Method for testing 4-(1, 1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole

Abstract

The invention belongs to the technical field of pharmaceutical analysis, particularly relates to a method for detecting 4-(1, 1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole, and provides a convenient, efficient and accurate detection method for solving the problem of detection of 4-(1, 1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole. The content of 4-(1, 1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole can be detected, so that the medication safety is effectively guaranteed, the quality control of voriconazole is facilitated, the method is convenient, efficient and accurate, the system applicability, repeatability, specificity and accuracy completely conform to the guidance principle verified by methods in Chinese Pharmacopoeia, and the method is suitable for popularization and application. The method can be used for quality control of voriconazole.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and specifically relates to a method for detecting 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole. Background Art

[0002] Voriconazole, chemical name (2R,3S)-2-(2,4-difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1-(1H-1,2,4-triazol-1-yl)-2-butanol, is an organic compound with the chemical formula C 16 H 14 F 3 N 5 O is a second-generation triazole antifungal compound that can act on severe fungal infections. Since voriconazole has a broad antifungal spectrum, strong antibacterial efficacy, good safety, and the domestic market demand for antifungal drugs is growing rapidly, it has huge market prospects.

[0003] 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine, (abbreviated as: dibromo impurity) Chemical structure: , is a potential by-product that may be produced during the synthesis of voriconazole and may remain in voriconazole. It is a genotoxic impurity. Therefore, its content needs to be strictly controlled during the production of voriconazole. Referring to the ICH M7 toxicology threshold TTC = 1.5µg / day, and the maximum daily dose of voriconazole in voriconazole tablets is 0.8g / day, according to the formula limit = TTC / maximum daily dose, it is calculated that the limit of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole raw materials is 1.875ppm.

[0004] At present, there are few literature reports on the detection method of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine. As a pharmaceutical industry, it is very necessary to strictly control the impurity content in the product. The study of material impurities is helpful for the optimization of drug synthesis process and quality control. The study of the inspection method of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole provides a good reference for evaluating the quality of voriconazole drugs, and also provides a technical basis for optimizing the synthesis process of voriconazole drugs. It is of great significance to determine the adverse reactions caused by 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole.

[0005] The invention discloses a detection method for determining 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole by using LC-MS. By using the LC-MS method for detection, a convenient, efficient and accurate detection method is provided to solve the detection problem of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole. The method can detect the content of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole, thereby effectively ensuring the safety of drug use and facilitating the quality control of voriconazole. Summary of the invention

[0006] The invention provides a detection method for determining 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole by using LC-MS. In order to solve the detection problem of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole, a convenient, efficient and accurate detection method is provided. The method can detect the content of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole, thereby effectively ensuring the safety of drug use and facilitating the quality control of voriconazole. The method is convenient, efficient and accurate, and fully complies with the guiding principles of method verification of the Chinese Pharmacopoeia in terms of system applicability, repeatability, specificity and accuracy, and can be used for the quality control of voriconazole.

[0007] To achieve the above object, the present invention provides the following technical solutions: A method for testing 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole, characterized in that the method is to perform pre-column derivatization of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine with a derivatization reagent ethylamine, and the derivatization product is detected in LC-MS, and the detection method comprises the following steps: (1) Prepare solutions, including blank solution, sensitivity solution, reference solution and test solution; (2) Determination method: Use LC-MS / MS to determine the content of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole. After the system is stable, add blank solution, sensitivity solution, reference solution and test solution respectively, and record the chromatogram; The chromatographic conditions are as follows: Chromatographic column: octadecylsilane bonded silica gel as filler, injection volume: 0.2-1.0µl, column temperature: 30±5℃, flow rate: 0.5±0.1ml / min, formic acid solution as mobile phase A, acetonitrile as mobile phase B, gradient elution; The mass spectrometry conditions were as follows: ion source type: ESI, drying gas temperature: 320 °C, drying gas flow rate: 8 L / min, nebulizer gas pressure: 40 psi, sheath gas temperature: 350 °C, sheath gas flow rate: 10 L / min, capillary voltage: 3500 V, polarity: positive, scan mode: MRM, EMV: 200 V, switching time setting of the switch valve: 0.0 min: to Waste; 6.0 min: to MS; 10.0 min: to Waste; .

