A method for determining the diethyl sulfate content in methyl 4-acetamido-2-ethoxybenzoate.
By combining gas chromatography with headspace sampling technology, the problems of simplicity and accuracy in the detection of diethyl sulfate content in methyl 4-acetamido-2-ethoxybenzoate have been solved in the existing technology, achieving efficient and accurate detection results and ensuring drug safety.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- 珠海润都制药股份有限公司
- Filing Date
- 2024-12-30
- Publication Date
- 2026-06-30
AI Technical Summary
There is a lack of a simple and accurate detection method for the content of diethyl sulfate in methyl 4-acetamido-2-ethoxybenzoate in the existing technology, especially since the method requires high-quality instruments and equipment and is not versatile.
Gas chromatography combined with headspace sampling was used, with sodium iodide and ascorbic acid as derivatizing agents. Derivatizing agent and reference solution were prepared, and the content of diethyl sulfate in methyl 4-acetamido-2-ethoxybenzoate was detected by gas chromatography. The specific steps included derivatizing agent preparation, test solution preparation, and chromatographic condition setting.
This provides a convenient, efficient, and accurate detection method to ensure the detection of diethyl sulfate content in methyl 4-acetamido-2-ethoxybenzoate, thus guaranteeing medication safety.
Smart Images
Figure CN122306966A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical analysis, specifically to a method for detecting the content of diethyl sulfate in methyl 4-acetamido-2-ethoxybenzoate. Background Technology
[0002] Genotoxic impurities, even at low levels, can directly cause DNA damage, leading to DNA mutations. Therefore, the content of genotoxic impurities in active pharmaceutical ingredients (APIs) is a key aspect of quality control, and the industry has established various standards to regulate the impurity content in drugs. Methyl 4-acetamido-2-ethoxybenzoate is a pharmaceutical synthesis intermediate that may generate diethyl sulfate impurities during its synthesis process. Diethyl sulfate is a typical genotoxic impurity, thus its content needs to be monitored.
[0003] Regarding the detection method of diethyl sulfate, Guo Zhiqiang et al., in their article "Determination of Two Genotoxic Impurities in Thioconazole Nitrate by Derivatization Combined with LC-MS" published in the journal Chemical Industry and Engineering, disclosed a method for determining the alkyl sulfate ester genotoxic impurities dimethyl sulfate and diethyl sulfate in thioconazole nitrate using HPLC-MS. The method employed a HILIC column and a 20 mmol·L⁻¹ aqueous phase. -1 The mixture consists of ammonium formate solution and formic acid (1000:1), with acetonitrile as the organic phase and an aqueous phase and organic phase (10:90). This method is used to detect thioconazole nitrate. It requires liquid chromatography-mass spectrometry (LC-MS) and a HILIC column, which places certain demands on the equipment and limits its versatility. Summary of the Invention
[0004] Based on the above technical background, we disclose a method for detecting the diethyl sulfate content in methyl 4-acetamido-2-ethoxybenzoate in this invention. This method is performed by gas chromatography and includes the following steps: preparing a solution. Derivatizing agent: Dissolve sodium iodide and ascorbic acid in purified water to prepare a derivatizing agent, wherein the sodium iodide concentration is preferably 600 mg / ml and the ascorbic acid concentration is preferably 10 mg / ml; Reference solution: Dissolve approximately ethyl sulfate reference standard in acetonitrile and dilute to the mark to prepare a reference solution, wherein the preferred concentration of diethyl sulfate is 2.5 μg / ml; Test solution: Take methyl 4-acetamido-2-ethoxybenzoate as the test sample, dissolve it in acetonitrile and derivatizing agent, and seal with a cap; wherein the methyl 4-acetamido-2-ethoxybenzoate is preferably 0.02 g / ml.
[0005] More specifically, the preparation process of a solution is as follows: Derivatizing agent: Weigh approximately 60g of sodium iodide and 1g of ascorbic acid accurately, place them in a 100ml volumetric flask, add purified water to dissolve and dilute to the mark, and shake well.
[0006] Blank solution: Accurately measure 4.0 ml of diluent and 1.0 ml of derivatizing agent into a 20 ml headspace vial and seal with a pressure cap.
[0007] Reference solution: Accurately weigh approximately 25 mg of diethyl sulfate reference standard, place it in a 100 ml volumetric flask, dissolve and dilute to the mark with diluent, and shake well; accurately measure 1.0 ml of the solution, place it in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well; accurately measure 4.0 ml of the reference standard stock solution and 1.0 ml of the derivatizing agent, place them in a 20 ml headspace vial, and seal with a cap.
[0008] Test solution: Weigh approximately 100 mg of methyl 4-acetamido-2-ethoxybenzoate accurately and place it in a 20 ml headspace vial. Accurately measure 4.0 ml of diluent and 1.0 ml of derivatizing agent into the vial and seal it with the cap.
[0009] Chromatographic conditions: Instrument: Gas chromatograph equipped with ECD detector & headspace sampler; Column: Agilent DB-624 30m × 0.53mm × 3μm or equivalent column; Injector temperature: 220℃; Detector temperature: 250℃; Split ratio: 40:1; Carrier gas: N2. 2， Flow rate: 1.5 mL / min, temperature program: initial column temperature 70 °C, increase to 150 °C at a rate of 5 °C / min and hold for 0 min; then run at 240 °C and hold for 5 min, flow rate 3 mL / min.
[0010] HS headspace conditions: Equilibrium temperature: 70℃, quantitative loop temperature: 80℃, transfer line temperature: 90℃, equilibrium time: 40 min, GC cycle time: 30 min, injection time: 0.5 min; after the system stabilizes, record the chromatogram. The RSD of the peak area of diethyl sulfate in the reference solution for 6 consecutive tests is ≤10.0%.
[0011] Calculation formula:
[0012] In the formula: A i : Peak area of diethyl sulfate in the test solution; A s :6 refers to the average peak area of diethyl sulfate in the reference solution; C s : Concentration of diethyl sulfate in the reference solution, µg / mL; C i : Concentration of the test solution, g / ml.
[0013] The verification of the method is summarized as follows:
[0014] The method provides a convenient, efficient, and accurate detection method for diethyl sulfate in methyl 4-acetamido-2-ethoxybenzoate. This method uses gas chromatography to detect the content of diethyl sulfate in methyl 4-acetamido-2-ethoxybenzoate, thereby effectively ensuring medication safety. Attached Figure Description
[0015] Figure 1 blank solution Figure 2 Reference solution Figure 3 For the test solution Figure 4 For the limit of quantitation solution Figure 5 For spiking solution Figure 6 For accuracy solutions, Figure 2-6 The peak text is Diethyl sulfate, and the numerical value is the same as the RT[min] value in the table below. Detailed Implementation Example 1
[0016] System Applicability System suitability was determined by measuring the RSD of the peak area of diethyl sulfate in the 6-pipe reference solution. The required peak area RSD of diethyl sulfate in the 6-pipe reference solution was ≤10.0%. Chromatographic conditions: The gas chromatograph is equipped with an ECD detector & headspace sampler, analytical balance, Agilent DB-624 30m × 0.53mm × 3μm column or equivalent performance column, injection port temperature: 220℃, detector temperature: 250℃, split ratio: 40:1, carrier gas: N2. 2， Flow rate: 1.5 mL / min, temperature program: initial column temperature 70 °C, increase to 150 °C at a rate of 5 °C / min and hold for 0 min; then run at 240 °C and hold for 5 min, flow rate 3 mL / min.
[0017] HS headspace conditions: Equilibrium temperature: 70℃, quantitative loop temperature: 80℃, transfer line temperature: 90℃, equilibrium time: 40 min, GC cycle time: 30 min, injection time: 0.5 min.
[0018]
[0019] The peak area RSD of diethyl sulfate in the reference solution was 2.1%, which meets the requirements of the protocol. (The standard stipulates that the peak area RSD of diethyl sulfate in the reference solution should be ≤10.0%).
[0020] Example 2 Specificity experiment Specificity was determined by examining whether the blank solution interfered with the detection of diethyl sulfate, whether other components interfered with the detection of diethyl sulfate, and the resolution between diethyl sulfate and adjacent peaks in the resolution solution. The blank solution should have minimal interference with the detection of diethyl sulfate; other components should have no interference with the detection of diethyl sulfate; and the resolution between diethyl sulfate and adjacent peaks in the resolution solution should be ≥1.5. Chromatographic conditions were the same as in Example 1.
[0021]
[0022] The blank solution showed no interference with the detection of diethyl sulfate; other components showed no interference with the detection of diethyl sulfate; the minimum resolution between diethyl sulfate and adjacent peaks in the resolution solution was 1.5. This meets the validation requirements. (Standard requirements: The blank solution should show no interference with the detection of diethyl sulfate; other components should show no interference with the detection of diethyl sulfate; the resolution between diethyl sulfate and adjacent peaks in the resolution solution should be ≥1.5.)
[0023] Example 3 Precision test Precision was achieved by determining the RSD of diethyl sulfate in six spiked solutions. The RSD of diethyl sulfate content in the six spiked solutions was required to be ≤10.0%. Chromatographic conditions were the same as in Example 1.
[0024]
[0025] The RSD of diethyl sulfate content in the 6 spiked solutions was 0.8%, which meets the requirements of the protocol (the standard stipulates that the RSD of diethyl sulfate content in 6 spiked solutions should be ≤10.0%).
Claims
1. A method for detecting the diethyl sulfate content in methyl 4-acetylamino-2- ethoxybenzoate, characterized in that the detection is carried out using gas chromatography, and in that Includes the following steps: (1) Preparation of solution Derivatizing agent: Dissolve sodium iodide and ascorbic acid in water to prepare a derivatizing agent; Reference solution: Dissolve diethyl sulfate reference standard in acetonitrile and dilute to the mark to prepare a reference solution; Test solution: Take methyl 4-acetamido-2-ethoxybenzoate as the test sample, add acetonitrile and derivatizing agent to dissolve it, and obtain the test solution; (2) Inject the reference solution and the test solution. The chromatographic conditions are as follows: a gas chromatograph equipped with an ECD detector and a headspace sampler is used. The column is an Agilent DB-624 30m×0.53mm×3μm. The injection port temperature is 220℃, the detector temperature is 250℃, the carrier gas is N2, the flow rate is 1.5mL / min, and the temperature program is as follows: the initial column temperature is 70℃, the temperature is increased to 150℃ at a rate of 5℃ / min and held for 0min; then the temperature is increased to 240℃ and held for 5min, and the flow rate is 3ml / min.
2. The headspace sampler as described in claim 1, wherein the headspace conditions are: equilibrium temperature: 70°C, quantitative loop temperature: 80°C, transfer line temperature: 90°C, equilibrium time: 40 min, GC cycle time: 30 min, and injection time: 0.5 min.