Method for determining potential genotoxic impurities in esomeprazole magnesium by HPLC (High Performance Liquid Chromatography)
The genotoxic impurities in esomeprazole magnesium were separated and detected by HPLC, which solved the problem of difficulty in controlling impurity content in existing technologies, and achieved efficient, rapid and low-cost detection results, thus ensuring drug safety.
Patent Information
- Application Number
- CN202510924872.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-04
- Publication Date
- 2025-11-21
AI Technical Summary
Existing technologies are insufficient to effectively control the content of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxides, a potential genotoxic impurity in esomeprazole magnesium, thus affecting drug safety.
The content of genotoxic impurities in esomeprazole magnesium was determined by HPLC using an Agilent Eclipse Plus C18 column and a mobile phase of pH 7.4 phosphate buffer (A) and acetonitrile (B), with gradient elution. The detection wavelength was 217-227 nm, the flow rate was 0.9-1.1 mL/min, the injection volume was 10 μL, and the external standard method was used.
It achieves efficient separation, rapid analysis, and low-cost detection of genotoxic impurities, controlling the impurity content to within 18.75 ppm to ensure drug quality and safety.
Smart Images
Figure CN120992784A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of substance detection, and relates to a method for determining a potential genotoxic impurity in esomeprazole magnesium by HPLC, in particular to a method for determining 4-methoxy-2,3,5-trimethylpyridine N-oxide (RC21) in esomeprazole magnesium by HPLC. BACKGROUND
[0002] Proton pump inhibitors (PPIs) are the first choice for treating acid-related diseases such as peptic ulcer, gastroesophageal reflux disease, etc. The commonly used proton pump inhibitors (PPIs) in clinical are omeprazole, lansoprazole, rabeprazole, pantoprazole and esomeprazole 5 kinds. Omeprazole, as the first proton pump inhibitor (PPI) drug, has been consistently recognized for its efficacy in treating acid-related diseases.
[0003] Esomeprazole is the S-optical isomer of omeprazole, and its chemical name is 5-methoxy-2-((S)-((4-methoxy-3,5-dimethyl-2-pyridyl) methyl) sulfinyl-1H-benzimidazole magnesium. Esomeprazole magnesium is the first isomer proton pump inhibitor (PPI) in the world, which can reduce gastric acid secretion by specifically inhibiting the proton pump in gastric wall cells, and is widely used in clinical applications. Indications: gastroesophageal reflux disease (GERD), healing of erosive esophagitis, symptomatic relief (such as heartburn, acid reflux) and prevention of recurrence; non-erosive reflux disease (NERD) symptom control, suitable for patients with typical reflux symptoms but no esophageal mucosa damage found by endoscopy; peptic ulcer, gastric ulcer and duodenal ulcer, used to promote ulcer healing and relieve symptoms such as abdominal pain, especially suitable for patients with excessive gastric acid secretion; combined with antibiotics to eradicate Helicobacter pylori (Hp), often combined with clarithromycin, amoxicillin and other antibiotics and bismuth agents to form a quadruple therapy for the eradication of Hp infection; Zollinger-Ellison syndrome, used to treat high gastric acid secretion state caused by gastrinoma, control the symptoms of severe peptic ulcer, diarrhea and other symptoms caused by excessive gastric acid secretion; other gastric acid-related diseases, functional dyspepsia (FD), which can improve the symptoms of patients with abnormal gastric acid secretion or reflux symptoms; prevention of non-steroidal anti-inflammatory drug (NSAIDs) related gastric mucosa damage: such as patients taking aspirin, ibuprofen and other drugs for a long time, which can reduce the risk of gastric ulcer and bleeding.
[0004] 4-methoxy-2,3,5-trimethylpyridine N-oxide (RC21, formula I) is a by-product of the initial material of esomeprazole magnesium, which has a warning structure of nitroxide. In order to ensure the safety of patients taking medicine, it should be considered as a potential genotoxic impurity.
