A method for detecting the content of the genotoxic impurity 6-chloro-2-hexanone in pentoxifylline bulk drugs and injections

Abstract

This application relates to the technical field of biomedical detection, and specifically discloses a method for detecting the content of the genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw materials and injection solutions. The detection method disclosed in this application uses gas chromatography-mass spectrometry (GC-MS) to detect samples; the gas chromatography parameters are: injection port temperature 190 - 210 °C; temperature programming: initial temperature 75 - 85 °C, heated to 150 - 170 °C at a rate of 15 - 25 °C / min, held for 1.5 - 2.5 min, then heated to 210 - 230 °C at a rate of 55 - 65 °C / min, held for 1.5 - 2.5 min; the mass spectrometry parameters are: solvent delay 3 - 4 min; transfer line temperature 240 - 260 °C; quadrupole temperature 140 - 160 °C; ion source temperature 240 - 260 °C. The method of this application is simple to operate, has a fast detection speed, and has the advantages of high sensitivity, high precision, and high repeatability.

Description

Technical Field

[0001] This application relates to the technical field of biomedical detection, and specifically relates to a method for detecting the content of the 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 achieving the effect of improving microcirculation; it can prevent the formation of thrombi by inhibiting platelet aggregation; it can also reduce blood lipid levels by reducing cholesterol synthesis and regulating lipid metabolism, and has a certain adjuvant therapeutic effect on patients with hyperlipidemia; it has a certain antioxidant stress ability and can scavenge free radicals, which helps to delay the aging process of the body. The metabolites of pentoxifylline also have the effects of improving blood viscosity and microcirculation.

[0003] 6-Chloro-2-hexanone is an important starting material in the synthesis of pentoxifylline. During the synthesis of pentoxifylline, 6-chloro-2-hexanone impurities are likely to remain, which may have an adverse impact on the safety of pentoxifylline use and seriously affect the quality of pentoxifylline and the health of patients. Therefore, the detection method for 6-chloro-2-hexanone in pentoxifylline is a currently concerned issue.

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

[0005] To control the impurities in 6-chloro-2-hexanone, ensure the quality of pentoxifylline synthesized using 6-chloro-2-hexanone as a starting material, and ensure the safety of patients' medication, developing a new detection method for related substances of 6-chloro-2-hexanone is a currently urgent technical problem. Summary of the Invention

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

[0007] This application provides a method for detecting the content of the 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;

[0008] The parameter conditions of the gas chromatography are as follows: inlet temperature: 190 - 210 °C; temperature programming: the initial temperature is 75 - 85 °C, heated at a heating rate of 15 - 25 °C / min to 150 - 170 °C, held for 1.5 - 2.5 min, then heated at a heating rate of 55 - 65 °C / min to 210 - 230 °C, held for 1.5 - 2.5 min;

[0009] The parameter conditions of the mass spectrometry 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; ion source temperature: 240 - 260 °C.

[0010] The detection method for the content of the genotoxic impurity 6-chloro-2-hexanone in the pentoxifylline raw material drug and injection adopted in this application is gas chromatography-mass spectrometry (GC-MS). This determination method uses gas chromatography as the separation system for 6-chloro-2-hexanone and other substances in the pentoxifylline raw material drug and injection to be tested, and mass spectrometry as the detection system for 6-chloro-2-hexanone. Gas chromatography-mass spectrometry combines the high separation ability of gas chromatography for the sample to be tested with the advantages of high selectivity, high sensitivity of mass spectrometry and the ability to provide relative molecular mass and structural information.

[0011] Gas chromatography has the characteristic of strong resolution for the sample to be tested. By selecting appropriate chromatographic columns, initial 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 test analysis, but also preliminarily separate the impurities that interfere with mass spectrometry, avoiding the influence of interfering impurities on the subsequent mass spectrometry, and then obtaining good peak type parameters, so as to effectively improve the accuracy and precision of the detection results.

[0012] By screening and optimizing the relevant test conditions of gas chromatography and mass spectrometry in gas chromatography-mass spectrometry, the detection method for the content of the genotoxic impurity 6-chloro-2-hexanone in the pentoxifylline raw material drug and injection provided in this application has the advantages of low detection limit, high precision, high repeatability and high accuracy.

[0013] Preferably, the temperature programming is as follows: the initial temperature is 78 - 82 °C, heated at a heating rate of 18 - 22 °C / min to 155 - 165 °C, held for 2 min, then heated at a heating rate of 57 - 62 °C / min to 215 - 225 °C, held for 2 min.

