Method for determining 4-nitrobenzenesulfonyl chloride in darunavir ethanol compound or preparation thereof by adopting ESI-LC-MS technology
By hydrolyzing 4-nitrobenzenesulfonyl chloride to 4-nitrobenzenesulfonic acid and combining it with ESI-LC-MS technology, the quantitative problem of 4-nitrobenzenesulfonyl chloride in darunavir ethanol compounds has been solved, achieving high sensitivity and high accuracy in detection and ensuring drug quality and safety.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANJING COMER BIOPHARMACEUTICAL CO LTD
- Filing Date
- 2024-11-14
- Publication Date
- 2026-05-15
AI Technical Summary
Existing technologies lack effective methods for accurately quantifying the content of 4-nitrobenzenesulfonyl chloride in darunavir ethanol or its formulations, especially since it cannot be ionized in ESI-LC-MS, making it difficult to achieve highly sensitive and accurate analysis.
By hydrolyzing 4-nitrobenzenesulfonyl chloride into 4-nitrobenzenesulfonic acid, which is easily ionized in ESI-LC-MS, and using ammonia water to carry out the hydrolysis reaction at room temperature, combined with ESI-LC-MS technology, reversed-phase chromatography column, specific mobile phase and mass spectrometry conditions, quantitative detection of 4-nitrobenzenesulfonyl chloride can be achieved.
This invention provides a highly sensitive and accurate analytical method that can accurately determine the content of 4-nitrobenzenesulfonyl chloride in darunavir ethanol or its preparations, meeting the requirements for drug quality and clinical drug safety.
Smart Images
Figure CN122042830A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for determining 4-nitrobenzenesulfonyl chloride in darunavir ethanol or its formulations using ESI-LC-MS technology. Background Technology
[0002] Darunavir, developed by Tibotec, a subsidiary of Johnson & Johnson in Iceland, is an HIV protease inhibitor indicated for patients infected with HIV who have not responded to existing antiretroviral drugs. Darunavir is an HIV-1 protease inhibitor that selectively inhibits the cleavage of the HIV-encoded Gag-Pol polyprotein in virus-infected cells, thereby preventing the formation of mature infectious viral particles. The structure of the darunavir ethanol compound is shown below:
[0003]
[0004] 4-Nitrobenzenesulfonyl chloride is a starting material in the synthesis of darunavir ethanol compounds. It contains a toxicity warning structure, the structural information of which is as follows:
[0005]
[0006] According to the ICH guideline "Assess and control DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risks [M7(R2)]", the TTC (threshold for toxicological concern) of 4-nitrobenzenesulfonyl chloride is 1.5 μg / day, and the limit for 4-nitrobenzenesulfonyl chloride in darunavir ethanol or its formulations is 1.25 ppm. There are no existing literature reports on methods for determining 4-nitrobenzenesulfonyl chloride in darunavir ethanol or its formulations using ESI-LC-MS technology.
[0007] To ensure drug quality and the safety and efficacy of clinical use, in-depth quantitative studies of genotoxic impurities in drugs are essential. Because genotoxic impurities are highly toxic and have low toxicity limits, accurate quantitative determination is extremely important. Summary of the Invention
[0008] The purpose of this invention is to solve the problem of quantitative determination of 4-nitrobenzenesulfonyl chloride, the starting material in darunavir ethanol or its preparations, and to provide an analytical method that is relatively simple to operate, highly sensitive, and accurate.
[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0010] This invention is based on the fact that 4-nitrobenzenesulfonyl chloride cannot be ionized in ESI-LC-MS. Ammonia is used to hydrolyze 4-nitrobenzenesulfonyl chloride into 4-nitrobenzenesulfonic acid, which is easily ionized in ESI-LC-MS. This establishes a highly sensitive, accurate and reliable method for the determination of 4-nitrobenzenesulfonyl chloride.
[0011] The principle of hydrolysis can be represented by the following reaction equation:
[0012]
[0013] This invention provides a method for determining 4-nitrobenzenesulfonyl chloride in darunavir ethanol or its formulations, the method being ESI-LC-MS technology, comprising:
[0014] Step (1): Hydrolyze 4-nitrobenzenesulfonyl chloride with ammonia at room temperature to generate 4-nitrobenzenesulfonic acid, which can be ionized in ESI-LC-MS;
[0015] Step (2): Using ESI-LC-MS, a reversed-phase column was used with aqueous phase A and organic phase B as mobile phase. The flow rate, column temperature, injection plate temperature, injection volume, gradient elution and mass spectrometry conditions were set. In MRM acquisition mode, 4-nitrobenzenesulfonic acid generated in step (1) was determined to achieve quantitative detection of 4-nitrobenzenesulfonyl chloride.
[0016] According to an embodiment of the present invention, the ammonia water in step (1) contains methanol, the reaction system is an ammonia water methanol solution, and the reaction is carried out at room temperature for 0~45 min;
[0017] Preferably, the concentration of the ammonia-methanol solution is 0-100%, and the reaction time at room temperature is 10 min;
[0018] More preferably, the concentration of the ammonia-methanol solution is 30%.
[0019] According to embodiments of the present invention, the reversed-phase chromatography column is a column packed with octadecylsilane-bonded silica gel (Pheromen, Titan C18, 2.1×50mm, 3µm) or a column packed with octadecylsilane-bonded silica gel (Pheromen, Titan C18, 2.1×100mm, 3µm). Preferably, the column is selected from a column packed with octadecylsilane-bonded silica gel (Pheromen, Titan C18, 2.1×50mm, 3µm).
[0020] According to an embodiment of the present invention, the mobile phase B is selected from acetonitrile or methanol.
[0021] Preferably, the mobile phase B is selected from acetonitrile.
[0022] According to embodiments of the present invention, in the liquid chromatography conditions disclosed herein, the flow rate is selected from 0.15~0.25 ml / min, the column temperature is selected from 25~35℃, the injection plate temperature is selected from room temperature, and the injection volume is selected from 1 μL.
[0023] Preferably, the flow rate is selected from 0.2 ml / min and the column temperature is selected from 30 °C.
[0024] According to an embodiment of the present invention, the gradient elution in the liquid chromatography conditions disclosed herein is as follows: Starting with 1-3% mobile phase B and 97-99% mobile phase A by volume ratio, maintain this mobile phase ratio for 3 min, gradually increase mobile phase B and decrease mobile phase A, until 3.1 min when mobile phase B is adjusted to 85% and mobile phase A to 15%, maintain this mobile phase ratio for 10 min, gradually decrease mobile phase B and increase mobile phase A, until 10.1 min when mobile phase B is adjusted to 2% and mobile phase A to 98%, maintain this mobile phase ratio for 20 min.
