flask, dilute to the mark with diluent, shake well, and incubate in a constant temperature water bath at 80°C for 2 hours. Allow to cool to room temperature. (1-Aminocyclopentylformitrile hydrochloride concentration: 33.3 μg / ml, equivalent to 1-aminocyclopentylformitrile concentration: 25.0 μg / ml) Test solution: Accurately weigh 2-butyl-1,3-diazaspirocyclic [4,4]non-1-en-4-one hydrochloride sample, place it in a 10 ml volumetric flask, add an appropriate amount of diluent to dissolve, then dilute to the mark, shake well, place in a constant temperature water bath at 80℃ for 2 hours, and cool to room temperature. (Concentration: 50 mg / ml); After the system stabilizes, inject the sample and record the chromatogram. Six injections of the reference solution and one injection of the test solution were used. The chromatographic conditions were as follows: Chromatographic column: The column is packed with octadecylsilane-bonded silica gel (e.g., YMC-Pack ODS-AQ, 4.6mm×150mm, 5μm or equivalent column). Trapping column: Welch Ghost-Buster Column, 4.6mm×50mm; Flow rate: 0.8 ml / min; Detection wavelength: 290 nm; Injection volume: 50 μl; Column temperature: 25℃; Mobile phase A: 0.1% aqueous phosphoric acid solution; Mobile phase B: methanol; Elute using the following gradient:

[0011] The result is calculated according to the following formula: 1-Aminocyclopentylformitrile content (%) = (Ru / Rs) × (Cs / Cu) × 0.001 × 110.16 / 146.62 × 100 Wherein: Ru: peak area of ​​1-aminocyclopentylformonitrile in the test solution; Rs: 6 represents the average peak area of ​​1-aminocyclopentylformonitrile in the reference solution; Cs: Concentration of 1-aminocyclopentylformonitrile in the reference solution (μg / ml); Cu: Concentration of the test solution (mg / ml); The verification of the method is summarized as follows:

[0012] Beneficial effects: The present invention provides a method for detecting the content of 1-aminocyclopentylformonitrile in 2-butyl-1,3-diazaspirocyclic[4,4]non-1-en-4-one hydrochloride. The method has high detection sensitivity, and the verification results show that the system applicability and specificity of this method meet the requirements and can meet the needs of drug production quality monitoring. Attached Figure Description

[0013] Figure 1 This is a chromatogram of a selective solution; Figure 2 This is the chromatogram of the reference solution; Figure 3 This is a repeatable solution chromatogram; Figure 4 This is a sensitivity solution chromatogram. Detailed Implementation Example 1

[0014] System Applicability Solution preparation: Diluent: 50% methanol aqueous solution; Blank solution: diluent.

[0015] 1-Aminocyclopentylformitrile stock solution: Accurately weigh approximately 33.92 mg of 1-aminocyclopentylformitrile hydrochloride reference standard, place it in a 50 mL volumetric flask, dissolve and dilute to the mark with diluent, and mix well. (Concentration of 1-aminocyclopentylformitrile: 507.0926 μg / mL) Reference solution: Accurately measure 1.0 ml of 1-aminocyclopentylformitrile stock solution into a 10 ml volumetric flask, dilute to the mark with diluent, shake well, and incubate in a constant temperature water bath at 80°C for 2 hours. Allow to cool to room temperature. (Concentration of 1-aminocyclopentylformitrile: 50.7093 μg / ml) Sensitivity solution: Accurately measure 1.0 ml of 1-aminocyclopentylformitrile stock solution into a 20 ml volumetric flask, dilute to the mark with diluent, shake well, and place in a constant temperature water bath at 80°C for 2 hours. Allow to cool to room temperature. (1-Aminocyclopentylformitrile concentration: 25.3546 μg / ml) The above solution was injected under the following chromatographic conditions: column: YMC-Pack ODS-AQ, 4.6 mm × 150 mm, 5 μm; UV detector, detection wavelength: 254 nm; column temperature: 25 ℃; flow rate: 0.8 ml / min; injection volume: 50 μl; gradient elution was used, with mobile phase A being 0.1% aqueous phosphoric acid solution and mobile phase B being methanol.

[0016] The elution gradient for gradient elution is:

[0017] The sensitivity solution chromatogram is as follows: Figure 4 As shown, the chromatogram of the reference solution is as follows: Figure 2 As shown.

[0018] System suitability test results Example 2

[0019] Specificity experiment Solution preparation: The 1-aminocyclopentylformitrile positioning solution (i.e., the reference solution) is the same as in Example 1; Test solution: Accurately weigh 495.0 mg of 2-butyl-1,3-diazaspirocyclic [4,4]non-1-en-4-one hydrochloride sample, place it in a 10 ml volumetric flask, add an appropriate amount of diluent to dissolve, then dilute to the mark, shake well, and place in a constant temperature water bath at 80℃ for 2 hours, then cool to room temperature. (Concentration: 49.5000 mg / ml) Selective solution: Measure 497.1 mg of 2-butyl-1,3-diazaspirocyclo[4,4]non-1-en-4-one hydrochloride and dissolve it in 1 ml of 1-aminocyclopentylformitrile stock solution, then dilute to 10 ml to obtain the solution. (Concentration: 2-butyl-1,3-diazaspirocyclo[4,4]non-1-en-4-one hydrochloride 49.7100 mg / ml, 1-aminocyclopentylformitrile 50.7093 μg / ml) The above solution was injected under the same chromatographic conditions as in Example 1; The chromatogram of the 1-aminocyclopentylformitrile positioning solution (i.e., the reference solution) is shown below. Figure 2 As shown, the selective solution chromatogram is as follows: Figure 1 As shown; Specificity detection results Example 3

[0020] Precision test Solution preparation: The diluent, test solution, and reference solution are the same as in Example 1; Preparation of reproducible solutions:

[0021] The above solution was injected under the same chromatographic conditions as in Example 1; the chromatogram of the repeatable solution (Precision-1) is shown in Figure 3.

[0022] The repeatability test results are shown below:

Claims

1. A method for detecting the content of 1-aminocyclopentylformonitrile in 2-butyl-1,3-diazaspirocyclic [4,4]non-1-en-4-one hydrochloride, comprising the following steps: (1) Solution preparation: Take 1-aminocyclopentylformitrile hydrochloride reference standard and prepare a reference standard solution with 50% methanol-water solution; take 2-butyl-1,3-diazaspirocyclo[4,4]non-1-en-4-one hydrochloride sample to be tested and prepare a test solution with 50% methanol-water solution. (2) The test solution and the reference solution were injected into a high performance liquid chromatograph for detection to determine the content of 1-aminocyclopentyl formonitrile in the test sample of 2-butyl-1,3-diazaspirocyclic [4,4]non-1-en-4-one hydrochloride. Chromatographic conditions: YMC-Pack ODS-AQ column, 4.6 mm × 150 mm, 5 μm; UV detector, detection wavelength: 254 nm, column temperature: 25 ℃, flow rate: 0.8 ml / min, injection volume: 50 μl; gradient elution was used, mobile phase A was 0.1% phosphoric acid aqueous solution; mobile phase B was methanol.

2. The method for detecting the content of 1-aminocyclopentylformonitrile in 2-butyl-1,3-diazaspirocyclic [4,4]non-1-en-4-one hydrochloride as described in claim 1, wherein the elution gradient of the gradient elution is: 。

Images