[0014] Preferably, the temperature programming is as follows: the initial temperature is 80 °C, heated at a heating rate of 20 °C / min to 160 °C, held for 2 min, then heated at a heating rate of 60 °C / min to 220 °C, held for 2 min.

[0015] Preferably, the chromatographic column for the gas chromatography conditions is VF-WAXms, with a specification of 30 m × 0.25 mm, 0.25 μm.

[0016] Preferably, the parameter conditions of the gas chromatography further include: flow rate: 0.8 - 1.2 ml / min; injection volume: 0.8 - 1.2 μl; split mode: split, split ratio of 4 - 6:1; control mode: constant flow; carrier gas: He.

[0017] Preferably, the parameter conditions of the gas chromatography further include: flow rate: 1.0 ml / min; injection volume: 1 μl; split mode: split, split ratio of 5:1; control mode: constant flow; carrier gas: He.

[0018] Preferably, the parameter conditions of the mass spectrometry are: acquisition type: MRM; ion source: EI; solvent delay: 3.2 - 3.8 min; transfer line temperature: 245 - 255 °C; quadrupole temperature: 145 - 155 °C; ion source temperature: 245 - 255 °C.

[0019] Preferably, the parameter conditions of the mass spectrometry are: acquisition type: MRM; ion source: EI; solvent delay: 3.5 min; transfer line temperature: 250 °C; quadrupole temperature: 150 °C; ion source temperature: 250 °C.

[0020] Preferably, the scanning parameters of the mass spectrometry are: analyte: 6-chloro-2-hexanone quantification ion; parent ion: 98 m / z; daughter ion: 43 m / z; dwell time: 220 ms; collision energy 10 eV.

[0021] Preferably, the preparation method of the pentoxifylline raw material drug test solution is: 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 and extract for 1 min, and take the supernatant to obtain;

[0022] The preparation method of the pentoxifylline injection test solution is: measure 5 ml of the pentoxifylline injection, place it in a 15 ml centrifuge tube, accurately add 2 ml of cyclohexane, vortex and extract for 1 min, and take the supernatant to obtain.

[0023] In summary, the technical solution of this application has the following effects:

[0024] In this experiment, a GC-MS / MS method was established to detect the content of 6-chloro-2-hexanone in pentoxifylline injection. Through the methodological evaluation of the system suitability, specificity, linearity and range, detection limit and quantitation limit, precision, accuracy, solution stability and durability of the method, the results all met the requirements of the "General Principles for Verification of Analytical Methods for Drug Quality Standards" in Part IV of the Chinese Pharmacopoeia 2020 Edition. Therefore, this method can be used to accurately detect the content of 6-chloro-2-hexanone in pentoxifylline raw material and injection.

[0025] By using gas chromatography-mass spectrometry (GC-MS) technology to detect the content of the genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw material and injection, this application provides a detection method with fast detection speed and simple operation method.

[0026] This application screens and optimizes the relevant test conditions of gas chromatography and mass spectrometry in GC-MS, and the detection method for the content of the genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw material and injection provided has the advantages of low detection limit, high precision, high repeatability and high accuracy. Description of the Drawings

[0027] Figure 1 It is the specificity determination result of the blank solution in the example.

[0028] Figure 2 It is the specificity determination result of the test solution in the example.

[0029] Figure 3 It is the specificity determination result of the reference solution at 100% limit concentration in the example.

[0030] Figure 4 It is the specificity determination result of the spiked test solution at 100% limit concentration in the example.

[0031] Figure 5 It is the linear graph of 6-chloro-2-hexanone in the example. Detailed Description of the Invention

[0032] The following further describes this application in detail with reference to examples, comparative examples and performance detection tests, and these examples should not be construed as limiting the scope claimed in this application.

[0033] According to the limit requirements of this application, the limit of chlorohexanone (hereinafter referred to as 6-chloro-2-hexanone) is 2.5 ppm. 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 verified this method.

[0034] Under the finally selected conditions, the system suitability, specificity, linearity and range, detection limit, quantitation limit, accuracy, stability and durability of the method were verified to confirm that the method is applicable to the determination of the content of 6-chloro-2-hexanone in pentoxifylline bulk drugs and injections.

[0035] The information on the main instruments, test drug samples, reagents and reference substances of this application is shown in Tables 1-4 respectively.