[0025] The beneficial effects of this invention are as follows: This invention is the first to report the determination of 4-nitrobenzenesulfonyl chloride in darunavir ethanol or its formulations using ESI-LC-MS technology. This invention can accurately determine 4-nitrobenzenesulfonyl chloride in darunavir ethanol or its formulations. This method is accurate, highly sensitive, and highly specific, providing a reliable quantitative method for the study of the genotoxic impurity 4-nitrobenzenesulfonyl chloride in darunavir ethanol or its formulations, and has positive effects and practical application value. Attached Figure Description
[0026] Figure 1 Blank image;
[0027] Figure 2 Compare the solution diagram;
[0028] Figure 3 Diagram of spiked solution for test sample;
[0029] Figure 4 Trends in the peak area of the product under different concentrations of ammonia solution;
[0030] Figure 5 Trend graph of product peak area variation at different reaction times;
[0031] Figure 6 Standard curve graph;
[0032] Figure 7 Comparison of peaks of the hydrolysis products of impurity 4-nitrobenzenesulfonyl chloride in Examples 1, 11 and 12;
[0033] Figure 8 Comparison of the peaks of the hydrolysis products of impurity 4-nitrobenzenesulfonyl chloride in Example 1 and Example 13. Detailed Implementation
[0034] Example 1
[0035] (1) Sample preparation: Weigh about 50 mg of darunavir ethanol compound accurately, place it in a 10 ml volumetric flask, add 1 ml of methanol and 1 ml of 30% ammonia methanol solution, sonicate to dissolve, let stand at room temperature for 10 minutes, dilute with methanol-water (50:50) to the mark, and shake well to obtain the sample.
[0036] (2) Blank: Take 1 ml of methanol, put it in a 10 ml volumetric flask, add 1 ml of 30% ammonia methanol solution, shake well, let stand at room temperature for 10 minutes, dilute with methanol-water (50:50) to the mark, and shake well.
[0037] (3) Reference stock solution: Take an appropriate amount of 4-nitrobenzenesulfonyl chloride reference standard, accurately weigh it, dissolve it in methanol and quantitatively dilute it to prepare a solution containing about 125 μg per 1 ml; accurately measure 1 ml, put it in a 20 ml volumetric flask, dilute it to the mark with methanol, and shake well; accurately measure 1 ml, put it in a 100 ml volumetric flask, dilute it to the mark with methanol, and shake well to obtain the solution.
[0038] (4) Reference solution: Accurately measure 1 ml of the reference stock solution, place it in a 10 ml volumetric flask, add 1 ml of 30% ammonia-methanol solution, shake well, let stand at room temperature for 10 minutes, dilute with methanol-water (50:50) to the mark, and shake well to obtain the solution.
[0039] (5) Sensitivity solution: Take 1 ml of (4) control solution, put it in a 10 ml volumetric flask, dilute it to the mark with methanol-water (50:50), and shake well.
[0040] (6) Spiked solution for test sample: Weigh about 50 mg of darunavir ethanol complex accurately, place it in a 10 ml volumetric flask, add 1 ml of (3) reference stock solution and 1 ml of 30% ammonia methanol solution, sonicate to dissolve, let stand at room temperature for 10 minutes, dilute to the mark with methanol-water (50:50), and shake well to obtain the solution.
[0041] (7) Brand and model of high performance liquid chromatograph: Agilent 1260 Infinity II;
[0042] (8) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;
[0043] (9) Chromatographic column: Phenomen Titan C18, 2.1×50mm, 3μm;
[0044] (10) Mobile phase A: ammonia-water (5:1000), mobile phase B: acetonitrile; elution is performed according to the following gradient: by volume ratio, start with 2% mobile phase B and 98% mobile phase A as the initial mobile phase, maintain this mobile phase ratio for 3 min, gradually increase mobile phase B and decrease mobile phase A, until 3.1 min when mobile phase B is adjusted to 85% and mobile phase A is adjusted to 15%, maintain this mobile phase ratio for 10 min, gradually decrease mobile phase B and increase mobile phase A, until 10.1 min when mobile phase B is adjusted to 2% and mobile phase A is adjusted to 98%, maintain this mobile phase ratio for 20 min.
[0045] (11) The flow rate is 0.2 ml per minute;
[0046] (12) The column temperature is 30℃;
[0047] (13) The temperature of the injection tray is room temperature;
[0048] (14) Injection volume 1 μL;
[0049] (15) The mass spectrometry conditions were as follows: detection was performed using the multiple reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, with electrospray ionization, collision gas at 8 psi, curtain gas at 35 psi, nebulizer 1 at 40 psi, nebulizer 2 at 45 psi, spray needle voltage at -4500 V, and drying temperature at 500 °C; quantitative ion pair was 202.0 / 138.1, declustering voltage (DP) was -60 V, and collision voltage (CE) was -35 V.
[0050] (16) Take blank, sensitivity solution, control solution and spiked solution of test sample and inject them.
[0051] (17) Figure 1 Blank space does not interfere with detection; Figure 2 In the control solution, the retention time of the hydrolysis product peak of 4-nitrobenzenesulfonyl chloride was 1.133 min; Figure 3 The elution retention time of the hydrolysis product peak of 4-nitrobenzenesulfonyl chloride in the spiked solution was consistent with that in the reference solution, indicating that other impurities in the darunavir ethanol compound did not interfere with the detection of the target peak, and the method has good specificity.
[0052] Example 2
[0053] (1) Preparation of ammonia-methanol solutions of different concentrations:
[0054] 1% ammonia-methanol solution: Measure 1 ml of ammonia water, place it in a 100 ml volumetric flask, dilute with methanol to the mark, and shake well.
[0055] 5% ammonia-methanol solution: Measure 1 ml of ammonia water, place it in a 20 ml volumetric flask, dilute with methanol to the mark, and shake well.
[0056] 10% ammonia-methanol solution: Measure 1 ml of ammonia water, place it in a 10 ml volumetric flask, dilute with methanol to the mark, and shake well.
[0057] 20% ammonia-methanol solution: Measure 2 ml of ammonia water, place it in a 10 ml volumetric flask, dilute with methanol to the mark, and shake well.
[0058] 30% ammonia-methanol solution: Measure 3 ml of ammonia water, place it in a 10 ml volumetric flask, dilute with methanol to the mark, and shake well.
[0059] 40% ammonia-methanol solution: Measure 4 ml of ammonia water, place it in a 10 ml volumetric flask, dilute with methanol to the mark, and shake well.
[0060] 50% ammonia-methanol solution: Measure 5 ml of ammonia water, place it in a 10 ml volumetric flask, dilute with methanol to the mark, and shake well.
[0061] 100% ammonia-methanol solution: Measure an appropriate amount of ammonia reagent.
[0062] (2) Reference stock solution: Take an appropriate amount of 4-nitrobenzenesulfonyl chloride reference standard, accurately weigh it, dissolve it in methanol and dilute it quantitatively to prepare a solution containing about 125 μg per 1 ml; accurately measure 1 ml, put it in a 20 ml volumetric flask, dilute it to the mark with methanol, and shake well; accurately measure 1 ml, put it in a 100 ml volumetric flask, dilute it to the mark with methanol, and shake well to obtain the solution.
[0063] (3) Preparation of test solutions for the amount of ammonia: Accurately measure 1 ml of the reference stock solution and place it in different 10 ml volumetric flasks. Add 1 ml of 1% ammonia methanol solution, 1 ml of 5% ammonia methanol solution, 1 ml of 10% ammonia methanol solution, 1 ml of 20% ammonia methanol solution, 1 ml of 30% ammonia methanol solution, 1 ml of 40% ammonia methanol solution, 1 ml of 50% ammonia methanol solution and 1 ml of 100% ammonia methanol solution to each flask. Shake well and let stand at room temperature for 10 minutes. Dilute to the mark with methanol-water (50:50) and shake well to obtain the solution.
