Method for detecting 2-amino-5-nitrothiazole genotoxic impurities in nitazoxanide
The detection of genotoxic impurities of 2-amino-5-nitrothiazole in nizonit was solved by high-performance liquid chromatography, and the problem of insufficient detection sensitivity in the prior art was solved, efficient and accurate detection results were achieved, and the safety of the drug was ensured.
Patent Information
- Application Number
- CN202110461147.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2021-04-27
- Publication Date
- 2025-03-11
- Estimated Expiration
- 2041-04-27
AI Technical Summary
The existing detection methods cannot efficiently and accurately detect genotoxic impurities of 2-amino-5-nitrothiazole in nizonit, especially in terms of sensitivity of 0.6 ppm.
High performance liquid chromatography was used to detect genotoxic impurities of 2-amino-5-nitrothiazole in nizonit. By selecting the appropriate chromatographic column, mobile phase and detection wavelength, efficient separation and quantification of the impurities were achieved.
This method is simple to operate, fast detection speed, high separation efficiency and high sensitivity. It can accurately detect 0.6ppm of 2-amino-5-nitrothiazole, ensuring the safety of the drug.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of analysis and detection, and in particular to a method for detecting 2-amino-5-nitrothiazole genotoxic impurities in nitazoxanide. Background Art
[0002] Genotoxic impurities are a type of impurity that can react with DNA, causing DNA damage, inducing gene mutations at very low levels, and may cause cancer. Due to their strong toxicity, they pose a strong threat to the safety of medication. In recent years, regulatory agencies in various countries, such as ICH, FDA, and EMA, have had clearer requirements for genotoxic impurities, and more and more pharmaceutical companies have focused on the control and detection of genotoxic impurities in the process of new drug development.
[0003] The structural formula of 2-amino-5-nitrothiazole is as follows:
[0004]
[0005] The structural analysis is: 1H NMR (600 MHz, DMSO-d6) δ8.80 (s, 2H), 8.26 (s, 1H); 13CNMR (150 MHz, DMSO-d6) δ173.87, 147.86, 135.70; m / z: calcd. for C3H3O2N3S, (M+H)+: 145.1, Found: 146.0.
[0006] 2-Amino-5-nitrothiazole is an important synthetic intermediate in dyes, medicines and pesticides. Due to its bactericidal effect, it can also be used as a bactericidal and disinfectant for poultry farming. 2-Amino-5-nitrothiazole contains a nitro group, a warning structure for genotoxic impurities, and has potential genotoxicity. According to the relevant ICH guidelines, the limit of genotoxic impurities is ≤1.5μg / Day, and there has been no relevant report on the detection method of this genotoxic impurity so far. Summary of the invention
[0007] The technical problem to be solved by the present invention is to provide a method for detecting 2-amino-5-nitrothiazole genotoxic impurities in nitazoxanide, which fills the blank of analyzing such genotoxic impurities by high performance liquid chromatography. Compared with the traditional liquid-to-mass spectrometry detection method, the method is simple to operate, convenient and efficient, has a wide range of applications, and has a higher sensitivity of up to 0.6 ppm, thereby ensuring the safety of the drug.
[0008] The method for detecting 2-amino-5-nitrothiazole genotoxic impurities in nitazoxanide is carried out by high performance liquid chromatography, 2-amino-5-nitrothiazole is used as a reference substance, acetonitrile is used as a diluent, and the content of 2-amino-5-nitrothiazole in the sample is calculated by an external standard method; wherein the chromatographic column uses octadecyl bonded silica gel as a filler, and the mobile phase uses a mixed solution of phosphoric acid aqueous solution and acetonitrile.
[0009] The pH value of phosphoric acid aqueous solution is 2.5-6.5.
[0010] In the mixed solution of phosphoric acid aqueous solution and acetonitrile, the volume percentage of phosphoric acid aqueous solution is 40-60%.
[0011] The liquid phase conditions are:
[0012] Chromatographic column: size 4.6mm×250mm, average particle size of filler particles 5μm;
[0013] Column temperature: 30-35°C;
[0014] Flow rate: 1.0ml / min;
[0015] Injection volume: 10-20 μl:
[0016] Detection wavelength: 350-390nm.
[0017] Preferably, the detection wavelength is 385 nm.
[0018] Preferably, the flow rate is 1.0 ml / min.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] The invention adopts a high performance liquid chromatography method to detect 2-amino-5-nitrothiazole genotoxic impurities in nitazoxanide, has the advantages of simple operation, fast detection speed, high separation efficiency, high sensitivity, strong specificity, high accuracy and good durability. By detecting the 2-amino-5-nitrothiazole genotoxic impurities in nitazoxanide, the 2-amino-5-nitrothiazole genotoxic impurities are controlled within 1.5 ppm, so as to better control the quality of the product. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 is the HPLC spectrum of the blank solvent of Example 1 of the present invention;
[0022] Figure 2 is the HPLC spectrum of the reference substance solution of Example 1 of the present invention;
[0023] Figure 3 is the mass spectrum of the reference solution of Comparative Example 1 of the present invention;
[0024] Figure 4 It is the HPLC spectrum of the blank solvent (KB), the reference solution (DZ) and the test solution (GSP) of Example 2 of the present invention. DETAILED DESCRIPTION
[0025] The present invention is further described below with reference to the embodiments.
[0026] Example 1
[0027] The high performance liquid chromatography method of the present invention is used to detect the 2-amino-5-nitrothiazole genotoxic impurity in nitazoxanide, wherein the liquid phase conditions are as follows:
[0028] Chromatographic column: size 4.6mm×250mm, filler is octadecyl bonded silica gel, average particle size of filler particles is 5μm;
[0029] Column temperature: 35°C;
[0030] Mobile phase: a mixed solution of phosphoric acid aqueous solution (pH 2.5) and acetonitrile in a volume ratio of 55:45;
[0031] Flow rate: 1.0ml / min;
[0032] Detection wavelength: 385nm.
[0033] The detection steps are as follows:
[0034] (1) Using 2-amino-5-nitrothiazole as a reference substance and acetonitrile as a solvent, a 2-amino-5-nitrothiazole reference substance solution with a concentration of 3 ng / ml was prepared;
[0035] (2) Take 20 μl of blank solvent and 20 μl of 2-amino-5-nitrothiazole reference solution respectively and detect them by high performance liquid chromatography. Record the chromatogram, such as Figure 1-2 As shown, the peak time and peak area of 2-amino-5-nitrothiazole in the reference solution were obtained.
[0036] from Figure 1-2 It can be seen that its quantitative limit is 0.6ppm and the limit is 1.5ppm. It has high sensitivity and meets the measurement requirements, that is, the quantitative limit is ≤1.5ppm.
[0037] Comparative Example 1
[0038] The genotoxic impurity of 2-amino-5-nitrothiazole in nitazoxanide was detected by high performance liquid chromatography-mass spectrometry, wherein the liquid chromatography-mass spectrometry coupling conditions are as follows:
[0039] Chromatographic column: size 4.6mm×250mm, filler is octadecyl bonded silica gel, average particle size of filler particles is 5μm;
[0040] Column temperature: 35°C;
[0041] Mobile phase: a mixed solution of 0.1% formic acid solution and acetonitrile in a volume ratio of 80:20;
[0042] Flow rate: 1.0ml / min;
[0043] Detection wavelength: 385nm.
[0044] The detection steps are as follows:
[0045] (1) Using 2-amino-5-nitrothiazole as a reference substance and acetonitrile as a solvent, a 2-amino-5-nitrothiazole reference substance solution with a concentration of 25 ng / ml was prepared;
[0046] (2) Take 10 μl of 2-amino-5-nitrothiazole reference solution and detect it by HPLC-MS, and record the spectrum. Figure 3 As shown, the peak time and peak area of 2-amino-5-nitrothiazole in the reference solution were obtained.
[0047] from Figure 3 As can be seen from the figure, its limit of quantification is 10ppm. The limit is 1.5ppm, which has a low sensitivity.
[0048] Examples 2-8 respectively test the specificity, quantitative limit and detection limit, linearity and range, accuracy, repeatability, intermediate precision and durability of the detection method, wherein the preparation method of the sample solution to be tested is as follows:
[0049] (1) Prepare blank solvent: acetonitrile.
