Method for detecting genotoxic impurities in fudosteine

Abstract

The invention discloses a method for detecting genotoxic impurities in fudosteine, which adopts a gas chromatography-mass spectrometry method and comprises the following steps: taking a test sample, putting the test sample into a headspace bottle, adding a diluent for dissolving, and then adding ethyl acetate for headspace extraction to obtain an upper ethyl acetate phase as a test sample solution; the method comprises the following steps: diluting reference substances 1, 3-dichloropropane and chloropropanol with a diluent to prepare a mixed solution, putting the mixed solution into a headspace bottle, and adding ethyl acetate for headspace extraction to obtain an upper ethyl acetate phase as a reference substance solution; sampling and detecting the test solution and the reference solution respectively; the genotoxic impurities are 1, 3-dichloropropane and chloropropanol. The detection method provided by the invention has excellent specificity, solution stability, accuracy, precision and durability, the chromatographic condition can effectively remove interfering substances in dosteine, and effective separation of impurities and main component chromatographic peaks is realized.

Description

Technical Field

[0001] The present application relates to the field of chemical detection, and in particular to a method for detecting genotoxic impurities in fudosteine. Background Art

[0002] In modern drug development and production, ensuring the purity and safety of drug ingredients is a crucial link, especially for the control of impurities with potential genotoxicity. Fudosteine ​​is a drug widely used to treat respiratory diseases such as asthma and chronic obstructive pulmonary disease. During its production process, genotoxic impurities such as chloropropanol and 1,3-dichloropropane may be accidentally introduced due to factors such as impure raw materials, synthetic byproducts or improper storage conditions. These impurities can cause DNA damage even at extremely low concentrations, and long-term exposure may lead to genetic mutations or increased cancer risks, seriously threatening the health and safety of patients.

[0003] Traditional detection methods, such as liquid chromatography (HPLC) combined with ultraviolet or fluorescence detectors, perform well in the separation and analysis of many compounds, but have limited sensitivity for the detection of low-concentration genotoxic impurities and are susceptible to interference from the sample matrix. Gas chromatography (GC) can provide higher separation efficiency and sensitivity, but it is challenging to analyze thermally unstable substances and has limited qualitative capabilities when used alone.

[0004] Gas chromatography-mass spectrometry (GC-MS) combines the high-resolution separation capability of gas chromatography with the high sensitivity and strong qualitative capability of mass spectrometry, and has become a powerful tool for detecting trace impurities in complex samples. However, the direct application of GC-MS to detect chloropropanols and 1,3-dichloropropane in drugs such as fudosteine ​​still faces challenges, including how to effectively remove interfering substances in the sample, optimize chromatographic conditions to achieve effective separation of impurities and main components, and develop reliable quantitative methods. However, there are currently no reports on the application of GC-MS detection to the detection of genotoxic impurities in fudosteine. Summary of the invention

[0005] The present application provides a method for detecting genotoxic impurities in fudosteine, which can achieve efficient and precise determination of the contents of chloropropanol and 1,3-dichloropropane in fudosteine ​​by gas chromatography-mass spectrometry.

[0006] The present application provides a method for detecting genotoxic impurities in fudosteine, which uses gas chromatography-mass spectrometry: Place the test sample in a headspace bottle, add diluent to dissolve it, then add ethyl acetate for headspace extraction to obtain the upper ethyl acetate phase as the test sample solution; Take reference substances 1,3-dichloropropane and chloropropanol, dilute with a diluent to prepare a mixed solution, put the mixed solution into a headspace bottle, add ethyl acetate to perform headspace extraction, and obtain the upper ethyl acetate phase as the reference substance solution; Take the test solution and the reference solution for testing respectively; The genotoxic impurities are 1,3-dichloropropane and chloropropanol.

[0007] The detection method of the present application is to vortex mix the ethyl acetate extractant with the test sample to enrich volatile impurities such as 1,3-dichloropropane and chloropropanol in the extractant; when left at rest, the impurities in the solvent evaporate and diffuse into the headspace area of ​​the headspace bottle for gas chromatography detection.

[0008] Preferably, the diluent is a 2,3-dichloro-1-propanol aqueous solution with a concentration of 60 to 65 ng / mL.

[0009] The diluent used in the present application has similar physical and chemical properties to 1,3-dichloropropane and can be used as an internal standard to correct errors and improve the accuracy and reproducibility of the detection results of 1,3-dichloropropane.

