Method for detecting nitrosamine impurities in levosalbutamol hydrochloride aerosol inhalation solution
The atomized inhalation solution of levosalbutamol hydrochloride was detected by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS/MS), which solved the problem of difficulty in detecting nitrosamine impurities in the prior art, and achieved efficient and accurate detection results.
Patent Information
- Application Number
- CN202411862042.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-17
- Publication Date
- 2025-03-21
AI Technical Summary
The prior art is difficult to quickly and accurately detect the nitrosamine impurity N-nitroso-albutamol in the atomized inhalation solution of levosalbutamol hydrochloride, and the detection efficiency is low.
Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS/MS) was used to detect the atomized inhalation solution of levolasbutamol hydrochloride. Through specific chromatography and mass spectrometry conditions, the rapid and accurate detection of N-nitroso-albutamol was achieved.
The rapid and accurate detection of the nitrosamine impurity N-nitroso-albutamol in the atomized inhalation solution of levosalbutamol hydrochloride was achieved, with the detection limit as low as 0.4% of the limit and the quantitative limit as low as 1.2% of the limit. The detection can be completed in only 10 minutes, improving the detection efficiency and accuracy.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of analysis and detection, and relates to a method for detecting nitrosamine impurities in a levosalbutamol hydrochloride atomized inhalation solution, and specifically relates to a UPLC-MS / MS method for detecting nitrosamine impurities N-nitroso-salbutamol in a levosalbutamol hydrochloride atomized inhalation solution. Background Art
[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.
[0003] Levalbuterol Hydrochloride has the following chemical structure: .
[0004] Levosalbutamol hydrochloride nebulized inhalation solution is a short-acting β2 receptor agonist used to treat or prevent bronchospasm caused by reversible airway obstructive disease. Nitrosamine compounds are known high-risk carcinogens. According to the latest FDA regulations, the recommended acceptable intake of N-nitrososalbutamol impurities is 1500 ng / day, and the maximum daily dose of levosalbutamol hydrochloride nebulized inhalation solution is 3.75 mg / day. The limit of nitrosamine impurities is calculated to be no more than 0.04%. After consulting the pharmacopoeia standards for salbutamol raw materials and related preparations, none of them contain the detection method of N-nitrososalbutamol impurities. After literature search, there is only a detection method for N-nitrososalbutamol impurities in levosalbutamol hydrochloride raw materials (patent CN116840386A). This method uses an ultraviolet detector, and the limit of quantification is equivalent to 19.30% of the limit, which does not reach below 10% of the limit. In addition, the analysis time of the method is 70 minutes, which is less efficient. Currently, no reports on the detection method of nitrosamine impurities in salbutamol hydrochloride preparations have been found.
[0005] Therefore, the rapid and accurate detection of the nitrosamine impurity N-nitroso-salbutamol in the nebulized inhalation solution of levosalbutamol hydrochloride is of great significance for the quality control of the nebulized inhalation solution of levosalbutamol hydrochloride and ensuring the safety of medication. Summary of the invention
[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for detecting nitrosamine impurities in a nebulized inhalation solution of salbutamol hydrochloride. The detection limit of the detection method is equivalent to 0.4% of the limit, and the quantitative limit is equivalent to 1.2% of the limit, thereby realizing rapid and accurate detection of the nitrosamine impurity N-nitroso-salbutamol in the nebulized inhalation solution of salbutamol hydrochloride.
[0007] In order to achieve the above object, the present invention is implemented through the following technical solutions: A method for detecting nitrosamine impurities in a levosalbuterol hydrochloride atomized inhalation solution comprises the following steps: The levosalbuterol hydrochloride nebulized inhalation solution was diluted to obtain a test solution; The test solution is detected by ultra-high performance liquid chromatography-mass spectrometry; The chromatographic conditions were as follows: the filler of the chromatographic column was octadecylsilane bonded silica gel; Mobile phase A is a formic acid aqueous solution, wherein the formic acid concentration is 0.09-0.11%, % is the volume percentage; Mobile phase B was acetonitrile; The nitrosamine impurity is N-nitroso-salbutamol.
