Method for detecting genotoxic impurities in doxofylline

Abstract

The present application provides a method for detecting genotoxic impurities in doxofylline, which effectively detects the genotoxic impurities in doxofylline by using gas chromatography-mass spectrometry. The detection method has good specificity, accuracy, durability and other system performance, and the detection limit is 3.75ppm. Moreover, the peak area and concentration of the genotoxic impurities within a specific concentration range show a good linear relationship.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, and in particular relates to a method for detecting genotoxic impurities in doxophylline. Background Technology

[0002] Genotoxic impurities are substances that can directly or indirectly damage DNA, leading to gene mutations or exhibiting carcinogenic potential. They are characterized by their ability to damage human genetic material even at very low concentrations, possessing mutagenic and carcinogenic properties, and posing a serious threat to human health during drug use.

[0003] The ICH M7 standard, "Assessing and Controlling DNA Reactive (Mutogenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risks," states that if a mutagenic substance (genotoxic impurity) is an impurity in a commercially available chemical, a synthetic intermediate, or a synthetic byproduct, the applicant should use risk-based reasoning to determine at which steps such a potential impurity should be assessed for hazard. If steps in the synthesis process that require assessment of the mutagenicity of these impurities and byproducts are identified, a risk assessment discussion is required.

[0004] Doxofylline, chemically named 7-[(1,3-dioxolane-2-yl)methyl]-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione, has the molecular formula C2. 11 H 14 N4O4, relative molecular mass: 266.26. Doxophylline is an oxycyclopentylmethyl derivative of theophylline, which has a significant inhibitory effect on phosphodiesterase. It can act directly on the bronchi, relaxing smooth muscle. Its effect is stronger than that of aminophylline, and it also has an antitussive effect with a long duration of action and no dependence. It has no central nervous system or gastrointestinal adverse reactions and does not affect cardiac function. It is used for bronchial asthma and other lung diseases accompanied by bronchospasm.

[0005] There are two types of mutagenic impurities that may exist in the process of producing doxofylline. The first type is toluene esters, such as methyl p-toluenesulfonate and ethyl p-toluenesulfonate. The second type includes haloalkane structures, namely dimethyl bromoacetaldehyde and its derivative ethylene glycol bromoacetaldehyde. Among them, the literature (Metabolic inactivation of mutagens in Drosophilamelanogaster. Zijlstra JA, Vogel EW; Mutation Research 198 (1): 73-83 (1988)) reported evidence of mutagenicity of methyl p-toluenesulfonate; the literature (MUTAGENICITY: MUTATION RESEARCH 87: 211(1981). DNA REPAIR-DEFICIENT BACTERIAL TESTS: POSITIVE.) reported evidence of mutagenicity of ethyl p-toluenesulfonate; the European Chemicals Agency (ECHA) provided evidence of mutagenicity of dimethyl bromoacetaldehyde and ethylene glycol bromoacetaldehyde. Controlling the content of these highly toxic impurities in the active pharmaceutical ingredient (API) at the source reduces the burden on subsequent processes to remove these impurities. A literature review has not found any reports of relevant detection methods, either domestically or internationally. Table 1 lists the mutagenic impurities that may be present in doxophylline.

[0006] Table 1. Potential mutagenic impurities in the theophylline process.

[0007]

[0008] Table 1 shows the possible pathways for the formation of the four types of impurities:

[0009] 1) Introduction of p-toluenesulfonate:

[0010] p-Toluenesulfonic acid, used as a catalyst in the second step of the reaction, may come into direct contact with ethanol, the refining solvent in both the second and third steps, to produce ethyl p-toluenesulfonate. Since methanol may be present in ethanol, there is also a risk of introducing methyl p-toluenesulfonate, as detailed below:

[0011]

[0012] 2) Introduction of haloalkanes:

[0013] Bromoacetaldehyde dimethyl acetal, as a starting material, may remain in subsequent steps; after acetal exchange, it reacts with ethylene glycol to potentially produce bromoacetaldehyde ethylene glycol acetal, as shown in the following pathway:

[0014] Summary of the Invention

[0015] The purpose of this invention is to provide a method for detecting genotoxic impurities in doxophylline with good system performance in terms of specificity, accuracy, and robustness.

[0016] To achieve the above technical objectives, the technical solution adopted in this application is as follows:

[0017] A method for detecting genotoxic impurities in doxophylline, wherein the genotoxic impurities are at least one of the following: dimethyl bromoacetaldehyde, ethylene glycol bromoacetaldehyde, methyl p-toluenesulfonate, and ethyl p-toluenesulfonate; the detection method employs gas chromatography-mass spectrometry; the gas chromatography detection conditions include: an initial temperature of 50-70℃, maintained for 1-3 min, increasing the temperature to 135-145℃ at a rate of 8-10℃ / min, maintaining for 1-3 min, and then increasing the temperature to 250-270℃ at a rate of 10-20℃ / min, maintaining for 8-12 min;

[0018] The mass spectrometry detection conditions include: using an EI ion source and performing quantitative analysis in SIM detection mode, wherein the quantitative ion m / z for bromoacetaldehyde dimethyl acetal is 75, the quantitative ion m / z for bromoacetaldehyde ethylene glycol acetal is 73, the quantitative ion m / z for methyl p-toluenesulfonate is 186, and the quantitative ion m / z for ethyl p-toluenesulfonate is 91.