[0010] Preferably, the content of 1,3-dichloropropane is calculated by an external standard method, and the content of chloropropanol is calculated by an internal standard method.

[0011] Preferably, in liquid chromatography detection, the chromatographic column is a polyethylene glycol column, a cyano column or an amino column, the injection mode is a split mode, the split ratio is 5:1, the injection volume is 1 μL, the injection port temperature is 220-250°C, the MS transmission line temperature is 220-250°C, and the column flow is 1-2 mL / min.

[0012] Preferably, the chromatographic column is a polyethylene glycol column.

[0013] Preferably, the split ratio is 4 to 6:1.

[0014] It should be noted that the "split ratio" refers to the ratio between the volume of sample gas entering the chromatographic column and the volume of gas being split (discharged).

[0015] Preferably, in liquid chromatography detection, the column temperature starts at 50-60°C, maintained for 3 minutes, increased to 120-140°C at a rate of 20-30°C per minute, maintained for 1 minute, increased to 240°C at a rate of 20-30°C per minute, and maintained for 3 minutes.

[0016] Preferably, in mass spectrometry detection, the ion source is an EI source, the ion source temperature is 230° C., and the solvent delay is 4 minutes.

[0017] Preferably, in mass spectrometry detection, the acquisition mode is SIM mode, 1,3-dichloropropane is collected from 4 to 6.5 minutes, m / z 76 is used as the quantitative ion, m / z 41, 63 and 78 are used as the qualitative ions, and the dwell time is 100 msec respectively; chloropropanol is collected from 6.5 to 8.8 minutes, m / z 58 is used as the quantitative ion, m / z 41 and 76 are used as the qualitative ions, and the dwell time is 100 msec respectively.

[0018] Preferably, in mass spectrometry detection, internal standard 2,3-dichloro-1-propanol is collected from 8.8 to the end, m / z 62 is used as the quantitative ion, m / z 64, 66 and 92 are used as the qualitative ions, and the dwell time is 100 msec respectively.

[0019] In summary, this application has the following beneficial effects: The detection method of the present application has excellent specificity, solution stability, accuracy, precision and durability. The chromatographic conditions can effectively remove interfering substances in the sample, realize the effective separation of impurities and main component chromatographic peaks, and have good linear correlation; the recovery rate of 1,3-dichloropropane at each concentration point is between 95.72% and 98.22%, and the RSD of the total recovery rate is ≤1.13%; the recovery rate of chloropropanol at each concentration point is between 98.31% and 104.73%, and the RSD of the total recovery rate is ≤2.46%. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the chromatogram of the test solution in the embodiment.

[0021] Figure 2 It is the chromatogram of the reference substance solution in the example.

[0022] Figure 3 This is a typical chromatogram of the limit of quantitation in Experiment 3.

[0023] Figure 4 This is the detection limit chromatogram of chloropropanol in experiment 3.

[0024] Figure 5 This is the detection limit chromatogram of 1,3-dichloropropane in Experiment 3.

[0025] Figure 6 This is the linear regression graph of the chloropropanol color in Experiment 4.

[0026] Figure 7 This is the linear regression plot of 1,3-dichloropropane from Experiment 4. DETAILED DESCRIPTION Example

[0027] A method for detecting genotoxic impurities in fudosteine, taking about 0.25 g of the test sample, accurately weighing, placing in a 20 mL headspace bottle, accurately adding 5 mL of diluent (containing an aqueous solution with a concentration of about 62.5 ng / mL of 2,3-dichloro-1-propanol), shaking to dissolve, accurately adding 3 mL of ethyl acetate, sealing with a cover, vortexing at 2500 rpm for 1 minute, standing for half an hour, taking the upper ethyl acetate phase as the test solution. Separately take an appropriate amount of 1,3-dichloropropane and chloropropanol reference substances, dilute with a diluent to prepare a mixed solution containing 62.5 ng of 1,3-dichloropropane and chloropropanol per 1 mL, accurately measuring 5 mL of the mixed solution and placing in a 20 mL headspace bottle, accurately adding 3 mL of ethyl acetate, sealing with a cover, vortexing at 2500 rpm for 1 minute, standing for half an hour, taking the upper ethyl acetate phase as the reference substance solution.