[0050] (2) Preparation of reference stock solution: Take 2-amino-5-nitrothiazole and add acetonitrile to prepare a solution containing 30 ng of 2-amino-5-nitrothiazole per 1 ml of acetonitrile.
[0051] (3) Preparation of reference solution: Take 2-amino-5-nitrothiazole and add acetonitrile to prepare a solution containing 3 ng of 2-amino-5-nitrothiazole per 1 ml of acetonitrile.
[0052] (4) Preparation of test solution: Take the test sample and add acetonitrile to prepare a 2 mg / ml solution.
[0053] (5) Prepare spiked test solution: accurately weigh 20 mg of the test sample and place it in a 10-ml volumetric flask. Accurately add 1 ml of the reference stock solution, add acetonitrile and ultrasonically dissolve and dilute to the mark. Shake well.
[0054] Unless otherwise specified, the liquid phase conditions are as follows:
[0055] Chromatographic column: size 4.6mm×250mm, filler is octadecyl bonded silica gel, average particle size of filler particles is 5μm;
[0056] Column temperature: 35°C;
[0057] Mobile phase: a mixed solution of phosphoric acid aqueous solution (pH 2.5) and acetonitrile in a volume ratio of 55:45;
[0058] Flow rate: 1.0ml / min;
[0059] Detection wavelength: 385nm.
[0060] Example 2: Specificity test
[0061] Take 10 μl of blank solvent, reference solution and test solution and inject them into HPLC respectively, and record the chromatogram. Figure 4 As shown, the results show that the blank solvent and the matrix in the test sample do not interfere with the determination of 2-amino-5-nitrothiazole, indicating that the detection method of the present invention has good specificity.
[0062] Example 3: Limit of Quantitation and Limit of Detection Tests
[0063] Take an appropriate amount of 2-amino-5-nitrothiazole, dissolve it in acetonitrile and dilute it to make a corresponding solution. Take 10μl and inject it into the high performance liquid chromatograph. Take the concentration of the solution when S / N≈10 as the quantitative limit concentration, dilute the quantitative limit solution 2 to 3 times, and take the concentration of the solution when S / N≈3 as the detection limit concentration. At the same time, examine the precision of the quantitative limit.
[0064] The results showed that the detection limit of 2-amino-5-nitrothiazole was 0.4 ng / ml and the quantification limit was 1.2 ng / ml.
[0065] Example 4: Linearity and range test
[0066] Accurately measure an appropriate amount of the reference substance stock solution, dilute it with acetonitrile to a series of reference substance solutions with concentrations of 1.5 ng / ml, 2.4 ng / ml, 3 ng / ml, 3.6 ng / ml, and 4.5 ng / ml, take 10 μl of each of the above series of reference substance solutions, inject them into a high performance liquid chromatograph, record the chromatogram, use the concentration as the abscissa, and the 2-amino-5-nitrothiazole peak area as the ordinate, and use the least squares method for linear regression. The results are shown in Table 1.
[0067] Table 1: 2-amino-5-nitrothiazole linear relationship test results
[0068]
[0069] The results showed that within the concentration range of 1.5 ng / ml to 4.5 ng / ml, there was a good linear relationship between the concentration and the peak area of 2-amino-5-nitrothiazole.
[0070] Example 5: Accuracy Test
[0071] Preparation of recovery solution:
[0072] Accurately weigh 20 mg of the test sample and place it in a 10 ml volumetric flask. Add an appropriate amount of 2-amino-5-nitrothiazole reference stock solution at a level of 50% of the 2-amino-5-nitrothiazole limit. Add acetonitrile to dissolve by ultrasound and dilute to the scale. Shake well and prepare 3 copies in parallel.
[0073] Accurately weigh 20 mg of the test sample and place it in a 10 ml volumetric flask. Add an appropriate amount of 2-amino-5-nitrothiazole reference stock solution according to the 100% level of the 2-amino-5-nitrothiazole limit. Add acetonitrile to dissolve by ultrasound and dilute to the scale. Shake well and prepare 3 copies in parallel.
[0074] Accurately weigh 20 mg of the test sample and place it in a 10 ml volumetric flask. Add an appropriate amount of 2-amino-5-nitrothiazole reference stock solution at a level of 150% of the 2-amino-5-nitrothiazole limit. Add acetonitrile to dissolve by ultrasound and dilute to the scale. Shake well and prepare 3 copies in parallel.