[0008] 0.09-0.11% formic acid aqueous solution is a solution containing 0.9~1.1 mL of formic acid per 1000 mL of solution.
[0009] The structure of N-nitroso-salbutamol is as follows: .
[0010] In some embodiments, during the detection process, the mass spectrometry conditions are: the ion source is an electrospray ion source, the scanning mode is a negative ion mode, and the detection mode is multiple reaction detection MRM.
[0011] In some embodiments, the diluent for diluting the levosalbutamol hydrochloride nebulized inhalation solution is a methanol-water solution.
[0012] Preferably, the volume percentage of methanol in the diluent is 25-35%, preferably 30%.
[0013] In some embodiments, the concentration of salbutamol hydrochloride in the test solution is 200-300 ng / mL, preferably 252 g / mL.
[0014] In some embodiments, the volume percentage of formic acid in mobile phase A is 0.1%.
[0015] In some embodiments, the gradient elution program is: From 0 to 3 min, mobile phase A was reduced from 95% to 70%, and mobile phase B was increased from 5% to 30%; 3-6 min, mobile phase A was reduced from 70% to 10%, and mobile phase B was increased from 30% to 90%; 8-8.1 min, mobile phase A increased from 10% to 95%, and mobile phase B decreased from 90% to 5%; 8.1~10 min, mobile phase A 95%, mobile phase B 5%, the above % are volume percentages.
[0016] In some embodiments, the chromatographic conditions are: the chromatographic column is filled with octadecylsilane bonded silica gel; the chromatographic column is Hypersil GOLD, 100 mm×2.1 mm, 1.9 μm; 0.1% formic acid aqueous solution is used as mobile phase A, acetonitrile solution is used as mobile phase B, and gradient elution is performed; the flow rate is 0.4 mL / min, the column temperature is 45°C, the injection volume is 10 μL, and the injector temperature is 4°C.
[0017] In some embodiments, the mass spectrometer is a triple quadrupole mass spectrometer, and after gradient elution, triple quadrupole mass spectrometer detection is used.
[0018] Preferably, the mass spectrometry conditions are: IS voltage is -4500 V; curtain gas CUR is 25 psi; nebulizer GAS1 is 55 psi; auxiliary gas GAS2 is 20 psi; ion source temperature is 550°C; collision gas: 8. The above mass spectrometry conditions are the optimal technical conditions in the technical solution of the present invention. Under these conditions, the detection effect is good. Those skilled in the art can make corresponding adjustments according to actual needs to obtain excellent detection effects.
[0019] The beneficial effects achieved by one or more embodiments of the present invention are as follows: The present invention provides a method for detecting nitrosamine impurity N-nitroso-salbutamol in a nebulized inhalation solution of levosalbutamol hydrochloride. The present invention proposes for the first time to use ultra-high performance liquid chromatography-mass spectrometry to analyze and detect the nitrosamine impurity N-nitroso-salbutamol in a nebulized inhalation solution of levosalbutamol hydrochloride, thereby providing a strong guarantee for effectively controlling the quality of drugs and ensuring the safety of drugs.
[0020] The ultra-high performance liquid chromatography tandem mass spectrometry technology of the present invention is used to detect the nitrosamine impurity N-nitroso-salbutamol mist in the levosalbutamol hydrochloride atomized inhalation solution, with high selectivity and good method specificity. The quantitative limit is as low as 1.2% of the limit, and the detection limit is as low as 0.4% of the limit. The detection can be completed in only 10 minutes. The method has high sensitivity and can quickly and accurately detect the content of trace nitrosamine impurity N-nitroso-salbutamol in the sample, thereby effectively reducing the analysis cost.