[0036] Table 1 Information Table of Main Instruments

[0037]

[0038] Table 2 Information Table of Samples

[0039] Name Sample Number Specification Storage Conditions Pentoxifylline YPT202401365 NA NA Pentoxifylline YPT202401366 NA NA Pentoxifylline YPT202401367 NA NA Pentoxifylline Injection YPT202401368 5ml: 100mg 0 days Pentoxifylline Injection YPT202401369 5ml: 100mg 0 days Pentoxifylline Injection YPT202401370 5ml: 100mg 0 days 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

[0040] Table 3 Information Table of Reagents

[0041] Name Specification Grade 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

[0042] Table 4 Information Table of Reference Substances

[0043] Compound Name Source Batch Number Content (%) 6-Chloro-2-hexanone CATO 0803-RD-0030 99.4

[0044] Examples

[0045] Example 1

[0046] Example 1 provides a method for detecting the content of the genotoxic impurity 6-chloro-2-hexanone in pentoxifylline bulk drugs and injections.

[0047] The method for detecting the content of the genotoxic impurity 6-chloro-2-hexanone in pentoxifylline bulk drugs and injections in Example 1 specifically includes the following steps in sequence:

[0048] 1. Gas chromatography conditions

[0049] Chromatographic column: VF-WAXms (30m × 0.25mm, 0.25μm);

[0050] Injection port 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 programming (see Table 5):

[0051] Table 5 Temperature Programming Table

[0052] Rate (℃ / min) Temperature (℃) Retention Time (min) / 80 0 20 160 2 60 220 2

[0053] 2. Mass spectrometry ion source parameters: As shown in Table 6.

[0054] Table 6 Mass Spectrometry Ion Source Parameters

[0055] Collection Type MRM Ion Source EI Solvent Delay 3.5min Transfer Line Temperature 250℃ Quadrupole Temperature 150℃ Ion Source Temperature 250℃

[0056] 3. Scanning parameters: as shown in Table 7.

[0057] Table 7 Scanning parameters

[0058]

[0059] 4. Solution preparation and acceptance criteria

[0060] 4.1 Solution preparation

[0061] HCl solution: Measure 1.6 ml of 36% - 38% hydrochloric acid and add it to 200 ml of water, mix well to obtain.

[0062] Blank solution: Precisely measure 0.04 ml of acetonitrile into a 15 - ml centrifuge tube, add 2 ml of HCl solution, then precisely add 2 ml of cyclohexane, vortex for 1 min, and take the supernatant to obtain.

[0063] The test solution of pentoxifylline raw material drug: Weigh about 0.1 g of pentoxifylline sample, precisely weigh it, place it in a 15 - ml centrifuge tube, precisely add 0.04 ml of acetonitrile and 2 ml of HCl solution, vortex until completely dissolved, then precisely add 2 ml of cyclohexane, vortex for 1 min, and take the supernatant to obtain.

[0064] The test solution of pentoxifylline injection: Measure about 5 ml of pentoxifylline injection, place it in a 15 - ml centrifuge tube, precisely add 2 ml of cyclohexane, vortex for 1 min, and take the supernatant to obtain.

[0065] Stock solution I of 6 - chloro - 2 - hexanone: Weigh an appropriate amount (17 mg) of 6 - chloro - 2 - hexanone reference substance, precisely weigh it, place it in a 10 - ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well. Thus, stock solution I of 6 - chloro - 2 - hexanone is obtained.

[0066] Stock solution II of 6 - chloro - 2 - hexanone: Measure 0.7 ml of stock solution I of 6 - chloro - 2 - hexanone, place it in a 10 - ml volumetric flask, dilute to the mark with acetonitrile, and shake well. Thus, stock solution II of 6 - chloro - 2 - hexanone is obtained. Detection limit stock solution: Precisely measure 0.05 ml of stock solution II of 6 - chloro - 2 - hexanone, place it in a 10 - ml volumetric flask, dilute to the mark with acetonitrile, and shake well to obtain the detection limit stock solution.

[0067] Stock solution for standard curve: Precisely measure appropriate amounts of stock solution II of 6 - chloro - 2 - hexanone into different volumetric flasks respectively, dilute to the mark with acetonitrile, and shake well to obtain a series of stock solutions for standard curve with different concentrations, as detailed in Table 8.

