Method for simultaneously determining multiple genotoxic impurities in voriconazole raw material medicine based on LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) technology

Abstract

The invention relates to the technical field of drug detection, in particular to a method for detecting five potential genotoxic impurities in a voriconazole bulk drug, and the method comprises the following steps: detecting the genotoxic impurities in the voriconazole bulk drug by adopting a high performance liquid chromatography-tandem mass spectrometry to obtain the content of the genotoxic impurities; wherein the high performance liquid chromatography takes a formic acid aqueous solution as a mobile phase A and a formic acid methanol solution as a mobile phase B. By adopting the high performance liquid chromatography-tandem mass spectrometry method, exclusive separation of voriconazole and five genotoxic impurities can be realized, and the content of each genotoxic impurity can be sensitively detected. According to the method, the sample treatment mode is simple, multiple genotoxic impurities can be detected at the same time, and the detection method is high in specificity, good in sensitivity and good in repeatability and durability.

Description

Technical Field

[0001] This application belongs to the field of drug detection technology, specifically relating to a method for simultaneously determining multiple genotoxic impurities in voriconazole raw material based on LC-MS / MS technology. Background Technology

[0002] Voriconazole is a triazole broad-spectrum antifungal drug suitable for adults and children over 2 years of age. It is used to treat serious fungal infections such as invasive aspergillosis (a fungal infection that begins in the lungs and spreads through the bloodstream to other organs), esophageal candidiasis (an infection caused by yeast fungi that can cause white patches in the mouth and throat), and candidemia (a fungal infection in the blood).

[0003] In the field of drug testing, from the perspective of facilitating research and maximizing safety, the presence of warning structures is generally considered highly correlated with determining whether a compound is genotoxic. For example, if certain functional groups or substructural units can react with certain functional macromolecules in organisms and cause gene mutations, these functional groups or substructural units are considered to have a warning effect on biological activity. Therefore, in practical research, using such warning structures to determine whether a compound is genotoxic makes the identification of genotoxic impurities in drugs faster, more targeted, and more convenient. Both the EMA and FDA guidelines consider structural assessment to be an effective method for studying genotoxic impurities in drugs, and the FDA recommends using structural assessment software to evaluate and measure the toxicity of compounds.

[0004] Lutz Müller et al. (A rationale for determining, testing, and controlling specific impurities in pharmaceuticals that possess potential forgenotoxicity, Regulatory Toxicology and Pharmacology, Vol 44, Issue 3, April 2006, pp. 198-211) combined relevant data and research to classify warning groups into 20 categories (see Fig. 1 on page 201), among which haloalkane groups and sulfonate groups are considered to be groups with strong potential for genotoxicity.

[0005] During the synthesis and storage of voriconazole active pharmaceutical ingredient (API), various impurities may be generated. Some of these impurities, containing warning structures such as haloalkane groups and sulfonate groups, are classified as genotoxic impurities. As shown in Table 1, impurities A, B, and C all contain haloalkane groups, which may damage DNA in vivo through nucleophilic substitution reactions. Impurities D and E are sulfonate compounds, which are mutagenic and may cause DNA alkylation. ToxTree, a commonly used structural evaluation software, showed that the above impurities are classified as genotoxic impurities due to the presence of haloalkyl groups and sulfonate groups. Analysis using another commonly used software, VenomPred, also showed that the impurities in Table 1 exhibit varying degrees of mutagenicity and carcinogenicity, indicating their association with genotoxicity.

[0006] Therefore, effective detection of genotoxic impurities in voriconazole raw materials is particularly important.

[0007] Table 1. Potential genotoxic impurities in voriconazole raw material

[0008]

[0009] Referring to the ICH M7 (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) guidelines, the limit for each impurity is set at the Threshold of Toxicological Concern (TC) of 1.5 μg / day. The maximum daily intake of voriconazole is 0.9 g / day, and the limit for a single impurity is (1.5 μg / day) / (0.9 g / day) = 1.67 ppm.

[0010] The generation pathways of various genotoxic impurities are as follows:

[0011] Impurity A generation path:

[0012] ;

[0013] Impurity B formation pathway:

[0014] ;

[0015] Impurity C generation pathway:

[0016] ;

[0017] Impurity D generation path:

[0018] ;

[0019] Impurity E generation pathway:

[0020] ;

[0021] Currently, research on genotoxic impurities in voriconazole is limited. Of the genotoxic impurities listed in Table 1, only the detection method for impurity A has been reported. For example, patent CN110441460A discloses a method for detecting (R / S)-4-(1-bromoethyl)-5-fluoro-6-chloro-pyrimidine (impurity A), using conventional high-performance liquid chromatography (HPLC). To achieve the required detection sensitivity, the sample requires cumbersome derivatization, which introduces certain difficulties to the operation and can easily lead to the loss or degradation of the target impurity during the process, failing to reflect the true content of genotoxic impurities in the product.

[0022] Patent CN114544843A also targets the study of 4-(1-bromoethyl)-5-fluoro-6-chloropyrimidine (impurity A). It uses high performance liquid chromatography-mass spectrometry (LC-MS) to determine the content of this impurity. The scanning mode is SIM. In order to improve the detection sensitivity, the test sample and the reference sample still need to be derivatized, which has the same problem as patent CN110441460A.