[0008] Further, the elution gradient is: .

[0009] Furthermore, the volume ratio of the mobile phase A: formic acid: water is 1:1000; For the blank solution: take an appropriate amount of ethylamine and place it in a volumetric flask, add diluent to dilute to the scale, shake well, heat in a 50-70°C water bath for 0.5-2h, and cool to room temperature; The reference substance solution: take an appropriate amount of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine reference substance ethylamine and place it in a volumetric flask, add diluent to dilute to the scale, shake well, heat in a water bath at 50-70°C for 0.5-2h, and cool to room temperature; Sensitivity solution: Take an appropriate amount of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine reference substance ethylamine and place it in a volumetric flask, add diluent to dilute to the scale, shake well, heat in a 50-70℃ water bath for 0.5-2h, and cool to room temperature; Test solution: Take an appropriate amount of the test sample and place it in a volumetric flask, add an appropriate amount of ethylamine and an appropriate amount of diluent to dissolve, then dilute to the scale with the diluent, shake well, heat in a 50-70℃ water bath for 0.5-2h, and cool to room temperature; the diluent is acetonitrile; the chromatographic column is PoroShell 120 EC-C18 2.7 μm, 4.6×100mm.

[0010] The invention discloses a method for detecting 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole. In order to solve the problem of detecting 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole, a convenient, efficient and accurate detection method is provided. The method can detect the content of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole, thereby effectively ensuring the safety of drug use. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the blank solution spectrum of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole; Figure 2This is the sensitivity solution spectrum of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole; Figure 3 It is the selective solution spectrum of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole; Figure 4 This is the spectrum of the reference solution of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole; Figure 5 This is the spectrum of the test solution of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole. DETAILED DESCRIPTION

[0012] The present invention is further illustrated by the following examples, but are not intended to limit the present invention.

[0013] Example 1: Determination of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole (1) Preparation of solution: Formic acid: AR and above; Water: ultrapure water; Ethylamine solution: 66.0-72.0%; Acetonitrile: HPLC and above; Diluent: acetonitrile; Blank solution: Accurately measure 250µl of ethylamine and place it in a 25ml volumetric flask. Add diluent to dilute to the mark, shake well, heat in a 60℃ water bath for 1h, and cool to room temperature.

[0014] Reference substance stock solution ①: Take about 20 mg of the dibromo impurity reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add diluent to dilute to the mark, and shake well; accurately measure 250 µl of the above solution, place it in a 20 ml volumetric flask, add diluent to dilute to the mark, and shake well. (Concentration: 12.5 µg / ml) Reference substance stock solution ②: Accurately measure 750µl of reference substance stock solution ①, place in a 100ml volumetric flask, add diluent to dilute to the mark, and shake well. (Concentration: 93.75ng / ml) Reference substance solution: Accurately measure 500µl of reference substance stock solution ② and 250µl of ethylamine, place in a 25ml volumetric flask, add diluent to dilute to the mark, shake well, heat in a 60℃ water bath for 1h, and cool to room temperature. (Concentration: 1.875ng / ml) Sensitivity solution: Accurately measure 100µl of reference stock solution ② and 250µl of ethylamine, place in a 25ml volumetric flask, add diluent to dilute to scale, shake well, heat in a 60℃ water bath for 1h, and cool to room temperature. (Concentration: 0.375ng / ml) Test solution: Take about 25 mg of the test sample, weigh accurately, place in a 25 ml volumetric flask, add 250 µl of ethylamine and an appropriate amount of diluent to dissolve, then dilute to scale with diluent, shake well, heat in a 60°C water bath for 1 hour, and cool to room temperature. (Concentration 1 mg / ml) (2) Chromatographic conditions: Instruments: Agilent InfinityII 1290 UHPLC+Agilent 6470 ESI-QQQ-MS, electronic analytical balance, constant temperature water bath.