[0068] Table 8 Stock solutions for standard curve

[0069]

[0070] Detection limit solution: Accurately measure 0.04 ml of the detection limit stock solution into a 15-ml centrifuge tube, add 2 ml of HCl solution, then accurately add 2 ml of cyclohexane, vortex for extraction for 1 min, and take the supernatant to obtain the detection limit solution. Quantitation limit solution: Accurately measure 0.04 ml of the STD1 (LOQ) stock solution into a 15-ml centrifuge tube, add 2 ml of HCl solution, then accurately add 2 ml of cyclohexane, vortex for extraction for 1 min, and take the supernatant to obtain the quantitation limit solution.

[0071] Standard curve solution: Accurately measure 0.04 ml of the standard curve stock solution into 15-ml centrifuge tubes respectively, add 2 ml of HCl solution, then accurately add 2 ml of cyclohexane, vortex for extraction for 1 min, and take the supernatants to obtain a series of standard curve solutions with different concentrations, as shown in Table 9 for details.

[0072] Table 9 Standard curve solution

[0073]

[0074] System suitability solution: Take the "100% limit concentration reference solution (STD4 solution)" under the item of "Standard curve", and inject it continuously for 6 times for analysis.

[0075] 50% limit concentration spiked test solution: Weigh about 0.1 g of the pentoxifylline sample, accurately weigh it, place it in a 15-ml centrifuge tube, accurately add 0.04 ml of the STD2 stock solution and 2 ml of HCl solution, vortex until completely dissolved, then accurately add 2 ml of cyclohexane, vortex for extraction for 1 min, and take the supernatant to obtain it. Prepare 3 portions in the same way;

[0076] 100% limit concentration spiked test solution: Weigh about 0.1 g of the pentoxifylline sample, accurately weigh it, place it in a 15-ml centrifuge tube, accurately add 0.04 ml of the STD4 stock solution and 2 ml of HCl solution, vortex until completely dissolved, then accurately add 2 ml of cyclohexane, vortex for extraction for 1 min, and take the supernatant to obtain it. Prepare 3 portions in the same way;

[0077] 150% limit concentration spiked test solution: Weigh about 0.1 g of the pentoxifylline sample, accurately weigh it, place it in a 15-ml centrifuge tube, accurately add 0.04 ml of the STD5 stock solution and 2 ml of HCl solution, vortex until completely dissolved, then accurately add 2 ml of cyclohexane, vortex for extraction for 1 min, and take the supernatant to obtain it. Prepare 3 portions in the same way.

[0078] 4.2 Acceptance criteria

[0079] Calculated by the standard curve method, 6-chloro-2-hexanone shall not exceed 2.5 ppm.

[0080] 5 Verification Results

[0081] 5.1 System Suitability

[0082] Take the STD4 solution and inject it continuously for 6 times to examine the instrument precision. It is required that the RSD (n = 6) of the target peak area in the system suitability solution for 6 consecutive injections should not exceed 10%, and the RSD (n = 6) of the retention time should not exceed 1%; the determination results of the system suitability solution are shown in Table 10.

[0083] Table 10 System Suitability Results Table

[0084]

[0085]

[0086] The results show that for the system suitability solution for 6 consecutive injections, the RSD (n = 6) of the peak area and retention time of 6-chloro-2-hexanone are 1.6% and 0.1% respectively, and the system suitability meets the requirements.

[0087] 5.2 Specificity

[0088] It is required that there is no obvious interference at the target peak in the chromatogram of the blank solution. If there is interference, the peak area of the interfering peak shall not be greater than 30% of the average peak area of the target compound at LOQ; in the chromatogram of the test solution, the resolution between the target peak and the adjacent peak shall be greater than 1.5; the target peak is shown in the chromatogram of the reference solution at 100% limit concentration; the resolution between the target peak and all adjacent peaks greater than LOQ in the chromatogram of the spiked test solution at 100% limit concentration shall be greater than 1.5. The determination results of each specificity solution (blank solution, test solution, reference solution at 100% limit concentration, spiked test solution at 100% limit concentration) are shown in Figures 1 - 4 .

[0089] The results show that the target peak is not detected in the chromatogram of the blank solution, and there is no interference with the detection; the target peak is shown in the chromatogram of the test solution, and no adjacent peak with a peak area greater than LOQ is detected; the target peak is shown in the chromatogram of the reference solution at 100% limit concentration; the target peak is shown in the chromatogram of the spiked test solution at 100% limit concentration, and no adjacent peak with a peak area greater than LOQ is detected; the results meet the requirements, and the method has good specificity.

[0090] 5.3 Linearity and Range

[0091] It is required that the correlation coefficient r of the linear regression equation is ≥ 0.990, and the ratio of the absolute value of the y-axis intercept to the response value at 100% limit concentration shall not exceed 20%. The determination results of the linear solution are shown in Table 11 and Figure 5 (Linear graph of 6-chloro-2-hexanone).

[0092] Table 11 Linear Results Table

[0093] 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

[0094] As shown by the above results, the linear and range results are shown in Table 12.

[0095] Table 12 Linear and Range Results

[0096]

[0097] The results show that within the range of 25.01 ng / ml to 250.06 ng / ml, 6-chloro-2-hexanone is approximately equivalent to 20% - 200% of the limit concentration. The peak area of 6-chloro-2-hexanone has a good linear relationship with the concentration, with a correlation coefficient r of 0.99994. The ratio of the absolute value of the y-axis intercept to the response value of the 100% limit concentration is 1.3%. The linear results meet the requirements.

[0098] 5.4 Detection Limit and Quantification Limit

[0099] It is required that the S / N of the target compound in the detection limit solution should not be less than 3, and the S / N of the target compound in the quantification limit solution should not be less than 10. The determination results of the detection limit and quantification limit solutions are shown in Tables 13 - 14:

[0100] Table 13 Detection Limit Solution Results Table

[0101] Name Concentration (ng / ml) Approximate Percentage of Limit Concentration (%) S / N 6-Chloro-2-hexanone 12.50 10 26.8

[0102] Table 14 Quantification Limit Solution Results Table

[0103] Name Concentration (ng / ml) Approximate Percentage of Limit Concentration (%) S / N 6-Chloro-2-hexanone 25.01 20 47.1

[0104] The results show that the concentration of the detection limit solution is 12.50 ng / ml, approximately equivalent to 10% of the limit concentration, and the S / N is 26.8; the concentration of the quantification limit solution is 25.01 ng / ml, approximately equivalent to 20% of the limit concentration, and the S / N is 70.1. The detection limit and quantification limit results of this method meet the requirements.

[0105] 5.5 Accuracy

[0106] It is required that the recovery rate of the target compound in 9 spiked test samples at 50%, 100%, and 150% of the limit concentration should be between 80% - 120%, and the RSD (n = 9) of the recovery rate should not be greater than 10%; the determination results of the accuracy solution are shown in Table 15.

[0107] Table 15 6-Chloro-2-Hexanone Accuracy Results Table

[0108]

[0109]

[0110] The results showed that the recovery rates of 6-chloro-2-hexanone in 9 spiked test samples at the concentration limits of 50%, 100% and 150% ranged from 84.2% to 97.6%, and the RSD (n = 9) of the recovery rates was 5.1%. The accuracy results met the requirements.

[0111] 5.6 Stability

[0112] It is required that at different time points, the ratio of the detected concentration of the target compound in each stability solution to the initial (0 h) detected concentration should be between 80% and 120%, indicating that the solution is stable within the inspection time. The determination results of each stability solution are shown in Table 16.

[0113] Table 16 Results of the stability of 6-chloro-2-hexanone

[0114]

[0115] The results showed that the test sample solution, the reference substance solution at the 100% limit concentration and the spiked test sample solution at the 100% limit concentration were stable when placed at room temperature for at least 27.0 hours.

[0116] 5.7 Robustness

[0117] The column temperature of the chromatographic column fluctuated within the range of 72 °C to 88 °C, and the flow rate fluctuated within the range of 0.9 ml / min to 1.1 ml / min. The recovery rate of the target compound in the spiked test sample solution at the 100% limit concentration was calculated. It is required that under each robustness condition, the system suitability meets the acceptance criteria; the correlation coefficient r of the standard curve is ≥ 0.990; the detection results of the target compound in the test sample solution should be consistent with the standard conditions (both less than the limit of quantitation, or when not less than the limit of quantitation, the change rate compared with the standard conditions does not exceed 30%); the recovery rate of the target compound in the spiked test sample solution at the 100% limit concentration should be between 75% and 120%. The determination results of robustness are shown in Tables 17 to 22.

[0118] Table 17 Results of the system suitability under the robustness conditions of 6-chloro-2-hexanone - peak area

[0119]

[0120] Table 18 Results of the system suitability under the robustness conditions of 6-chloro-2-hexanone - retention time

[0121]

[0122] Table 19 Results of the linearity under the robustness conditions of 6-chloro-2-hexanone

[0123]

[0124]

[0125] Table 20 Results Table of Linearity and Range of 6-Chloro-2-hexanone under Durability Conditions

[0126]

[0127] Table 21 Results Table of Test Samples of 6-Chloro-2-hexanone under Durability Conditions

[0128]

[0129] Table 22 Results Table of Recovery Rate of 6-Chloro-2-hexanone under Durability Conditions

[0130]

[0131] The results show that the initial column temperature of the chromatographic column fluctuates within the range of 72 °C to 88 °C, and the flow rate fluctuates within the range of 0.9 ml / min to 1.1 ml / min. The above results all meet the durability requirements, and the durability of this method is good.

[0132] 6 Sample Detection

[0133] Prepare the corresponding batches of test sample solutions according to the following test sample solution preparation method, and perform injection analysis. The results are shown in Table 23.

[0134] Table 23 Results Table of Sample Tests

[0135]

[0136]

[0137] Note: The detection limit of 6-chloro-2-hexanone is 12.50 ng / ml, that is, 0.25 ppm (μg / g), and the quantification limit is 25.00 ng / ml, that is, 0.5 ppm (μg / g).

[0138] Example 2

[0139] Example 2 provides a method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw material drugs and injection solutions.

[0140] The difference between Example 2 and Example 1 lies in: the temperature programming in the gas chromatography conditions is different, as specifically shown below.

[0141] The gas chromatography conditions are as follows:

[0142] Chromatographic column: VF-WAXms (30 m × 0.25 mm, 0.25 μm);

[0143] Injector temperature: 190 °C; Flow rate: 1.2 ml / min; Injection volume: 0.8 μl; Split mode: Split, split ratio 4:1; Control mode: Constant flow; Carrier gas: He; The temperature program is as follows: Initial temperature is 75 °C, heated at a rate of 25 °C / min to 150 °C, held for 1.5 min, then heated at a rate of 55 °C / min to 230 °C, held for 2.5 min.

[0144] Specificity test results:

[0145] The results showed that no target peak was detected in the chromatogram of the blank solution, indicating no interference with the detection; the chromatogram of the test solution showed the target peak, and no adjacent peak with a peak area greater than the LOQ was detected; the chromatogram of the reference solution at 100% limit concentration showed the target peak; the chromatogram of the spiked test solution at 100% limit concentration showed the target peak, and no adjacent peak with a peak area greater than the LOQ was detected; the results met the requirements, indicating good specificity of the method.

[0146] Example 3

[0147] Example 3 provides a method for detecting the content of the genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw materials and injections.

[0148] The difference between Example 3 and Example 1 is that the temperature program in the gas chromatography conditions is different, as shown below.

[0149] Gas chromatography conditions are as follows:

[0150] Chromatographic column: VF-WAXms (30 m × 0.25 mm, 0.25 μm);

[0151] Injector temperature: 210 °C; Flow rate: 0.8 ml / min; Injection volume: 1.2 μl; Split mode: Split, split ratio 6:1; Control mode: Constant flow; Carrier gas: He; The temperature program is as follows: Initial temperature is 85 °C, heated at a rate of 15 °C / min to 170 °C, held for 2.5 min, then heated at a rate of 65 °C / min to 210 °C, held for 1.5 min.

[0152] Specificity test results:

[0153] The results showed that no target peak was detected in the chromatogram of the blank solution, indicating no interference with the detection; the chromatogram of the test solution showed the target peak, and no adjacent peak with a peak area greater than the LOQ was detected; the chromatogram of the reference solution at 100% limit concentration showed the target peak; the chromatogram of the spiked test solution at 100% limit concentration showed the target peak, and no adjacent peak with a peak area greater than the LOQ was detected; the results met the requirements, indicating good specificity of the method.

[0154] Comparative example

[0155] Comparative Example 1

[0156] Comparative Example 1 provides a method for detecting the content of the genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw materials and injection solutions.

[0157] The difference between Comparative Example 1 and Example 1 lies in that the temperature programming in the gas chromatography conditions is different, as shown below.

[0158] The gas chromatography conditions are as follows:

[0159] Column: VF-WAXms (30m × 0.25mm, 0.25μm);

[0160] Injection port 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 programming: Initial temperature is 50°C, heated at a rate of 10°C / min to 220°C, and held for 7 min.