[0023] CN110308212A uses high-performance liquid chromatography (HPLC) to detect voriconazole-related substances, including only genotoxic impurity A (FLKZ-V4 in Table 1, pages 4-5 of the specification). In this patent, impurity A is studied as a common impurity, with a control limit set at 0.1%, which is far higher than the control requirements for genotoxic impurities. This limitation stems from the HPLC detection method used in this patent, which cannot achieve the required higher detection sensitivity. Furthermore, the research in this application shows that this method cannot separate all impurities in Table 1, indicating poor specificity.

[0024] Current research on genotoxic impurities of voriconazole faces the following challenges:

[0025] The genotoxic impurity profile is incomplete, failing to adequately assess potential genotoxic impurities in conjunction with process and stability considerations; furthermore, there is a lack of methods for the simultaneous detection of multiple genotoxic impurities. Existing techniques involve complex sample processing, and during derivatization, the poor chemical stability of genotoxic impurities can easily lead to their loss during extraction.

[0026] The aforementioned issues pose challenges to the quality control and safety evaluation of voriconazole, making it unable to meet the requirements of the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) guidelines on limits for genotoxic impurities. Summary of the Invention

[0027] This application provides a method for simultaneously separating multiple genotoxic impurities from voriconazole active pharmaceutical ingredient (API). By using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) combined with optimized mobile phase, sensitive detection of genotoxic impurities can be achieved without complex sample derivatization. Risk assessments can be performed on each genotoxic impurity, and reasonable limit standards can be established. An effective extraction, accurate quantification, and control system covering five genotoxic impurities that may be present under synthetic processes and storage conditions can be constructed to ensure the safety of voriconazole API.

[0028] This application focuses on the aforementioned genotoxic impurities, filling gaps in related fields through innovative technological means, providing support for the high-quality production and safe clinical application of voriconazole, meeting the stringent requirements of drug regulatory regulations for the control of genotoxic impurities, and possessing significant scientific value and application prospects.

[0029] To achieve the purpose of this application, the following technical solution is adopted:

[0030] This application provides a method for detecting genotoxic impurities in voriconazole raw material, the method comprising: detecting genotoxic impurities in voriconazole raw material using high performance liquid chromatography-tandem mass spectrometry to obtain the content of the genotoxic impurities; wherein the high performance liquid chromatography uses formic acid aqueous solution as mobile phase A and formic acid methanol solution as mobile phase B;

[0031] The genotoxic impurities include any one or a combination of at least two of the following compounds, preferably a combination of at least three, more preferably a combination of at least four, and even more preferably a combination of five:

[0032] Impurity A Impurity B Impurity C Impurity D Impurity E.

[0033] Preferably, the method specifically includes the following steps:

[0034] (1) Mix and dissolve voriconazole raw material and diluent one to obtain the test solution; mix and dissolve genotoxic impurity and diluent two to obtain the control solution;

[0035] (2) The test solution and the control solution were detected by high performance liquid chromatography-tandem mass spectrometry, and the content of the genotoxic impurities was calculated by external standard method.

[0036] Preferably, the first diluent and the second diluent each independently comprise any one or a combination of at least two of acetonitrile, water, or formic acid, and more preferably a combination of acetonitrile, water, and formic acid.

[0037] Preferably, the volume ratio of acetonitrile, water and formic acid is 500:500:1.

[0038] Preferably, the ultraviolet detection wavelength of the high-performance liquid chromatography is 260 nm.

[0039] Preferably, the injection volume of the high-performance liquid chromatography is 30~50μL, and more preferably 40μL.

[0040] Preferably, the chromatographic column of the high performance liquid chromatography uses octadecylsilane-bonded silica gel as the packing material.

[0041] Preferably, the chromatographic column has an inner diameter of 4.6 mm, a length of 150 mm, and a particle size of 3.5 µm.

[0042] The high-performance liquid chromatography column used in this application can be, for example, an Agilent ZORBAX Eclipse XDB-C18 column, 4.6 × 150 mm, 3.5 µm, or a column with equivalent performance.

[0043] Preferably, the flow rate of the mobile phase in the high-performance liquid chromatography is 0.40 mL / min to 0.60 mL / min, more preferably 0.48 mL / min to 0.52 mL / min, and even more preferably 0.5 mL / min.

[0044] Preferably, the column temperature of the high-performance liquid chromatography is 20~30℃, more preferably 24~26℃, and even more preferably 25℃.

[0045] Preferably, the volume percentage of formic acid in the mobile phase A or B of the high performance liquid chromatography is 0.05% to 0.15%, more preferably 0.09% to 0.11%, and even more preferably 0.10%.

[0046] Preferably, the high-performance liquid chromatography employs a gradient elution method.

[0047] Preferably, the gradient elution conditions are as follows:

[0048] From 0 to 13 min, the volume percentage of mobile phase A was 40% and that of mobile phase B was 60%. Then, the volume percentage changed uniformly to 19 min, where the volume percentage of mobile phase A was 29% and that of mobile phase B was 71%. Then, the volume percentage changed uniformly to 21 min, where the volume percentage of mobile phase A was 20% and that of mobile phase B was 80%, and remained unchanged until 31 min. Then, the volume percentage changed uniformly to 32 min, where the volume percentage of mobile phase A was 10% and that of mobile phase B was 90%, and remained unchanged until 37 min. Then, the volume percentage changed uniformly to 38 min, where the volume percentage of mobile phase A was 40% and that of mobile phase B was 60%, and remained unchanged until 43 min.