[0021] The present invention also verifies the accuracy of nitrosamine impurities in the range of 50% to 150% of the limit concentration (0.04%), and the recovery rate of N-nitroso-salbutamol is between 90% and 108%, with good accuracy; the linearity of N-nitroso-salbutamol in the range of 1.2% to 200% of the limit concentration is also verified, and N-nitroso-salbutamol has r>0.999 in the above range, with good linearity. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.
[0023] Figure 1 This is the blank solution spectrum of Example 1 of the present invention; Figure 2 The N-nitroso-salbutamol impurity reference solution spectrum of Example 1 of the present invention; Figure 3 The sample solution spectrum of Example 1 of the present invention; Figure 4 This is the system applicability solution spectrum of Example 1 of the present invention. DETAILED DESCRIPTION
[0024] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.
[0025] The mass spectrometry conditions are as follows: the mass spectrometer is a triple quadrupole mass spectrometer, the ion source is an electrospray ion source, the scanning mode is a negative ion mode, and the detection mode is a multiple reaction detection MRM; the IS voltage is -4500 V; the curtain gas CUR is 25 psi; the nebulizer GAS1 is 55 psi; the auxiliary gas GAS2 is 20 psi; the ion source temperature is 550°C; the collision gas: 8; for qualitative detection, Q1 is 267.0Da, Q3 is 218.9Da, DP is -87V, and CE is -23V; for quantitative detection, Q1 is 267.0 Da, Q3 is 151.0 Da, DP is -88 V, and CE is -31V.
[0026] In quantitative detection, Q1 was 267.0Da, Q3 was 151.0Da, DP was -88V, and CE was -31V.
[0027] It should be noted that the content of trace N-nitroso-salbutamol in the salbutamol hydrochloride nebulized inhalation solution can be calculated by the external standard method, and the calculation formula is as follows: N-nitroso-salbutamol (%) = ×100 Where: A U --N-nitroso-salbutamol peak area in the test solution; A S --N-nitroso-salbutamol peak area in reference solution; C S --N-nitroso-salbutamol concentration in reference solution, ng / mL; C U --Concentration of the test sample solution calculated based on the labeled amount (in terms of salbutamol), ng / mL.
[0028] The present invention will be further described below in conjunction with the embodiments.
[0029] Example 1: Specificity 1-1 Solution Preparation The molecular formula of N-nitroso-salbutamol is C 13 H 20 N2O4, the reference substance was purchased from ANTU Company, Cat No.: S230563.
[0030] Dilution solution (blank solution): Measure 300 mL of methanol and 700 mL of water and mix well.
[0031] N-nitroso-salbutamol impurity reference solution: Accurately weigh an appropriate amount of N-nitroso-salbutamol impurity reference, dissolve it in diluent and quantitatively dilute it to make a 10 ng / mL solution.
[0032] Test solution: Take an appropriate amount of salbutamol hydrochloride nebulizer inhalation solution, dissolve it in diluent and quantitatively dilute it to make a solution containing approximately 25 μg of salbutamol per 1 mL.
[0033] System suitability solution: Take an appropriate amount of salbutamol hydrochloride nebulized inhalation solution, add an appropriate amount of N-nitroso-salbutamol impurity, and dilute with diluent to prepare a mixed solution containing approximately 25 μmg of salbutamol and 10 ng of N-nitroso-salbutamol per 1 mL.
[0034] 1-2 Chromatographic and mass spectrometry conditions Column: Hypersil GOLD, 100 mm × 2.1 mm, 1.9 μm; Mobile Phase: Mobile phase A was 0.1% formic acid in water; Mobile phase B was acetonitrile; Elution method: gradient elution;
[0035] Flow rate: 0.4 mL / min; The column temperature was 45°C; The injection volume was 10 μL; The injector temperature was 4 °C.