[0005]
[0006] Esomeprazole magnesium is the main active ingredient in esomeprazole magnesium-related formulations. The acceptable limits for genotoxic impurities are generally calculated based on the threshold for toxicological concern (TTC) limits.
[0007]
[0008] Table 1. TTC Limits
[0009] Treatment period ≤ 1 month 1-12 months 1-10 years ≥ 10 years Daily intake (μg / d) 120 20 10 1.5
[0010] Esomeprazole magnesium, as the main active ingredient in oral formulations, has a maximum daily dose of 80 mg. Based on the strictest daily intake of 1.5 μg / d, the acceptable limit for genotoxic impurities is 17.85 ppm. Summary of the Invention
[0011] The purpose of this invention is to establish an HPLC (high performance liquid chromatography) method for determining the potential genotoxic impurity 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide (RC21) in esomeprazole magnesium, so as to better control the quality of esomeprazole magnesium.
[0012] The objective of this invention is achieved through the following technical solution:
[0013] A method for determining potential genotoxic impurities in esomeprazole magnesium by HPLC includes:
[0014] Preparation of test solution: Weigh 30-90 mg of esomeprazole magnesium accurately, place it in a 10 mL volumetric flask, add an appropriate amount of methanol, sonicate to dissolve the sample, dilute to the mark with methanol, and shake well.
[0015] Preparation of reference solution: Weigh an appropriate amount of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide accurately, quantify with methanol, and prepare a reference solution containing 14-170 ng of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide per 1 mL.
[0016] The reference solution and the test solution were analyzed by HPLC, and the content of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxides, a genotoxic impurity, in esomeprazole magnesium was determined by external standard method.
[0017] Preferably, the test solution is prepared as follows: Weigh approximately 60-90 mg of esomeprazole magnesium accurately, place it in a 10 mL volumetric flask, add an appropriate amount of methanol, sonicate to dissolve the sample, dilute to the mark with methanol, and shake well.
[0018] More preferably, prepare the test solution: accurately weigh about 60 mg of esomeprazole magnesium, place it in a 10 mL volumetric flask, add an appropriate amount of methanol, sonicate to dissolve the sample, dilute to the mark with methanol, and shake well.
[0019] Preferably, the reference solution is prepared by accurately weighing an appropriate amount of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide, quantifying it with methanol, and preparing a reference solution containing 110-170 ng of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide per 1 mL of solution.
[0020] More preferably, a reference solution is prepared by accurately weighing an appropriate amount of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide and quantitatively dissolving it in methanol to prepare a reference solution containing 110 ng of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide per 1 mL of solution.
[0021] The HPLC detection conditions were as follows: the column was packed with octadecylsilane-bonded silica gel; mobile phase A was pH 7.4 phosphate buffer; mobile phase B was acetonitrile; gradient elution was performed; the detection wavelength was 217–227 nm; the flow rate was 0.9–1.1 mL / min; the injection volume was 10 μL; and the gradient elution program was as follows:
[0022]
[0023] The chromatographic column is selected from Agilent Eclipse Plus C18 (4.6×150mm, 5μm) or Agilent Eclipse Plus C18 (4.6×150mm, 3.5μm).
[0024] Preferably, the HPLC detection conditions are as follows: an Agilent Eclipse Plus C18 column (4.6 × 150 mm, 5 μm) or an Agilent Eclipse Plus C18 column (4.6 × 150 mm, 3.5 μm) is used; gradient elution is performed using pH 7.4 phosphate buffer as mobile phase A and acetonitrile as mobile phase B; the detection wavelength is 217–227 nm; the flow rate is 0.9–1.1 mL / min; the injection volume is 10 μL; and the gradient elution program is as follows:
[0025]
[0026] More preferably, the HPLC detection conditions are as follows: the chromatographic column is an Agilent Eclipse Plus C18 (4.6 × 150 mm, 5 μm) or an Agilent Eclipse Plus C18 (4.6 × 150 mm, 3.5 μm); the mobile phase is pH 7.4 phosphate buffer (A), and the mobile phase is acetonitrile (B); gradient elution is performed; the detection wavelength is 217 nm, 222 nm, or 227 nm; the flow rate is 0.9 mL / min, 1.0 mL / min, or 1.1 mL / min; the injection volume is 10 μL; and the gradient elution program is as follows:
[0027]
[0028]
[0029] The pH 7.4 phosphate buffer solution is prepared by weighing 3.36 g of sodium dihydrogen phosphate (dihydrate) and 1.11 g of disodium hydrogen phosphate (dodecanoate) into 1000 mL of water and adjusting the pH to 7.4 with 1 mol / L sodium hydroxide solution.