[0049] Preferably, the mass spectrometer uses a triple quadrupole mass spectrometer detector and an electrospray ion source, with detection performed in positive ion mode.

[0050] Preferably, in the mass spectrometry detection, the m / z of the ion pairs of impurities A, B, C, D, and E are 240.5 / 159.1, 319.2 / 240.0, 285.0 / 205.0, 247.2 / 107.2, and 261.2 / 107.2, respectively.

[0051] Preferably, in the mass spectrometry detection, the declustering voltages DP of impurities A, B, C, D, and E are 86.86V, 80.52V, 86.15V, 100.99V, and 108.44V, respectively.

[0052] Preferably, in the mass spectrometry detection, the collision energies CE of impurities A, B, C, D, and E are 37.19V, 26.40V, 38.56V, 23.36V, and 26.66V, respectively.

[0053] Compared with the prior art, this application has at least the following beneficial effects:

[0054] On the one hand, by directly dissolving and preparing test samples and reference standards using conventional solvents, and selecting 0.05%–0.15% formic acid aqueous solution and 0.05%–0.15% formic acid methanol solution as mobile phases, combined with targeted screening of precursor and daughter ion parameters, the detection sensitivity of genotoxic impurities was significantly improved, especially for impurities A, B, and C. This application found that using acetonitrile as the mobile phase affects the ionization process of impurities A, B, and C, with the most significant ionization inhibition effect on impurities A and B. However, using methanol as the mobile phase significantly improved the response of each impurity. Methanol, as a protonating solvent, provides a better protonation environment for sample detection, improving ionization efficiency. Based on the optimization of the underrivatized system, this application addresses the detection sensitivity problem of impurities containing haloalkane groups from another technical dimension by enhancing the protonation effect through the methanol mobile phase.

[0055] On the other hand, by screening specific precursor / daughter ion pairs based on the chemical structure characteristics of each genotoxic impurity, and combining this with multiple reaction monitoring (MRM) mode, the target analytes were precisely located. This effectively avoided the influence of the voriconazole main component and other interfering substances in the matrix on the detection signal, significantly reducing background noise. This optimization further improved the detection sensitivity of the five genotoxic impurities. Furthermore, combined with separation by a liquid chromatography system, it enabled the dedicated detection of the five genotoxic impurities, meeting the ICH M7 requirements for trace analysis of genotoxic impurities.

[0056] Compared with existing technologies, the method of this application can effectively measure and control genotoxic impurities in voriconazole raw materials and simplifies the sample preparation process. It features high specificity, high sensitivity (limit of quantitation as low as 0.345 ng / mL, limit of detection as low as 0.173 ng / mL), high accuracy, good precision, and good robustness, enabling accurate quantitative determination of five genotoxic impurities in voriconazole raw materials. The method of this application exhibits high specificity, low limits of quantitation and detection, and good repeatability and separation.

[0057] This method can accurately determine the content of five genotoxic impurities in voriconazole raw materials, which is beneficial to the quality control of voriconazole raw materials and has the prospect of promotion and application from a safety perspective. Attached Figure Description

[0058] Figure 1 This is the mass spectrum of the blank solvent (ion pair: m / z 247.2 / 107.2, corresponding to impurity D).

[0059] Figure 2 This is the mass spectrum of the blank solvent (ion pair: m / z 261.2 / 107.2, corresponding to impurity E).

[0060] Figure 3 This is the mass spectrum of the blank solvent (ion pair: m / z 240.5 / 159.1, corresponding to impurity A).

[0061] Figure 4 This is the mass spectrum of the blank solvent (ion pair: m / z 319.2 / 240.0, corresponding to impurity B).

[0062] Figure 5 This is the mass spectrum of the blank solvent (ion pair: m / z 285.0 / 205.0, corresponding to impurity C).

[0063] Figure 6 Total ion chromatogram for detecting genotoxic impurities in voriconazole raw material.

[0064] Figure 7 This is a linear relationship graph for impurity A.

[0065] Figure 8 This is a linear relationship graph for impurity B.

[0066] Figure 9 This is a linear relationship graph for impurity C.

[0067] Figure 10 This is a linear relationship graph for impurity D.

[0068] Figure 11 This is a linear relationship graph for impurity E.

[0069] Figure 12 Mass spectrum of impurity A for the limit of quantitation of genotoxic impurities in voriconazole raw material.

[0070] Figure 13 Mass spectrum of impurity B for the limit of quantitation of genotoxic impurities in voriconazole raw material.

[0071] Figure 14 Detection of genotoxic impurities in voriconazole raw material at the limit of quantitation - impurity C mass spectrum.

[0072] Figure 15 Detection of genotoxic impurities in voriconazole raw material at the limit of quantitation - mass spectrum of impurity D.

[0073] Figure 16 Detection of genotoxic impurities in voriconazole raw material using mass spectrometry - Impurity E.

[0074] Figure 17 Selected ion chromatograms (impurity D) for Example 1 and Comparative Example 1.

[0075] Figure 18 Selected ion chromatograms (impurity E) for Example 1 and Comparative Example 1.

[0076] Figure 19 Selected ion chromatograms (impurity A) for Example 1 and Comparative Example 1.

[0077] Figure 20 Selected ion chromatograms (impurity B) for Example 1 and Comparative Example 1.