[0028] The determination was carried out according to gas chromatography (Chinese Pharmacopoeia General Chapter 0521) and mass spectrometry (Chinese Pharmacopoeia General Chapter 0431). Among them, the gas chromatography method used a chromatographic column (DB-WAX, 30m×0.25mm, 0.25μm) filled with polyethylene glycol; the injection was carried out in split mode, the split ratio was 6:1, the injection volume was 1μL, the injection port temperature was 220℃, the MS transmission line temperature was 220℃, the column flow was 1mL / min, the column temperature was 50℃, maintained for 3 minutes, increased to 130℃ at a rate of 30℃ per minute, maintained for 1 minute, increased to 240℃ at a rate of 20℃ per minute, and maintained for 3 minutes.

[0029] The mass spectrometry method uses an EI source, an ion source temperature of 230°C, a solvent delay of 4 minutes, and acquisition in SIM mode. 1,3-dichloropropane is collected from 4 to 6.5 minutes, with m / z 76 as the quantitative ion, m / z 41, 63 and 78 as the qualitative ions, and the dwell time is 100 msec respectively; chloropropanol is collected from 6.5 to 8.8 minutes, with m / z 58 as the quantitative ion, m / z 41 and 76 as the qualitative ions, and the dwell time is 100 msec respectively; from 8.8 to the end of the acquisition of the internal standard 2,3-dichloro-1-propanol, with m / z 62 as the quantitative ion, m / z 64, 66 and 92 as the qualitative ions, and the dwell time is 100 msec respectively. Take the test solution and the reference solution and inject them separately, and record the chromatogram (such as Figure 1 , Figure 2 As shown in the table), 1,3-dichloropropane was calculated using the external standard method, and chloropropanol was calculated using the internal standard method.

[0030] See also Figure 1 , Figure 2 It can be seen that 1,3-dichloropropane and chloropropanol were not detected in the test solution of this application. Verification test

[0031] Test 1: System suitability Before extraction: 2,3-Dichloro-1-propanol stock solution: Take about 20 mg of 2,3-dichloro-1-propanol, accurately weigh it, put it into a 10 mL volumetric flask, dilute it to the scale with isopropanol, shake well, and use it as the 2,3-dichloro-1-propanol stock solution.

[0032] Dilution: Accurately measure 156.2 μL of 2,3-dichloro-1-propanol stock solution and place it in a 50 mL volumetric flask, dilute to the scale with pure water, and shake well; accurately measure 5 mL and place it in a 500 mL volumetric flask, dilute to the scale with pure water, and shake well.

[0033] 1,3-Dichloropropane stock solution: Take about 20 mg of 1,3-dichloropropane, accurately weigh it, put it into a 10 mL volumetric flask, dilute it to the scale with diluent, shake well, and use it as the 1,3-dichloropropane stock solution.

[0034] Chloropropanol stock solution: Take about 20 mg of chloropropanol, weigh accurately, place in a 10 mL volumetric flask, dilute to the scale with diluent, shake well, and use as the chloropropanol stock solution.

[0035] Reference substance stock solution: Accurately measure 0.781 mL of 1,3-dichloropropane stock solution and 0.781 mL of chloropropanol stock solution and place them in the same 50 mL volumetric flask, dilute to the scale with the diluent, and shake well; Accurately measure 1 mL and place it in a 50 mL volumetric flask, dilute to the scale with the diluent, and shake well.

[0036] Reference solution: Accurately measure 5 mL of reference stock solution and place it in a 50 mL volumetric flask, dilute to the scale with diluent, and shake well.

[0037] extraction: Blank solution: Accurately measure 5 mL of the diluent and place it in a headspace bottle. Accurately add 3 mL of ethyl acetate, cover and seal, vortex at 2500 rpm for 1 minute, let stand for half an hour, and take the upper ethyl acetate phase.

[0038] Reference solution: Place 5 mL of the reference solution before precision extraction in a headspace bottle, accurately add 3 mL of ethyl acetate, cover and seal, vortex at 2500 rpm for 1 minute, let stand for half an hour, and take the upper ethyl acetate phase.

[0039] Six injections of the extracted reference solution were made to examine the peak area of ​​1,3-dichloropropane and the relative correction factor (RSD%) of chloropropanol.

[0040] Requirements: For 6 consecutive calibration curves of the reference solution, the RSD of the 1,3-dichloropropane peak area and the relative correction factor of chloropropanol should be ≤20.0%.

