Method for detecting content of genotoxic impurity 6-chloro-2-hexanone in pentoxifylline bulk drug and injection

Abstract

The invention relates to the technical field of biological medicine detection, and particularly discloses a method for detecting the content of a genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw material medicine and injection. According to the detection method disclosed by the invention, a sample is detected by adopting gas chromatography-mass spectrometry (GC-MS); the gas chromatographic parameters are as follows: the temperature of a sample inlet is 190-210 DEG C; according to the heating procedure, the initial temperature ranges from 75 DEG C to 85 DEG C, the temperature is increased to 150 DEG C to 170 DEG C at the speed of 15 DEG C / min to 25 DEG C / min and kept for 1.5 min to 2.5 min, and the temperature is increased to 210 DEG C to 230 DEG C at the speed of 55 DEG C / min to 65 DEG C The mass spectrum parameters are as follows: the solvent delay is 3-4 minutes; the temperature of the transmission line is 240-260 DEG C; the temperature of a quadrupole rod is 140-160 DEG C; the temperature of the ion source is 240-260 DEG C; the method disclosed by the invention is simple to operate and high in detection speed, and has the advantages of high sensitivity, high precision and high repeatability.

Description

Technical Field

[0001] The present application relates to the technical field of biomedical detection, and in particular to a method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw materials and injections. Background Art

[0002] As a vasodilator, pentoxifylline has multiple effects: it can promote blood circulation and increase the permeability of capillaries, thereby improving microcirculation; it can prevent the formation of blood clots by inhibiting platelet aggregation; it can also reduce blood lipid levels by reducing cholesterol synthesis and regulating lipid metabolism, and has a certain auxiliary therapeutic effect on patients with hyperlipidemia; it has a certain ability to resist oxidative stress and can remove free radicals, which helps to delay the aging process of the body. The metabolites of pentoxifylline also have the effect of improving blood viscosity and improving microcirculation.

[0003] 6-chloro-2-hexanone is an important starting material in the synthesis of pentoxifylline. In the process of synthesizing pentoxifylline, 6-chloro-2-hexanone impurities are easily left, which may have adverse effects on the safety of pentoxifylline, seriously affecting the quality of pentoxifylline and the health of patients. Therefore, the detection method of 6-chloro-2-hexanone in pentoxifylline is currently a problem of concern.

[0004] At present, a method for detecting 6-chloro-2-hexanone in pentoxifylline using gas chromatography has been developed. Gas chromatography has some disadvantages: it has low sensitivity for some genotoxic impurities and may not be able to perform effective analysis, which reduces the sensitivity and accuracy of the detection method for 6-chloro-2-hexanone in pentoxifylline.

[0005] In order to control the impurities in 6-chloro-2-hexanone, ensure the quality of pentoxifylline synthesized using 6-chloro-2-hexanone as the starting material, and ensure the safety of medication for patients, developing a new detection method for 6-chloro-2-hexanone-related substances is a technical problem that needs to be solved urgently. Summary of the invention

[0006] In order to solve the above technical problems, the present application provides a method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw materials and injections.

[0007] The present application provides a method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw materials and injections, using gas chromatography-mass spectrometry GC-MS to detect the sample to be tested; The parameters of the gas chromatography are as follows: injection port temperature: 190-210°C; heating program: initial temperature 75-85°C, heating to 150-170°C at a heating rate of 15-25°C / min, maintaining for 1.5-2.5min, then heating to 210-230°C at a heating rate of 55-65°C / min, maintaining for 1.5-2.5min; The mass spectrometry parameters are as follows: acquisition type: MRM; ion source: EI; solvent delay: 3-4 min; transfer line temperature: 240-260° C.; quadrupole temperature: 140-160° C.; and ion source temperature: 240-260° C.

[0008] The method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in pentoxifylline API and injection adopted in the present application is gas chromatography-mass spectrometry (GC-MS), which uses gas chromatography as a separation system for 6-chloro-2-hexanone and other substances in the pentoxifylline API and injection samples to be tested, and mass spectrometry as a detection system for 6-chloro-2-hexanone. GC-MS combines the high separation ability of gas chromatography for the samples to be tested with the advantages of mass spectrometry, which has high selectivity, high sensitivity and can provide relative molecular mass and structural information.