[0078] Figure 21 Selected ion chromatograms (impurity C) for Example 1 and Comparative Example 1. Detailed Implementation

[0079] The technical solution of this application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of this application and should not be construed as limiting the scope of this application.

[0080] The specific implementation method of this application is described in detail below:

[0081] The high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) method was used, and the specific conditions and methods are as follows:

[0082] (1) High performance liquid chromatography conditions: Octadecylsilane bonded silica gel was used as the packing material (Agilent ZORBAXEclipse XDB-C18, 4.6×150mm, 3.5µm or equivalent column); column temperature was 20~30℃, flow rate was 0.4-0.6mL / min; injection volume was 30~50μL; 0.05%~0.15% formic acid aqueous solution was used as mobile phase A, and 0.05%~0.15% formic acid methanol solution was used as mobile phase B, and linear gradient elution was performed according to the table below.

[0083] Table 2 Gradient elution program

[0084]

[0085] Mass spectrometry conditions:

[0086] Table 3 Mass Spectrometry Conditions

[0087]

[0088] (2) Preparation of diluent. Take 500 mL of acetonitrile, dilute with water to 1000 mL, add 1 mL of formic acid, mix well, and the solution is ready.

[0089] (3) Preparation of the test solution. Weigh approximately 1.2 g of voriconazole accurately, place it in a 10 mL volumetric flask, add diluent, sonicate to dissolve and dilute to the mark (10 mL) of the volumetric flask, and shake well.

[0090] (4) Preparation of control solutions. Weigh approximately 3.0 mg each of impurity A, impurity B, impurity C, impurity D and impurity E into different 5 mL volumetric flasks, dissolve and dilute to the mark (5 mL) with acetonitrile, and shake well to prepare control solution 1. Accurately measure 0.3 mL of each of control solution 1 into the same 100 mL volumetric flask, dilute to the mark (100 mL) with diluent, and shake well to prepare control solution 2. Accurately measure 1 mL of control solution 2 into a 10 mL volumetric flask, dilute to the mark (10 mL) with diluent, and shake well.

[0091] (5) Calculation method for genotoxic impurity content.

[0092] The result is obtained by calculating the peak area using the external standard method.

[0093] Impurity content (ppm)

[0094] In the formula:

[0095] Ws represents the sample weight of the reference standard, in mg;

[0096] Ps represents the content of the reference standard;

[0097] Ns is the dilution factor of the reference standard;

[0098] As represents the peak area of ​​the reference standard;

[0099] At represents the peak area of ​​the analyte in the test sample;

[0100] Nt is the dilution factor of the test sample;

[0101] Wt is the sample weight of the test sample, in mg.

[0102] Example 1: Specificity Experiment

[0103] 1-1. Information on the test sample and impurities

[0104] Table 4 Summary of Information on Test Samples and Impurities

[0105]

[0106] 1-2. Solution preparation

[0107] Diluent: Measure 500 mL of acetonitrile, dilute with water to 1000 mL, add 1 mL of formic acid, and mix well.

[0108] Test solution: Weigh approximately 1.2 g of voriconazole accurately, place it in a 10 mL volumetric flask, add diluent, sonicate to dissolve and dilute to the mark (10 mL) on the volumetric flask, and shake well;

[0109] Reference solutions: Take approximately 3.0 mg each of impurity A, impurity B, impurity C, impurity D, and impurity E reference standards, place them in separate 5 mL volumetric flasks, dissolve and dilute to the mark (5 mL) with acetonitrile, and mix well to prepare reference solution 1. Accurately measure 0.3 mL of each of reference solution 1, place them in the same 100 mL volumetric flask, dilute to the mark (100 mL) with diluent, and mix well to prepare reference solution 2. Accurately measure 1 mL of reference solution 2, place it in a 10 mL volumetric flask, dilute to the mark (10 mL) with diluent, and mix well.

[0110] 1-3. Test Conditions

[0111] Instrument: High Performance Liquid Chromatography-Mass Spectrometry

[0112] The high-performance liquid chromatography (HPLC) conditions were as follows: octadecylsilane-bonded silica gel (Agilent ZORBAXEclipse XDB-C18, 4.6 × 150 mm, 3.5 µm) was used as the stationary phase; the column temperature was 25 °C, the flow rate was 0.5 mL / min, the injection volume was 40 μL, and 0.1% formic acid aqueous solution was used as mobile phase A, and 0.1% formic acid methanol solution was used as mobile phase B. Linear gradient elution was performed according to the table below, and the UV detection wavelength was 260 nm.

[0113] Table 5 Gradient elution program

[0114]

[0115] The mass spectrometry conditions are as follows:

[0116] Table 6 Mass Spectrometry Conditions

[0117]

[0118] 1-4. Experimental Procedures and Results

[0119] Accurately measure 40 μL each of the above solutions (diluent, reference solution, and test solution) and inject them separately into a liquid chromatography-tandem triple quadrupole mass spectrometer. Record the results as follows: .

[0120] Table 7. Results of specificity study of genotoxic impurities in voriconazole active pharmaceutical ingredient.

[0121]

[0122] The above samples were separated using liquid chromatography-tandem triple quadrupole mass spectrometry. The experimental results show that:

[0123] When the blank solvent was tested, no characteristic peaks were observed in the spectrum under the detection channels for specific ion pairs of each impurity, indicating that the blank solvent did not interfere with the detection of impurities in the test solution. Figures 1-5 ).