[0019] Preferably, in the gas chromatography detection conditions, the initial temperature is 55~65℃, maintained for 2~3 min, then increased to 135~145℃ at a rate of 8~10℃ / min, maintained for 2~3 min, and then increased to 255~265℃ at a rate of 10~20℃ / min, maintained for 8~12 min.

[0020] Preferably, in the gas chromatography detection conditions, the initial temperature is 60±2℃, maintained for 2 min, then increased to 140±2℃ at a rate of 10℃ / min, maintained for 2 min, and then increased to 260±2℃ at a rate of 20℃ / min, maintained for 10 min.

[0021] Preferably, the detection conditions of the gas chromatography further include: an injection port temperature of 180~220℃; helium as the carrier gas; a column flow rate of 1.0 ml / min ± 0.1 ml / min; a split ratio of 10:1; and an injection volume of 1~2 μL.

[0022] Preferably, the detection conditions of the gas chromatography further include: an injection port temperature of 200±2℃; a carrier gas of helium; a column flow rate of 1.0 ml / min; a split ratio of 10:1; and an injection volume of 1 μL.

[0023] Preferably, the detection conditions of the gas chromatography further include: using a capillary column with 5% phenyl-95% methyl polysiloxane as the stationary phase; preferably, the capillary column is SH-Rxi-5Sil MS, 0.25mm×30m, 0.25μm.

[0024] Preferably, the mass spectrometry conditions further include: a voltage of 70 eV, an ion source temperature of 230 °C, a gas chromatography-mass spectrometry interface temperature of 250 °C, and a solvent delay time of 2.5 min.

[0025] Preferably, the detection method specifically includes the following steps:

[0026] Step 1, Solution preparation: This includes impurity reference solution and test solution, wherein:

[0027] Impurity reference solution: Take appropriate amounts of bromoacetaldehyde dimethyl acetal, bromoacetaldehyde ethylene glycol acetal, methyl p-toluenesulfonate, and ethyl p-toluenesulfonate reference standards, accurately weigh them, and add chloroform to prepare a single solution, a pairwise mixed solution, a tri-triple mixed solution, or a total mixed solution containing 1 μg of each per ml.

[0028] Test solution: Weigh 500 mg of doxofylline accurately, place it in a volumetric flask, add 2 ml of chloroform accurately to dissolve it, shake for 5 min, filter it through a 0.22 μm filter, and take the filtrate as the test solution;

[0029] Step 2: The impurity reference solution and the test solution are detected by gas chromatography-mass spectrometry.

[0030] Step 3: Calculate the impurity content in theophylline based on the test results. The formula for calculating the impurity content is as follows:

[0031] ,

[0032] Among them, A 供 A represents the peak area of ​​the target component in the test sample. 对 C represents the peak area of ​​the target component in the reference standard. 对 The concentration of the reference standard is μg / ml; W 供 The sample weight is mg; V 供 The volume of the test sample diluted is in ml.

[0033] Preferably, the genotoxic impurities are: dimethyl bromide, ethylene glycol bromide, methyl p-toluenesulfonate, and ethyl p-toluenesulfonate.

[0034] The technical effects of this invention are as follows:

[0035] The detection method of the present invention has good specificity, accuracy, repeatability and robustness, with a detection limit of 3.75 ppm; the system suitability meets the requirements; and the peak area of ​​bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol acetal (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P) shows a good linear relationship with concentration within a specific concentration range. Attached Figure Description

[0036] Figure 1 The image shows the GCMS diagrams of four toxic impurities in doxophylline in Example 1 of this invention: sample, blank, and chromatogram.

[0037] Figure 2 This is a GCMS diagram of four toxic impurities in doxophylline in Example 1 of the present invention: sample-control.

[0038] Figure 3 This is a GCMS diagram of four toxic impurities in doxophylline in Example 1 of the present invention: sample-control.

[0039] Figure 4 The image shows the GCMS graph of four toxic impurities in doxophylline in Example 1 of this invention – sample – test sample 3532004001.

[0040] Figure 5 The image shows the GCMS graph of four toxic impurities in doxophylline in Example 1 of this invention – sample – test sample 3532004002.

[0041] Figure 6 The image shows the GCMS graph of four toxic impurities in doxophylline in Example 1 of this invention – sample – test sample 3532004003.