[0009] Gas chromatography has the characteristic of high separation for the samples to be tested. By selecting the appropriate chromatographic column, starting column temperature, heating rate, column flow rate and other parameter conditions, the separation effect of 6-chloro-2-hexanone and other substances can be effectively improved. It can not only provide sufficient ions for mass spectrometry testing and analysis, but also preliminarily separate impurities that interfere with the mass spectrum, avoid the influence of interfering impurities on subsequent mass spectrometry, and then obtain good peak parameters, which can effectively improve the accuracy and precision of the test results.

[0010] The present application screens and optimizes the relevant test conditions of gas chromatography and mass spectrometry in gas chromatography-mass spectrometry, and provides a method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw materials and injections, which has the advantages of low detection limit, high precision, high repeatability and high accuracy.

[0011] Preferably, the heating program is: the initial temperature is 78-82°C, the temperature is increased to 155-165°C at a heating rate of 18-22°C / min, maintained for 2 minutes, and then the temperature is increased to 215-225°C at a heating rate of 57-62°C / min, and maintained for 2 minutes.

[0012] Preferably, the heating program is: the initial temperature is 80°C, the temperature is increased to 160°C at a heating rate of 20°C / min, maintained for 2 minutes, and then the temperature is increased to 220°C at a heating rate of 60°C / min, and maintained for 2 minutes.

[0013] Preferably, the chromatographic column of the gas chromatography condition is VF-WAXms, with specifications of 30m×0.25mm, 0.25μm.

[0014] Preferably, the gas chromatography parameter conditions also include: flow rate: 0.8-1.2 ml / min; injection volume: 0.8-1.2 μl; split mode: split, split ratio is 4-6:1; control mode: constant flow; carrier gas: He.

[0015] Preferably, the gas chromatography parameter conditions also include: flow rate: 1.0 ml / min; injection volume: 1 μl; split mode: split, split ratio is 5:1; control mode: constant flow; carrier gas: He.

[0016] Preferably, the mass spectrometry parameters are as follows: acquisition type: MRM; ion source: EI; solvent delay: 3.2-3.8 min; transmission line temperature: 245-255° C.; quadrupole temperature: 145-155° C.; and ion source temperature: 245-255° C.

[0017] Preferably, the mass spectrometry parameters are as follows: acquisition type: MRM; ion source: EI; solvent delay: 3.5 min; transmission line temperature: 250° C.; quadrupole temperature: 150° C.; and ion source temperature: 250° C.

[0018] Preferably, the scanning parameters of the mass spectrometer are: analyte: 6-chloro-2-hexanone quantitative ion; parent ion: 98 m / z; daughter ion: 43 m / z; dwell time: 220 ms; collision energy 10 eV.

[0019] Preferably, the preparation method of the pentoxifylline bulk drug test solution is as follows: weigh 0.1 g of the pentoxifylline sample, accurately weigh it, place it in a 15 ml centrifuge tube, accurately add 0.04 ml of acetonitrile and 2 ml of HCl solution, vortex until completely dissolved, then accurately add 2 ml of cyclohexane, vortex extract for 1 min, and take the supernatant; The preparation method of the test solution of pentoxifylline injection is as follows: measure 5 ml of pentoxifylline injection, place it in a 15 ml centrifuge tube, accurately add 2 ml of cyclohexane, vortex extract for 1 minute, and take the supernatant.

[0020] In summary, the technical solution of this application has the following effects: In this experiment, a GC-MS / MS method was established to detect the content of 6-chloro-2-hexanone in pentoxifylline injection. The system applicability, specificity, linearity and range, detection limit and quantification limit, precision, accuracy, solution stability and durability of the method were evaluated. The results were in line with the requirements of the "9101 Guidelines for Validation of Analytical Methods for Pharmaceutical Quality Standards" in Part IV of the 2020 edition of the "Chinese Pharmacopoeia". Therefore, this method can be used to accurately detect the content of 6-chloro-2-hexanone in pentoxifylline raw materials and injection.