[0064] (4) Brand and model of high performance liquid chromatograph: Agilent 1260 Infinity II;
[0065] (5) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;
[0066] (6) Chromatographic column: Phenomen Titan C18, 2.1×50mm, 3μm;
[0067] (7) Mobile phase A: ammonia-water (5:1000), mobile phase B: acetonitrile; elution is performed according to the following gradient: by volume ratio, start with 2% mobile phase B and 98% mobile phase A as the initial mobile phase, maintain this mobile phase ratio for 3 min, gradually increase mobile phase B and decrease mobile phase A, until 3.1 min when mobile phase B is adjusted to 85% and mobile phase A is adjusted to 15%, maintain this mobile phase ratio for 10 min, gradually decrease mobile phase B and increase mobile phase A, until 10.1 min when mobile phase B is adjusted to 2% and mobile phase A is adjusted to 98%, maintain this mobile phase ratio for 20 min.
[0068] (8) The flow rate is 0.2 ml per minute;
[0069] (9) The column temperature is 30℃;
[0070] (10) The temperature of the injection tray is room temperature;
[0071] (11) Injection volume 1 μL;
[0072] (12) The mass spectrometry conditions were as follows: multiple reaction monitoring (MRM) mode of triple quadrupole mass spectrometer, electrospray ionization, collision gas 8 psi, curtain gas 35 psi, nebulizer 1 40 psi, nebulizer 2 45 psi, spray needle voltage -4500V, drying temperature 500℃; quantitative ion pair 202.0 / 138.1, declustering voltage (DP) -60V, collision voltage (CE) -35V.
[0073] (13) Take samples of each test solution used to investigate the amount of ammonia water.
[0074] (14) Table 1 shows the rate of change of the peak area of the product with different concentrations of ammonia water and the peak area of the product in the previous test solution; the rate of change = (peak area of the product in the current test solution - peak area of the product in the previous test solution) / peak area of the product in the previous test solution × 100%.
[0075] Table 1. Results of the change rate of product peak area with different concentrations of ammonia solution compared to the peak area of the previous test solution.
[0076]
[0077] (15) Figure 4 This is a graph showing the trend of product peak area variation with different concentrations of ammonia water.
[0078] (16) Through Table 1 and Figure 4 It can be seen that the optimal concentration of ammonia and methanol is 30%.
[0079] Example 3
[0080] (1) Reference stock solution: Take an appropriate amount of 4-nitrobenzenesulfonyl chloride reference standard, accurately weigh it, dissolve it in methanol and quantitatively dilute it to prepare a solution containing about 125 μg per 1 ml; accurately measure 1 ml, put it in a 20 ml volumetric flask, dilute it to the mark with methanol, and shake well; accurately measure 1 ml, put it in a 100 ml volumetric flask, dilute it to the mark with methanol, and shake well to obtain the solution.
[0081] (2) Preparation of test solution for reaction time: Accurately measure 1 ml of the reference stock solution and place it in different 10 ml volumetric flasks. Add 1 ml of 30% ammonia-methanol solution to each flask, shake well, and place at room temperature for 5 minutes, 10 minutes, 20 minutes, 30 minutes and 45 minutes respectively. Dilute to the mark with methanol-water (50:50) and shake well to obtain the solution.
[0082] (3) Brand and model of high performance liquid chromatograph: Agilent 1260 Infinity II;
[0083] (4) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;
[0084] (5) Chromatographic column: Phenomen Titan C18, 2.1×50mm, 3μm;
[0085] (6) Mobile phase A: ammonia-water (5:1000), mobile phase B: acetonitrile; elution is performed according to the following gradient: by volume ratio, start with 2% mobile phase B and 98% mobile phase A as the initial mobile phase, maintain this mobile phase ratio for 3 min, gradually increase mobile phase B and decrease mobile phase A, until 3.1 min when mobile phase B is adjusted to 85% and mobile phase A is adjusted to 15%, maintain this mobile phase ratio for 10 min, gradually decrease mobile phase B and increase mobile phase A, until 10.1 min when mobile phase B is adjusted to 2% and mobile phase A is adjusted to 98%, maintain this mobile phase ratio for 20 min.
[0086] (7) The flow rate is 0.2 ml per minute;
[0087] (8) The column temperature is 30℃;
[0088] (9) The temperature of the injection tray is room temperature;
[0089] (10) Injection volume 1 μL;
[0090] (11) The mass spectrometry conditions were as follows: detection was performed using the multiple reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, with electrospray ionization, collision gas at 8 psi, curtain gas at 35 psi, nebulizer 1 at 40 psi, nebulizer 2 at 45 psi, spray needle voltage at -4500 V, and drying temperature at 500 °C; quantitative ion pair was 202.0 / 138.1, declustering voltage (DP) was -60 V, and collision voltage (CE) was -35 V.
[0091] (12) Take samples of each test solution for the reaction time to be investigated.
[0092] (13) Table 2 shows the rate of change of the product peak area of the test solution at different reaction times compared with the product peak area of the test solution at the previous reaction time; the rate of change = (product peak area of the test solution at the current reaction time - product peak area of the test solution at the previous reaction time) / product peak area of the test solution at the previous reaction time × 100%.
[0093] Table 2. Results of the change rate of product peak area at different reaction times compared with the product peak area of the test solution at the previous reaction time.
[0094]
[0095] (14) Figure 5 This is a graph showing the trend of product peak area changes at different reaction times.
[0096] (15) Through Table 2 and Figure 5 It can be seen that a reaction time of 10 minutes is optimal.
[0097] Example 4
[0098] (1) Linear solution stock solution: Take an appropriate amount of 4-nitrobenzenesulfonyl chloride reference standard, accurately weigh it, dissolve it in methanol and quantitatively dilute it to prepare a solution containing about 125 μg per 1 ml; accurately measure 1 ml, put it in a 20 ml volumetric flask, dilute it to the mark with methanol, and shake well; accurately measure 5 ml, put it in a 50 ml volumetric flask, dilute it to the mark with methanol, and shake well to obtain the solution.
[0099] (2) Linear solution 1: Accurately measure 0.2 ml of (1) reference stock solution, place it in a 100 ml volumetric flask, dilute with methanol to the mark, and shake well; accurately measure 1 ml, place it in a 10 ml volumetric flask, add 1 ml of 30% ammonia methanol solution, shake well, place at room temperature for 10 minutes, dilute with methanol-water (50:50) to the mark, and shake well to obtain the solution.
[0100] (3) Linear solution 2: Accurately measure 1 ml of (1) reference stock solution, place it in a 100 ml volumetric flask, dilute with methanol to the mark, and shake well; accurately measure 1 ml, place it in a 10 ml volumetric flask, add 1 ml of 30% ammonia methanol solution, shake well, place at room temperature for 10 minutes, dilute with methanol-water (50:50) to the mark, and shake well to obtain the solution.