[0042] Figure 7 This is a linear relationship graph of KDS-bromoacetal dimethyl acetal in Example 2 of the present invention.

[0043] Figure 8 This is a linear relationship graph of KDS-bromoacetaldehyde ethylene glycol in Example 2 of the present invention.

[0044] Figure 9 This is a linear relationship graph of KDS-p-toluenesulfonate in Example 2 of the present invention.

[0045] Figure 10 This is a linear relationship graph of KDS-ethyl p-toluenesulfonate in Example 2 of the present invention. Detailed Implementation

[0046] The experimental instruments, apparatus, and reagents used in this embodiment of the invention are shown in Tables 2-4, respectively:

[0047] Table 2 Experimental Instruments

[0048]

[0049] Table 3 Experimental Apparatus

[0050]

[0051] Table 4 Experimental reagents and reagents

[0052]

[0053] Furthermore, it should be noted in the description of this invention that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0054] In a specific embodiment of the present invention, a method for detecting genotoxic impurities in doxophylline is provided. The method includes: simultaneously detecting four impurities in the doxophylline using gas chromatography-mass spectrometry (GC-MS).

[0055] 1) The structural formula of bromoacetaldehyde dimethyl acetal (KDS-SM-B) is:

[0056] ;

[0057] 2) The structural formula of bromoacetaldehyde ethylene glycol condensate (KDS-IM-O) is:

[0058] ;

[0059] 3) The structural formula of methyl p-toluenesulfonate (KDS-IM-Q) is:

[0060] ;

[0061] 4) The structural formula of ethyl p-toluenesulfonate (KDS-IM-P) is:

[0062] .

[0063] This invention can also detect single impurities, mixtures of two or three impurities, and is not limited to detecting four impurities simultaneously.

[0064] The detection conditions are as follows: Chromatographic conditions include:

[0065] Chromatographic conditions: A capillary column (SH-Rxi-5Sil MS 0.25mm×30m, 0.25μm) with 5% phenyl-95% methylpolysiloxane as stationary phase was used. The initial temperature was 60℃, held for 2 minutes, then increased to 140℃ at a rate of 10℃ / min, held for 2 minutes, and then increased to 260℃ at a rate of 20℃ / min, held for 10 minutes. The injection port temperature was 200℃; helium was used as the carrier gas; the column flow rate was 1.0 ml / min; the split ratio was 10:1; and the injection volume was 1 μl.

[0066] Mass spectrometry conditions include:

[0067] The mass spectrometry conditions used were as follows: EI ion source, voltage 70 eV, ion source temperature 230℃; gas chromatography-mass spectrometry interface temperature 250℃; solvent delay time 2.5 min; detection mode SIM; quantitative ion m / z values ​​for bromoacetaldehyde dimethyl acetal 75, bromoacetaldehyde ethylene glycol acetal 73, methyl p-toluenesulfonate 186, and ethyl p-toluenesulfonate 91.

[0068] For system suitability testing, accurately weigh appropriate amounts of reference standards for bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol acetal (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P), and dissolve them in chloroform to prepare a mixed solution containing approximately 1 μg of each standard per 1 ml. This solution serves as the system suitability test solution. Accurately measure an appropriate amount of the system suitability test solution and quantitatively dilute it with chloroform to prepare a solution containing approximately 0.2 μg each of bromoacetaldehyde dimethyl acetal, bromoacetaldehyde ethylene glycol acetal, methyl p-toluenesulfonate, and ethyl p-toluenesulfonate per 1 ml. Shake well to obtain the sensitivity test solution. Accurately inject 1 μl each of the system suitability test solution and the sensitivity test solution into the chromatograph and record the chromatograms. In the system suitability solution chromatogram, the elution order is as follows: dimethyl bromoacetaldehyde, ethylene glycol bromoacetaldehyde, methyl p-toluenesulfonate, and ethyl p-toluenesulfonate. The resolution between each peak should meet the requirements. In the sensitivity solution chromatogram, the signal-to-noise ratio of the peak height of each main peak should be greater than 10. When the reference solution is injected continuously for 6 times, the relative standard deviation of the peak area of ​​each peak should not be greater than 10%.

[0069] For the assay, accurately weigh an appropriate amount of this product, add chloroform to prepare a solution containing approximately 100 mg per ml, and shake well to obtain the test solution. Accurately weigh appropriate amounts of bromoacetaldehyde dimethyl acetal, bromoacetaldehyde ethylene glycol acetal, methyl p-toluenesulfonate, and ethyl p-toluenesulfonate reference standards, add chloroform to prepare a solution containing approximately 0.4 μg of each per ml, and shake well to obtain the reference solutions. Accurately measure 1 μl of each of the test solution and reference solutions, and inject them separately into the gas chromatograph, recording the chromatograms. If the chromatogram of the test solution contains a peak with the same retention time as bromoacetaldehyde dimethyl acetal, bromoacetaldehyde ethylene glycol acetal, methyl p-toluenesulfonate, or methyl p-toluenesulfonate, calculate the concentration of bromoacetaldehyde dimethyl acetal, bromoacetaldehyde ethylene glycol acetal, methyl p-toluenesulfonate, and ethyl p-toluenesulfonate using the external standard method. The concentration of each of these substances in this product must not exceed 3.75 ppm.