[0041] Results: For 6 consecutive tests on the reference solution, the RSD of the peak area of ​​1,3-dichloropropane was 1.17%, and the RSD of the relative correction factor of chloropropanol was 2.11%. The results are shown in Tables 1 and 2.

[0042] Table 1. 1,3-Dichloropropane system suitability test results

[0043] Table 2. Test results of chloropropanol system suitability

[0044] Conclusion: This method has good system applicability.

[0045] Test 2: Exclusivity Solution preparation: Blank solution and reference solution: Take from the solution prepared in Test 1.

[0046] Before extraction: Test solution: Take about 0.25g of the product, weigh accurately, place in a 20mL headspace bottle, accurately add 5mL of diluent, shake to dissolve, and obtain.

[0047] 100% limit spiked test solution: Take about 0.25g of this product, weigh accurately, place in a 20mL headspace bottle, accurately add 5mL of reference solution, shake to dissolve, and obtain.

[0048] extraction: Test solution: Take the test solution before extraction, accurately add 3 mL of ethyl acetate, seal with a lid, vortex at 2500 rpm for 1 minute, let stand for half an hour, and take the upper ethyl acetate phase.

[0049] 100% limit spiked test solution: Take the spiked test solution before extraction, accurately add 3 mL of ethyl acetate, seal with a lid, vortex at 2500 rpm for 1 minute, let stand for half an hour, and take the upper ethyl acetate phase.

[0050] Take the blank solution after extraction, the reference solution, the test solution, and the 100% limit spiked test solution and inject them respectively, and record the chromatograms.

[0051] Requirements: The blank solution should have no interference; the test solution should have no interference with the detection of chloropropanol and 1,3-dichloropropane.

[0052] Results: The blank solution and the test solution had no interference with the detection of chloropropanols and 1,3-dichloropropane.

[0053] Conclusion: This method has good specificity.

[0054] Experiment 3: Limit of Quantitation and Limit of Detection Take the reference stock solution, prepare the quantification limit solution and the detection limit solution, take the concentration when S / N≥3 as the detection limit, take the concentration when S / N≥10 as the quantification limit, prepare 6 quantification limit solutions in parallel. Take the quantification limit solution and the detection limit solution and inject them separately, and record the chromatogram.

[0055] Requirements: The S / N of the chromatographic peak at the detection limit concentration is ≥3; the S / N of the chromatographic peak at the quantification limit concentration is ≥10, and the RSD of the peak area of ​​1,3-dichloropropane and the RSD of the relative correction factor of chloropropanol at the 6 quantitative limit concentrations are both ≤20.0%.

[0056] Results: The 10% limit concentration is the detection limit of 1,3-dichloropropane, and the 25% limit concentration is the detection limit of chloropropanol. At the detection limit concentration, the S / N of chloropropanol is 7.30, and the S / N of 1,3-dichloropropane is 13.27. The 50% limit concentration is the quantitative limit, and the S / N of chloropropanol is 35.12 and the S / N of 1,3-dichloropropane is 42.37 at the quantitative limit concentration; the relative correction factor RSD of chloropropanol in the six quantitative limit solutions is 17.67%, and the peak area RSD of 1,3-dichloropropane is 11.80%; the results are detailed in Tables 3, 4, 5, 6 and Figure 3 , Figure 4 , Figure 5 .

[0057] Table 3. Results of the quantitative limit and detection limit test for 1,3-dichloropropane

[0058] Table 4. Results of repeatability test of quantitation limit of 1,3-dichloropropane

[0059] Table 5. Results of the quantification limit and detection limit tests for chloropropanol

[0060] Table 6. Results of repeatability test on quantification limit of chloropropanol

[0061] Conclusion: The percentage content corresponding to the quantitative limit of chloropropanol and 1,3-dichloropropane in this method is 0.625ppm, the percentage content corresponding to the detection limit of chloropropanol is 0.313ppm, and the percentage content corresponding to the detection limit of 1,3-dichloropropane is 0.125ppm, which can meet the detection sensitivity requirements.

[0062] Experiment 4: Linear Correlation Accurately measure an appropriate amount of reference substance stock solution, prepare 200% limit, 150% limit, 100% limit, 75% limit, 50% limit and LOQ concentration solutions respectively, process them according to the extraction method, and take the ethyl acetate phase for sampling and analysis.

[0063] Solution preparation: 200% linear solution: Accurately measure 2 mL of the reference solution and place it in a 10 mL volumetric flask. Dilute to the scale with the diluent and shake well.