[0101] (4) Linear solution 3: Accurately measure 3 ml of (1) reference standard stock solution, place it in a 100 ml volumetric flask, dilute with methanol to the mark, and shake well; accurately measure 1 ml, place it in a 10 ml volumetric flask, add 1 ml of 30% ammonia methanol solution, shake well, place at room temperature for 10 minutes, dilute with methanol-water (50:50) to the mark, and shake well to obtain the solution.
[0102] (5) Linear solution 4: Accurately measure 1 ml of (1) reference stock solution, place it in a 20 ml volumetric flask, dilute with methanol to the mark, and shake well; accurately measure 1 ml, place it in a 10 ml volumetric flask, add 1 ml of 30% ammonia methanol solution, shake well, place at room temperature for 10 minutes, dilute with methanol-water (50:50) to the mark, and shake well to obtain the solution.
[0103] (6) Linear solution 5: Accurately measure 2 ml of (1) reference stock solution, place it in a 20 ml volumetric flask, dilute with methanol to the mark, and shake well; accurately measure 1 ml, place it in a 10 ml volumetric flask, add 1 ml of 30% ammonia methanol solution, shake well, place at room temperature for 10 minutes, dilute with methanol-water (50:50) to the mark, and shake well to obtain the solution.
[0104] (7) Linear solution 6: Accurately measure 3 ml of (1) reference stock solution, place it in a 20 ml volumetric flask, dilute with methanol to the mark, and shake well; accurately measure 1 ml, place it in a 10 ml volumetric flask, add 1 ml of 30% ammonia methanol solution, shake well, place at room temperature for 10 minutes, dilute with methanol-water (50:50) to the mark, and shake well to obtain the solution.
[0105] (8) Linear solution 7: Accurately measure 4 ml of (1) reference stock solution, place it in a 20 ml volumetric flask, dilute with methanol to the mark, and shake well; accurately measure 1 ml, place it in a 10 ml volumetric flask, add 1 ml of 30% ammonia methanol solution, shake well, place at room temperature for 10 minutes, dilute with methanol-water (50:50) to the mark, and shake well to obtain the solution.
[0106] (9) Brand and model of high performance liquid chromatograph: Agilent 1260 Infinity II;
[0107] (10) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;
[0108] (11) Column: Phenomen Titan C18, 2.1×50mm, 3μm;
[0109] (12) Mobile phase A: ammonia-water (5:1000), mobile phase B: acetonitrile; elution is performed according to the following gradient: by volume ratio, start with 2% mobile phase B and 98% mobile phase A as the initial mobile phase, maintain this mobile phase ratio for 3 min, gradually increase mobile phase B and decrease mobile phase A, until 3.1 min when mobile phase B is adjusted to 85% and mobile phase A is adjusted to 15%, maintain this mobile phase ratio for 10 min, gradually decrease mobile phase B and increase mobile phase A, until 10.1 min when mobile phase B is adjusted to 2% and mobile phase A is adjusted to 98%, maintain this mobile phase ratio for 20 min.
[0110] (13) The flow rate is 0.2 ml per minute;
[0111] (14) The column temperature is 30℃;
[0112] (15) The temperature of the injection tray is room temperature;
[0113] (16) Injection volume 1 μL;
[0114] (17) The mass spectrometry conditions were as follows: detection was performed using the multiple reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, with electrospray ionization, collision gas at 8 psi, curtain gas at 35 psi, nebulizer 1 at 40 psi, nebulizer 2 at 45 psi, spray needle voltage at -4500 V, and drying temperature at 500 °C; quantitative ion pair was 202.0 / 138.1, declustering voltage (DP) was -60 V, and collision voltage (CE) was -35 V.
[0115] (18) Inject linear solutions 1, 2, 3, 4, 5, 6 and 7.
[0116] (19) Figure 6 This is a standard linear regression diagram. As shown in the diagram, within the theoretical concentration range of 0.125~12.5 ng / ml, the linear regression between the concentration of 4-nitrobenzenesulfonyl chloride and the peak area of the hydrolysis product yields a correlation coefficient R² of 0.9996, which is greater than 0.990 and meets the requirements of the Chinese Pharmacopoeia.
[0117] Example 5
[0118] (1) Reference stock solution: Take an appropriate amount of 4-nitrobenzenesulfonyl chloride reference standard, accurately weigh it, dissolve it in methanol and dilute it quantitatively to prepare a solution containing about 125 μg per 1 ml; accurately measure 1 ml, put it in a 20 ml volumetric flask, dilute it to the mark with methanol, and shake well; accurately measure 1 ml, put it in a 100 ml volumetric flask, dilute it to the mark with methanol, and shake well to obtain the solution.
[0119] (2) Limit of Quantification Solution: Accurately measure 1 ml of (1) reference standard stock solution, place it in a 50 ml volumetric flask, dilute with methanol to the mark, and shake well; accurately measure 1 ml, place it in a 10 ml volumetric flask, add 1 ml of 30% ammonia methanol solution, shake well, place at room temperature for 10 minutes, dilute with methanol-water (50:50) to the mark, and shake well to obtain the solution.
[0120] (3) Detection limit solution: Accurately measure 1 ml of (1) reference stock solution, place it in a 50 ml volumetric flask, dilute with methanol to the mark, and shake well; accurately measure 3 ml, place it in a 10 ml volumetric flask, dilute with methanol to the mark, and shake well; accurately measure 1 ml, place it in a 10 ml volumetric flask, add 1 ml of 30% ammonia methanol solution, shake well, place at room temperature for 10 minutes, dilute with methanol-water (50:50) to the mark, and shake well to obtain the solution.
[0121] (4) Brand and model of high performance liquid chromatograph: Agilent 1260 Infinity II;
[0122] (5) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;
[0123] (6) Chromatographic column: Phenomen Titan C18, 2.1×50mm, 3μm;
[0124] (7) Mobile phase A: ammonia-water (5:1000), mobile phase B: acetonitrile; elution is performed according to the following gradient: by volume ratio, start with 2% mobile phase B and 98% mobile phase A as the initial mobile phase, maintain this mobile phase ratio for 3 min, gradually increase mobile phase B and decrease mobile phase A, until 3.1 min when mobile phase B is adjusted to 85% and mobile phase A is adjusted to 15%, maintain this mobile phase ratio for 10 min, gradually decrease mobile phase B and increase mobile phase A, until 10.1 min when mobile phase B is adjusted to 2% and mobile phase A is adjusted to 98%, maintain this mobile phase ratio for 20 min.
[0125] (8) The flow rate is 0.2 ml per minute;
[0126] (9) The column temperature is 30℃;
[0127] (10) The temperature of the injection tray is room temperature;
[0128] (11) Injection volume 1 μl;
[0129] (12) The mass spectrometry conditions were as follows: multiple reaction monitoring (MRM) mode of triple quadrupole mass spectrometer, electrospray ionization, collision gas 8 psi, curtain gas 35 psi, nebulizer 1 40 psi, nebulizer 2 45 psi, spray needle voltage -4500V, drying temperature 500℃; quantitative ion pair 202.0 / 138.1, declustering voltage (DP) -60V, collision voltage (CE) -35V.
[0130] (13) Inject the solution with the limit of detection and the limit of quantitation.