[0070] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These descriptions are for illustrative purposes only and are not intended to limit the scope of the invention.

[0071] The process of exploring the conditions:

[0072] 1) Exploration of the starting temperature of gas chromatography

[0073] Generally, the initial temperature in gas chromatography detection does not significantly affect the sample detection results. However, the inventors found in practice that the separation of bromoacetaldehyde dimethyl acetal and bromoacetaldehyde ethylene glycol was poor at an initial temperature of 40℃. Therefore, a series of initial temperatures were set: 40℃, 50℃, 60℃, 70℃, and 80℃. The initial temperature was maintained for 3 minutes at an initial heating rate of 10℃ / min. The temperature was then increased to 150℃ and maintained for 3 minutes. Finally, the temperature was increased to 260℃ at a heating rate of 20℃ / min and maintained for 15 minutes. The injection port temperature was 200℃. Helium was used as the carrier gas. The column flow rate was 1.0 ml / min with a split ratio of 10:1. The injection volume was 1 μL. The capillary column was an SH-Rxi-5Sil MS, 0.25 mm × 30 m, 0.25 μm. The effects of different initial temperatures on the test results were investigated, and the results are shown in Table 5. It is best to control the initial temperature between 50 and 70℃.

[0074] 2) Heating rate

[0075] The initial temperature was set to 60℃, and the effect of different heating rates was investigated. The heating rates were 8℃ / min, 10℃ / min, 15℃ / min and 20℃ / min, and other conditions were the same as in 1). The results are shown in Table 5, which shows that the heating rate has no significant effect on the detection results. Therefore, a heating rate of 8~10℃ / min is acceptable.

[0076] 3) Duration of maintaining the final temperature (260℃)

[0077] The duration of the final temperature may have a significant impact on the degree of impurity separation. Therefore, the duration was set to 5 min, 8 min, 10 min, 12 min, and 15 min to examine its impact on the detection results. The results are shown in Table 5. It is best to control the duration to 8-12 min.

[0078] Table 5 Results of the investigation on initial temperature, heating rate, and holding time

[0079]

[0080] Example 1

[0081] This embodiment provides a method for detecting genotoxic impurities in doxophylline, as detailed below:

[0082] Sample solution preparation

[0083] <Blank Reagent>: Chloroform

[0084] <Reference Solution>: Take appropriate amounts of reference standards of bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol acetal (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P), accurately weigh them, and add chloroform to prepare a mixed solution containing 1 μg of each per 1 ml.

[0085] <Test Solution>: Accurately weigh approximately 500 mg of this product, place it in a volumetric flask, accurately add 2 ml of chloroform to dissolve, shake for 5 min, filter (using a 0.22 μm filter), and use the filtrate as the test solution. This product represents 3 batches of doxofylline raw materials from 4 different sources.

[0086] Parameters of chromatography and mass spectrometry

[0087] Chromatographic conditions include:

[0088] Chromatographic conditions: A capillary column (SH-Rxi-5Sil MS 0.25mm×30m, 0.25μm) with 5% phenyl-95% methylpolysiloxane as stationary phase was used. The initial temperature was 60℃, held for 2 minutes, then increased to 140℃ at a rate of 10℃ / min, held for 2 minutes, and then increased to 260℃ at a rate of 20℃ / min, held for 10 minutes. The injection port temperature was 200℃; helium was used as the carrier gas; the column flow rate was 1.0 ml / min; the split ratio was 10:1; and the injection volume was 1 μl.

[0089] Mass spectrometry conditions include:

[0090] The mass spectrometry conditions used were as follows: EI ion source, voltage 70 eV, ion source temperature 230℃; gas chromatography-mass spectrometry interface temperature 250℃; solvent delay time 2.5 min; detection mode SIM; quantitative ion m / z values ​​for bromoacetaldehyde dimethyl acetal 75, bromoacetaldehyde ethylene glycol acetal 73, methyl p-toluenesulfonate 186, and ethyl p-toluenesulfonate 91.

[0091] According to the method for detecting genotoxic impurities of doxophylline provided by the present invention, Table 6 below lists the peak order and determination parameters of the target compound.

[0092] Table 6. Elution order and measurement parameters of the target compounds

[0093]

[0094] The determination method is as follows:

[0095] Accurately measure 1 μl of each of the test solution and the reference solution, inject them into the chromatograph, and record the chromatogram.