[0021] The content of genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw materials and injection is detected by gas chromatography-mass spectrometry technology. The present application provides a detection method with fast detection speed and simple operation method.

[0022] The present application screens and optimizes the relevant test conditions of gas chromatography and mass spectrometry in gas chromatography-mass spectrometry, and provides a method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw materials and injections, which has the advantages of low detection limit, high precision, high repeatability and high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The specificity determination results of the blank solution in the examples are shown in Table 2.

[0024] Figure 2 It is the specificity determination result of the test solution in the embodiment.

[0025] Figure 3 It is the specificity determination result of the 100% limit concentration reference substance solution in the example.

[0026] Figure 4 The specificity test results of the test solution spiked with 100% limit concentration in the example are shown.

[0027] Figure 5 It is the linear graph of 6-chloro-2-hexanone in Example. DETAILED DESCRIPTION

[0028] The present application is further described in detail below in conjunction with examples, comparative examples and performance testing experiments. These examples should not be construed as limiting the scope of protection claimed in the present application.

[0029] According to the limit requirements of this application, the limit of chlorohexanone (hereinafter referred to as 6-chloro-2-hexanone) is 2.5ppm. Based on the properties of the raw material and the target substance, this study established a GC-MS / MS method to detect the content of 6-chloro-2-hexanone in pentoxifylline injection, and validated the method.

[0030] Under the finally selected conditions, the system suitability, specificity, linearity and range, detection limit, quantification limit, accuracy, stability and durability of the method were verified, confirming that the method is suitable for the determination of 6-chloro-2-hexanone in pentoxifylline bulk drug and injection.

[0031] The main instruments, test samples, reagents and reference materials information of this application are shown in Tables 1-4 respectively.

[0032] Table 1 Main instrument information Table 2 Sample information table name Inspection number Specification Placement conditions Pentoxifylline YPT202401365 NA NA Pentoxifylline YPT202401366 NA NA Pentoxifylline YPT202401367 NA NA Pentoxifylline Injection YPT202401368 5ml: 100mg Day 0 Pentoxifylline Injection YPT202401369 5ml: 100mg Day 0 Pentoxifylline Injection YPT202401370 5ml: 100mg Day 0 Pentoxifylline Injection YPT202401371 5ml: 100mg Long term 18 months Pentoxifylline Injection YPT202401372 5ml: 100mg Long term 18 months Pentoxifylline Injection YPT202401373 5ml: 100mg Long term 18 months Table 3 Reagent information name Specification level batch number source Acetonitrile 4L / bottle HPLC O8331440 CNW Cyclohexane 4L / bottle HPLC Z0190144 CNW hydrochloric acid 500ml / bottle AR 20240105 Guangzhou Chemical Reagent Factory Table 4 Reference Material Information Compound Name source batch number content(%) 6-Chloro-2-hexanone CATO 0803-RD-0030 99.4 Example

[0033] Example 1 Example 1 provides a method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw materials and injections.

[0034] The method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in the pentoxifylline bulk drug and injection in Example 1 specifically comprises the following steps in sequence: 1. Gas chromatography conditions Chromatographic column: VF-WAXms (30m×0.25mm, 0.25μm); Inlet temperature: 200°C; flow rate: 1.0 ml / min; injection volume: 1 μl; split mode: split, split ratio 5:1; control mode: constant flow; carrier gas: He; temperature program (see Table 5): Table 5 Heating schedule Rate (℃ / min) Temperature(℃) Holding time (min) / 80 0 20 160 2 60 220 2 2. Mass spectrometry ion source parameters: as shown in Table 6.

[0035] Table 6 Mass spectrometry ion source parameters Collection Type MRM Ion source EI Solvent delay 3.5min Transfer line temperature 250℃ Quadrupole temperature 150℃ Ion source temperature 250℃ 3. Scanning parameters: as shown in Table 7.

[0036] Table 7 Scanning parameters 4. Solution preparation and acceptance criteria 4.1 Solution preparation HCl solution: Measure 1.6 ml of 36%-38% hydrochloric acid into 200 ml of water and mix well.

[0037] Blank solution: Accurately measure 0.04 ml of acetonitrile in a 15 ml centrifuge tube, add 2 ml of HCl solution, then accurately add 2 ml of cyclohexane, vortex extract for 1 min, and take the supernatant.

[0038] Pentoxifylline API test solution: Weigh about 0.1 g of pentoxifylline sample, accurately weigh it, place it in a 15 ml centrifuge tube, accurately add 0.04 ml of acetonitrile and 2 ml of HCl solution, vortex until completely dissolved, then accurately add 2 ml of cyclohexane, vortex extraction for 1 min, and take the supernatant.

[0039] Pentoxifylline injection test solution: measure about 5 ml of pentoxifylline injection, place it in a 15 ml centrifuge tube, accurately add 2 ml of cyclohexane, vortex extract for 1 min, and take the supernatant.

[0040] 6-Chloro-2-hexanone stock solution I: Weigh 17 mg of 6-chloro-2-hexanone reference substance accurately, place in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to scale, shake well to obtain 6-chloro-2-hexanone stock solution I.

[0041] 6-Chloro-2-hexanone stock solution II: Measure 0.7 ml of 6-chloro-2-hexanone stock solution I, place it in a 10 ml volumetric flask, dilute to the mark with acetonitrile, and shake well to obtain 6-chloro-2-hexanone stock solution II. Detection limit stock solution: Accurately measure 0.05 ml of 6-chloro-2-hexanone stock solution II, place it in a 10 ml volumetric flask, dilute to the mark with acetonitrile, and shake well to obtain the detection limit stock solution.

[0042] Standard curve stock solution: Accurately measure appropriate amounts of 6-chloro-2-hexanone stock solution II into different volumetric bottles, dilute to the scale with acetonitrile, and shake well to obtain a series of standard curve stock solutions of different concentrations. See Table 8 for details.

[0043] Table 8 Standard Curve Stock Solution Detection limit solution: Accurately measure 0.04 ml of the detection limit stock solution in a 15 ml centrifuge tube, add 2 ml of HCl solution, then accurately add 2 ml of cyclohexane, vortex extract for 1 min, and take the supernatant to obtain the detection limit solution. Quantification limit solution: Accurately measure 0.04 ml of STD1 (LOQ) stock solution in a 15 ml centrifuge tube, add 2 ml of HCl solution, then accurately add 2 ml of cyclohexane, vortex extract for 1 min, and take the supernatant to obtain the quantification limit solution.

[0044] Standard curve solution: Accurately measure 0.04 ml of the standard curve stock solution in a 15 ml centrifuge tube, add 2 ml of HCl solution, then accurately add 2 ml of cyclohexane, vortex extract for 1 min, and take the supernatant to obtain a series of standard curve solutions of different concentrations, see Table 9 for details.

[0045] Table 9 Standard curve solution System suitability solution: Take the "100% limit concentration reference solution (STD4 solution)" under the "Standard curve" item and inject it 6 times continuously for analysis.

[0046] 50% limit concentration spiked test solution: weigh about 0.1g of pentoxifylline sample, accurately weigh, place in a 15ml centrifuge tube, accurately add 0.04ml STD2 stock solution and 2ml HCl solution, vortex until completely dissolved, then accurately add 2ml cyclohexane, vortex extraction for 1min, take the supernatant and prepare 3 portions in the same way; 100% limit concentration spiked test solution: weigh about 0.1g of pentoxifylline sample, accurately weigh, place in a 15ml centrifuge tube, accurately add 0.04ml STD4 stock solution and 2ml HCl solution, vortex until completely dissolved, then accurately add 2ml cyclohexane, vortex extraction for 1min, take the supernatant and prepare 3 portions in the same way; 150% limit concentration spiked test solution: weigh about 0.1 g of pentoxifylline sample, accurately weigh, place in a 15 ml centrifuge tube, accurately add 0.04 ml of STD5 stock solution and 2 ml of HCl solution, vortex until completely dissolved, then accurately add 2 ml of cyclohexane, vortex extraction for 1 min, take the supernatant and prepare 3 portions in the same way.

[0047] 4.2 Acceptance criteria According to the standard curve method, the concentration of 6-chloro-2-hexanone shall not exceed 2.5 ppm.