[0064] 150% linear solution: Accurately measure 1.5mL of reference solution and place it in a 10mL volumetric flask. Dilute to the scale with diluent and shake well.

[0065] 100% linear solution: Accurately measure 1 mL of the reference solution and place it in a 10 mL volumetric flask. Dilute to the scale with the diluent and shake well.

[0066] 75% linear solution: Accurately measure 0.75mL of reference solution and place it in a 10mL volumetric flask. Dilute to the scale with diluent and shake well.

[0067] 50% linear solution: Accurately measure 0.5mL of reference solution and place it in a 10mL volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0068] LOQ solution: Prepared according to the concentration in Test 3.

[0069] extraction: Accurately measure 5 mL of each linear solution and place it in a headspace bottle. Accurately add 3 mL of ethyl acetate. Vortex at 2500 rpm for 1 min. Let stand for half an hour and take the upper ethyl acetate phase.

[0070] Take the above extracted linear solutions and inject them respectively, record the chromatograms. Linearly regress the chloropropanol peak area ratio (y) to the solution concentration ratio (x), and linearly regress the 1,3-dichloropropane peak area (y) to the solution concentration (x) to obtain the regression equation, correlation coefficient and intercept ratio.

[0071] Requirements: Linear correlation coefficient r≥0.990; 1,3-dichloropropane unit concentration peak area response value RSD≤20.0%; chloropropanol relative correction factor RSD≤20.0%; the ratio of the absolute value of the intercept to the 100% limit ≤25.0%.

[0072] Results: The linear correlation coefficient r of 1,3-dichloropropane was 0.9903, the RSD of the peak area response per unit concentration was 10.43%, and the ratio of the absolute value of the intercept to the 100% limit response was 13.8%; the linear correlation coefficient r of chloropropanol was 0.9930, the RSD of the relative correction factor was 7.26%, and the ratio of the absolute value of the intercept to the 100% limit response was 3.16%. The results are shown in Tables 7, 8 and Figure 6 , Figure 7 .

[0073] Table 7. 1,3-Dichloropropane linear test results

[0074] Table 8. Chloropropanol linearity test results

[0075] Conclusion: This method has good linearity in the concentration range of 31.96 ng / mL to 127.85 ng / mL for chloropropanol and 29.96 ng / mL to 119.85 ng / mL for 1,3-dichloropropane.

[0076] Test 5: Accuracy Take this product and design spiked samples at three concentrations of 50%, 100%, and 150% of the quantitative limit and limit concentration, with three copies for each concentration, totaling n=9.

[0077] Solution preparation: 2,3-Dichloro-1-propanol stock solution: Take about 20 mg of 2,3-dichloro-1-propanol, accurately weigh it, put it into a 10 mL volumetric flask, dilute it to the scale with isopropanol, shake well, and use it as the 2,3-dichloro-1-propanol stock solution.

[0078] Dilution: Accurately measure 156.2 μL of 2,3-dichloro-1-propanol stock solution and place it in a 50 mL volumetric flask, dilute to the scale with pure water, and shake well; accurately measure 5 mL and place it in a 500 mL volumetric flask, dilute to the scale with pure water, and shake well.

[0079] Blank solution: Accurately measure 5 mL of the diluent and place it in a headspace bottle. Accurately add 3 mL of ethyl acetate, cover and seal, vortex at 2500 rpm for 1 minute, let stand for half an hour, and take the upper ethyl acetate phase.

[0080] 1,3-Dichloropropane stock solution: Take about 20 mg of 1,3-dichloropropane, accurately weigh it, put it into a 10 mL volumetric flask, dilute it to the scale with diluent, shake well, and use it as the 1,3-dichloropropane stock solution.

[0081] Chloropropanol stock solution: Take about 20 mg of chloropropanol, weigh accurately, place in a 10 mL volumetric flask, dilute to the scale with diluent, shake well, and use as the chloropropanol stock solution.

[0082] Reference substance stock solution: Accurately measure 0.781 mL of 1,3-dichloropropane stock solution and 0.781 mL of chloropropanol stock solution and place them in the same 50 mL volumetric flask, dilute to the scale with the diluent, and shake well; Accurately measure 1 mL and place it in a 50 mL volumetric flask, dilute to the scale with the diluent, and shake well.

[0083] Reference substance solution: Accurately measure 5 mL of reference substance stock solution and place it in a 50 mL volumetric flask, dilute the diluent to the scale, shake well, accurately measure 5 mL and place it in a headspace bottle, accurately add 3 mL of ethyl acetate, cover and seal, vortex at 2500 rpm for 1 minute, let stand for half an hour, and take the upper ethyl acetate phase.

[0084] 50% limit spike solution: Accurately measure 2.5mL of reference substance stock solution and place it in a 50mL volumetric flask. Dilute to the scale with diluent and shake well.

[0085] 100% limit spike solution: Accurately measure 5 mL of reference stock solution and place it in a 50 mL volumetric flask, dilute to the scale with diluent, shake well, and the solution is ready.

[0086] 150% limit spike solution: Accurately measure 7.5mL of reference substance stock solution and place it in a 50mL volumetric flask. Dilute to the scale with diluent and shake well.

[0087] Test solution: Take about 0.25g of the product, weigh accurately, place in a 20mL headspace bottle, accurately add 5mL of diluent, shake to dissolve, accurately add 3mL of ethyl acetate, cover and seal, vortex at 2500rpm for 1 minute, let stand for half an hour, take the upper ethyl acetate phase, and obtain.

[0088] 50% limit spiked test solution: Take about 0.25g of this product, weigh accurately, place in a 20mL headspace bottle, accurately add 5mL of 50% limit spiked solution, shake to dissolve, accurately add 3mL of ethyl acetate, cover and seal, vortex at 2500rpm for 1 minute, let stand for half an hour, take the upper ethyl acetate phase, and prepare three times in parallel.

[0089] 100% limit spiked test solution: Take about 0.25g of this product, weigh accurately, place in a 20mL headspace bottle, accurately add 5mL of 100% limit spiked solution, shake to dissolve, accurately add 3mL of ethyl acetate, cover and seal, vortex at 2500rpm for 1 minute, let stand for half an hour, take the upper ethyl acetate phase, and prepare three times in parallel.

[0090] 150% limit spiked test solution: Take about 0.25g of this product, weigh accurately, place in a 20mL headspace bottle, accurately add 5mL of 150% limit spiked solution, shake to dissolve, accurately add 3mL of ethyl acetate, cover and seal, vortex at 2500rpm for 1 minute, let stand for half an hour, take the upper ethyl acetate phase, and prepare three replicates in parallel.

[0091] Take blank solution, reference solution, test solution and test solution spiked with different limits, determine according to the law, and calculate the recoveries of impurities 1,3-dichloropropane and chloropropanol by external standard method and internal standard method respectively.

[0092] Requirements: The recovery rate should be between 70% and 130%; the RSD of three replicate recoveries at each concentration point and the RSD of the total recovery rate should be ≤20.0%.

[0093] Results: The recovery rates of 1,3-dichloropropane at each concentration point were between 95.72% and 98.22%, and the RSD of the three recoveries at each concentration point and the RSD of the total recovery rate were both ≤1.13%; the recovery rates of chloropropanol at each concentration point were between 98.31% and 104.73%, and the RSD of the three recoveries at each concentration point and the RSD of the total recovery rate were both ≤2.46%. The results are shown in Tables 9 and 10.

[0094] Table 9. 1,3-Dichloropropane accuracy test results

[0095] Table 10. Chloropropanol accuracy test results

[0096] Conclusion: This method has good accuracy.

[0097] Test 6: Precision 6.1 Repeatability Prepare reference solution and test solution, prepare 6 portions of 100% limit spiked test solution under the accuracy item, determine according to the law, and calculate the recovery, average recovery and RSD of the recovery of the 6 portions of 100% limit spiked test solution.

[0098] Requirements: The recovery rate of 6 100% limit spiked test solutions should be between 70% and 130%; the recovery rate RSD should be ≤20.0%.

[0099] Results: The recoveries of 1,3-dichloropropane in the 6 100% limit spiked test solutions were all between 93.95% and 97.98%, with an RSD of 1.91%; the recoveries of chloropropanols were all between 99.47% and 104.68%, with an RSD of 1.78%. The results are shown in Tables 11 and 12.

[0100] Table 11. 1,3-Dichloropropane repeatability test results

[0101] Table 12. Chloropropanol repeatability test results

[0102] Conclusion: This method has good reproducibility.