[0096] Calculation formula: , where A 供 —Peak area of ​​the target component in the test sample; A 对 —Peak area of ​​the target component in the reference standard; C 对 —Reference concentration, μg / ml; W 供 —Sample weight of the test sample, mg; V 供 —Dilution volume of the test sample, ml;

[0097] Limits: If the chromatogram of the test solution contains a peak with the same retention time as the reference solution, the content of bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol acetal (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P) shall not exceed 3.75 μg / g (ppm) according to the peak area calculation method using the external standard method.

[0098] The measurement results are as follows:

[0099] <Methodological Validation Results>: As shown in Table 7.

[0100] Table 7. Summary of Methodological Validation and Results

[0101]

[0102]

[0103]

[0104] Figure 1 The detection chromatogram for the blank solvent shows no chromatographic peak signal for the blank reagent, indicating that the four toxic impurities—bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol acetal (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P)—were not detected.

[0105] Figure 2 and Figure 3 The peak areas, retention times, and peak heights of four basic toxic impurity reference standards (parallel control solutions) are shown. Figure 2 and Figure 3 The information from the quantitative results table is extracted as shown in Tables 8 and 9 below:

[0106] Table 8 Figure 2 Quantitative results

[0107]

[0108] Table 9 Figure 3 Quantitative results

[0109]

[0110] Figure 4 The chromatogram for batch 3532004001 of doxofylline raw material shows the peak signals of four colorless impurities in the sample, indicating that the four toxic impurities, namely bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol acetal (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P), were not detected.

[0111] Figure 5 The chromatogram for batch 3532004002 of doxofylline raw material shows the peak signals of four colorless impurities in the sample, indicating that the four toxic impurities, namely bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol acetal (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P), were not detected.

[0112] Figure 6 The chromatogram for batch 3532004003 of doxofylline raw material shows the peak signals of four colorless impurities in the sample, indicating that the four toxic impurities, namely bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol acetal (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P), were not detected.

[0113] In summary, neither the blank solvent nor the test solution interfered with the detection of the four basic toxic impurities, and none of the four basic toxic impurities were detected in the three batches (3532004001, 3532004002, and 3532004003) of the test solution.

[0114] The detection results of each impurity are shown in Table 10:

[0115] Table 10 Results of Genotoxic Impurity Detection

[0116]

[0117] Example 2

[0118] The specific methodological verification process is as follows:

[0119] 1) Specificity test

[0120] Sample solution preparation

[0121] <Blank Reagent>: Chloroform

[0122] <Reference Solution>: Take appropriate amounts of reference standards of bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol acetal (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P), accurately weigh them, and add chloroform to prepare a mixed solution containing 1 μg of each per 1 ml.

[0123] <Test Solution>: Weigh approximately 500 mg of this product accurately, place it in a volumetric flask, add 2 ml of chloroform accurately to dissolve it, shake for 5 min, filter (using a 0.22 μm filter), and take the filtrate as the test solution.

[0124] Accurately measure 1 μl each of blank solvent, reference solution and test solution, inject them into the chromatograph, record the chromatograms, and the results are shown in Table 11 below.

[0125] Table 11 Results of specificity tests for four basic toxic impurities of doxophylline

[0126]

[0127] Conclusion: The blank solvent does not interfere with the detection of dimethyl bromide (KDS-SM-B), ethylene glycol bromide (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P), indicating that the method has good specificity.

[0128] 2) Limit of Quantification

[0129] Reference solution: Take appropriate amounts of reference standards of bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol acetal (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P), accurately weigh them, and add chloroform to prepare a mixed solution containing about 1 μg of each in 1 ml.

[0130] Limit of quantitation solution: Take the reference solution and dilute it stepwise with chloroform to prepare a solution with an S / N ratio of approximately 10:1 for bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol acetal (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P). This solution is used as the limit of quantitation solution.

[0131] Accurately measure 1 μl of the limit of quantitation solution and inject it into the chromatograph. The results are shown in Table 12 below.

[0132] Table 12 Results of Quantification Limits for Four Toxic Impurities in Doxophylline

[0133]

[0134] Conclusion: With six consecutive injections of the limit of quantitation (LOQ) solution, the retention time RSD was less than 1.0%, the peak area RSD was less than 15%, and the signal-to-noise ratio (S / N) was ≥10. The LOQs (equivalent to the limits of the test samples) for four impurities—bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol acetal (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P)—were 0.79 μg / g, 0.85 μg / g, 0.80 μg / g, and 0.79 μg / g, respectively, with a limit of 3.75 μg / g for all. This indicates that under these conditions, LOQs can be accurately detected for bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol acetal (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P).