[0131] (14) Table 3 Results of detection limit and quantitation limit. As shown in Table 3, the detection limit is 0.077 ppm and the quantitation limit is 0.0256 ppm.
[0132] Table 3 Results of Limit of Detection and Limit of Quantification
[0133]
[0134] Example 6
[0135] (1) Sample preparation: Weigh about 50 mg of darunavir ethanol compound accurately, place it in a 10 ml volumetric flask, add 1 ml of methanol and 1 ml of 30% ammonia methanol solution, sonicate to dissolve, let stand at room temperature for 10 minutes, dilute with methanol-water (50:50) to the mark, and shake well to obtain the sample.
[0136] (2) 30% recovery stock solution: Take an appropriate amount of 4-nitrobenzenesulfonyl chloride reference standard, accurately weigh it, dissolve it in methanol and quantitatively dilute it to prepare a solution containing about 125 μg per 1 ml; accurately measure 1 ml, put it in a 20 ml volumetric flask, dilute it to the mark with methanol and shake well; accurately measure 1 ml, put it in a 10 ml volumetric flask, dilute it to the mark with methanol and shake well; accurately measure 3 ml, put it in a 100 ml volumetric flask, dilute it to the mark with methanol and shake well to obtain the solution.
[0137] (3) 100% recovery stock solution: Take an appropriate amount of 4-nitrobenzenesulfonyl chloride reference standard, accurately weigh it, dissolve it in methanol and quantitatively dilute it to prepare a solution containing about 125 μg per 1 ml; accurately measure 1 ml, put it in a 20 ml volumetric flask, dilute it to the mark with methanol, and shake well; accurately measure 1 ml, put it in a 100 ml volumetric flask, dilute it to the mark with methanol, and shake well to obtain the solution.
[0138] (4) 150% recovery stock solution: Take an appropriate amount of 4-nitrobenzenesulfonyl chloride reference standard, accurately weigh it, dissolve it in methanol and quantitatively dilute it to prepare a solution containing about 125 μg per 1 ml; accurately measure 1 ml, put it in a 20 ml volumetric flask, dilute it to the mark with methanol and shake well; accurately measure 1 ml, put it in a 10 ml volumetric flask, dilute it to the mark with methanol and shake well; accurately measure 3 ml, put it in a 20 ml volumetric flask, dilute it to the mark with methanol and shake well to obtain the solution.
[0139] (5) Control solution: Accurately measure 1 ml of (3) 100% recovery stock solution, place it in a 10 ml volumetric flask, add 1 ml of 30% ammonia methanol solution, shake well, let stand at room temperature for 10 minutes, dilute with methanol-water (50:50) to the mark, shake well to obtain the solution.
[0140] (6) 30% recovery rate solution: Weigh about 50 mg of darunavir ethanol compound accurately, place it in a 10 ml volumetric flask, add 1 ml of (2) 30% recovery rate stock solution and 1 ml of 30% ammonia methanol solution, sonicate to dissolve, let stand at room temperature for 10 minutes, dilute to the mark with methanol-water (50:50), and shake well to obtain the solution.
[0141] (7) 100% recovery solution: Weigh about 50 mg of darunavir ethanol compound accurately, place it in a 10 ml volumetric flask, add 1 ml of (3) 100% recovery stock solution and 1 ml of 30% ammonia methanol solution, sonicate to dissolve, let stand at room temperature for 10 minutes, dilute to the mark with methanol-water (50:50), and shake well to obtain the solution.
[0142] (8) 150% recovery rate solution: Weigh about 50 mg of darunavir ethanol compound accurately, place it in a 10 ml volumetric flask, add 1 ml of (4) 150% recovery rate stock solution and 1 ml of 30% ammonia methanol solution, sonicate to dissolve, let stand at room temperature for 10 minutes, dilute to the mark with methanol-water (50:50), and shake well to obtain the solution.
[0143] (9) Brand and model of high performance liquid chromatograph: Agilent 1260 Infinity II;
[0144] (10) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;
[0145] (11) Column: Phenomen Titan C18, 2.1×50mm, 3μm;
[0146] (12) Mobile phase A: ammonia-water (5:1000), mobile phase B: acetonitrile; elution is performed according to the following gradient: by volume ratio, start with 2% mobile phase B and 98% mobile phase A as the initial mobile phase, maintain this mobile phase ratio for 3 min, gradually increase mobile phase B and decrease mobile phase A, until 3.1 min when mobile phase B is adjusted to 85% and mobile phase A is adjusted to 15%, maintain this mobile phase ratio for 10 min, gradually decrease mobile phase B and increase mobile phase A, until 10.1 min when mobile phase B is adjusted to 2% and mobile phase A is adjusted to 98%, maintain this mobile phase ratio for 20 min.
[0147] (13) The flow rate is 0.2 ml per minute;
[0148] (14) The column temperature is 30℃;
[0149] (15) The temperature of the injection tray is room temperature;
[0150] (16) Injection volume 1 μL;
[0151] (17) The mass spectrometry conditions were as follows: detection was performed using the multiple reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, with electrospray ionization, collision gas at 8 psi, curtain gas at 35 psi, nebulizer 1 at 40 psi, nebulizer 2 at 45 psi, spray needle voltage at -4500 V, and drying temperature at 500 °C; quantitative ion pair was 202.0 / 138.1, declustering voltage (DP) was -60 V, and collision voltage (CE) was -35 V.
[0152] (18) Inject the reference solution and the recovery solution.
[0153] (19) Table 4 shows the recovery rate results. As can be seen from Table 4, the recovery rate is 95%~105%, and the RSD (n=9) is 2.3%, which meets the requirements of the Chinese Pharmacopoeia.
[0154] Table 4 Recovery and Precision Results
[0155]
[0156] Example 7
[0157] (1) Reference stock solution: Take an appropriate amount of 4-nitrobenzenesulfonyl chloride reference standard, accurately weigh it, dissolve it in methanol and dilute it quantitatively to prepare a solution containing about 125 μg per 1 ml; accurately measure 1 ml, put it in a 20 ml volumetric flask, dilute it to the mark with methanol, and shake well; accurately measure 1 ml, put it in a 100 ml volumetric flask, dilute it to the mark with methanol, and shake well to obtain the solution.
[0158] (2) Reference solution: Accurately measure 1 ml of the reference stock solution, place it in a 10 ml volumetric flask, add 1 ml of 30% ammonia-methanol solution, shake well, let stand at room temperature for 10 minutes, dilute to the mark with methanol-water (50:50), and shake well to obtain the solution.
[0159] (3) Sensitivity solution: Take 1 ml of (2) reference solution, put it in a 10 ml volumetric flask, dilute it to the mark with methanol-water (50:50), and shake well.
[0160] (4) Brand and model of high performance liquid chromatograph: Agilent 1260 Infinity II;
[0161] (5) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;
[0162] (6) Chromatographic column: Phenomen Titan C18, 2.1×50mm, 3μm;
[0163] (7) Mobile phase A: ammonia-water (5:1000), mobile phase B: acetonitrile; elution is performed according to the following gradient: by volume ratio, start with 2% mobile phase B and 98% mobile phase A as the initial mobile phase, maintain this mobile phase ratio for 3 min, gradually increase mobile phase B and decrease mobile phase A, until 3.1 min when mobile phase B is adjusted to 85% and mobile phase A is adjusted to 15%, maintain this mobile phase ratio for 10 min, gradually decrease mobile phase B and increase mobile phase A, until 10.1 min when mobile phase B is adjusted to 2% and mobile phase A is adjusted to 98%, maintain this mobile phase ratio for 20 min.
[0164] (8) The flow rate is 0.2 ml per minute;
[0165] (9) The column temperature is 25℃;
[0166] (10) The temperature of the injection tray is room temperature;
[0167] (11) Injection volume 1 μl;
[0168] (12) The mass spectrometry conditions were as follows: detection was performed using the multiple reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, with electrospray ionization, collision gas at 8 psi, curtain gas at 35 psi, nebulizer 1 at 40 psi, nebulizer 2 at 45 psi, spray needle voltage at -4500 V, and drying temperature at 500 °C; quantitative ion pair was 202.0 / 138.1, declustering voltage (DP) was -60 V, and collision voltage (CE) was -35 V.
[0169] (13) Take a sensitivity solution for injection.
[0170] Example 8
[0171] (1) Reference stock solution: Take an appropriate amount of 4-nitrobenzenesulfonyl chloride reference standard, accurately weigh it, dissolve it in methanol and dilute it quantitatively to prepare a solution containing about 125 μg per 1 ml; accurately measure 1 ml, put it in a 20 ml volumetric flask, dilute it to the mark with methanol, and shake well; accurately measure 1 ml, put it in a 100 ml volumetric flask, dilute it to the mark with methanol, and shake well to obtain the solution.
[0172] (2) Reference solution: Accurately measure 1 ml of the reference stock solution, place it in a 10 ml volumetric flask, add 1 ml of 30% ammonia-methanol solution, shake well, let stand at room temperature for 10 minutes, dilute to the mark with methanol-water (50:50), and shake well to obtain the solution.
[0173] (3) Sensitivity solution: Take 1 ml of (2) reference solution, put it in a 10 ml volumetric flask, dilute it to the mark with methanol-water (50:50), and shake well.
[0174] (4) Brand and model of high performance liquid chromatograph: Agilent 1260 Infinity II;
[0175] (5) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;
[0176] (6) Chromatographic column: Phenomen Titan C18, 2.1×50mm, 3μm;
[0177] (7) Mobile phase A: ammonia-water (5:1000), mobile phase B: acetonitrile; elution is performed according to the following gradient: by volume ratio, start with 2% mobile phase B and 98% mobile phase A as the initial mobile phase, maintain this mobile phase ratio for 3 min, gradually increase mobile phase B and decrease mobile phase A, until 3.1 min when mobile phase B is adjusted to 85% and mobile phase A is adjusted to 15%, maintain this mobile phase ratio for 10 min, gradually decrease mobile phase B and increase mobile phase A, until 10.1 min when mobile phase B is adjusted to 2% and mobile phase A is adjusted to 98%, maintain this mobile phase ratio for 20 min.
[0178] (8) The flow rate is 0.2 ml per minute;
[0179] (9) The column temperature is 35℃;
[0180] (10) The temperature of the injection tray is room temperature;
[0181] (11) Injection volume 1 μL;
[0182] (12) The mass spectrometry conditions were as follows: multiple reaction monitoring (MRM) mode of triple quadrupole mass spectrometer, electrospray ionization, collision gas 8 psi, curtain gas 35 psi, nebulizer 1 40 psi, nebulizer 2 45 psi, spray needle voltage -4500V, drying temperature 500℃; quantitative ion pair 202.0 / 138.1, declustering voltage (DP) -60V, collision voltage (CE) -35V.
[0183] (13) Take a sensitivity solution for injection.
[0184] Example 9
[0185] (1) Reference stock solution: Take an appropriate amount of 4-nitrobenzenesulfonyl chloride reference standard, accurately weigh it, dissolve it in methanol and dilute it quantitatively to prepare a solution containing about 125 μg per 1 ml; accurately measure 1 ml, put it in a 20 ml volumetric flask, dilute it to the mark with methanol, and shake well; accurately measure 1 ml, put it in a 100 ml volumetric flask, dilute it to the mark with methanol, and shake well to obtain the solution.
[0186] (2) Reference solution: Accurately measure 1 ml of the reference stock solution, place it in a 10 ml volumetric flask, add 1 ml of 30% ammonia-methanol solution, shake well, let stand at room temperature for 10 minutes, dilute to the mark with methanol-water (50:50), and shake well to obtain the solution.
[0187] (3) Sensitivity solution: Take 1 ml of (2) reference solution, put it in a 10 ml volumetric flask, dilute it to the mark with methanol-water (50:50), and shake well.
[0188] (4) Brand and model of high performance liquid chromatograph: Agilent 1260 Infinity II;
[0189] (5) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;
[0190] (6) Chromatographic column: Phenomen Titan C18, 2.1×50mm, 3μm;
[0191] (7) Mobile phase A: ammonia-water (5:1000), mobile phase B: acetonitrile; elution is performed according to the following gradient: by volume ratio, start with 2% mobile phase B and 98% mobile phase A as the initial mobile phase, maintain this mobile phase ratio for 3 min, gradually increase mobile phase B and decrease mobile phase A, until 3.1 min when mobile phase B is adjusted to 85% and mobile phase A is adjusted to 15%, maintain this mobile phase ratio for 10 min, gradually decrease mobile phase B and increase mobile phase A, until 10.1 min when mobile phase B is adjusted to 2% and mobile phase A is adjusted to 98%, maintain this mobile phase ratio for 20 min.
[0192] (8) The flow rate is 0.15 ml per minute;
[0193] (9) The column temperature is 30℃;
[0194] (10) The temperature of the injection tray is room temperature;
[0195] (11) Injection volume 1 μl;
[0196] (12) The mass spectrometry conditions were as follows: multiple reaction monitoring (MRM) mode of triple quadrupole mass spectrometer, electrospray ionization, collision gas 8 psi, curtain gas 35 psi, nebulizer 1 40 psi, nebulizer 2 45 psi, spray needle voltage -4500V, drying temperature 500℃; quantitative ion pair 202.0 / 138.1, declustering voltage (DP) -60V, collision voltage (CE) -35V.
[0197] (13) Take a sensitivity solution for injection.
[0198] Example 10
[0199] (1) Reference stock solution: Take an appropriate amount of 4-nitrobenzenesulfonyl chloride reference standard, accurately weigh it, dissolve it in methanol and dilute it quantitatively to prepare a solution containing about 125 μg per 1 ml; accurately measure 1 ml, put it in a 20 ml volumetric flask, dilute it to the mark with methanol, and shake well; accurately measure 1 ml, put it in a 100 ml volumetric flask, dilute it to the mark with methanol, and shake well to obtain the solution.
[0200] (2) Reference solution: Accurately measure 1 ml of the reference stock solution, place it in a 10 ml volumetric flask, add 1 ml of 30% ammonia-methanol solution, shake well, let stand at room temperature for 10 minutes, dilute to the mark with methanol-water (50:50), and shake well to obtain the solution.
[0201] (3) Sensitivity solution: Take 1 ml of (2) reference solution, put it in a 10 ml volumetric flask, dilute it to the mark with methanol-water (50:50), and shake well.
[0202] (4) Brand and model of high performance liquid chromatograph: Agilent 1260 Infinity II;
[0203] (5) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;
[0204] (6) Chromatographic column: Phenomen Titan C18, 2.1×50mm, 3μm;
[0205] (7) Mobile phase A: ammonia-water (5:1000), mobile phase B: acetonitrile; elution is performed according to the following gradient: by volume ratio, start with 2% mobile phase B and 98% mobile phase A as the initial mobile phase, maintain this mobile phase ratio for 3 min, gradually increase mobile phase B and decrease mobile phase A, until 3.1 min when mobile phase B is adjusted to 85% and mobile phase A is adjusted to 15%, maintain this mobile phase ratio for 10 min, gradually decrease mobile phase B and increase mobile phase A, until 10.1 min when mobile phase B is adjusted to 2% and mobile phase A is adjusted to 98%, maintain this mobile phase ratio for 20 min.
[0206] (8) The flow rate is 0.25 ml per minute;
[0207] (9) The column temperature is 30℃;
[0208] (10) The temperature of the injection tray is room temperature;
[0209] (11) Injection volume 1 μL;
[0210] (12) The mass spectrometry conditions were as follows: multiple reaction monitoring (MRM) mode of triple quadrupole mass spectrometer, electrospray ionization, collision gas 8 psi, curtain gas 35 psi, nebulizer 1 40 psi, nebulizer 2 45 psi, spray needle voltage -4500V, drying temperature 500℃; quantitative ion pair 202.0 / 138.1, declustering voltage (DP) -60V, collision voltage (CE) -35V.
[0211] (13) Take a sensitivity solution for injection.
[0212] (14) Table 5 shows the signal-to-noise ratio results of the peak of the hydrolysis product of impurity 4-nitrobenzenesulfonyl chloride in Examples 1, 7, 8, 9 and 10. As can be seen from Table 5, at a flow rate of 0.15~0.25 ml / min and a column temperature of 25~35℃, the signal-to-noise ratio of the peak of the hydrolysis product of 4-nitrobenzenesulfonyl chloride in the solution is greater than 10, which meets the requirements of the Chinese Pharmacopoeia.
[0213] Table 5. Product peak signal-to-noise ratio results
[0214]
[0215] Example 11
[0216] (1) Reference stock solution: Take an appropriate amount of 4-nitrobenzenesulfonyl chloride reference standard, accurately weigh it, dissolve it in methanol and dilute it quantitatively to prepare a solution containing about 125 μg per 1 ml; accurately measure 1 ml, put it in a 20 ml volumetric flask, dilute it to the mark with methanol, and shake well; accurately measure 1 ml, put it in a 100 ml volumetric flask, dilute it to the mark with methanol, and shake well to obtain the solution.
[0217] (2) Reference solution: Accurately measure 1 ml of the reference stock solution, place it in a 10 ml volumetric flask, add 1 ml of 30% ammonia-methanol solution, shake well, let stand at room temperature for 10 minutes, dilute to the mark with methanol-water (50:50), and shake well to obtain the solution.
[0218] (3) Brand and model of high performance liquid chromatograph: Agilent 1260 Infinity II;
[0219] (4) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;
[0220] (5) Chromatographic column: Phenomen Titan C18, 2.1×50mm, 3μm;
[0221] (6) Mobile phase A: ammonia-water (5:1000), mobile phase B: acetonitrile; elution is performed according to the following gradient: by volume ratio, start with 1% mobile phase B and 99% mobile phase A as the initial mobile phase, maintain this mobile phase ratio for 3 min, gradually increase mobile phase B and decrease mobile phase A, until 3.1 min when mobile phase B is adjusted to 85% and mobile phase A is adjusted to 15%, maintain this mobile phase ratio for 10 min, gradually decrease mobile phase B and increase mobile phase A, until 10.1 min when mobile phase B is adjusted to 1% and mobile phase A is adjusted to 99%, maintain this mobile phase ratio for 20 min.
[0222] (7) The flow rate is 0.2 ml per minute;
[0223] (8) The column temperature is 30℃;
[0224] (9) The temperature of the injection tray is room temperature;
[0225] (10) Injection volume 1 μL;
[0226] (11) The mass spectrometry conditions were as follows: detection was performed using the multiple reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, with electrospray ionization, collision gas at 8 psi, curtain gas at 35 psi, nebulizer 1 at 40 psi, nebulizer 2 at 45 psi, spray needle voltage at -4500 V, and drying temperature at 500 °C; quantitative ion pair was 202.0 / 138.1, declustering voltage (DP) was -60 V, and collision voltage (CE) was -35 V.
[0227] (12) Take the control solution and inject it.
[0228] Example 12
[0229] (1) Reference stock solution: Take an appropriate amount of 4-nitrobenzenesulfonyl chloride reference standard, accurately weigh it, dissolve it in methanol and dilute it quantitatively to prepare a solution containing about 125 μg per 1 ml; accurately measure 1 ml, put it in a 20 ml volumetric flask, dilute it to the mark with methanol, and shake well; accurately measure 1 ml, put it in a 100 ml volumetric flask, dilute it to the mark with methanol, and shake well to obtain the solution.
[0230] (2) Reference solution: Accurately measure 1 ml of the reference stock solution, place it in a 10 ml volumetric flask, add 1 ml of 30% ammonia-methanol solution, shake well, let stand at room temperature for 10 minutes, dilute to the mark with methanol-water (50:50), and shake well to obtain the solution.
[0231] (3) Brand and model of high performance liquid chromatograph: Agilent 1260 Infinity II;
[0232] (4) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;
[0233] (5) Chromatographic column: Phenomen Titan C18, 2.1×50mm, 3μm;
[0234] (6) Mobile phase A: ammonia-water (5:1000), mobile phase B: acetonitrile; elution is performed according to the following gradient: by volume ratio, start with 3% mobile phase B and 97% mobile phase A as the initial mobile phase, maintain this mobile phase ratio for 3 min, gradually increase mobile phase B and decrease mobile phase A, until 3.1 min when mobile phase B is adjusted to 85% and mobile phase A is adjusted to 15%, maintain this mobile phase ratio for 10 min, gradually decrease mobile phase B and increase mobile phase A, until 10.1 min when mobile phase B is adjusted to 3% and mobile phase A is adjusted to 97%, maintain this mobile phase ratio for 20 min.
[0235] (7) The flow rate is 0.2 ml per minute;
[0236] (8) The column temperature is 30℃;
[0237] (9) The temperature of the injection tray is room temperature;
[0238] (10) Injection volume 1 μL;
[0239] (11) The mass spectrometry conditions were as follows: detection was performed using the multiple reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, with electrospray ionization, collision gas at 8 psi, curtain gas at 35 psi, nebulizer 1 at 40 psi, nebulizer 2 at 45 psi, spray needle voltage at -4500 V, and drying temperature at 500 °C; quantitative ion pair was 202.0 / 138.1, declustering voltage (DP) was -60 V, and collision voltage (CE) was -35 V.
[0240] (12) Take the control solution and inject it.
[0241] (13) Figure 7 The peak results for the hydrolysis products of impurity 4-nitrobenzenesulfonyl chloride in Examples 1, 11, and 12 are obtained from... Figure 7 It can be seen that in mobile phase A: ammonia-water (5:1000), and mobile phase B: acetonitrile; the elution gradient is as follows: Starting with 1%–3% mobile phase B and 99%–97% mobile phase A by volume ratio, maintain this mobile phase ratio for 3 min, gradually increase mobile phase B and decrease mobile phase A, until 3.1 min when mobile phase B is adjusted to 85% and mobile phase A to 15%, maintain this mobile phase ratio for 10 min, gradually decrease mobile phase B and increase mobile phase A, until 10.1 min when mobile phase B is adjusted to 1%–3% and mobile phase A to 99%–97%, maintain this mobile phase ratio for 20 min. The hydrolysis product of impurity 4-nitrobenzenesulfonyl chloride shows a good peak shape.
[0242] Example 13
[0243] (1) Reference stock solution: Take an appropriate amount of 4-nitrobenzenesulfonyl chloride reference standard, accurately weigh it, dissolve it in methanol and dilute it quantitatively to prepare a solution containing about 125 μg per 1 ml; accurately measure 1 ml, put it in a 20 ml volumetric flask, dilute it to the mark with methanol, and shake well; accurately measure 1 ml, put it in a 100 ml volumetric flask, dilute it to the mark with methanol, and shake well to obtain the solution.
[0244] (2) Reference solution: Accurately measure 1 ml of the reference stock solution, place it in a 10 ml volumetric flask, add 1 ml of 30% ammonia-methanol solution, shake well, let stand at room temperature for 10 minutes, dilute to the mark with methanol-water (50:50), and shake well to obtain the solution.
[0245] (3) Brand and model of high performance liquid chromatograph: Agilent 1260 Infinity II;
[0246] (4) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000; (5) Chromatographic column: Phenomen Titan C18, 2.1×50mm, 3μm;
[0247] (6) Mobile phase A: ammonia-water (5:1000), mobile phase B: methanol; elution is carried out according to the following gradient: by volume ratio, start with 2% mobile phase B and 98% mobile phase A as the initial mobile phase, maintain this mobile phase ratio for 3 min, gradually increase mobile phase B and decrease mobile phase A, until 3.1 min when mobile phase B is adjusted to 85% and mobile phase A is adjusted to 15%, maintain this mobile phase ratio for 10 min, gradually decrease mobile phase B and increase mobile phase A, until 10.1 min when mobile phase B is adjusted to 2% and mobile phase A is adjusted to 98%, maintain this mobile phase ratio for 20 min.
[0248] (7) The flow rate is 0.2 ml per minute;
[0249] (8) The column temperature is 30℃;
[0250] (9) The temperature of the injection tray is room temperature;
[0251] (10) Injection volume 1 μL;
[0252] (11) The mass spectrometry conditions were as follows: detection was performed using the multiple reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, with electrospray ionization, collision gas at 8 psi, curtain gas at 35 psi, nebulizer 1 at 40 psi, nebulizer 2 at 45 psi, spray needle voltage at -4500 V, and drying temperature at 500 °C; quantitative ion pair was 202.0 / 138.1, declustering voltage (DP) was -60 V, and collision voltage (CE) was -35 V.
[0253] (12) Take the control solution and inject it.
[0254] (13) Figure 8 The peak results for the hydrolysis products of impurity 4-nitrobenzenesulfonyl chloride in Examples 1 and 13 are obtained from... Figure 8 It can be seen that when the mobile phase B is acetonitrile, the peak shape of the hydrolysis product of impurity 4-nitrobenzenesulfonyl chloride is good.
[0255] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The detection method described in this invention has been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.
Claims
1. A method for determining 4-nitrobenzenesulfonyl chloride in darunavir ethanol or its formulations, characterized in that, The method described is ESI-LC-MS technology, which includes: Step (1): Hydrolyze 4-nitrobenzenesulfonyl chloride with ammonia at room temperature to generate 4-nitrobenzenesulfonic acid, which can be ionized in ESI-LC-MS; Step (2): Using ESI-LC-MS, a reversed-phase column was used with aqueous phase A and organic phase B as mobile phase. The flow rate, column temperature, injection plate temperature, injection volume, gradient elution and mass spectrometry conditions were set. In MRM acquisition mode, 4-nitrobenzenesulfonic acid generated in step (1) was determined to achieve quantitative detection of 4-nitrobenzenesulfonyl chloride.
2. The method for determining 4-nitrobenzenesulfonyl chloride in darunavir ethanol or its formulations according to claim 1, characterized in that, The ammonia water in step (1) contains methanol, and the reaction system is an ammonia water-methanol solution, which is reacted at room temperature for 0-45 minutes.
3. The method for determining 4-nitrobenzenesulfonyl chloride in darunavir ethanol or its formulations according to claim 2, characterized in that, The concentration of the ammonia-methanol solution is 0-100%, and the reaction time is 10 min.
4. The method for determining 4-nitrobenzenesulfonyl chloride in darunavir ethanol or its formulations according to claim 3, characterized in that, The concentration of the ammonia-methanol solution is 30%.
5. The method for determining 4-nitrobenzenesulfonyl chloride in darunavir ethanol or its formulations according to claim 1, characterized in that, The mobile phase B is selected from acetonitrile or methanol.
6. The method for determining 4-nitrobenzenesulfonyl chloride in darunavir ethanolate or its formulations according to claim 5, characterized in that, The mobile phase B is selected from acetonitrile.
7. The method for determining 4-nitrobenzenesulfonyl chloride in darunavir ethanol or its formulations according to claim 1, characterized in that, The flow rate is selected from 0.15~0.25ml / min, the column temperature is selected from 25~35℃, the injection plate temperature is selected from room temperature, and the injection volume is selected from 1μL.
8. The method for determining 4-nitrobenzenesulfonyl chloride in darunavir ethanolate or its formulations according to claim 1, characterized in that, The gradient elution is as follows: using a volume ratio of 1-3% mobile phase B and 97-99% mobile phase A as the initial mobile phase, maintain this mobile phase ratio for 3 minutes, gradually increase mobile phase B and decrease mobile phase A, until 3.1 minutes when mobile phase B is adjusted to 85% and mobile phase A to 15%, maintain this mobile phase ratio for 10 minutes, gradually decrease mobile phase B and increase mobile phase A, until 10.1 minutes when mobile phase B is adjusted to 2% and mobile phase A to 98%, maintain this mobile phase ratio for 20 minutes.
9. A method for determining 4-nitrobenzenesulfonyl chloride in darunavir ethanol complex or its formulations according to any one of claims 1-8, characterized in that, The mass spectrometry conditions were as follows: detection was performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, with electrospray ionization and negative ion mode scanning; collision gas was 8 psi, curtain gas was 35 psi, nebulizer 1 was 40 psi, nebulizer 2 was 45 psi, spray needle voltage was -4500 V, and drying temperature was 500 °C; quantitative ion pair was 202.0 / 138.1, declustering voltage (DP) was -60 V, and collision voltage (CE) was -35 V.