[0048] 5. Verify the results 5.1 System suitability Take STD4 solution and inject it 6 times continuously to examine the precision of the instrument. It is required that the RSD (n=6) of the peak area of ​​the target substance in 6 consecutive injections of the system suitability solution should not exceed 10%, and the RSD (n=6) of the retention time should not exceed 1%; the results of the system suitability solution test are shown in Table 10.

[0049] Table 10 System suitability results The results showed that after 6 consecutive injections of system suitability solution, the RSDs (n=6) of the peak area and retention time of 6-chloro-2-hexanone were 1.6% and 0.1%, respectively, and the system suitability met the requirements.

[0050] 5.2 Exclusivity It is required that there is no obvious interference at the target peak in the blank solution chromatogram. If there is interference, the peak area of ​​the interference peak shall not be greater than 30% of the average peak area of ​​the target compound LOQ; the separation degree between the target peak and the adjacent peaks in the test solution chromatogram should be greater than 1.5; the target peak is displayed in the chromatogram of the 100% limit concentration reference solution; the separation degree between the target peak and all adjacent peaks greater than LOQ in the chromatogram of the 100% limit concentration spiked test solution should be greater than 1.5. The specificity of each solution (blank solution, test solution, 100% limit concentration reference solution, 100% limit concentration spiked test solution) is shown in Figure 1 to Figure 4 .

[0051] The results showed that the target peak was not detected in the chromatogram of the blank solution, and there was no interference with the detection; the target peak was shown in the chromatogram of the test solution, and the adjacent peak with a peak area greater than LOQ was not detected; the target peak was shown in the chromatogram of the 100% limit concentration reference solution; the target peak was shown in the chromatogram of the 100% limit concentration spiked test solution, and the adjacent peak with a peak area greater than LOQ was not detected; the results were in compliance with the regulations, and the method had good specificity.

[0052] 5.3 Linearity and range The correlation coefficient of the linear regression equation is required to be r≥0.990, and the ratio of the absolute value of the y-axis intercept to the 100% limit concentration response value shall not exceed 20%. The linear solution determination results are shown in Table 11 and Figure 5 (Linear plot for 6-chloro-2-hexanone).

[0053] Table 11 Linearity results name Concentration (ng / ml) Peak area STD1 25.01 610 STD2 62.51 1520 STD3 100.02 2356 STD4 125.03 2998 STD5 187.54 4438 STD6 250.06 5882 From the above results, the linearity and range results are shown in Table 12.

[0054] Table 12 Linearity and range results The results showed that: 6-chloro-2-hexanone was in the range of 25.01ng / ml to 250.06ng / ml, which was approximately equivalent to 20% to 200% of the limit concentration. The peak area and concentration of 6-chloro-2-hexanone showed good linearity, with a correlation coefficient r of 0.99994. The ratio of the absolute value of the y-axis intercept to the 100% limit concentration response value was 1.3%, and the linear results were in compliance with the regulations.

[0055] 5.4 Limit of detection and limit of quantification It is required that the S / N of the target compound in the detection limit solution should be no less than 3, and the S / N of the target compound in the quantification limit solution should be no less than 10. The results of the detection limit and quantification limit solution are shown in Table 13 and Table 14: Table 13 Detection limit solution results name Concentration (ng / ml) Approximately equivalent to the limit concentration percentage (%) S / N 6-Chloro-2-hexanone 12.50 10 26.8 Table 14 Quantitative Limit Solution Results name Concentration (ng / ml) Approximately equivalent to the limit concentration percentage (%) S / N 6-Chloro-2-hexanone 25.01 20 47.1 The results showed that the detection limit solution concentration was 12.50 ng / ml, approximately equivalent to 10% of the limit concentration, and the S / N was 26.8; the quantification limit solution concentration was 25.01 ng / ml, approximately equivalent to 20% of the limit concentration, and the S / N was 70.1. The detection limit and quantification limit results of this method met the requirements.

[0056] 5.5 Accuracy The recovery rate of the target compound in 9 spiked test solutions with limit concentrations of 50%, 100% and 150% is required to be between 80% and 120%, and the RSD (n=9) of the recovery rate is not greater than 10%; the accuracy solution determination results are shown in Table 15.

[0057] Table 15 6-chloro-2-hexanone accuracy results The results showed that the recovery of 6-chloro-2-hexanone in 9 spiked test solutions at limit concentrations of 50%, 100% and 150% ranged from 84.2% to 97.6%, the RSD of the recovery (n=9) was 5.1%, and the accuracy results met the requirements.

[0058] 5.6 Stability It is required that at different time points, the ratio of the detection concentration of the target compound in each stability solution to the initial (0h) detection concentration should be between 80% and 120%, and the solution is stable during the observation time. The stability test results of each solution are shown in Table 16.

[0059] Table 16 6-chloro-2-hexanone stability results The results showed that the test solution, 100% limit concentration reference solution and 100% limit concentration spiked test solution were stable at room temperature for at least 27.0 hours.

[0060] 5.7 Durability The column temperature fluctuates within the range of 72℃~88℃, and the flow rate fluctuates within the range of 0.9ml / min~1.1ml / min. The recovery rate of the target compound in the 100% limit concentration spiked test solution is calculated. It is required that the system suitability meets the acceptance criteria under each durability condition; the standard curve correlation coefficient r≥0.990; the detection results of the target compound in the test solution should be consistent with the standard conditions (all less than the quantitative limit, or when not less than the quantitative limit, the change rate compared with the standard conditions does not exceed 30%); the recovery rate of the target compound in the 100% limit concentration spiked test solution should be between 75% and 120%. The durability test results are shown in Tables 17 to 22.

[0061] Table 17 System suitability results under 6-chloro-2-hexanone durability conditions - Peak area Table 18 System suitability results under 6-chloro-2-hexanone durability conditions - retention time Table 19 Linearity results of 6-chloro-2-hexanone under durability conditions Table 20 Linearity and range results of 6-chloro-2-hexanone under durability conditions Table 21 Test results of 6-chloro-2-hexanone under durability conditions Table 22 Recovery results of 6-chloro-2-hexanone under durability conditions The results showed that the initial column temperature of the chromatographic column fluctuated in the range of 72℃~88℃, and the flow rate fluctuated in the range of 0.9ml / min~1.1ml / min. The above results all met the durability requirements, and this method had good durability.

[0062] 6. Sample testing Prepare the corresponding batches of test solution according to the following test solution preparation method, inject and analyze, and the results are shown in Table 23.

[0063] Table 23 Sample test results Note: The detection limit of 6-chloro-2-hexanone is 12.50 ng / ml, i.e. 0.25 ppm (μg / g), and the quantification limit is 25.00 ng / ml, i.e. 0.5 ppm (μg / g).

[0064] Example 2 Example 2 provides a method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw materials and injections.

[0065] The difference between Example 2 and Example 1 is that the temperature increase program in the gas chromatography conditions is different, as shown below.

[0066] The gas chromatography conditions were: Chromatographic column: VF-WAXms (30m×0.25mm, 0.25μm); Inlet temperature: 190℃; flow rate: 1.2ml / min; injection volume: 0.8μl; split mode: split, split ratio 4:1; control mode: constant flow; carrier gas: He; heating program: initial temperature is 75℃, heated to 150℃ at a heating rate of 25℃ / min, maintained for 1.5min, then heated to 230℃ at a heating rate of 55℃ / min, maintained for 2.5min.

[0067] Specificity test results: The results showed that the target peak was not detected in the chromatogram of the blank solution, and there was no interference with the detection; the target peak was shown in the chromatogram of the test solution, and the adjacent peak with a peak area greater than LOQ was not detected; the target peak was shown in the chromatogram of the 100% limit concentration reference solution; the target peak was shown in the chromatogram of the 100% limit concentration spiked test solution, and the adjacent peak with a peak area greater than LOQ was not detected; the results were in compliance with the regulations, and the method had good specificity.

[0068] Example 3 Example 3 provides a method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw materials and injections.

[0069] The difference between Example 3 and Example 1 is that the temperature increase program in the gas chromatography conditions is different, as shown below.