[0161] Specificity test results:

[0162] The results show that the test sample interferes with the detection of the target peak.

[0163] Comparative Example 2

[0164] Comparative Example 2 provides a method for detecting the content of the genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw materials and injection solutions.

[0165] The difference between Comparative Example 2 and Example 1 lies in that the temperature programming in the gas chromatography conditions is different, as shown below.

[0166] The gas chromatography conditions are as follows:

[0167] Column: VF-WAXms (30m × 0.25mm, 0.25μm);

[0168] Injection port 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 programming: Initial temperature is 95°C, heated at a rate of 20°C / min to 160°C, held for 2 min, then heated at a rate of 60°C / min to 220°C, and held for 2 min.

[0169] Specificity test results:

[0170] The results show that the test sample interferes with the detection of the target peak.

[0171] Comparative Example 3

[0172] Comparative Example 3 provides a method for detecting the content of the genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw material drug and injection.

[0173] The difference between Comparative Example 3 and Example 1 lies in that the temperature programming in the gas chromatography conditions is different, which is specifically as follows.

[0174] The gas chromatography conditions are as follows:

[0175] Chromatographic column: VF-WAXms (30m×0.25mm, 0.25μm);

[0176] Injector temperature: 180°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 programming is: The initial temperature is 80°C, the temperature is increased at a rate of 10°C / min to 180°C, held for 1 min, and then the temperature is increased at a rate of 75°C / min to 240°C, held for 5 min.

[0177] Specificity test results:

[0178] The results show that the test sample interferes with the detection of the target peak.

[0179] Comparative Example 4

[0180] Comparative Example 4 provides a method for detecting the content of the genotoxic impurity 6-chloro-2-hexanone in pentoxifylline raw material drug and injection.

[0181] The difference between Comparative Example 4 and Example 1 lies in that the temperature programming in the gas chromatography conditions is different, which is specifically as follows.

[0182] The gas chromatography conditions are as follows:

[0183] Chromatographic column: VF-WAXms (30m×0.25mm, 0.25μm);

[0184] Injector temperature: 220°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 programming is: The initial temperature is 80°C, the temperature is increased at a rate of 30°C / min to 140°C, held for 5 min, and then the temperature is increased at a rate of 45°C / min to 200°C, held for 1 min.

[0185] Specificity test results:

[0186] The results show that the elution time of the target peak is too early and the blank solvent interferes with the detection of the target peak.

[0187] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made thereto based on the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. A method for detecting the content of the genotoxic impurity 6-chloro-2-hexanone in pentoxifylline bulk drug and injection, which is characterized in that, The test sample is detected by gas chromatography - mass spectrometry; The parameter conditions of the gas chromatography are as follows: inlet temperature: 190 - 210 °C; temperature programming: the initial temperature is 75 - 85 °C, and it is heated to 150 - 170 °C at a heating rate of 15 - 25 °C / min, held for 1.5 - 2.5 min, then heated to 210 - 230 °C at a heating rate of 55 - 65 °C / min, and held for 1.5 - 2.5 min; The parameter conditions of the mass spectrometry 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; ion source temperature: 240 - 260 °C; The chromatographic column for the gas chromatography conditions is VF - WAXms, with a specification of 30 m × 0.25 mm, 0.25 μm; The preparation method of the test solution of pentoxifylline raw material drug is as follows: weigh 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 extract for 1 min, and take the supernatant to obtain; 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 min, and take the supernatant to obtain.

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

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

4. The method for detecting the content of genotoxic impurity 6-chloro-2-hexanone in pentoxifylline bulk drug and injection according to claim 1, wherein, The parameter conditions of the gas chromatography 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.

5. The detection method for the content of genotoxic impurity 6-chloro-2-hexanone in pentoxifylline bulk drug and injection according to claim 1, wherein, The parameter conditions of the gas chromatography 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.

6. The detection method for the content of the genotoxic impurity 6-chloro-2-hexanone in pentoxifylline bulk drug and injection according to claim 1, characterized in that, The parameter conditions of the mass spectrometry 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; ion source temperature: 245 - 255 °C.

7. The detection method for the content of genotoxic impurity 6-chloro-2-hexanone in pentoxifylline bulk drug and injection according to claim 6, characterized in that, The parameter conditions 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; ion source temperature: 250 °C.

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

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