[0135] 3) Detection limit

[0136] Take the reference solution under the limit of quantitation test and dilute it stepwise with chloroform to prepare a solution with an S / N ratio of approximately 3:1 for bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol acetal (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P), which will be used as the limit of detection solution.

[0137] Accurately measure 1 μl of the detection limit solution and inject it into the chromatograph. The results are shown in Table 13 below.

[0138] Table 13 Results of the detection limits for four toxic impurities in doxophylline

[0139]

[0140] Note: Methyl p-toluenesulfonate (KDS-IM-Q) has the lowest signal-to-noise ratio, so methyl p-toluenesulfonate (KDS-IM-Q) is used as the standard.

[0141] Conclusion: The signal-to-noise ratio (S / N) of the detection limit solution is ≥3. The detection limits (equivalent to the limits of the test samples) for bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol acetal (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P) are 0.40 μg / g, 0.42 μg / g, 0.40 μg / g, and 0.40 μg / g, respectively, with a limit of 3.75 μg / g for all samples. This indicates that the chromatographic conditions have high detection sensitivity and meet the detection requirements for bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol acetal (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P).

[0142] 4) Linearity and Range

[0143] Bromoacetaldehyde dimethyl acetal (KDS-SM-B) stock solution: Weigh approximately 10 mg of bromoacetaldehyde dimethyl acetal (KDS-SM-B) reference standard accurately, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with chloroform, and shake well to obtain the solution.

[0144] Bromoacetaldehyde ethylene glycol condensate (KDS-IM-O) stock solution: Weigh approximately 10 mg of bromoacetaldehyde ethylene glycol condensate (KDS-IM-O) reference standard accurately, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with chloroform, and shake well to obtain the solution.

[0145] Methyl p-toluenesulfonate (KDS-IM-Q) stock solution: Weigh approximately 10 mg of methyl p-toluenesulfonate (KDS-IM-Q) reference standard accurately, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with chloroform, and shake well to obtain the solution.

[0146] Ethyl p-toluenesulfonate (KDS-IM-P) stock solution: Weigh approximately 10 mg of ethyl p-toluenesulfonate (KDS-IM-P) reference standard accurately, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with chloroform, and shake well to obtain the solution.

[0147] 10 μg / ml mixed standard solution: Accurately measure 200 μl each of the stock solutions of bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P), place them in a 20 ml volumetric flask, dilute to the mark with chloroform, and shake well to obtain the solution.

[0148] Linear solutions: Preparation methods are shown in Table 14 below.

[0149] Accurately measure 1 μl of each linear solution, inject it into the chromatograph, record the chromatogram, and the results are shown in Table 15 below.

[0150] Table 14 Preparation methods of linear solutions for four basic toxic impurities of doxophylline

[0151]

[0152] Table 15 Linearity results of four toxic impurities in doxophylline

[0153]

[0154]

[0155] Conclusion: Within the measured concentration range, regression equations were constructed with the concentrations of bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol acetal (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P) as the x-axis and the peak areas as the y-axis. The linear relationship graph is shown below. Figures 7-10 As shown.

[0156] The regression equation for bromoacetaldehyde diacetate (KDS-SM-B) is: y=26877x-2174.6, r=0.9984>0.99, which means that the concentration of bromoacetaldehyde diacetate (KDS-SM-B) and the peak area are linearly related in the range of 0.1984~1.9835μg / ml.

[0157] The regression equation for ethylene glycol bromoacetaldehyde (KDS-IM-O) is: y=21185x-3368.7, r=0.9971>0.99, which means that the concentration of ethylene glycol bromoacetaldehyde (KDS-IM-O) and the peak area are linearly related in the range of 0.2115~2.1148μg / ml.

[0158] The regression equation for methyl p-toluenesulfonate (KDS-IM-Q) is: y=19283x-4625.9, r=0.9911>0.99, which means that the concentration of methyl p-toluenesulfonate (KDS-IM-Q) and the peak area are linearly related in the range of 0.1993~1.9933μg / ml.

[0159] The regression equation for ethyl p-toluenesulfonate (KDS-IM-P) is: y=21527x-4454.8, r=0.9972>0.99, which means that the concentration of ethyl p-toluenesulfonate (KDS-IM-P) and the peak area are linearly related in the range of 0.1980~1.9796μg / ml.

[0160] 5) Repeatability

[0161] Blank solvent: chloroform.

[0162] 100% spiked solution: Accurately measure 2 ml of the 10 μg / ml mixed standard solution under the 2.5 linearity and range section, place it in a 20 ml volumetric flask, dilute to the mark with chloroform, and shake well.

[0163] Reference solution (prepare two parallel portions and take the average value for calculation): same as 100% spiked solution.