[0030] The beneficial effects of this invention are:
[0031] This invention employs HPLC to determine 4-methoxy-2,3,5-trimethylpyridine nitrogen oxides (RC21), a potential genotoxic impurity in esomeprazole magnesium. It features high separation efficiency, fast analysis speed, high detection sensitivity, and low detection cost.
[0032] Based on the method of this invention, by detecting potential genotoxic impurities in esomeprazole magnesium and controlling the potential genotoxic impurity 4-methoxy-2,3,5-trimethylpyridine N-oxide in esomeprazole magnesium to be no more than 18.75 ppm, the quality of esomeprazole magnesium can be effectively controlled. Attached Figure Description
[0033] Figure 1 The HPLC comparison chromatograms of the RC21 positioning solution, spiked test solution, test solution, reference solution, and blank solvent in Example 1 are shown below. From top to bottom, the HPLC chromatograms are of the RC21 positioning solution, spiked test solution, test solution, reference solution, and blank solvent.
[0034] Figure 2 This is the HPLC spectrum of the detection limit solution in Example 2.
[0035] Figure 3 This is the HPLC chromatogram of the limit-of-quantity solution in Example 3.
[0036] Figure 4The first image shows the HPLC chromatogram of the reference solution in Example 4; from top to bottom, the second image shows the HPLC chromatograms of the reference solutions at detection wavelengths of 222 nm, 217 nm, and 227 nm.
[0037] Figure 5 The HPLC chromatogram of the reference solution in Example 5 (Agilent Eclipse Plus C18 column (4.6 mm × 150 mm, 3.5 μm)).
[0038] Figure 6 The image shows the HPLC chromatogram of the reference solution in Example 6 (flow rate 0.9 mL / min).
[0039] Figure 7 The image shows the HPLC chromatogram of the reference solution in Example 6 (flow rate 1.1 mL / min).
[0040] Figure 8 This is the HPLC chromatogram of the accuracy solution for the quantitation limit in Example 7.
[0041] Figure 9 This is the HPLC chromatogram of the 80% accuracy solution in Example 7.
[0042] Figure 10 This is the HPLC chromatogram of the 100% accuracy solution in Example 7.
[0043] Figure 11 This is the HPLC chromatogram of the 120% accuracy solution in Example 7.
[0044] Figure 12 The HPLC chromatogram of the limit-of-quantity solution under HPLC detection conditions in Comparative Example 1 is shown. Detailed Implementation
[0045] The technical solution of the present invention is further illustrated by the following embodiments, but the embodiments are not intended to limit the present invention.
[0046] All reagents used in the examples are commercially available or synthesized using existing methods.
[0047] HPLC (Liquid Chromatography) Model:
[0048]
[0049] Reagents used:
[0050]
[0051] Example 1
[0052] Methods for HPLC determination of potentially genotoxic impurities in esomeprazole magnesium include:
[0053] Preparation of test solution: Weigh approximately 60 mg of esomeprazole magnesium accurately, place it in a 10 mL volumetric flask, add an appropriate amount of methanol (blank solvent), sonicate to dissolve the sample, dilute to the mark with methanol, and shake well.
[0054] Preparation of reference solution: Weigh an appropriate amount of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide (RC21) accurately, quantify with methanol, and prepare a solution containing approximately 110 ng of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide per 1 mL.