[0070] The gas chromatography conditions were: Chromatographic column: VF-WAXms (30m×0.25mm, 0.25μm); Inlet temperature: 210℃; flow rate: 0.8ml / min; injection volume: 1.2μl; split mode: split, split ratio 6:1; control mode: constant flow; carrier gas: He; heating program: initial temperature is 85℃, heated to 170℃ at a heating rate of 15℃ / min, maintained for 2.5min, then heated to 210℃ at a heating rate of 65℃ / min, maintained for 1.5min.

[0071] Specificity test results: The results showed that the target peak was not detected in the chromatogram of the blank solution, and there was no interference with the detection; the target peak was shown in the chromatogram of the test solution, and the adjacent peak with a peak area greater than LOQ was not detected; the target peak was shown in the chromatogram of the 100% limit concentration reference solution; the target peak was shown in the chromatogram of the 100% limit concentration spiked test solution, and the adjacent peak with a peak area greater than LOQ was not detected; the results were in compliance with the regulations, and the method had good specificity.

[0072] Comparative Example Comparative Example 1 Comparative Example 1 provides a method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw materials and injections.

[0073] The difference between Comparative Example 1 and Example 1 is that the temperature increase program in the gas chromatography conditions is different, as shown below.

[0074] The gas chromatography conditions were: Chromatographic column: VF-WAXms (30m×0.25mm, 0.25μm); Inlet temperature: 200℃; flow rate: 1.0ml / min; injection volume: 1μl; split mode: split, split ratio 5:1; control mode: constant flow; carrier gas: He; heating program: initial temperature of 50℃, heating to 220℃ at a heating rate of 10℃ / min, and maintaining for 7min.

[0075] Specificity test results: The results showed that the test sample interfered with the detection of the target peak.

[0076] Comparative Example 2 Comparative Example 2 provides a method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw materials and injections.

[0077] The difference between Comparative Example 2 and Example 1 is that the temperature increase program in the gas chromatography conditions is different, as shown below.

[0078] The gas chromatography conditions were: Chromatographic column: VF-WAXms (30m×0.25mm, 0.25μm); Inlet temperature: 200℃; flow rate: 1.0ml / min; injection volume: 1μl; split mode: split, split ratio 5:1; control mode: constant flow; carrier gas: He; heating program: initial temperature is 95℃, heated to 160℃ at a heating rate of 20℃ / min, maintained for 2min, then heated to 220℃ at a heating rate of 60℃ / min, maintained for 2min.

[0079] Specificity test results: The results showed that the test sample interfered with the detection of the target peak.

[0080] Comparative Example 3 Comparative Example 3 provides a method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw materials and injections.

[0081] The difference between Comparative Example 3 and Example 1 is that the temperature increase program in the gas chromatography conditions is different, as shown below.

[0082] The gas chromatography conditions were: Chromatographic column: VF-WAXms (30m×0.25mm, 0.25μm); Inlet temperature: 180℃; flow rate: 1.0ml / min; injection volume: 1μl; split mode: split, split ratio 5:1; control mode: constant flow; carrier gas: He; heating program: initial temperature is 80℃, heated to 180℃ at a heating rate of 10℃ / min, maintained for 1min, then heated to 240℃ at a heating rate of 75℃ / min, maintained for 5min.

[0083] Specificity test results: The results showed that the test sample interfered with the detection of the target peak.

[0084] Comparative Example 4 Comparative Example 4 provides a method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw materials and injections.

[0085] The difference between Comparative Example 4 and Example 1 is that the temperature increase program in the gas chromatography conditions is different, as shown below.

[0086] The gas chromatography conditions were: Chromatographic column: VF-WAXms (30m×0.25mm, 0.25μm); Inlet temperature: 220℃; flow rate: 1.0ml / min; injection volume: 1μl; split mode: split, split ratio 5:1; control mode: constant flow; carrier gas: He; heating program: initial temperature is 80℃, heated to 140℃ at a heating rate of 30℃ / min, maintained for 5min, then heated to 200℃ at a heating rate of 45℃ / min, maintained for 1min.

[0087] Specificity test results: The results showed that the target peak eluted too early and the blank solvent interfered with the detection of the target peak.