[0164] 100% spiked test solution (prepare 6 parallel portions): Weigh approximately 500 mg of this product accurately, place it in a volumetric flask, accurately measure 2 ml of 100% spiked solution to dissolve it, shake for 5 min, filter (using a 0.22 μm filter), and take the filtrate as the 100% spiked test solution.

[0165] Accurately measure 1 μl of each of the above solutions, inject them into the chromatograph, record the chromatograms, and the results are shown in Table 16 below.

[0166] Table 16 Repeatability results of four toxic impurities of doxophylline

[0167]

[0168] Conclusion: The RSDs of the contents of bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol acetal (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P) in the six spiked test solutions were all less than 15%, indicating good method repeatability.

[0169] 6) Accuracy

[0170] Blank solvent: chloroform.

[0171] Bromoacetaldehyde dimethyl acetal (KDS-SM-B) stock solution: Weigh approximately 10 mg of bromoacetaldehyde dimethyl acetal (KDS-SM-B) reference standard accurately, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with chloroform, and shake well to obtain the solution.

[0172] Bromoacetaldehyde ethylene glycol condensate (KDS-IM-O) stock solution: Weigh approximately 10 mg of bromoacetaldehyde ethylene glycol condensate (KDS-IM-O) reference standard accurately, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with chloroform, and shake well to obtain the solution.

[0173] Methyl p-toluenesulfonate (KDS-IM-Q) stock solution: Weigh approximately 10 mg of methyl p-toluenesulfonate (KDS-IM-Q) reference standard accurately, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with chloroform, and shake well to obtain the solution.

[0174] Ethyl p-toluenesulfonate (KDS-IM-P) stock solution: Weigh approximately 10 mg of ethyl p-toluenesulfonate (KDS-IM-P) reference standard accurately, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with chloroform, and shake well to obtain the solution.

[0175] 10 μg / ml mixed standard solution: Accurately measure 100 μl each of the stock solutions of bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P), place them in a 10 ml volumetric flask, dilute to the mark with chloroform, and shake well to obtain the solution.

[0176] 50% limit solution: Accurately measure 1 ml of 10 μg / ml mixed standard solution, place it in a 20 ml volumetric flask, dilute to the mark with chloroform, and shake well to obtain the solution.

[0177] 100% limit solution: Accurately measure 1 ml of 10 μg / ml mixed standard solution, place it in a 10 ml volumetric flask, dilute to the mark with chloroform, and shake well to obtain the solution.

[0178] 150% limit solution: Accurately measure 3 ml of 10 μg / ml mixed standard solution, place it in a 20 ml volumetric flask, dilute to the mark with chloroform, and shake well to obtain the solution.

[0179] Reference solution: Same as 100% limit solution.

[0180] Test solution: Weigh approximately 500 mg of this product accurately, place it in a volumetric flask, accurately measure 2 ml of chloroform to dissolve it, shake for 5 min, filter (using a 0.22 μm filter), and take the filtrate as the test solution.

[0181] 50% spiked test solution (prepare 3 parallel portions): Weigh approximately 500 mg of this product accurately, place it in a volumetric flask, accurately measure 2 ml of 50% limit solution to dissolve it, shake for 5 min, filter (using a 0.22 μm filter), and take the filtrate as the 50% spiked test solution.

[0182] 100% spiked test solution (prepare 3 parallel portions): Weigh approximately 500 mg of this product accurately, place it in a volumetric flask, accurately measure 2 ml of 100% limit solution to dissolve it, shake for 5 min, filter (using a 0.22 μm filter), and take the filtrate as the 100% spiked test solution.

[0183] 150% spiked test solution (prepare 3 parallel portions): Weigh approximately 500 mg of this product accurately, place it in a volumetric flask, accurately measure 2 ml of 150% limit solution to dissolve it, shake for 5 min, filter (using a 0.22 μm filter), and take the filtrate as the 150% spiked test solution.

[0184] Accurately measure 1 μl of each of the above solutions, inject them into the chromatograph, record the chromatograms, and the results are shown in Table 17 below.

[0185] Table 17. Accuracy Results of Four Basic Toxic Impurities in Doxophylline

[0186]

[0187]

[0188] Conclusion: The average recovery rate of bromoacetaldehyde dimethyl acetal (KDS-SM-B) was 84.68%, with an RSD (n=9) of 5.59%; the average recovery rate of bromoacetaldehyde ethylene glycol acetal (KDS-IM-O) was 109.5%, with an RSD (n=9) of 8.21%; the average recovery rate of methyl p-toluenesulfonate (KDS-IM-Q) was 85.37%, with an RSD (n=9) of 12.38%; and the average recovery rate of ethyl p-toluenesulfonate (KDS-IM-P) was 83.38%, with an RSD (n=9) of 9.57%. The recoveries were all between 70% and 120%, and the RSDs were all less than 15%, indicating that this method is accurate in determining the content of bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol acetal (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P).