[0124] The total ion current spectrum of the reference solution ( Figure 6 As can be seen, impurities A, B, C, D and E eluted at 19.39 min, 28.26 min, 20.18 min, 6.83 min and 9.14 min, respectively.

[0125] The above experimental results show that the detection method provided in this application exhibits good specificity.

[0126] Example 2 System Suitability Experiment

[0127] Preparation of reference solution: Prepared according to the method in Example 1.

[0128] Using the detection conditions of Example 1, 40 μL of the reference solution was precisely measured and injected six times consecutively. The results were recorded and are shown in the table below. As shown in Figure 8.

[0129] Table 8 Results of System Applicability Assessment

[0130]

[0131] The reference solution was injected six times consecutively, and the RSD of the peak area of ​​each impurity was less than 10.0%, indicating good injection precision. The recovery rates of the working control and the test control were in the range of 90% to 110%, which met the requirements, and the system suitability met the requirements.

[0132] Example 3: Linearity and Range Experiment

[0133] 3-1. Solution Preparation

[0134] Diluent: Measure 500 mL of acetonitrile, dilute with water to 1000 mL, add 1 mL of formic acid, and mix well.

[0135] Linear stock solution: Weigh approximately 3.0 mg each of impurity A, impurity B, impurity C, impurity D and impurity E reference standards, place them in different 5 mL volumetric flasks, dissolve and dilute to the mark (5 mL) with acetonitrile, and shake well to obtain reference standard stock solution 1; accurately measure 0.3 mL of each of reference standard stock solution 1, place them in the same 100 mL volumetric flask, dilute to the mark (100 mL) with diluent, and shake well to obtain reference standard stock solution 2, which is used as the linear stock solution.

[0136] Linear solutions: Measure the linear stock solution according to the table below, add diluent to prepare solutions of the corresponding concentrations, and prepare linear solutions of L1-L6.

[0137] Table 9 Preparation of linear solutions

[0138]

[0139] 3-2. Experimental Procedures and Results

[0140] The above solutions (L1-L6) were tested, and the results are shown in Tables 10-14.

[0141] Table 10 Linearity results for impurity A

[0142]

[0143] Table 11 Linearity results for impurity B

[0144]

[0145] Table 12 Linearity results for impurity C

[0146]

[0147] Table 13 Linearity results for impurity D

[0148]

[0149] Table 14 Linearity Results for Impurity E

[0150]

[0151] in conclusion:

[0152] Impurity A showed a linear relationship between peak area and concentration within the concentration range of 18.930 ng / mL to 378.592 ng / mL, with the linear equation y = 924.3963 x - 2818.0379 and a correlation coefficient r of 0.997, which is greater than 0.990, indicating good linearity. The percentage of the absolute value of the y-intercept to the peak area of ​​the 100% solution was 1.7%, which is less than 25%, meeting the requirements and indicating a good linear relationship. The sum of squared residuals was 217017501.

[0153] Impurity B showed a linear relationship between peak area and concentration within the concentration range of 36.072 ng / mL to 360.723 ng / mL, with the linear equation y = 239.9917 x - 381.6307 and a correlation coefficient r of 0.998, which is greater than 0.990, indicating good linearity. The percentage of the absolute value of the y-intercept to the peak area of ​​the 100% solution was 0.89%, less than 25%, meeting the requirements and indicating a good linear relationship. The sum of squared residuals was 6,556,750.

[0154] Impurity C showed a linear relationship between peak area and concentration within the range of 9.341 ng / mL to 373.643 ng / mL, with the linear equation y = 2651.5797 x + 2865.1102 and a correlation coefficient r of 0.9992, which is greater than 0.990, indicating good linearity. The percentage of the absolute value of the y-intercept to the peak area of ​​the 100% solution was 0.57%, less than 25%, which meets the requirements and indicates a good linear relationship. The sum of squared residuals was 572525042.

[0155] Impurity D showed a linear relationship between peak area and concentration within the range of 0.345 ng / mL to 345.048 ng / mL, with the linear equation y = 616020.1029x + 9418936.7235 and a correlation coefficient r of 0.996, which is greater than 0.990, indicating good linearity. The percentage of the absolute value of the y-intercept to the peak area of ​​the 100% solution was 7.9%, less than 25%, which meets the requirements and indicates a good linear relationship. The sum of squared residuals was 157702156282518.

[0156] Impurity E showed a linear relationship between peak area and concentration within the concentration range of 0.351 ng / mL to 351.480 ng / mL. The linear equation was y = 830221.4038x + 13508102.1952, with a correlation coefficient r of 0.996, which is greater than 0.990, indicating good linearity. The percentage of the absolute value of the y-intercept to the peak area of ​​the 100% solution was 8.2%, which is less than 25%, meeting the requirements and indicating a good linear relationship. The sum of squared residuals was 324100094509334.

[0157] Example 4: Limit of Quantitation and Limit of Detection Experiment

[0158] 4-1. Preparation of Limit of Quantitation Solutions

[0159] Impurity A Quantitation Limit Solution: Accurately measure 0.1 mL of the linear stock solution and place it in a 10 mL volumetric flask. Dilute to the mark (10 mL) with diluent and mix well. Prepare 6 parallel aliquots (i.e., prepared under the same conditions and methods).

[0160] Impurity B Quantitation Limit Solution: Accurately measure 0.2 mL of the linear stock solution and place it in a 10 mL volumetric flask. Dilute to the mark (10 mL) with diluent and mix well. Prepare 6 parallel aliquots.