[0088] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in pentoxifylline bulk drug and injection, characterized in that: Gas chromatography-mass spectrometry GC-MS is used to detect the samples to be tested; The parameters of the gas chromatography are as follows: injection port temperature: 190-210°C; heating program: initial temperature 75-85°C, heating to 150-170°C at a heating rate of 15-25°C / min, maintaining for 1.5-2.5min, then heating to 210-230°C at a heating rate of 55-65°C / min, maintaining for 1.5-2.5min; The mass spectrometry parameters are as follows: acquisition type: MRM; ion source: EI; solvent delay: 3-4 min; transfer line temperature: 240-260° C.; quadrupole temperature: 140-160° C.; and ion source temperature: 240-260° C.

2. The method for detecting the content of 6-chloro-2-hexanone, a genotoxic impurity, in the pentoxifylline bulk drug and injection according to claim 1, characterized in that: The heating program is as follows: the initial temperature is 78-82°C, the temperature is increased to 155-165°C at a heating rate of 18-22°C / min, and maintained for 2 minutes, and then the temperature is increased to 215-225°C at a heating rate of 57-62°C / min, and maintained for 2 minutes.

3. The method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in the pentoxifylline bulk drug and injection according to claim 2, characterized in that: The heating program is as follows: the initial temperature is 80°C, the temperature is increased to 160°C at a heating rate of 20°C / min, maintained for 2 minutes, and then the temperature is increased to 220°C at a heating rate of 60°C / min, and maintained for 2 minutes.

4. The method for detecting the content of 6-chloro-2-hexanone, a genotoxic impurity, in the pentoxifylline bulk drug and injection according to claim 1, characterized in that: The chromatographic column of the gas chromatography condition is VF-WAXms, with specifications of 30m×0.25mm, 0.25μm.

5. The method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in the pentoxifylline bulk drug and injection according to claim 1, characterized in that: The gas chromatography parameters are also Including: flow rate: 0.8-1.2ml / min; injection volume: 0.8-1.2μl; split mode: split, split ratio is 4-6:1; control mode: constant flow; carrier gas: He.

6. The method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in the pentoxifylline bulk drug and injection according to claim 1, characterized in that: The gas chromatography parameters are also Including: flow rate: 1.0ml / min; injection volume: 1μl; split mode: split, split ratio is 5:1; control mode: constant flow; carrier gas: He.

7. The method for detecting the content of 6-chloro-2-hexanone, a genotoxic impurity, in the pentoxifylline bulk drug and injection according to claim 1, characterized in that: The mass spectrometry parameters are as follows: acquisition type: MRM; ion source: EI; solvent delay: 3.2-3.8 min; transfer line temperature: 245-255° C.; quadrupole temperature: 145-155° C.; and ion source temperature: 245-255° C.

8. The method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in the pentoxifylline bulk drug and injection according to claim 7, characterized in that: The parameters of the mass spectrometry are as follows: acquisition type: MRM; ion source: EI; solvent delay: 3.5 min; transfer line temperature: 250° C.; quadrupole temperature: 150° C.; and ion source temperature: 250° C.

9. The method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in the pentoxifylline bulk drug and injection according to claim 1, characterized in that: The scanning parameters of the mass spectrometer are: analyte: 6-chloro-2-hexanone quantitative ion; parent ion: 98 m / z; daughter ion: 43 m / z; dwell time: 220 ms; collision energy: 10 eV.

10. The method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in the pentoxifylline bulk drug and injection according to claim 1, characterized in that: The preparation method of the pentoxifylline bulk drug test solution is as follows: weigh 0.1 g of the pentoxifylline sample, accurately weigh it, place it in a 15 ml centrifuge tube, accurately add 0.04 ml of acetonitrile and 2 ml of HCl solution, vortex until completely dissolved, then accurately add 2 ml of cyclohexane, vortex extract for 1 min, and take the supernatant; The preparation method of the test solution of pentoxifylline injection is as follows: measure 5 ml of pentoxifylline injection, place it in a 15 ml centrifuge tube, accurately add 2 ml of cyclohexane, vortex extract for 1 minute, and take the supernatant.

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