[0103] 6.2 Intermediate Precision At different times and with different personnel, repeat the test operation. Prepare 6 portions of reference solution, test solution and 100% limit spiked test solution according to the law, and calculate the recovery rate, average recovery rate and RSD of the recovery rate of 1,3-dichloropropane and chloropropanol in the 6 portions of 100% limit spiked test solution.

[0104] Requirements: The recovery rate of 6 samples of 100% limit spiked test solution should be between 70% and 130%; the recovery rate RSD of 6 samples of 100% limit spiked test solution should be ≤20.0%. The total recovery rate RSD of 12 samples of 100% limit spiked test solution should be ≤20.0%.

[0105] Results: The recoveries of 1,3-dichloropropane in the 6 100% limit spiked test solutions were between 93.61% and 98.52%, with an RSD of 1.72%; the recoveries of chloropropanol were between 99.93% and 104.67%, with an RSD of 1.76%. The RSDs of the recoveries of 1,3-dichloropropane and chloropropanol in the 12 100% limit spiked test solutions were 1.74% and 1.68%, respectively. The results are shown in Tables 13, 14, 15, and 16.

[0106] Table 13. Results of intermediate precision test of 1,3-dichloropropane

[0107] Table 14. Results of intermediate precision test of chloropropanol

[0108] Table 15. 1,3-Dichloropropane precision test results

[0109] Table 16. Chloropropanol precision test results

[0110] Conclusion: This method has good precision.

[0111] The above verification shows that this method meets the requirements of various verification indicators and is suitable for the detection of genotoxic impurities 1,3-dichloropropane and chloropropanol in fudosteine.

[0112] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for detecting genotoxic impurities in fudosteine, characterized in that: Using gas chromatography-mass spectrometry: Place the sample in a headspace bottle, add diluent to dissolve it, then add ethyl acetate for headspace extraction to obtain the upper ethyl acetate phase as the sample solution; Take reference substances 1,3-dichloropropane and chloropropanol, dilute with a diluent to prepare a mixed solution, put the mixed solution into a headspace bottle, add ethyl acetate to perform headspace extraction, and obtain the upper ethyl acetate phase as the reference substance solution; Take the test solution and the reference solution for testing respectively; The genotoxic impurities are 1,3-dichloropropane and chloropropanol.

2. The detection method according to claim 1, characterized in that: The diluent is a 2,3-dichloro-1-propanol aqueous solution with a concentration of 60-65 ng / mL.

3. The detection method according to claim 1, characterized in that: The content of 1,3-dichloropropane was calculated by the external standard method, and the content of chloropropanol was calculated by the internal standard method.

4. The detection method according to claim 1, characterized in that: In liquid chromatography detection, the chromatographic column is a polyethylene glycol column, a cyano column or an amino column, the injection mode is a split mode, the split ratio is 5:1, the injection volume is 1 μL, the injection port temperature is 220-250°C, the MS transmission line temperature is 220-250°C, and the column flow is 1-2 mL / min.

5. The detection method according to claim 4, characterized in that: The chromatographic column is a polyethylene glycol column.

6. The detection method according to claim 4, characterized in that: The split ratio is 4 to 6:

1.

7. The detection method according to claim 1, characterized in that: In liquid chromatography detection, the column temperature starts at 50-60°C, maintained for 3 minutes, increased to 120-140°C at a rate of 20-30°C per minute, maintained for 1 minute, increased to 240°C at a rate of 20-30°C per minute, and maintained for 3 minutes.

8. The detection method according to claim 1, characterized in that: In mass spectrometry detection, the ion source is an EI source, the ion source temperature is 220 to 240° C., and the solvent delay is 3 to 5 minutes.

9. The detection method according to claim 1, characterized in that: In mass spectrometry detection, the acquisition mode was SIM mode, 1,3-dichloropropane was collected from 4 to 6.5 minutes, m / z 76 was used as the quantitative ion, m / z 41, 63 and 78 were used as the qualitative ions, and the dwell time was 100 msec respectively; chloropropanol was collected from 6.5 to 8.8 minutes, m / z 58 was used as the quantitative ion, m / z 41 and 76 were used as the qualitative ions, and the dwell time was 100 msec respectively.

10. The detection method according to claim 1, characterized in that: In mass spectrometry detection, internal standard 2,3-dichloro-1-propanol was collected from 8.8 to the end, with m / z 62 as the quantitative ion, and m / z 64, 66, and 92 as the qualitative ions, and the dwell time was 100 msec, respectively.

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