[0036] Ion source: ESI source, negative ion scanning; Scanning mode: MRM acquisition mode; IS voltage: 4500 V (-); Curtain gas CUR is 25 psi; Nebulizer GAS1 is 55 psi; Auxiliary gas GAS2 is 20 psi; Ion source temperature is 550°C; Collision gas: 8; Mass spectrometry parameters
[0037] 1-3 Experimental conclusion: Accurately measure the above solution, inject it into the ultra-high performance liquid chromatography-mass spectrometer, and record the chromatogram. The results are shown in Table 1, and the blank solution spectrum is shown in Figure 1 , N-nitroso-salbutamol impurity reference solution see Figure 2 , test solution see Figure 3 , System Suitability Solutions See Figure 4 .
[0038] Table 1 System suitability and impurity localization results
[0039] Conclusion: The blank solvent and the test solution do not interfere with the detection of N-nitroso-salbutamol impurities, and the specificity of the present invention is good.
[0040] Example 2: Linearity and range experiments 2-1 Solution preparation Accurately weigh an appropriate amount of N-nitroso-salbutamol impurity reference substance and prepare a series of concentration linearity test solutions with concentrations ranging from the quantitative limit (1.2% in Example 3) to about 200%.
[0041] 2-2 Chromatographic and mass spectrometric conditions are the same as those in Example 1.
[0042] 2-3 Conclusion: The above solution was accurately measured and injected into an ultra-high performance liquid chromatography-mass spectrometer to record the chromatogram. The results are shown in Table 2. A linear regression equation was made with concentration as the abscissa and peak area as the ordinate.
[0043] Table 2 N-nitroso-salbutamol impurity linearity test results
[0044] Conclusion: Within the determination range (equivalent to 1.2%-200% of the limit), the linear relationship of N-nitroso-salbutamol impurity is good and meets the methodological requirements.
[0045] Example 3: Limit of Quantitation and Limit of Detection 3-1 Solution preparation The linear solution in Example 2 was diluted step by step, and the concentration when the signal-to-noise ratio S / N≈3 was taken as the detection limit, and the concentration when S / N≈10 was taken as the quantification limit.
[0046] 3-2 Chromatographic and mass spectrometric conditions are the same as those in Example 1.
[0047] 3-3 Conclusion: The above solution was accurately measured and injected into the ultra-high performance liquid chromatography-mass spectrometer to record the chromatogram. The results are shown in Table 3.
[0048] Table 3 Results of limit of quantitation and limit of detection tests
[0049] Conclusion: The quantitative limit of N-nitroso-salbutamol impurity is lower than 10% of the impurity limit of this product. The detection limit (equivalent to 0.4% of the limit) and the quantitative limit (equivalent to 1.2% of the limit) of the present invention both meet the requirements for quantitative detection of N-nitroso-salbutamol impurities in the test sample, and the method has high sensitivity.
[0050] Example 4: Accuracy Experiment The accuracy was investigated through recovery test, and the study covered the limit concentration range of 50%~150%.
[0051] 4-1 Solution preparation N-nitroso-salbutamol impurity stock solution (100 μg / mL): Take an appropriate amount of N-nitroso-salbutamol impurity reference substance, accurately weigh it, add 30% methanol to dissolve it and dilute it to make a 100 μg / mL solution.
[0052] N-nitroso-salbutamol impurity stock solution (10 μg / mL): Accurately measure 1 mL of N-nitroso-salbutamol impurity stock solution (100 μg / mL), place it in a 10 mL volumetric flask, dilute to the scale with 30% methanol, and shake well.
[0053] N-nitroso-salbutamol impurity stock solution (100 ng / mL): Accurately measure 1 mL of N-nitroso-salbutamol impurity stock solution (10 μg / mL), place it in a 100 mL volumetric flask, dilute to the scale with 30% methanol, and shake well.
[0054] Reference solution: Accurately measure 1 mL of N-nitroso-salbutamol impurity stock solution (100 ng / mL), place in a 10 mL volumetric flask, dilute to the mark with 30% methanol, and shake well.