[0055] Preparation of RC21 positioning solution: Weigh 11.48 mg of RC21 reference standard and place it in a 100 mL volumetric flask. Add an appropriate amount of blank solvent, sonicate to dissolve, and dilute to volume with the blank solvent. Accurately measure 1.0 mL of the impurity stock solution and place it in a 100 mL volumetric flask. Dilute to the mark with the blank solvent and mix well.
[0056] Preparation of spiked test solution: Weigh 60.01 mg of esomeprazole magnesium trihydrate and place it in a 10 mL volumetric flask. Add an appropriate amount of blank solvent and dissolve by sonication. Accurately measure 1.0 mL of impurity stock solution and place it in the same volumetric flask. Dilute the blank solvent to the mark and shake well.
[0057] The reference solution, test solution, RC21 positioning solution, spiked test solution, and blank solvent were analyzed by HPLC. HPLC detection conditions: the column was an Agilent Eclipse Plus C18 liquid chromatography column (4.6 mm inner diameter × 150 mm length, 5 μm particle size), mobile phase A was pH 7.4 phosphate buffer (3.36 g of sodium dihydrogen phosphate (dihydrate) and 1.11 g of disodium hydrogen phosphate (dodecanoate) were weighed into 1000 mL of water, and the pH was adjusted to 7.4 with 1 mol / L sodium hydroxide solution), and acetonitrile was used as mobile phase B for gradient elution; the detection wavelength was 222 nm; the flow rate was 1.0 mL / min; the injection volume was 10 μL; the gradient elution program is shown in Table 2.
[0058] Table 2. Gradient elution program
[0059]
[0060]
[0061] Figure 1 The HPLC comparison chromatograms from top to bottom show the RC21 positioning solution, spiked test solution, test solution, reference solution, and blank solvent. This indicates that the blank solvent does not interfere with the detection of 4-methoxy-2,3,5-trimethylpyridine N-oxides.
[0062] Example 2
[0063] Preparation of detection limit solution: Weigh an appropriate amount of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide (RC21) accurately, quantify with methanol, and prepare a solution containing approximately 14 ng of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide per 1 mL.
[0064] The limit of detection solution was analyzed by HPLC. The HPLC detection conditions were as follows: the column was an Agilent EclipsePlus C18 (4.6 mm × 150 mm, 5 μm); the mobile phase A was pH 7.4 phosphate buffer (3.36 g of sodium dihydrogen phosphate (dihydrate) and 1.11 g of disodium hydrogen phosphate (dodecanoate) were weighed into 1000 mL of water and the pH was adjusted to 7.4 with 1 mol / L sodium hydroxide solution); the mobile phase B was acetonitrile; the detection wavelength was 222 nm; the flow rate was 1.0 mL / min; the injection volume was 10 μL; and the gradient elution program is shown in Table 2.
[0065] The HPLC chromatogram of the detection limit solution is shown below. Figure 2 It can be seen that the detection limit of the method of the present invention is 2.3 ppm, which is equivalent to 12% of the limit of 18.75 ppm. This indicates that the detection sensitivity of the method of the present invention is very high.
[0066] Example 3
[0067] Prepare a solution with a specified volume limit: Weigh an appropriate amount of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide (RC21) accurately, quantify with methanol, and prepare a solution containing approximately 34 ng of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide per 1 mL.
[0068] The limit of quantitation solution was detected by HPLC. The HPLC detection conditions were as follows: the column was an Agilent EclipsePlus C18 (4.6 mm × 150 mm, 5 μm); the mobile phase A was pH 7.4 phosphate buffer (3.36 g of sodium dihydrogen phosphate (dihydrate) and 1.11 g of disodium hydrogen phosphate (dodecanoate) were weighed into 1000 mL of water and the pH was adjusted to 7.4 with 1 mol / L sodium hydroxide solution); the mobile phase B was acetonitrile; the detection wavelength was 222 nm; the flow rate was 1.0 mL / min; the injection volume was 10 μL; and the gradient elution program is shown in Table 2.
[0069] The HPLC chromatogram of the limit-of-quantity solution is shown below. Figure 3 It can be seen that the limit of quantitation of the method of the present invention is 5.7 ppm, which is 30% of the limit of 18.75 ppm. This indicates that the method of the present invention has very high quantitative sensitivity.
