Method for detecting 1-amino-4-cyclopentyl piperazine in rifapentine
The detection of 1-amino-4-cyclopentylpiperazine in rifapentine by liquid-mass junction is solved, and the problem of lack of detection methods in the prior art is achieved, effective control of the quality of rifapentine products is achieved, and the sensitivity and accuracy of detection are improved.
Patent Information
- Application Number
- CN202510055310.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-14
- Publication Date
- 2025-04-15
AI Technical Summary
The prior art lacks methods for detecting 1-amino-4-cyclopentylpiperazine, which makes it difficult to control the product quality of rifopentine.
The 1-amino-4-cyclopentylpiperazine in rifopentin was detected by gradient elution using a Waters XBridge C18 chromatography column, combined with a trifluoroacetic acid solution and a mixed solution of methanol or methanol acetonitrile as the mobile phase.
Effective detection of 1-amino-4-cyclopentylpiperazine in rifapentin is achieved, with a detection limit of less than 5ppm and a quantitative limit of less than 10ppm. It has good specificity, sensitivity and accuracy, and is suitable for controlling the relevant impurity content in rifapentin and improving product quality.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of drug analysis, and in particular to a method for detecting 1-amino-4-cyclopentylpiperazine. Background Art
[0002] Rifapentine is an antibiotic used to treat tuberculosis, which inhibits the activity of DNA-dependent RNA polymerase in susceptible cells. Rifapentine has a bactericidal effect and has a very broad spectrum of activity against most Gram-positive and Gram-negative bacteria (including Pseudomonas aeruginosa), especially Mycobacterium tuberculosis. 1-Amino-4-cyclopentylpiperazine is an impurity introduced and degraded during the synthesis of rifapentine, which can further generate the N-nitrosamine impurity 1-cyclopentyl-4-nitrosopiperazine. Therefore, the detection of 1-amino-4-cyclopentylpiperazine is of great significance for the quality control of rifapentine raw materials and preparations. However, there is currently no literature or other information disclosing the determination method of 1-amino-4-cyclopentylpiperazine. In order to control the product quality of rifapentine, it is necessary to develop a method for detecting 1-amino-4-cyclopentylpiperazine in rifapentine.
[0003] Summary of the invention
[0004] In view of the shortcomings of the current prior art, the object of the present invention is to provide a method for detecting 1-amino-4-cyclopentylpiperazine in rifapentine, which is beneficial to the quality control of rifapentine products.
[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: A method for detecting 1-amino-4-cyclopentylpiperazine in rifapentine, the method using liquid chromatography-mass spectrometry, characterized in that it comprises the following chromatographic conditions: Column: Waters XBridge C18; Mobile phase A: 0.2% trifluoroacetic acid solution; Mobile phase B: methanol or a mixture of methanol and acetonitrile; Elution mode: gradient elution.
[0006] Furthermore, in the above detection method of the present invention, the elution gradient of the gradient elution is as follows:
[0007] Furthermore, in the detection method of the present invention, the volume ratio of methanol to acetonitrile in the mixture solution of methanol and acetonitrile is 3:(3-1), preferably 3:1.
[0008] Furthermore, in the above detection method of the present invention, the column temperature in the chromatographic conditions is 20-25°C.
[0009] Furthermore, in the above detection method of the present invention, the flow rate in the chromatographic conditions is 0.4~0.6ml / min.