[0161] Impurity C Quantitation Limit Solution: Accurately measure 50 μL of the linear stock solution and place it in a 10 mL volumetric flask. Dilute to the mark (10 mL) with diluent and mix well. Prepare 6 parallel aliquots.

[0162] Impurity D and Impurity E Quantitation Limit Solution: Accurately measure 2 μL of the linear stock solution and place it in a 10 mL volumetric flask. Dilute to the mark (10 mL) with diluent and mix well. Prepare 6 parallel aliquots.

[0163] 4-2. Preparation of Detection Limit Solution

[0164] Accurately measure 2.5 mL of the above-mentioned limit of quantitation solution and place it in a 5 mL volumetric flask. Dilute to the mark (5 mL) with diluent and mix well. Prepare three parallel aliquots.

[0165] 4-3. Experimental Procedures and Results

[0166] Accurately measure 40 μL of each of the above solutions and perform detection. The chromatographic conditions are the same as in Example 1. The results are shown in [Figure 1]. Tables 15 and 16. See Table 15 for the limit of quantitation mass spectra. Figures 12-16 .

[0167] Table 15 Results of Limit of Detection and Limit of Quantification - 1

[0168]

[0169] Table 16 Results of Limit of Detection and Limit of Quantification - 2

[0170]

[0171] Using the above detection conditions, the limits of quantitation and detection of various genotoxic impurities in voriconazole raw material were determined, and the results are as follows:

[0172] The limit of quantitation for impurity A is 18.930 ng / mL, equivalent to 10% of the limit concentration, with an S / N value of 15.6–19.3 and a peak area RSD of 6 needles, which is less than 15.0%. The limit of detection is 9.465 ng / mL, equivalent to 5% of the limit concentration, with an S / N value of 7.7–9.7, which meets the requirements.

[0173] The limit of quantitation for impurity B is 36.072 ng / mL, equivalent to 20% of the limit concentration, with an S / N value of 17.2–20.0 and a peak area RSD of 7.9% for the 6-needle quantitation limit, which is less than 15.0%. The limit of detection is 18.036 ng / mL, equivalent to 10% of the limit concentration, with an S / N value of 7.7–8.5, which meets the requirements.

[0174] The limit of quantitation for impurity C is 9.341 ng / mL, equivalent to 5% of the limit concentration, with an S / N value of 14.1–19.0 ​​and a peak area RSD of 6 needles, which is less than 15.0%. The limit of detection is 4.671 ng / mL, equivalent to 2.5% of the limit concentration, with an S / N value of 8.4–9.7, which meets the requirements.

[0175] The limit of quantitation for impurity D is 0.345 ng / mL, which is equivalent to 0.2% of the limit concentration, with an S / N value of 11.6~16.6. The RSD value of the peak area of ​​the quantitation limit for the 6 needles is 1.5%, which is less than 15.0%. The limit of detection is 0.173 ng / mL, which is equivalent to 0.1% of the limit concentration, with an S / N value of 4.4~5.1, which meets the requirements.

[0176] The limit of quantitation for impurity E is 0.351 ng / mL, equivalent to 0.2% of the limit concentration, with an S / N value of 13.3–19.7. The RSD value of the peak area of ​​the quantitation limit for 6 needles is 1.4%, which is less than 15.0%. The limit of detection is 0.176 ng / mL, equivalent to 0.1% of the limit concentration, with an S / N value of 4.4–8.0, which meets the requirements.

[0177] Experimental results show that this method can meet the sensitive detection requirements of various genotoxic impurities in voriconazole raw material, and the method has good sensitivity.

[0178] Example 5 Recovery rate experiment

[0179] 5-1. Solution Preparation

[0180] Reference stock solution: Accurately weigh approximately 3.0 mg each of impurity A, impurity B, impurity C, impurity D, and impurity E, and place them in separate 5 mL volumetric flasks. Dissolve and dilute each flask to the mark (5 mL) with acetonitrile, and mix well. This is reference stock solution 1. Accurately measure 0.3 mL of each of reference stock solution 1, place them in the same 100 mL volumetric flask, and dilute to the mark (100 mL) with diluent. Mix well. This is reference stock solution 2. Prepare two parallel solutions.

[0181] Reference solution: Accurately measure 1 mL of reference stock solution 2 and place it in a 10 mL volumetric flask. Dilute to the mark (10 mL) with diluent and mix well. Prepare two parallel portions.

[0182] Test solution: Accurately weigh approximately 1.2 g of voriconazole, place it in a 10 mL volumetric flask, add diluent, sonicate to dissolve and dilute to the mark (10 mL) on the volumetric flask, shake well, and this is the test solution. Prepare two parallel solutions.

[0183] Accuracy solution:

[0184] Preparation of R1-30% solution

[0185] Accurately weigh approximately 1.2 g of voriconazole and place it in a 10 mL volumetric flask. Add an appropriate amount of diluent and sonicate to dissolve. Then accurately add 0.3 mL of the reference stock solution and dilute to the mark (10 mL) with diluent. Shake well. Prepare three parallel batches.

[0186] Preparation of R2-100% solution

[0187] Accurately weigh approximately 1.2 g of voriconazole and place it in a 10 mL volumetric flask. Add an appropriate amount of diluent and sonicate to dissolve. Then, accurately add 2.1 mL of the reference stock solution and dilute to the mark (10 mL) on the volumetric flask with diluent. Shake well. Prepare three parallel batches.

[0188] Preparation of R3-150% solution

[0189] Accurately weigh approximately 1.2 g of voriconazole and place it in a 10 mL volumetric flask. Add an appropriate amount of diluent and sonicate to dissolve. Then, accurately add 1.5 mL of the reference stock solution and dilute to the mark (10 mL) on the volumetric flask with diluent. Shake well. Prepare three parallel batches.

[0190] 5-2. Experimental Procedures and Results

[0191] Accurately measure 40 μL of each of the above solutions and perform analysis. The chromatographic conditions are the same as in Example 1. The results are shown in the table below. The formula for calculating the relative standard deviation is as follows:

[0192]

[0193] Table 17 Accuracy Results for Impurity A

[0194]

[0195] Table 18 Accuracy Results for Impurity B

[0196]

[0197] Table 19 Accuracy Results for Impurity C

[0198]

[0199] Table 20 Accuracy Results for Impurity D

[0200]

[0201] Table 21 Accuracy Results for Impurity E

[0202]

[0203] By testing 30%, 100%, and 150% accuracy solutions (equivalent to limit concentrations), the recoveries of the samples were between 75% and 88%, 77% and 94%, 75% and 85%, 84% and 100%, and 99% and 113%, respectively, all within the range of 75% to 120%. The RSDs of the recoveries of the nine samples were 6.7%, 7.9%, 4.7%, 6.9%, and 5.3%, respectively, all less than 15.0%, indicating good accuracy.

[0204] Example 6 Precision Experiment

[0205] The method repeatability is verified and obtained by repeatedly testing the same batch of samples to examine the changes in the impurity content.

[0206] 6-1. Repeatability

[0207] Six spiking solutions of the test sample were prepared in parallel according to the preparation method of the 100% recovery solution in Example 5. The control solution was prepared according to the test method described in Example 1. The content of each genotoxic impurity was determined by the external standard method.

[0208] 6-2. Experimental Procedures and Results

[0209] Different researchers conducted the experiment repeatedly on different dates, using different instruments, to examine the intermediate precision. The results are shown below. twenty two.

[0210] Table 22 Precision Results

[0211]

[0212] The RSD values ​​of impurity content in the 6 repeatability samples were 2.6%, 3.2%, 1.5%, 1.6%, and 1.3%, respectively. The RSD values ​​of impurity content in the 6 intermediate precision samples were 4.7%, 4.0%, 2.2%, 2.5%, and 1.9%, respectively, all less than 10.0%. The RSD values ​​of impurity content in the 12 precision samples were 8.2%, 3.5%, 3.3%, 2.9%, and 2.6%, respectively, all less than 15.0%, indicating good precision.

[0213] Example 7 Durability Test

[0214] The system's applicability and tolerance to changes in detection results were examined by appropriately modifying method parameters. The robustness of this product was investigated by varying the column flow rate (±0.1 mL / min), column temperature (±5°C), and formic acid concentration in the mobile phase (±0.05%), based on the detection conditions of Example 1. The effects of these variations on the detection results of various impurities were analyzed. The preparation of the spiked solution and other chromatographic conditions were the same as in Example 1; the changes in robustness parameters and the results are shown below. twenty three, 24 and 25.

[0215] Table 23 Durability Parameter Variation Table

[0216]

[0217] Table 24 Durability-System Suitability Results

[0218]

[0219] Table 25 Durability Test Results

[0220]

[0221] The durability test results showed that, based on Example 1, with adjusted parameters and under normal conditions, the maximum RSD values ​​of the peak areas of impurities A, B, C, D, and E in the reference solution were 4.2%, 5.7%, 3.5%, 2.3%, and 1.6%, respectively, all not exceeding 10.0%. The recoveries of the two reference standards were 98.8%~106.2%, 97.4%~107.1%, 102.0%~109.8%, 98.3%~10.7%, and 96.0%~99.1%, respectively, all within the range of 90%~110%, which met the requirements.

[0222] Under adjusted parameters, the maximum RSD values ​​of impurity A, impurity B, impurity C, impurity D and impurity E compared with the results under normal conditions were 13.9%, 13.5%, 13.8%, 14.6% and 10.3%, respectively, all less than 15.0%, indicating good durability.

[0223] Comparative Example 1

[0224] Comparative Example 1 used LC-MS to detect genotoxic impurities in voriconazole raw material. The difference between Comparative Example 1 and Example 1 is that the mobile phase B organic phase system used in Comparative Example 1 is acetonitrile, the elution method is slightly different from that in Example 1, and other conditions are the same as in Example 1.

[0225] The chromatographic conditions of Example 1 and Comparative Example 1 are compared as shown below.

[0226] Table 26 Comparison of differences between the method in Example 1 and the method in Comparative Example 1

[0227]

[0228] The impurity reference solution of Example 1 of this application was investigated using the method in Comparative Example 1 (Table 26). The selected ion chromatograms (i.e., extracted ion chromatograms of mass spectrometry) of Example 1 and Comparative Example 1 are shown below. Figures 17-21 As shown in the figure, under the detection conditions of Comparative Example 1, the response values ​​of impurities A, B, and C are low, with impurities A and B almost matching the baseline noise. This indicates that using acetonitrile as the mobile phase has a significant impact on the ionization of these impurities. The method in Example 1 can significantly improve the response of the above impurities and can specifically detect five genotoxic impurities.

[0229] Comparative Example 2

[0230] Comparative Example 2 was analyzed using the method described in patent CN110308212A. The difference between Comparative Example 2 and Example 1 lies in the different high-performance liquid chromatography (HPLC) conditions, as detailed in Table 27. Solution L4 from "Example 3" was used as the limit concentration reference, and 20 μL was injected. All other conditions were the same as in Example 1.

[0231] Table 27 Detection methods for Comparative Example 2

[0232]

[0233] The results showed that the response values ​​of each impurity were low and could not meet the detection sensitivity requirements under the limit requirements (the minimum quantitative detection concentration is about 0.03%). In addition, impurity A and impurity C overlapped. Using Comparative Example 2 to detect the impurities in this application patent, the specificity could not meet the requirements.

[0234] The method described in this application is characterized by high specificity, high sensitivity (limit of quantification as low as 0.345 ng / mL, limit of detection as low as 0.173 ng / mL), high accuracy, good precision, and good robustness. It can accurately quantify five genotoxic impurities in voriconazole raw material.

[0235] This application illustrates the method of this application through the above embodiments, but this application is not limited to the above process steps, that is, it does not mean that this application must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to this application, the addition of reagents used in this application, and the adjustment of parameters, etc., all fall within the protection scope and disclosure scope of this application.

Claims

1. A method for detecting genotoxic impurities in voriconazole raw material, characterized in that, The method includes: detecting genotoxic impurities in voriconazole raw material using high performance liquid chromatography-tandem mass spectrometry to obtain the content of the genotoxic impurities; wherein the high performance liquid chromatography uses formic acid aqueous solution as mobile phase A and formic acid methanol solution as mobile phase B; The genotoxic impurities include any one or a combination of at least two of the following compounds: Impurity A Impurity B Impurity C Impurity D Impurity E.

2. The method according to claim 1, characterized in that, The method specifically includes the following steps: (1) Mix and dissolve voriconazole raw material and diluent one to obtain the test solution; mix and dissolve genotoxic impurity and diluent two to obtain the control solution; (2) The test solution and the control solution were detected by high performance liquid chromatography-tandem mass spectrometry, and the content of the genotoxic impurities was calculated by the external standard method. Preferably, the first diluent and the second diluent each independently comprise any one or a combination of at least two of acetonitrile, water, or formic acid, and more preferably a combination of acetonitrile, water, and formic acid; Preferably, the volume ratio of acetonitrile, water and formic acid is 500:500:

1.

3. The method according to claim 1 or 2, characterized in that, The ultraviolet detection wavelength of the high-performance liquid chromatography is 260 nm.

4. The method according to any one of claims 1-3, characterized in that, The injection volume for the high-performance liquid chromatography is 30~50μL.

5. The method according to any one of claims 1-4, characterized in that, The high-performance liquid chromatography column is packed with octadecylsilane-bonded silica gel. Preferably, the chromatographic column has an inner diameter of 4.6 mm, a length of 150 mm, and a particle size of 3.5 µm.

6. The method according to any one of claims 1-5, characterized in that, The mobile phase flow rate of the high-performance liquid chromatography is 0.40 mL / min to 0.60 mL / min, preferably 0.48 mL / min to 0.52 mL / min.

7. The method according to any one of claims 1-6, characterized in that, The column temperature of the high-performance liquid chromatography is 20~30℃, preferably 24~26℃.

8. The method according to any one of claims 1-7, characterized in that, The volume percentage of formic acid in the mobile phases A and B of the high-performance liquid chromatography is independently 0.05% to 0.15%, preferably 0.09% to 0.11%.

9. The method according to any one of claims 1-8, characterized in that, The high-performance liquid chromatography employs a gradient elution method. Preferably, the gradient elution conditions are as follows: From 0 to 13 min, the volume percentage of mobile phase A was 40% and that of mobile phase B was 60%. Then, the volume percentage changed uniformly to 19 min, where the volume percentage of mobile phase A was 29% and that of mobile phase B was 71%. Then, the volume percentage changed uniformly to 21 min, where the volume percentage of mobile phase A was 20% and that of mobile phase B was 80%, and remained unchanged until 31 min. Then, the volume percentage changed uniformly to 32 min, where the volume percentage of mobile phase A was 10% and that of mobile phase B was 90%, and remained unchanged until 37 min. Then, the volume percentage changed uniformly to 38 min, where the volume percentage of mobile phase A was 40% and that of mobile phase B was 60%, and remained unchanged until 43 min.

10. The method according to any one of claims 1-9, characterized in that, The mass spectrometer uses a triple quadrupole mass spectrometer detector and an electrospray ion source, with detection in positive ion mode. Preferably, in the mass spectrometry detection, the ion pairs m / z of impurities A, B, C, D, and E are 240.5 / 159.1, 319.2 / 240.0, 285.0 / 205.0, 247.2 / 107.2, and 261.2 / 107.2, respectively. Preferably, in the mass spectrometry detection, the declustering voltages for impurities A, B, C, D, and E are 86.86V, 80.52V, 86.15V, 100.99V, and 108.44V, respectively. Preferably, in the mass spectrometry detection, the collision energies of impurities A, B, C, D, and E are 37.19V, 26.40V, 38.56V, 23.36V, and 26.66V, respectively.

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