[0055] Accuracy solution: Accurately measure 1.2 ml of levosalbutamol hydrochloride nebulized inhalation solution (3 ml: 0.63 mg) into 10 mL volumetric flasks, accurately measure 0.5 mL, 1.0 mL and 1.5 mL of N-nitroso-salbutamol impurity stock solution (100 ng / mL), respectively, dissolve and dilute to the scale with diluent, shake well, and prepare 3 replicates in parallel for each spiked concentration level.
[0056] 4-2 Chromatographic and mass spectrometric conditions are the same as those in Example 1.
[0057] 4-3 Conclusion: The above solution was accurately measured and injected into the ultra-high performance liquid chromatography-mass spectrometer. The chromatogram was recorded and calculated by the peak area according to the external standard method. The accuracy results are shown in Table 4.
[0058] Table 4 Accuracy test results
[0059] Conclusion: Under 50%, 100% and 150% accuracy solutions, the recoveries of N-nitroso-salbutamol impurities were between 90% and 108%, with an RSD of 1.35%. The test results showed that the accuracy of the present invention was good.
[0060] Example 5: Precision Experiment 5-1 Solution preparation Accurately measure 1.2 ml of salbutamol hydrochloride nebulized inhalation solution, place it in a 10 mL volumetric flask, add 1.0 mL of N-nitroso-salbutamol impurity stock solution (100 ng / mL), add diluent to dissolve and dilute to the scale, shake well, and prepare 6 copies in parallel.
[0061] Control solution: same as Example 4.
[0062] 5-2 Chromatographic and mass spectrometric conditions are the same as those in Example 1.
[0063] 5-3 Conclusion: Accurately measure the above solution, inject it into the ultra-high performance liquid chromatography-mass spectrometer, record the chromatogram, calculate the impurity content, and the precision results are shown in Table 5.
[0064] Table 5 Precision test results
[0065] Conclusion: The test results of 6 replicate sample solutions prepared in parallel showed that the RSD value of N-nitroso-salbutamol impurity was less than 10%, which proved that the present invention had good repeatability.
[0066] Example 6: Solution stability experiment 6-1 Solution preparation Take the test solution and the reference solution in Example 1, place them in a sample injector at 4°C, and inject and test them at different time points.
[0067] 6-2 Chromatographic and mass spectrometric conditions are the same as those in Example 1.
[0068] 6-3 Conclusion: The above solution was accurately measured and injected into the ultra-high performance liquid chromatography-mass spectrometer to record the chromatogram. The solution stability results are shown in Table 6.
[0069] Table 6 Solution stability test results
[0070] Conclusion: After the test solution and the reference solution were placed at 4°C for 12 hours, the peak area of N-nitroso-salbutamol impurity did not change significantly, indicating that the test solution and the reference solution were stable within 12 hours.
[0071] Example 7: Durability Test Durability refers to the degree to which the test results are not affected by slight changes in the chromatographic conditions. The preparation of the spiked test solution (system suitability solution), test solution and reference solution is the same as in Example 1. On the basis of the test conditions of Example 1, the durability of the test method is changed under the conditions of temperature, flow rate and initial ratio of mobile phase B. The test results are shown in Table 7.
[0072] Table 7 Durability test results
[0073] Conclusion: When the column temperature was changed by ±5℃, the flow rate was ±0.05 mL / min, and the initial proportion of mobile phase B was ±2%, the blank solution and the test solution did not interfere with the determination of N-nitroso-salbutamol impurities. No impurities were detected in the test samples, and the recovery rate of the spiked test samples met the requirements. The method of the present invention has good durability.
[0074] Based on the above experimental results, the detection method of the present invention is used to detect the nitrosamine impurity N-nitroso-salbutamol in the levosalbutamol hydrochloride atomized inhalation solution, and the blank solvent and the main component do not interfere with the detection of nitrosamine impurities. The method has strong specificity and high sensitivity, and the detection limit is as low as 0.4% of the limit and the quantitative limit is as low as 1.2% of the limit, and the quantitative determination is accurate. It is of great significance for the quality control of the levosalbutamol hydrochloride atomized inhalation solution and its preparations and for ensuring the safety of medication.
[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A method for detecting nitrosamine impurities in a levosalbuterol hydrochloride atomized inhalation solution, characterized in that: The steps include: The levosalbuterol hydrochloride nebulized inhalation solution was diluted to obtain a test solution; The test solution is detected by ultra-high performance liquid chromatography-mass spectrometry; The chromatographic conditions were as follows: the filler of the chromatographic column was octadecylsilane bonded silica gel; Mobile phase A is a formic acid aqueous solution, wherein the formic acid concentration is 0.09-0.11%, % is the volume percentage; Mobile phase B was acetonitrile; The nitrosamine impurity is N-nitroso-salbutamol.
2. The method for detecting nitrosamine impurities in the salbutamol hydrochloride atomized inhalation solution according to claim 1, characterized in that: During the detection process, the mass spectrometry conditions were as follows: the ion source was an electrospray ion source, the scanning mode was a negative ion mode, and the detection mode was multiple reaction detection MRM.
3. The method for detecting nitrosamine impurities in the salbutamol hydrochloride atomized inhalation solution according to claim 1, characterized in that: The diluent for diluting the salbutamol hydrochloride nebulized inhalation solution is a methanol aqueous solution.
4. The method for detecting nitrosamine impurities in the salbutamol hydrochloride atomized inhalation solution according to claim 3, characterized in that: In the diluent, the volume percentage of methanol is 25-35%, preferably 30%.
5. The method for detecting nitrosamine impurities in the salbutamol hydrochloride atomized inhalation solution according to claim 1, characterized in that: In the test solution, the concentration of salbutamol hydrochloride is 200-300 ng / mL, preferably 252 g / mL.
6. The method for detecting nitrosamine impurities in the levosalbuterol hydrochloride atomized inhalation solution according to claim 1, characterized in that: The volume percentage of formic acid in mobile phase A is 0.1%.
7. The method for detecting nitrosamine impurities in the salbutamol hydrochloride atomized inhalation solution according to claim 1, characterized in that: The gradient elution program was: From 0 to 3 min, mobile phase A was reduced from 95% to 70%, and mobile phase B was increased from 5% to 30%; 3-6 min, mobile phase A was reduced from 70% to 10%, and mobile phase B was increased from 30% to 90%; 8-8.1 min, mobile phase A increased from 10% to 95%, and mobile phase B decreased from 90% to 5%; 8.1~10 min, mobile phase A 95%, mobile phase B 5%, the above % are volume percentages.
8. The method for detecting nitrosamine impurities in the levosalbuterol hydrochloride atomized inhalation solution according to claim 7, characterized in that: The chromatographic conditions were as follows: the chromatographic column was filled with octadecylsilane bonded silica gel; the chromatographic column was HypersilGOLD, 100 mm×2.1 mm, 1.9 μm; 0.1% formic acid aqueous solution was used as mobile phase A, acetonitrile solution was used as mobile phase B, and gradient elution was performed; the flow rate was 0.4 mL / min, the column temperature was 45°C, the injection volume was 10 μL, and the injector temperature was 4°C.
9. The method for detecting nitrosamine impurities in the levosalbuterol hydrochloride atomized inhalation solution according to claim 1, characterized in that: The mass spectrometer is a triple quadrupole mass spectrometer, and after gradient elution, triple quadrupole mass spectrometer detection is adopted.
10. The method for detecting nitrosamine impurities in the levosalbuterol hydrochloride atomized inhalation solution according to claim 9, characterized in that: The mass spectrometry conditions were as follows: IS voltage, −4500 V; curtain gas CUR, 25 psi; nebulizer GAS1, 55 psi; Auxiliary gas GAS2 is 20 psi; ion source temperature is 550℃; collision gas: 8.