[0189] 7) Solution stability

[0190] The 100% spiked test solution was left to stand at room temperature for 20 hours. At 0h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, and 20h, 1 μl of the solution was accurately injected into the instrument, and the chromatograms were recorded. The results are shown in Table 18 below.

[0191] Table 18. Injection results of solutions containing four toxic impurities of doxophylline.

[0192]

[0193]

[0194] Conclusion: After the spiked test solution was left at room temperature for 20 hours, the RSD values ​​of the contents of dimethyl bromoacetaldehyde (KDS-SM-B), ethylene glycol bromoacetaldehyde (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P) were all less than 15%, indicating that the test solution was stable after being left at room temperature for 20 hours.

[0195] The results above demonstrate that the detection method for genotoxic impurities of doxophylline provided by this invention exhibits good specificity, accuracy, repeatability, and robustness. Furthermore, the detection limit of this method is 3.75 ppm; the system suitability meets the requirements; the peak areas of bromoacetaldehyde dimethyl acetal (KDS-SM-B), bromoacetaldehyde ethylene glycol acetal (KDS-IM-O), methyl p-toluenesulfonate (KDS-IM-Q), and ethyl p-toluenesulfonate (KDS-IM-P) show a good linear relationship with concentration within a specific concentration range; regarding solution stability, 4-dimethylaminopyridine exhibits good stability within 20 hours.

[0196] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for detecting genotoxic impurities in doxophylline, characterized in that, The genotoxic impurities are: dimethyl bromide, ethylene glycol bromide, methyl p-toluenesulfonate, and ethyl p-toluenesulfonate; the detection method is gas chromatography-mass spectrometry (GC-MS); the GC detection conditions include: an initial temperature of 55-65℃, maintained for 2-3 min, increasing the temperature at a rate of 8-10℃ / min to 135-145℃, maintained for 2-3 min, and then increasing the temperature at a rate of 10-20℃ / min to 255-265℃, maintained for 8-12 min; and an injection port temperature of 180-220℃. The carrier gas was helium; the column flow rate was 1.0 ml / min ± 0.1 ml / min, and the split ratio was 10:1; the injection volume was 1~2 μL; a capillary column with 5% phenyl-95% methyl polysiloxane as the stationary phase was used; the detection conditions of the mass spectrometry included: using an EI ion source and performing quantitative analysis in SIM detection mode, wherein the quantitative ion m / z of bromoacetaldehyde dimethyl acetal was 75, the quantitative ion m / z of bromoacetaldehyde ethylene glycol acetal was 73, the quantitative ion m / z of methyl p-toluenesulfonate was 186, and the quantitative ion m / z of ethyl p-toluenesulfonate was 91.

2. The detection method according to claim 1, characterized in that, In the gas chromatography detection conditions, the initial temperature is 60±2℃, maintained for 2 min, then increased to 140±2℃ at a rate of 10℃ / min, maintained for 2 min, and then increased to 260±2℃ at a rate of 20℃ / min, maintained for 10 min.

3. The detection method according to claim 1 or 2, characterized in that, The gas chromatography detection conditions are as follows: injection port temperature is 200±2℃; carrier gas is helium; column flow rate is 1.0 ml / min; split ratio is 10:1; injection volume is 1 μL.

4. The detection method according to claim 1, characterized in that, The capillary column was an SH-Rxi-5Sil MS, 0.25mm × 30m, 0.25μm.

5. The detection method according to claim 1, characterized in that, The mass spectrometry conditions also include: a voltage of 70 eV, an ion source temperature of 230 °C, a gas chromatography-mass spectrometry interface temperature of 250 °C, and a solvent delay time of 2.5 min.

6. The detection method according to claim 1, characterized in that, Specifically, the steps include the following: Step 1, Solution preparation: This includes impurity reference solution and test solution, wherein: Impurity reference solution: Take appropriate amounts of bromoacetaldehyde dimethyl acetal, bromoacetaldehyde ethylene glycol acetal, methyl p-toluenesulfonate, and ethyl p-toluenesulfonate reference standards, accurately weigh them, and add chloroform to prepare a total mixed solution containing 1 μg of each per 1 ml. Test solution: Weigh 500 mg of doxofylline accurately, place it in a volumetric flask, add 2 ml of chloroform accurately to dissolve it, shake for 5 min, filter it through a 0.22 μm filter, and take the filtrate as the test solution; Step 2: The impurity reference solution and the test solution are detected by gas chromatography-mass spectrometry. Step 3: Calculate the impurity content in theophylline based on the test results. The formula for calculating the impurity content is as follows: ， Among them, A 供 A represents the peak area of ​​the target component in the test sample. 对 C represents the peak area of ​​the target component in the reference standard. 对 The concentration of the reference standard is μg / ml; W 供 The sample weight is mg; V 供 The volume of the test sample diluted is in ml.

Images