[0015] Chromatographic column: PoroShell 120 EC-C18 2.7 μm, 4.6×100 mm or equivalent Injection volume: 0.5 μl Column temperature: 30°C Flow rate: 0.5ml / min Mobile phase A: Formic acid: Water = 1:1000 (V / V) Mobile phase B: acetonitrile

[0016]

[0017] Switching time setting of on / off valve: 0min: to Waste; 6.0min: to MS; 10.0min:to Waste。

[0018]

[0019] Note: 1) * represents quantitative ion.

[0020] 2) The switching time of the mass spectrometer switch valve can be appropriately adjusted according to the actual situation of the instrument. The purpose is to avoid collecting voriconazole signals and ensure the response of the dibromo impurity derivative signals.

[0021] (3) Steps After the system is stable, inject 1 injection of blank solution, 1 injection of sensitivity solution, 6 injections of reference solution, 1 injection of blank solution, and 1 injection of test solution, and record the spectrum.

[0022] Require The S / N value of the dibrominated impurity derivative in the sensitivity solution should not be less than 10; the RSD of the dibrominated impurity derivative peak area for the reference solution should not be greater than 10.0%.

[0023] calculate

[0024] Where: R U : The measured peak area of ​​the dibromo impurity derivative in the spectrum of the test solution; R S : 6 is the average peak area of ​​the dibromo impurity derivative in the reference solution spectrum; C S : Concentration of dibromo impurity in reference solution (ng / ml); n: dilution factor of the test sample, 25; M: Sample volume of the test product, mg limit

[0025] The detection method of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole also includes method validation before detection. The method validation is based on the chromatographic conditions of the formal detection, and the measurement results are as follows:

[0026] Example 2: System Applicability System suitability is achieved by evaluating the S / N value of dibrominated impurity derivatives in the sensitivity solution and the RSD of the peak area of ​​dibrominated impurity derivatives in the reference solution. The S / N value of dibrominated impurity derivatives in the sensitivity solution should be no less than 10; the RSD of dibrominated impurity derivatives in the reference solution should be no more than 10.0% in the peak area. In order to confirm the stability of the system during the sequence operation, the reference solution is tested every 8 hours and at the end of the sequence. The RSD of dibrominated impurity derivatives in the reference solution for the next 6 consecutive times should be no more than 10.0%; if it exceeds this range, an evaluation investigation should be conducted.

[0027]

[0028] Example 3: Specificity The specificity of the method is achieved by determining whether the blank solution interferes with the detection and the separation degree between the dibrominated impurity derivative peak and the adjacent impurity peak in the selective solution. It is required that the blank solution should have no interference with the detection and the separation degree between the dibrominated impurity derivative peak and the adjacent impurity peak in the selective solution should be no less than 1.5.

[0029]

[0030] Example 4: Precision Repeatability is achieved by measuring the RSD of the results of the dibrominated impurities in 6 test solutions (spiked), and it is required that the RSD of the results of the dibrominated impurities in 6 test solutions (spiked) should not be greater than 10.0%.

[0031]

[0032] Example 5: Limit of Detection and Limit of Quantitation The detection limit is obtained by testing the ratio of its response signal to noise is not less than 3:1; the quantification limit is obtained by testing the ratio of its response signal to noise is not less than 10:1. At the LOQ concentration level, 6 quantification limit test solutions are repeatedly examined, requiring that the RSD of the peak area per unit concentration of the dibromo impurity derivative in the 6 LOQ solutions should be no more than 20.0%, and the LOQ 双溴杂质 It should be no greater than 0.5625ppm, S / N≥10, S / N of dibromo impurity derivatives in LOD solution≥3, and LOD<LOQ.

[0033]

[0034]

[0035] Example 6: Linearity and Range Six points were evenly selected within the range of LOQ concentration to 150% of the limit concentration, and the concentration was used as the horizontal axis and the peak area as the vertical axis to draw a curve. It is required that the dibromo impurity should be linear within the range of LOQ concentration to 150% of the limit concentration, and the square of the linear correlation coefficient R (R 2 ) should be no less than 0.99, and the absolute value of the y-intercept should be within 25% of the 100% concentration peak area.

[0036]

[0037] Example 7: Accuracy Accuracy is achieved through the recovery rate between the actual concentration and theoretical concentration of the measured component and the total RSD of the recovery rate (n=9). It is required that when the accuracy solution with LOQ concentration, 100% limit concentration and 150% limit concentration is added, the recovery rate of dibromo impurities should be between 70.0% and 130.0%, and the total RSD of the recovery rate (n=9) should not be greater than 20.0%.

[0038]

[0039] Example 8: Durability The reference solution, test solution and selective solution were placed at room temperature for a period of time and then injected, and the change pattern of the test results over time was investigated, so as to provide a reference for the placement time of the reference solution and the test solution during the test.

[0040] Requirements: Compared with the reference solution at 0 hr, the recovery rate of the dibromo impurity in the reference solution during the room temperature investigation period should be between 80.0% and 120.0%, and there should be no obvious trend of change. The reference solution is stable during the room temperature investigation period. If dibromo impurities are detected in the test solution at 0hr, and the test solution is placed at room temperature for a period of time, the change value of the test result is within 20% of the limit, and there is no obvious change trend, then the test solution is stable during the room temperature investigation; if dibromo impurities are not detected in the test solution at 0hr, and the test solution is placed at room temperature for a period of time, and no dibromo impurities are detected, then the test solution is stable during the room temperature investigation; When the selective solution is left at room temperature for a period of time, the recovery rate of the dibromo impurity should be between 80.0% and 120.0%, and there should be no obvious trend of change, then the selective solution is stable during the room temperature investigation.

[0041]

[0042]

[0043]

Claims

1. A method for testing 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole, characterized in that: The method is to perform pre-column derivatization of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine with a derivatization reagent ethylamine, and the derivatization product is detected in LC-MS. The detection method comprises the following steps: (1) Prepare solutions, including blank solution, sensitivity solution, reference solution and test solution; (2) Determination method: Use LC-MS to determine the content of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine in voriconazole. After the system is stable, add blank solution, sensitivity solution, reference solution and test solution respectively, and record the chromatogram; The chromatographic conditions are as follows: Chromatographic column: octadecylsilane bonded silica gel as filler, injection volume: 0.2-1.0µl, column temperature: 30±5℃, flow rate: 0.5±0.1ml / min, formic acid solution as mobile phase A, acetonitrile as mobile phase B, gradient elution; The mass spectrometry conditions were as follows: ion source type: ESI, drying gas temperature: 320 °C, drying gas flow rate: 8 L / min, nebulizer gas pressure: 40 psi, sheath gas temperature: 350 °C, sheath gas flow rate: 10 L / min, capillary voltage: 3500 V, polarity: positive, scan mode: MRM, EMV: 200 V, switching time setting of the switch valve: 0.0 min: to Waste; 6.0 min: to MS; 10.0 min: to Waste; 。 2. The inspection method according to claim 1, characterized in that: The elution gradient is: 。 3. The inspection method according to claim 2, characterized in that: The volume ratio of the mobile phase A: formic acid: water is 1:1000; For the blank solution: take an appropriate amount of ethylamine and place it in a volumetric flask, add diluent to dilute to the scale, shake well, heat in a 50-70°C water bath for 0.5-2h, and cool to room temperature; The reference substance solution: take an appropriate amount of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine reference substance ethylamine and place it in a volumetric flask, add diluent to dilute to the scale, shake well, heat in a water bath at 50-70°C for 0.5-2h, and cool to room temperature; Sensitivity solution: Take an appropriate amount of 4-(1,1-dibromoethyl)-6-chloro-5-fluoropyrimidine reference substance ethylamine and place it in a volumetric flask, add diluent to dilute to the scale, shake well, heat in a 50-70℃ water bath for 0.5-2h, and cool to room temperature; Test solution: Take an appropriate amount of the test sample and place it in a volumetric flask, add an appropriate amount of ethylamine and an appropriate amount of diluent to dissolve, then dilute to the scale with the diluent, shake well, heat in a 50-70℃ water bath for 0.5-2h, and cool to room temperature; the diluent is acetonitrile; the chromatographic column is PoroShell 120 EC-C18 2.7 μm, 4.6×100mm.

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