[0010] Furthermore, the detection method of the present invention adopts the following mass spectrometry parameters:
[0011] The beneficial technical effect of the present invention is that the present invention adopts a liquid chromatography-mass spectrometry method, selects a Waters XBridge C18 chromatographic column, and uses a trifluoroacetic acid solution and methanol or a methanol-acetonitrile mixed solution as a mobile phase to detect 1-amino-4-cyclopentylpiperazine in rifapentine, and the content of 1-amino-4-cyclopentylpiperazine in the test solution can be calculated using a reference substance external standard method. The method of the present invention is simple, the detection limit is less than 5ppm, the quantitative limit is less than 10ppm, and it has good specificity, sensitivity and accuracy. It is suitable for the determination of 1-amino-4-cyclopentylpiperazine in rifapentine, which is beneficial to the control of the content of related impurities in rifapentine and improves the product quality of rifapentine. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 The chromatograms of the reference solution, the test solution and the blank solvent in Example 1 when the chromatographic column is Waters XBridge C18; Figure 2 The chromatograms of the reference solution and the test solution in Example 1 when the chromatographic column is Waters XBridge Shield RP18; Figure 3 The chromatograms of the reference solution and the test solution in Example 2 when the mobile phase A is 10 mM ammonium formate (pH=9.0); Figure 4 The chromatograms of the reference solution and the test solution in Example 2 when the mobile phase A is 10 mM ammonium formate (pH=6.0); Figure 5 The chromatograms of the reference solution and the test solution when the mobile phase A is 0.1% formic acid water in Example 2 are shown; Figure 6 The chromatograms of the reference solution, the test solution and the blank solvent when the mobile phase B is a mixture solution of methanol and acetonitrile (3:1) in Example 3; Figure 7 : is the chromatogram of LOD solution and LOQ solution in Example 6; Figure 8 The chromatograms are of the reference solution, the test solution and the medium level accuracy solution in Example 7. DETAILED DESCRIPTION
[0013] The following examples are used to further illustrate and understand the spirit of the present invention, but are not intended to limit the scope of protection of the present invention in any way.
[0014] Example 1
[0015] The content of 1-amino-4-cyclopentylpiperazine in rifapentine was determined by liquid chromatography-mass spectrometry, and the chromatographic conditions were as follows: Chromatographic column: Waters XBridge C18 (150×4.6mm, 3.5μm) or Waters XBridge ShieldRP18 (150×4.6mm, 3.5μm); Mobile phase A: 0.2% trifluoroacetic acid solution; Mobile phase B: methanol; Elution method: gradient elution, the elution gradient is as follows:
[0016] Flow rate: 0.5ml / min; Column temperature: 20°C; Injection volume: 5 μl; Collection time: 30min; Ion source: ESI (+); Mass spectrometry parameters:
[0017] Experimental steps: (1) Preparation of mobile phase A: Accurately measure 500 ml of ultrapure water, add 1 ml of trifluoroacetic acid, mix well and sonicate for 15 min to obtain a 0.2% trifluoroacetic acid solution; (2) Preparation of reference solution: Take an appropriate amount of 1-amino-4-cyclopentylpiperazine reference substance, accurately weigh it, and quantitatively dilute it with methanol to prepare a solution containing about 100 ng of 1-amino-4-cyclopentylpiperazine per 1 ml; (3) Preparation of test solution: Accurately weigh about 10 mg of rifapentine and place it in a 10 ml volumetric flask. Add an appropriate amount of methanol and sonicate to dissolve it. Then add solvent to make the volume to the mark to prepare a solution containing about 1 mg of rifapentine per 1 ml. (4) Detection: Take the blank solvent methanol, the above-mentioned reference solution and the test solution and inject them into the liquid chromatography-mass spectrometer respectively, measure under the above-mentioned chromatographic conditions, and record the chromatogram. Calculate the content of 1-amino-4-cyclopentylpiperazine in the test solution according to the reference external standard method.
[0018] See the detection spectrum Figure 1~Figure 2The results showed that when the Waters XBridge Shield RP18 column was used, the peak shape of the analyte in the test sample was abnormal; the Waters XBridge C18 column could be used to effectively separate and detect 1-amino-4-cyclopentylpiperazine in rifapentine.
[0019] Example 2
[0020] The content of 1-amino-4-cyclopentylpiperazine in rifapentine was determined by liquid chromatography-mass spectrometry, and the chromatographic conditions were as follows: Chromatographic column: Waters XBridge C18 (150×4.6mm, 3.5μm); Mobile phase A: 10 mM ammonium formate (pH = 9.0), 10 mM ammonium formate (pH = 6.0), or 0.1% formic acid in water; The remaining chromatographic conditions were the same as in Example 1.
[0021] Experimental steps: (1) Preparation of reference solution and test solution: same as in Example 1; (2) Detection: Take the above-mentioned reference solution and test solution and inject them into the liquid chromatography-mass spectrometer respectively, measure them under the above-mentioned chromatographic conditions, and record the chromatogram. Calculate the content of 1-amino-4-cyclopentylpiperazine in the test solution according to the reference external standard method.
[0022] See the detection spectrum Figure 3~Figure 5 The results showed that when 10 mM ammonium formate (pH = 9.0), 10 mM ammonium formate (pH = 6.0) or 0.1% formic acid water was used as mobile phase A, the on-column degradation in the test solution was obvious and the detection of 1-amino-4-cyclopentylpiperazine could not be achieved.
[0023] Example 3
[0024] The content of 1-amino-4-cyclopentylpiperazine in rifapentine was determined by liquid chromatography-mass spectrometry, and the chromatographic conditions were as follows: Chromatographic column: Waters XBridge C18 (150×4.6mm, 3.5μm); Mobile phase B: methanol: acetonitrile = 3:1; The remaining chromatographic conditions were the same as in Example 1.
[0025] Experimental steps: (1) Preparation of reference solution and test solution: same as in Example 1; (2) Detection: Take the blank solvent methanol, the above-mentioned reference solution and the test solution and inject them into the liquid chromatography-mass spectrometer respectively, measure under the above-mentioned chromatographic conditions, and record the chromatogram. Calculate the content of 1-amino-4-cyclopentylpiperazine in the test solution according to the reference external standard method.
[0026] See the detection spectrum Figure 6 The results showed that when a methanol-acetonitrile (3:1) mixed solution was used as mobile phase B, the peak shape of 1-amino-4-cyclopentylpiperazine in the reference solution and the test solution was good, and this chromatographic condition could realize the detection of 1-amino-4-cyclopentylpiperazine in rifapentine.
[0027] Example 4
[0028] Chromatographic column: Waters XBridge C18 (150×4.6mm, 3.5μm); Flow rate: 0.4ml / min, 0.5ml / min or 0.6ml / min; The remaining chromatographic conditions were the same as in Example 1.
[0029] Experimental steps: (1) Preparation of reference solution and test solution: same as in Example 1; (2) Detection: Take the blank solvent methanol, the above-mentioned reference solution and the test solution and inject them into the liquid chromatography-mass spectrometer respectively, measure under the above-mentioned chromatographic conditions, and record the chromatogram. Calculate the content of 1-amino-4-cyclopentylpiperazine in the test solution according to the reference external standard method.
[0030] The test results show that when the flow rate is 0.4-0.6 ml / min in the chromatographic conditions, the peak shape of 1-amino-4-cyclopentylpiperazine in the reference solution and the test solution is good, and the chromatographic conditions can better realize the detection of 1-amino-4-cyclopentylpiperazine in rifapentine.
[0031] Example 5
[0032] Chromatographic column: Waters XBridge C18 (150×4.6mm, 3.5μm); Column temperature: 20℃, 22℃ or 25℃; The remaining chromatographic conditions were the same as in Example 1.
[0033] Experimental steps: (1) Preparation of reference solution and test solution: same as in Example 1; (2) Detection: Take the blank solvent methanol, the above-mentioned reference solution and the test solution and inject them into the liquid chromatography-mass spectrometer respectively, measure under the above-mentioned chromatographic conditions, and record the chromatogram. Calculate the content of 1-amino-4-cyclopentylpiperazine in the test solution according to the reference external standard method.
[0034] The test results show that when the column temperature is 20-25°C in the chromatographic conditions, 1-amino-4-cyclopentylpiperazine in rifapentine can be well detected.
[0035] Example 6
[0036] Chromatographic column: Waters XBridge C18 (150×4.6mm, 3.5μm); The remaining chromatographic conditions were the same as in Example 1.
[0037] Experimental steps: (1) Preparation of reference solution: same as in Example 1; (2) Preparation of LOD solution: Accurately pipette 0.5 ml of the reference solution into a 10 ml volumetric flask, add methanol to dissolve and dilute to the mark, mix well, and the LOD solution (containing about 5 ng / ml of 1-amino-4-cyclopentylpiperazine, i.e. 5 ppm) is obtained. (3) Preparation of LOQ solution: Accurately pipette 1 ml of the reference solution into a 10 ml volumetric flask, add methanol to dissolve and dilute to the mark, mix well, and the LOQ solution (containing about 10 ng / ml of 1-amino-4-cyclopentylpiperazine, i.e. 10 ppm) is obtained. (4) Detection: Take the above LOD solution and LOQ solution and inject them into the liquid chromatography-mass spectrometer respectively, perform the measurement under the above chromatographic conditions, and record the chromatogram and signal-to-noise ratio.
[0038] See the detection spectrum Figure 7 The results showed that the signal-to-noise ratios of the LOD solution and the LOQ solution were 5.74 and 14.26, respectively, when the chromatographic conditions were used to detect the LOD solution and the LOQ solution. This method met the quality control requirements for drug analysis.
[0039] Example 7
[0040] The chromatographic conditions were the same as in Example 6.
[0041] Experimental steps: (1) Preparation of reference solution and test solution: same as in Example 1.
[0042] (2) Preparation of reference stock solution: Take an appropriate amount of 1-amino-4-cyclopentylpiperazine reference substance, accurately weigh it, and quantitatively dilute it with methanol to prepare a solution containing about 1 μg of 1-amino-4-cyclopentylpiperazine per 1 ml; (3) Preparation of spiked test solution (i.e., medium-level accuracy solution): Accurately weigh approximately 10 mg of rifapentine and place in a 10-ml volumetric flask. Add an appropriate amount of methanol and sonicate to dissolve. Accurately add 1 ml of reference stock solution (1 μg / ml) and dilute to volume with methanol to prepare a solution containing approximately 1 mg of rifapentine and approximately 100 ng of 1-amino-4-cyclopentylpiperazine per 1 ml. (4) Detection: Take the above-mentioned reference solution, test solution, and spiked test solution (medium level accuracy solution) and inject them into the liquid chromatography-mass spectrometer respectively, measure under the above-mentioned chromatographic conditions, and record the chromatogram. Calculate the content of 1-amino-4-cyclopentylpiperazine in the test solution according to the reference external standard method.
[0043] The test results are shown in the table below:
[0044] See the detection spectrum Figure 8 The results showed that when the chromatographic conditions were used to detect 1-amino-4-cyclopentylpiperazine in rifapentine, the recovery rate was 91.5%, and the method met the quality control requirements for drug analysis.
Claims
1. A method for detecting 1-amino-4-cyclopentylpiperazine in rifapentine, wherein the method adopts liquid chromatography-mass spectrometry, characterized in that The following chromatographic conditions were included: Column: Waters XBridge C18; Mobile phase A: 0.2% trifluoroacetic acid solution; Mobile phase B: methanol or a mixture of methanol and acetonitrile; Elution mode: gradient elution.
2. The detection method according to claim 1, wherein the elution gradient of the gradient elution is as follows: 。 3. The detection method according to claim 1, wherein the volume ratio of methanol to acetonitrile in the mixture solution of methanol and acetonitrile is 3:(3~1).
4. The detection method according to claim 3, wherein the volume ratio of methanol to acetonitrile is 3:
1.
5. The detection method according to claim 1, wherein the column temperature in the chromatographic conditions is 20-25°C.
6. The detection method according to claim 1, wherein the flow rate in the chromatographic conditions is 0.4-0.6 ml / min.
7. The detection method according to any one of claims 1 to 6, wherein the chromatographic conditions include the following mass spectrometry parameters: 。