[0070] Example 4
[0071] Preparation of reference solution: Take an appropriate amount of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide (RC21), accurately weigh it, and quantify it with methanol to prepare a solution containing approximately 110 ng of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide per 1 mL.
[0072] The reference solution was analyzed by HPLC. The HPLC detection conditions were as follows: the column was an Agilent EclipsePlus C18 (4.6 mm × 150 mm, 5 μm); the mobile phase A was pH 7.4 phosphate buffer (3.36 g of sodium dihydrogen phosphate (dihydrate) and 1.11 g of disodium hydrogen phosphate (dodecanoate) were weighed into 1000 mL of water, and the pH was adjusted to 7.4 with 1 mol / L sodium hydroxide solution); and the mobile phase B was acetonitrile. Gradient elution was performed. The detection wavelengths were 217 nm, 222 nm, and 227 nm; the flow rate was 1.0 mL / min; and the injection volume was 10 μL. The gradient elution program is shown in Table 2.
[0073] The HPLC chromatogram of the reference solution is shown below. Figure 4 This indicates that the wavelength has no significant impact on the detection results.
[0074] Example 5
[0075] Preparation of reference solution: Take an appropriate amount of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide (RC21), accurately weigh it, and quantify it with methanol to prepare a solution containing approximately 110 ng of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide per 1 mL.
[0076] The reference solution was analyzed by HPLC. The HPLC detection conditions were as follows: the column was an Agilent Eclipse Plus C18 column (inner diameter 4.6 mm × length 150 mm, particle size 3.5 μm); the mobile phase A was pH 7.4 phosphate buffer (3.36 g of sodium dihydrogen phosphate (dihydrate) and 1.11 g of disodium hydrogen phosphate (dodecanoate) were weighed into 1000 mL of water, and the pH was adjusted to 7.4 with 1 mol / L sodium hydroxide solution); the mobile phase B was acetonitrile; the detection wavelength was 222 nm; the flow rate was 1.0 mL / min; the injection volume was 10 μL; and the gradient elution program is shown in Table 2.
[0077] The HPLC chromatogram of the reference solution is shown below. Figure 5 This indicates that different column specifications have no significant impact on the detection results.
[0078] Example 6
[0079] Preparation of reference solution: Take an appropriate amount of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide (RC21), accurately weigh it, and quantify it with methanol to prepare a solution containing approximately 110 ng of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide per 1 mL.
[0080] The reference solution was analyzed by HPLC. The HPLC detection conditions were as follows: the column was an Agilent Eclipse Plus C18 column (4.6 mm × 150 mm, 5 μm); the mobile phase A was pH 7.4 phosphate buffer (3.36 g of sodium dihydrogen phosphate (dihydrate) and 1.11 g of disodium hydrogen phosphate (dodecanoate) were weighed into 1000 mL of water and the pH was adjusted to 7.4 with 1 mol / L sodium hydroxide solution); the mobile phase B was acetonitrile; the detection wavelength was 222 nm; the flow rate was 0.9 mL / min or 1.1 mL / min; the injection volume was 10 μL; the gradient elution program is shown in Table 2.
[0081] The HPLC chromatogram of the reference solution is shown below. Figure 6 (flow rate of 0.9 mL / min) and Figure 7 (Flow rate was 1.1 mL / min), indicating that changes in flow rate had no significant impact on the detection results.
[0082] Example 7
[0083] Examine the accuracy of the method of the present invention.
[0084] Methods for HPLC determination of potentially genotoxic impurities in esomeprazole magnesium include:
[0085] Preparation of test solution: Weigh approximately 60 mg of esomeprazole magnesium accurately, place it in a 10 mL volumetric flask, add an appropriate amount of methanol, sonicate to dissolve the sample, dilute to the mark with methanol, and shake well.
[0086] Preparation of reference solution: Take an appropriate amount of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide (RC21), accurately weigh it, and quantify it with methanol to prepare a solution containing approximately 110 ng of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide per 1 mL.