[0075] Take 10 μl of blank solvent, reference solution, test solution, and recovery solution, respectively, and inject them into the high performance liquid chromatograph to record the chromatogram. Calculate the total amount of 2-amino-5-nitrothiazole measured by the peak area according to the external standard method, and calculate the recovery rate. The test results are shown in Table 2.
[0076] Table 2: 2-amino-5-nitrothiazole recovery test results
[0077]
[0078] The results showed that the recovery rates of 2-amino-5-nitrothiazole were between 80% and 115%, and the accuracy met the requirements.
[0079] Example 6: Repeatability test
[0080] Prepare 6 spiked test solutions in parallel, take 10 μl of each of the blank solution, reference solution, and spiked test solution, inject them into the HPLC, and record the chromatogram. Calculate the content of 2-amino-5-nitrothiazole by peak area according to the external standard method, and calculate the RSD. The test results are shown in Table 3.
[0081] Table 3: Repeatability test results of 2-amino-5-nitrothiazole
[0082]
[0083] The results show that the detection method of the present invention has good repeatability.
[0084] Example 7: Intermediate precision test
[0085] Prepare 6 spiked test solutions in parallel at different times, using different instruments and different personnel.
[0086] Take 10 μl of each of the blank solution, reference solution, and spiked test solution, inject them into the HPLC, and record the chromatogram. Calculate the content of 2-amino-5-nitrothiazole by peak area according to the external standard method, calculate RSD (n=12), and examine the intermediate precision of the method. The test results are shown in Table 4.
[0087] Table 4: Intermediate precision test results of 2-amino-5-nitrothiazole
[0088]
[0089] The results show that the detection method of the present invention has good intermediate precision.
[0090] Example 8: Durability Test
[0091] In order to investigate the tolerance of the method of the present invention to the detection of the product when the chromatographic conditions undergo slight changes, the durability of the chromatographic conditions was investigated, and the specific parameter changes are shown in Table 5.
[0092] Table 5: Chromatographic condition variation parameters
[0093] Chromatographic parameters Standard conditions Scope of verification Flow rate 1.0ml / min 0.8ml / min~1.2ml / min Mobile phase pH 2.5 2.3～2.7 Chromatographic column brand C18 Chromatographic columns of different brands
[0094] Take 10 μl of each of the blank solution, reference solution, and spiked test solution, inject them into the high performance liquid chromatograph, and record the chromatogram. Calculate the content of 2-amino-5-nitrothiazole by the peak area according to the external standard method. The test results are shown in Table 6.
[0095] Table 6: Durability test results of 2-amino-5-nitrothiazole
[0096]
[0097]
[0098] From the above results, it can be seen that fine-tuning the chromatographic conditions has no effect on the determination of 2-amino-5-nitrothiazole genotoxic impurities, indicating that the chromatographic conditions of the detection method of the present invention are durable.
Claims
1. A method for detecting 2-amino-5-nitrothiazole genotoxic impurities in nitazoxanide, characterized in that: The 2-amino-5-nitrothiazole content in the sample was calculated by the external standard method using 2-amino-5-nitrothiazole as a reference substance and acetonitrile as a diluent by high performance liquid chromatography; wherein the chromatographic column used octadecyl bonded silica gel as a filler, and the mobile phase used a mixed solution of phosphoric acid aqueous solution and acetonitrile, in which the volume percentage of the phosphoric acid aqueous solution and the acetonitrile mixed solution was 55%, and the pH value of the phosphoric acid aqueous solution was 2.3-2.7; the liquid phase conditions were: Chromatographic column: size 4.6mm×250mm, average particle size of filler particles 5μm; Column temperature: 30-35°C; Flow rate: 1.0ml / min; Injection volume: 10-20 μl; Detection wavelength: 350-390nm; The detection limit concentration of the 2-amino-5-nitrothiazole is 0.4 ng / mL, and the quantification limit concentration is 1.2 ng / mL.
2. The method for detecting 2-amino-5-nitrothiazole genotoxic impurities in nitazoxanide according to claim 1, wherein: The detection wavelength is 385 nm.