[0087] Impurity reference solution: Weigh an appropriate amount of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide (RC21) accurately, quantify with methanol, and prepare a solution containing approximately 1.1 μg of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide per 1 mL.
[0088] Quantification limit accuracy solution: Accurately weigh approximately 60 mg of esomeprazole magnesium, place it in a 10 mL volumetric flask, add an appropriate amount of methanol, and sonicate to dissolve the sample; accurately measure 0.3 mL of impurity reference solution, place it in a volumetric flask, dilute to the mark with methanol, and shake well.
[0089] 80% accuracy solution: Weigh approximately 60 mg of esomeprazole magnesium accurately, place it in a 10 mL volumetric flask, add an appropriate amount of methanol, and sonicate to dissolve the sample; accurately measure 0.8 mL of impurity reference solution, place it in a volumetric flask, dilute to the mark with methanol, and shake well.
[0090] 100% accuracy solution: Weigh approximately 60 mg of esomeprazole magnesium accurately, place it in a 10 mL volumetric flask, add an appropriate amount of methanol, and sonicate to dissolve the sample; accurately measure 1 mL of impurity reference solution, place it in a volumetric flask, dilute to the mark with methanol, and shake well.
[0091] 120% accuracy solution: Weigh approximately 60 mg of esomeprazole magnesium accurately, place it in a 10 mL volumetric flask, add an appropriate amount of methanol, and sonicate to dissolve the sample; accurately measure 1.2 mL of impurity reference solution, place it in a volumetric flask, dilute to the mark with methanol, and shake well.
[0092] The reference solution, test solution, limit of quantitation (LOQ) accuracy solution, 80% accuracy solution, 100% accuracy solution, and 120% accuracy solution were analyzed by HPLC. HPLC detection conditions: the column was an Agilent EclipsePlus C18 (4.6 mm × 150 mm, 5 μm); mobile phase A was pH 7.4 phosphate buffer (3.36 g of sodium dihydrogen phosphate (dihydrate) and 1.11 g of disodium hydrogen phosphate (dodecanoic acid) were weighed into 1000 mL of water, and the pH was adjusted to 7.4 with 1 mol / L sodium hydroxide solution); acetonitrile was used as mobile phase B; gradient elution was performed; the detection wavelength was 222 nm; the flow rate was 1.0 mL / min; the injection volume was 10 μL; the gradient elution program is shown in Table 2.
[0093] Figure 8 HPLC chromatogram of the solution for the limit of quantitation accuracy; Figure 9 HPLC chromatogram of an 80% accuracy solution; Figure 10 HPLC chromatogram of a 100% accuracy solution; Figure 11 This is the HPLC chromatogram of a 120% accuracy solution.
[0094] The content of 4-methoxy-2,3,5-trimethylpyridine nitride (RC21) in the limit of quantitation (LOQ) accuracy solution, 80% accuracy solution, 100% accuracy solution, and 120% accuracy solution was determined using the external standard method. The recovery rate of 4-methoxy-2,3,5-trimethylpyridine nitride (RC21) in the LOQ accuracy solution was 94.6%, the recovery rate in the 80% accuracy solution was 104.1%, the recovery rate in the 100% accuracy solution was 103.2%, and the recovery rate in the 120% accuracy solution was 101.9%. Therefore, the method of this invention can effectively and accurately reflect the quality of the product.
[0095] Comparative Example 1
[0096] The inventors used the preferred HPLC detection conditions of this invention (Example 1), only adjusting the elution gradient.
[0097] Prepare a solution with a specified volume limit: Weigh an appropriate amount of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide (RC21) accurately, quantify with methanol, and prepare a solution containing approximately 100 ng of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide per mL.
[0098] Table 3. Gradient elution program
[0099]
[0100] See results Figure 12 Under these HPLC detection conditions, the concentration of the phase with the limit of quantitation is three times that in Example 3. The method has low sensitivity.
Claims
1. A method for determining potential genotoxic impurities in esomeprazole magnesium by HPLC, characterized in that: include: Preparation of test solution: Weigh 30-90 mg of esomeprazole magnesium accurately, place it in a 10 mL volumetric flask, add an appropriate amount of methanol, sonicate to dissolve the sample, dilute to the mark with methanol, and shake well. Preparation of reference solution: Weigh an appropriate amount of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide accurately, quantify with methanol, and prepare a reference solution containing 14-170 ng of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide per 1 mL. The reference solution and the test solution were analyzed by HPLC, and the content of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxides, a genotoxic impurity, in esomeprazole magnesium was determined by external standard method.
2. The method for determining potential genotoxic impurities in esomeprazole magnesium by HPLC according to claim 1, characterized in that: Preparation of test solution: Weigh approximately 60-90 mg of esomeprazole magnesium accurately, place it in a 10 mL volumetric flask, add an appropriate amount of methanol, sonicate to dissolve the sample, dilute to the mark with methanol, and shake well.
3. The method for determining potential genotoxic impurities in esomeprazole magnesium by HPLC according to claim 1 or 2, characterized in that: Preparation of test solution: Weigh approximately 60 mg of esomeprazole magnesium accurately, place it in a 10 mL volumetric flask, add an appropriate amount of methanol, sonicate to dissolve the sample, dilute to the mark with methanol, and shake well.
4. The method for determining potential genotoxic impurities in esomeprazole magnesium by HPLC according to claim 1, characterized in that: Preparation of reference solution: Weigh an appropriate amount of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide accurately, quantify with methanol, and prepare a reference solution containing 110-170 ng of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide per 1 mL of solution.
5. The method for determining potential genotoxic impurities in esomeprazole magnesium by HPLC according to claim 1 or 4, characterized in that: Preparation of reference solution: Weigh an appropriate amount of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide accurately and prepare a reference solution containing 110 ng of 4-methoxy-2,3,5-trimethylpyridine nitrogen oxide per 1 mL of methanol.
6. The method for determining potential genotoxic impurities in esomeprazole magnesium by HPLC according to claim 1, characterized in that: The HPLC detection conditions were as follows: the column was packed with octadecylsilane-bonded silica gel; mobile phase A was pH 7.4 phosphate buffer; mobile phase B was acetonitrile; gradient elution was performed; the detection wavelength was 217–227 nm; the flow rate was 0.9–1.1 mL / min; the injection volume was 10 μL; and the gradient elution program was as follows:
7. The method for determining potential genotoxic impurities in esomeprazole magnesium by HPLC according to claim 6, characterized in that: The chromatographic column is selected from Agilent Eclipse Plus C18 (4.6×150mm, 5μm) or Agilent Eclipse Plus C18 (4.6×150mm, 3.5μm).
8. The method for determining potential genotoxic impurities in esomeprazole magnesium by HPLC according to claim 6, characterized in that: The HPLC detection conditions were as follows: An Agilent Eclipse Plus C18 column (4.6 × 150 mm, 5 μm) or Agilent Eclipse Plus C18 column (4.6 × 150 mm, 3.5 μm) was used; gradient elution was performed using pH 7.4 phosphate buffer as mobile phase A and acetonitrile as mobile phase B; the detection wavelength was 217–227 nm; the flow rate was 0.9–1.1 mL / min; the injection volume was 10 μL; and the gradient elution program was as follows:
9. The method for determining potential genotoxic impurities in esomeprazole magnesium by HPLC according to claim 8, characterized in that: The HPLC detection conditions were as follows: Agilent Eclipse Plus C18 column (4.6 × 150 mm, 5 μm) or Agilent Eclipse Plus C18 column (4.6 × 150 mm, 3.5 μm) was used; pH 7.4 phosphate buffer was used as mobile phase A, and acetonitrile was used as mobile phase B, with gradient elution; the detection wavelength was 217 nm, 222 nm, or 227 nm; the flow rate was 0.9 mL / min, 1.0 mL / min, or 1.1 mL / min; the injection volume was 10 μL; and the gradient elution program was as follows: