Method for determining potential genotoxic impurities in esomeprazole sodium by HPLC (High Performance Liquid Chromatography)

Abstract

The invention discloses a method for determining potential genotoxic impurities in esomeprazole sodium by HPLC (High Performance Liquid Chromatography), which comprises the following steps: preparing a test solution: taking 20-100mg of esomeprazole sodium, precisely weighing, putting into a 10mL measuring flask, adding methanol, carrying out ultrasonic treatment to dissolve a sample, diluting to a scale by using methanol, and shaking uniformly; a proper amount of 4-methoxy-3, 5-dimethyl pyridine nitrogen oxide is taken, precisely weighed and quantified with methyl alcohol, and the reference substance solution containing 73-360 ng of 4-methoxy-3, 5-dimethyl pyridine nitrogen oxide in 1 mL of the solution is prepared; respectively carrying out HPLC (High Performance Liquid Chromatography) detection on the reference substance solution and the test solution, and determining the content of the impurity 4-methoxy-3, 5-dimethyl pyridine nitrogen oxide in the esomeprazole sodium by adopting an external standard method. The method has the advantages of high separation efficiency, high analysis speed, high detection sensitivity and low detection cost, and can effectively control the quality of esomeprazole sodium.

Description

Technical Field

[0001] The present invention belongs to the field of substance detection and relates to a method for determining potential genotoxic impurities in esomeprazole sodium by HPLC, and in particular to a method for determining 4-methoxy-3,5-dimethylpyridine nitrogen oxide (RC27) in esomeprazole sodium by HPLC. Background Art

[0002] Proton pump inhibitors (PPIs) are the first choice for treating acid-related diseases such as peptic ulcers and gastroesophageal reflux disease. Five PPIs are commonly used clinically: omeprazole, lansoprazole, rabeprazole, pantoprazole, and esomeprazole. As the first PPI, omeprazole has been widely recognized for its efficacy in treating acid-related diseases. Esomeprazole, the S-optical isomer of omeprazole, is the world's first isomeric PPI, reducing gastric acid secretion by specifically inhibiting the proton pump in gastric parietal cells.

[0003] Esomeprazole sodium, chemical name is 5-methoxy-2-((S)-((4-methoxy-3,5-dimethyl-2-pyridyl)methyl)sulfinyl-1H-benzimidazole sodium. A large number of clinical experiments and drug studies have confirmed that it maintains gastric pH>4 for a longer time, has higher acid suppression efficiency, and has better efficacy than the previous two generations of proton pump inhibitors (PPIs), with small individual differences. As a new generation of proton pump inhibitors (PPI), it is now widely used in the clinical treatment of many gastric acid-related diseases, with indications: gastroesophageal reflux disease (GERD), treatment of erosive reflux esophagitis, long-term maintenance treatment to prevent recurrence of patients with cured esophagitis, symptom control of gastroesophageal reflux disease (GERD), and appropriate Combination therapy with antibacterial therapy can eradicate Helicobacter pylori, treat duodenal ulcers associated with Helicobacter pylori infection, and prevent recurrence of Helicobacter pylori-related peptic ulcers. Due to its metabolic advantages, esomeprazole has higher bioavailability and more consistent pharmacokinetics than omeprazole, increasing drug delivery to the proton pump and resulting in superior acid suppression compared to other proton pump inhibitors. While oral esomeprazole can achieve good clinical results, for some patients, such as those with dysphagia, vomiting, acute upper gastrointestinal bleeding, and those recovering from major surgery, oral administration becomes an unfeasible route of administration, making intravenous administration an inevitable option. Therefore, esomeprazole sodium for injection is suitable for patients who require PPIs but cannot take them orally.

[0004] 4-Methoxy-3,5-dimethylpyridine nitrogen oxide (RC27, Formula I) is a by-product of the starting material of esomeprazole sodium. It has a warning structure of nitrogen and oxygen. To ensure the safety of patients' medication, it should be regarded as a potential genotoxic impurity.

[0005]

[0006] Esomeprazole sodium is the main active ingredient in esomeprazole sodium-related preparations. The acceptable limit of genotoxic impurities is generally calculated based on the threshold of toxicological concern (TTC) limit.

[0007]

[0008] Table 1. TTC limits

[0009] Treatment period ≤1 month 1 to 12 months 1 to 10 years ≥10 years Daily intake (μg / d) 120 20 10 1.5

[0010] Esomeprazole sodium is the main active ingredient of the oral preparation, and its maximum daily dose is 40 mg. Based on the strictest daily intake of 1.5 μg / d, the acceptable limit of genotoxic impurities is 37.5 ppm. Summary of the Invention

[0011] The purpose of the present invention is to establish a HPLC (high performance liquid chromatography) method for determining the potential genotoxic impurity 4-methoxy-3,5-dimethylpyridine nitrogen oxide (RC27) in esomeprazole sodium, so as to better control the quality of esomeprazole sodium.

[0012] The purpose of the present invention is achieved through the following technical solutions:

[0013] A method for determining potential genotoxic impurities in esomeprazole sodium by HPLC, comprising:

[0014] Prepare the test solution: take 20-100 mg of esomeprazole sodium, accurately weigh it, place it in a 10 mL volumetric flask, add an appropriate amount of methanol, sonicate to dissolve the sample, dilute to the scale with methanol, and shake well;

[0015] Prepare a reference solution: accurately weigh an appropriate amount of 4-methoxy-3,5-dimethylpyridine N-oxide and quantify it with methanol to prepare a reference solution containing 73-360 ng of 4-methoxy-3,5-dimethylpyridine N-oxide per 1 mL of solution.

[0016] The reference solution and the test solution were respectively subjected to HPLC detection, and the content of the impurity 4-methoxy-3,5-dimethylpyridine nitrogen oxide in esomeprazole sodium was determined by external standard method.

[0017] Preferably, the test solution is prepared as follows: about 70-100 mg of esomeprazole sodium is accurately weighed and placed in a 10 mL volumetric flask, an appropriate amount of methanol is added, ultrasonically dissolved, diluted to the mark with methanol, and shaken well.

[0018] More preferably, the test solution is prepared as follows: about 100 mg of esomeprazole sodium is accurately weighed and placed in a 10 mL volumetric flask, an appropriate amount of methanol is added, ultrasonically dissolved, diluted to the mark with methanol, and shaken well.

[0019] Preferably, a reference solution is prepared by taking an appropriate amount of 4-methoxy-3,5-dimethylpyridine N-oxide, accurately weighing it, and quantitatively preparing a reference solution containing 360 ng of 4-methoxy-3,5-dimethylpyridine N-oxide per 1 mL of solution with methanol.

[0020] The HPLC detection conditions are as follows: the chromatographic column uses octadecylsilane bonded silica as the filler, pH 2.8-3.0 phosphate buffer as the mobile phase A, acetonitrile as the mobile phase B, and gradient elution; the detection wavelength is 258-262 nm; the column temperature is 25-35°C; the flow rate is 0.9-1.1 mL / min; the injection volume is 20 μL; and the gradient elution program is:

[0021]

[0022] The chromatographic column is selected from Kromasil-C18 chromatographic column (150×4.6 mm, 5 μm) and YMC-Triart C18 chromatographic column (4.6 mm×150 mm, 5 μm).

[0023] Preferably, the HPLC detection conditions are as follows: Kromasil-C18 column (4.6 mm × 150 mm, 5 μm), pH 3.0 phosphate buffer as mobile phase A, acetonitrile as mobile phase B, gradient elution; detection wavelength 260 nm; column temperature 30° C.; flow rate 1.0 mL / min; injection volume 20 μL, gradient elution program:

[0024]

[0025] The pH 2.8-3.0 phosphate buffer solution is prepared by dissolving 2.72 g of potassium dihydrogen phosphate in 900 mL of water, adding 1 mL of triethylamine, and then adding water to 1000 mL. The pH value is adjusted to 2.8-3.0 with phosphoric acid.

[0026] The pH 3.0 phosphate buffer solution was prepared by dissolving 2.72 g of potassium dihydrogen phosphate in 900 mL of water, adding 1 mL of triethylamine, and then adding water to 1000 mL. The pH value was adjusted to 3.0 with phosphoric acid.

[0027] Beneficial effects of the present invention:

[0028] The present invention adopts HPLC to determine the potential genotoxic impurity 4-methoxy-3,5-dimethylpyridine nitrogen oxide (RC27) of esomeprazole sodium, with high separation efficiency, fast analysis speed, high detection sensitivity and low detection cost.

[0029] Based on the method of the present invention, by detecting the potential genotoxic impurities of esomeprazole sodium, the potential genotoxic impurity 4-methoxy-3,5-dimethylpyridine nitrogen oxide of esomeprazole sodium is controlled to be no more than 37.5 ppm, thereby effectively controlling the quality of esomeprazole sodium. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 HPLC comparison chart of blank solution, reference solution and test solution in Example 1; from top to bottom are HPLC spectra of blank solvent, reference solution and test solution.

[0031] Figure 2 This is the HPLC spectrum of the detection limit solution in Example 2.

[0032] Figure 3 HPLC spectrum of the quantitative limit solution in Example 3.

[0033] Figure 4 The HPLC spectrum of the reference solution in Example 4 (detection wavelength 258 nm) is shown.

[0034] Figure 5 The HPLC spectrum of the reference solution in Example 4 (detection wavelength 260 nm) is shown.

[0035] Figure 6 The HPLC spectrum of the reference solution in Example 4 (detection wavelength 262 nm) is shown.

[0036] Figure 7 The HPLC spectrum (YMC-C18 column) of the reference solution in Example 5 is shown.

[0037] Figure 8 This is the HPLC spectrum of the reference solution in Example 6 (flow rate 0.9 mL / min).

[0038] Figure 9 This is the HPLC spectrum of the reference solution in Example 6 (flow rate 1.1 mL / min).

[0039] Figure 10 This is the HPLC spectrum of the reference solution in Example 7 (column temperature 25°C).

[0040] Figure 11 This is the HPLC spectrum of the reference solution in Example 7 (column temperature 35°C).

[0041] Figure 12 This is the HPLC spectrum of the reference solution in Example 8 (mobile phase A is 91%, mobile phase B is 9%).

[0042] Figure 13 This is the HPLC spectrum of the reference solution in Example 8 (mobile phase A is 89%, mobile phase B is 11%).

[0043] Figure 14 This is the HPLC spectrum of the reference solution in Example 9 (mobile phase A is pH 2.8).

[0044] Figure 15 This is the HPLC spectrum of the 50% accuracy solution in Example 10.

[0045] Figure 16 This is the HPLC spectrum of the 100% accuracy solution in Example 10.

[0046] Figure 17 This is the HPLC spectrum of the 150% accuracy solution in Example 10.

[0047] Figure 18 This is the HPLC spectrum of the 100% accuracy solution under the HPLC detection conditions in Comparative Example 1.

[0048] Figure 19 This is the HPLC spectrum of the reference solution under the HPLC detection conditions in Comparative Example 2. DETAILED DESCRIPTION

[0049] The technical solutions of the present invention are further illustrated by the following examples, but the examples are not intended to limit the present invention.

[0050] The reagents used in the examples are all commercially available or simply synthesized according to existing methods.

[0051] HPLC (liquid chromatography) model:

[0052]

[0053] Reagents used:

[0054]

[0055] Example 1

[0056] The HPLC method for the determination of potential genotoxic impurities in esomeprazole sodium includes:

[0057] Prepare the test solution: take about 100 mg of esomeprazole sodium, accurately weigh it, place it in a 10 mL volumetric flask, add an appropriate amount of methanol, sonicate to dissolve the sample, dilute to the scale with methanol, and shake well.

[0058] Prepare reference solution: Take an appropriate amount of 4-methoxy-3,5-dimethylpyridine N-oxide (RC27), accurately weigh it, and quantitatively prepare a solution containing approximately 360 ng of 4-methoxy-3,5-dimethylpyridine N-oxide (RC27) per 1 mL of solution with methanol.

[0059] The reference solution and the test solution were detected by HPLC respectively. The HPLC detection conditions were as follows: the chromatographic column was a Kromasil-C18 liquid chromatography column (inner diameter 4.6 mm × length 150 mm, particle size 5 μm), pH 3.0 phosphate buffer (take 2.72 g potassium dihydrogen phosphate, add 900 mL water to dissolve, then add 1 mL triethylamine, add water to 1000 mL, and adjust the pH value to 3.0 with phosphoric acid) as the mobile phase A, acetonitrile as the mobile phase B, and gradient elution was performed; the detection wavelength was 260 nm; the column temperature was 30°C; the flow rate was 1.0 mL / min; the injection volume was 20 μL; the gradient elution program is shown in Table 2.

[0060] Table 2. Gradient elution program

[0061]

[0062] Figure 1 The HPLC comparison chart of blank solvent, reference solution, and test solution shows that the blank solvent has no interference with the detection of 4-methoxy-3,5-dimethylpyridine nitrogen oxide.

[0063] Example 2

[0064] Preparation of detection limit solution: Take appropriate amount of 4-methoxy-3,5-dimethylpyridine N-oxide (RC27), accurately weigh it, and quantify it with methanol to prepare a solution containing approximately 22 ng of 4-methoxy-3,5-dimethylpyridine N-oxide per 1 mL of solution.

[0065] The detection limit solution was subjected to HPLC detection. The HPLC detection conditions were as follows: a Kromasil-C18 column (4.6 mm × 150 mm, 5 μm), pH 3.0 phosphate buffer (2.72 g of potassium dihydrogen phosphate was dissolved in 900 mL of water, 1 mL of triethylamine was added, and the mixture was made up to 1000 mL with water, and the pH value was adjusted to 3.0 with phosphoric acid) as the mobile phase A, and acetonitrile as the mobile phase B, with gradient elution; the detection wavelength was 260 nm; the column temperature was 30°C; the flow rate was 1.0 mL / min; the injection volume was 20 μL; the gradient elution program is shown in Table 2.

[0066] The HPLC spectrum of the detection limit solution is shown in Figure 2 It can be seen that the detection limit of the method of the present invention is 2.2 ppm, which is equivalent to 6% of the limit of 37.5 ppm, indicating that the detection sensitivity of the method of the present invention is very high.

[0067] Example 3

[0068] Prepare the quantitative limit solution: Take an appropriate amount of 4-methoxy-3,5-dimethylpyridine N-oxide (RC27), accurately weigh it, and quantify it with methanol to prepare a solution containing approximately 73 ng of 4-methoxy-3,5-dimethylpyridine N-oxide per 1 mL of solution.

[0069] The quantification limit solution was detected by HPLC. The HPLC detection conditions were as follows: a Kromasil-C18 column (4.6 mm × 150 mm, 5 μm), pH 3.0 phosphate buffer (2.72 g of potassium dihydrogen phosphate was dissolved in 900 mL of water, 1 mL of triethylamine was added, and the mixture was made up to 1000 mL with water, and the pH value was adjusted to 3.0 with phosphoric acid) as the mobile phase A, and acetonitrile as the mobile phase B, with gradient elution; the detection wavelength was 260 nm; the column temperature was 30°C; the flow rate was 1.0 mL / min; the injection volume was 20 μL; the gradient elution program is shown in Table 2.

[0070] The HPLC spectrum of the quantitative limit solution is shown in Figure 3 It can be seen that the quantitative limit of the method of the present invention is 7.3 ppm, which is equivalent to 20% of the limit of 37.5 ppm, indicating that the quantitative sensitivity of the method of the present invention is very high.

[0071] Example 4

[0072] Prepare reference solution: Take appropriate amount of 4-methoxy-3,5-dimethylpyridine N-oxide (RC27), accurately weigh it, and quantify it with methanol to prepare a solution containing approximately 360 ng of 4-methoxy-3,5-dimethylpyridine N-oxide per 1 mL of solution.

[0073] The reference solution was subjected to HPLC detection. The HPLC detection conditions were as follows: a Kromasil-C18 column (4.6 mm × 150 mm, 5 μm), pH 3.0 phosphate buffer (2.72 g of potassium dihydrogen phosphate was dissolved in 900 mL of water, 1 mL of triethylamine was added, and the mixture was made up to 1000 mL with water, and the pH value was adjusted to 3.0 with phosphoric acid) as the mobile phase A, acetonitrile as the mobile phase B, and gradient elution was performed; the detection wavelengths were 258 nm, 260 nm, and 262 nm, respectively; the column temperature was 30°C; the flow rate was 1.0 mL / min; the injection volume was 20 μL; the gradient elution program is shown in Table 2.

[0074] The HPLC spectrum of the reference solution is shown in Figure 4 (Detection wavelength 258nm), Figure 5 (Detection wavelength 260nm), Figure 6 (Detection wavelength 260nm), indicating that the wavelength has no significant effect on the detection results.

[0075] Example 5

[0076] Prepare reference solution: Take appropriate amount of 4-methoxy-3,5-dimethylpyridine N-oxide (RC27), accurately weigh it, and quantitatively prepare a solution containing approximately 360 ng of 4-methoxy-3,5-dimethylpyridine N-oxide per 1 mL of solution with methanol.

[0077] The reference solution was tested by HPLC. The HPLC test conditions were as follows: a YMC-Triart C18 column (4.6 mm × length 150 mm, particle size 5 μm), pH 3.0 phosphate buffer (2.72 g of potassium dihydrogen phosphate was dissolved in 900 mL of water, 1 mL of triethylamine was added, and the mixture was made up to 1000 mL with water, and the pH value was adjusted to 3.0 with phosphoric acid) as mobile phase A, acetonitrile as mobile phase B, and gradient elution was performed; the detection wavelength was 260 nm; the column temperature was 30°C; the flow rate was 1.0 mL / min; the injection volume was 20 μL; the gradient elution program is shown in Table 2.

[0078] HPLC spectrum of reference solution is shown in Figure 7 , indicating that chromatographic columns from different manufacturers have no significant effect on the test results.

[0079] Example 6

[0080] Prepare reference solution: Take appropriate amount of 4-methoxy-3,5-dimethylpyridine N-oxide (RC27), accurately weigh it, and quantitatively prepare a solution containing approximately 360 ng of 4-methoxy-3,5-dimethylpyridine N-oxide per 1 mL of solution with methanol.

[0081] The reference solution was subjected to HPLC detection. The HPLC detection conditions were as follows: a Kromasil-C18 column (4.6 mm × 150 mm, 5 μm), pH 3.0 phosphate buffer (2.72 g of potassium dihydrogen phosphate was dissolved in 900 mL of water, 1 mL of triethylamine was added, and the mixture was made up to 1000 mL with water, and the pH value was adjusted to 3.0 with phosphoric acid) as the mobile phase A, acetonitrile as the mobile phase B, and gradient elution was performed; the detection wavelength was 260 nm; the column temperature was 30°C; the flow rate was 0.9 mL / min or 1.1 mL / min; the injection volume was 20 μL; the gradient elution program is shown in Table 2.

[0082] HPLC spectrum of reference solution is shown in Figure 8 (flow rate of 0.9 mL / min) and Figure 9 (flow rate is 1.1 mL / min), indicating that the change in flow rate has no significant effect on the test results.

[0083] Example 7

[0084] Prepare reference solution: Take appropriate amount of 4-methoxy-3,5-dimethylpyridine N-oxide (RC27), accurately weigh it, and quantitatively prepare a solution containing approximately 360 ng of 4-methoxy-3,5-dimethylpyridine N-oxide per 1 mL of solution with methanol.

[0085] The reference solution was subjected to HPLC detection. The HPLC detection conditions were as follows: a Kromasil-C18 column (4.6 mm × 150 mm, 5 μm), pH 3.0 phosphate buffer (2.72 g of potassium dihydrogen phosphate was dissolved in 900 ml of water, 1 ml of triethylamine was added, and the mixture was made up to 1000 ml with water, and the pH value was adjusted to 3.0 with phosphoric acid) as the mobile phase A, acetonitrile as the mobile phase B, and gradient elution was performed; the detection wavelength was 260 nm; the column temperature was 25°C or 35°C; the flow rate was 1.0 mL / min; the injection volume was 20 μL; the gradient elution program is shown in Table 2.

[0086] HPLC spectrum of reference solution is shown in Figure 10 (column temperature was 25°C) and Figure 11 (column temperature is 35℃), indicating that changes in column temperature have no significant effect on the test results.

[0087] Example 8

[0088] Prepare reference solution: Take appropriate amount of 4-methoxy-3,5-dimethylpyridine N-oxide (RC27), accurately weigh it, and quantify it with methanol to prepare a solution containing approximately 360 ng of 4-methoxy-3,5-dimethylpyridine N-oxide per 1 mL of solution.

[0089] The reference solution was subjected to HPLC detection. The HPLC detection conditions were as follows: a Kromasil-C18 column (4.6 mm × 150 mm, 5 μm), pH 3.0 phosphate buffer (2.72 g of potassium dihydrogen phosphate was dissolved in 900 mL of water, 1 mL of triethylamine was added, and the mixture was made up to 1000 mL with water, and the pH was adjusted to 3.0 with phosphoric acid) as the mobile phase A, and acetonitrile as the mobile phase B, with gradient elution; the detection wavelength was 260 nm; the column temperature was 30° C.; the flow rate was 1.0 mL / min; the injection volume was 20 μL; the gradient elution program was shown in Table 3, with an initial gradient of 91% mobile phase A, 9% mobile phase B; or 89% mobile phase A, 11% mobile phase B.

[0090] Table 3. Gradient elution program

[0091]

[0092] The HPLC spectrum of the reference solution is shown in Figure 12 (Starting gradient: 91% mobile phase A, 9% mobile phase B) and Figure 13(Starting gradient: 89% mobile phase A, 11% mobile phase B), indicating that the change in the starting mobile phase ratio has no significant effect on the detection results.

[0093] Example 9

[0094] Prepare reference solution: Take appropriate amount of 4-methoxy-3,5-dimethylpyridine N-oxide (RC27), accurately weigh it, and quantify it with methanol to prepare a solution containing approximately 360 ng of 4-methoxy-3,5-dimethylpyridine N-oxide per 1 mL of solution.

[0095] The reference solution was subjected to HPLC detection. The HPLC detection conditions were as follows: a Kromasil-C18 column (4.6 mm × 150 mm, 5 μm), pH 2.8 phosphate buffer (2.72 g of potassium dihydrogen phosphate was dissolved in 900 mL of water, 1 mL of triethylamine was added, and the mixture was made up to 1000 mL with water, and the pH value was adjusted to 2.8 with phosphoric acid) as the mobile phase A, acetonitrile as the mobile phase B, and gradient elution was performed; the detection wavelength was 260 nm; the column temperature was 30°C; the flow rate was 1.0 mL / min; the injection volume was 20 μL; the gradient elution program is shown in Table 2.

[0096] The HPLC spectrum of the reference solution is shown in Figure 14 (The pH value of mobile phase A is 2.8), indicating that the change of mobile phase pH value has no significant effect on the detection results.

[0097] Example 10

[0098] Investigate the accuracy of the method of the present invention

[0099] The HPLC method for the determination of potential genotoxic impurities in esomeprazole sodium includes:

[0100] Prepare the test solution: take about 100 mg of esomeprazole sodium, accurately weigh it, place it in a 10 mL volumetric flask, add an appropriate amount of methanol, sonicate to dissolve the sample, dilute to the scale with methanol, and shake well.

[0101] Prepare reference solution: Take appropriate amount of 4-methoxy-3,5-dimethylpyridine N-oxide (RC27), accurately weigh it, and quantify it with methanol to prepare a solution containing approximately 360 ng of 4-methoxy-3,5-dimethylpyridine N-oxide per 1 mL of solution.

[0102] Impurity reference solution: Take an appropriate amount of 4-methoxy-3,5-dimethylpyridine N-oxide (RC27), accurately weigh it, and quantify it with methanol to prepare a solution containing approximately 3.6 μg of 4-methoxy-3,5-dimethylpyridine N-oxide per 1 mL of solution.

[0103] 50% accuracy solution: Take approximately 100 mg of esomeprazole sodium, accurately weigh it, place it in a 10 mL volumetric flask, add an appropriate amount of methanol and sonicate to dissolve the sample; accurately measure 0.5 mL of impurity reference solution, place it in a volumetric flask, dilute to the scale with methanol, and shake well.

[0104] 100% accuracy solution: Take approximately 100 mg of esomeprazole sodium, accurately weigh it, 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 scale with methanol, and shake well.

[0105] 150% accuracy solution: Take approximately 100 mg of esomeprazole sodium, accurately weigh it, place it in a 10 mL volumetric flask, add an appropriate amount of methanol and sonicate to dissolve the sample; accurately measure 1.5 mL of impurity reference solution, place it in a volumetric flask, dilute to the scale with methanol, and shake well.

[0106] The reference solution, test solution, 50% accuracy solution, 100% accuracy solution, and 150% accuracy solution were subjected to HPLC detection. The HPLC detection conditions were as follows: a Kromasil-C18 column (4.6 mm × 150 mm, 5 μm), pH 3.0 phosphate buffer (2.72 g of potassium dihydrogen phosphate was dissolved in 900 ml of water, 1 ml of triethylamine was added, and water was added to 1000 ml, and the pH was adjusted to 3.0 with phosphoric acid) as the mobile phase A, acetonitrile as the mobile phase B, and gradient elution was performed; the detection wavelength was 260 nm; the column temperature was 30°C; the flow rate was 1.0 mL / min; the injection volume was 20 μL; the gradient elution program is shown in Table 2.

[0107] Figure 15 HPLC spectrum of 50% accuracy solution; Figure 16 HPLC spectrum of the solution with 100% accuracy; Figure 17 This is the HPLC spectrum of the 150% accuracy solution.

[0108] The external standard method was used to determine the content of 4-methoxy-3,5-dimethylpyridine N-oxide (RC27) in 50% accuracy solutions, 100% accuracy solutions, and 150% accuracy solutions. The recovery rate of 4-methoxy-3,5-dimethylpyridine N-oxide (RC27) was 85.2% at 50% accuracy, 95.3% at 100% accuracy in pH 3.0 phosphate buffer, and 98.3% at 150% accuracy. This demonstrates that the method of the present invention can effectively and accurately reflect product quality.

[0109] Comparative Example 1

[0110] The inventors adopted the preferred HPLC detection conditions of the present invention (Example 1) and only adjusted the pH value of mobile phase A.

[0111] Mobile phase A was pH 3.1 phosphate buffer (dissolve 2.72 g of potassium dihydrogen phosphate in 900 mL of water, add 1 mL of triethylamine, add water to 1000 mL, and adjust the pH to 3.1 with phosphoric acid).

[0112] See the results Figure 18 Under the HPLC detection conditions, after the 100% accuracy solution (Example 10) was injected, unknown impurities interfered with the detection of 4-methoxy-3,5-dimethylpyridine nitrogen oxide.

[0113] Comparative Example 2

[0114] The inventors adopted the preferred HPLC detection conditions of the present invention (Example 1), and only adjusted the injection volume to 40 μL.

[0115] See the results Figure 19 Under the HPLC detection conditions, after the reference solution (prepared with reference to Example 1) was injected, the peak shape of 4-methoxy-3,5-dimethylpyridine nitrogen oxide was abnormal.

Claims

1. A method for determining potential genotoxic impurities in esomeprazole sodium by HPLC, characterized in that: include: Prepare the test solution: take 20-100 mg of esomeprazole sodium, accurately weigh it, place it in a 10 mL volumetric flask, add an appropriate amount of methanol, sonicate to dissolve the sample, dilute to the scale with methanol, and shake well; Prepare a reference solution: accurately weigh an appropriate amount of 4-methoxy-3,5-dimethylpyridine N-oxide and quantify it with methanol to prepare a reference solution containing 73-360 ng of 4-methoxy-3,5-dimethylpyridine N-oxide per 1 mL of solution. The reference solution and the test solution were respectively subjected to HPLC detection, and the content of the impurity 4-methoxy-3,5-dimethylpyridine nitrogen oxide in esomeprazole sodium was determined by external standard method.

2. The method for determining potential genotoxic impurities in esomeprazole sodium by HPLC according to claim 1, characterized in that: Prepare the test solution: Take 70-100 mg of esomeprazole sodium, accurately weigh it, place it in a 10 mL volumetric flask, add an appropriate amount of methanol, sonicate to dissolve the sample, dilute to the scale with methanol, and shake well.

3. The method for determining potential genotoxic impurities in esomeprazole sodium by HPLC according to claim 2, characterized in that: Prepare the test solution: take 100 mg of esomeprazole sodium, accurately weigh it, place it in a 10 mL volumetric flask, add an appropriate amount of methanol, sonicate to dissolve the sample, dilute to the scale with methanol, and shake well.

4. The method for determining potential genotoxic impurities in esomeprazole sodium by HPLC according to claim 1, characterized in that: Prepare reference solution: Take an appropriate amount of 4-methoxy-3,5-dimethylpyridine N-oxide, accurately weigh it, and quantitatively prepare a reference solution with methanol containing 360 ng of 4-methoxy-3,5-dimethylpyridine N-oxide per 1 mL of solution.

5. The method for determining potential genotoxic impurities in esomeprazole sodium by HPLC according to claim 1, characterized in that: The HPLC detection conditions are as follows: the chromatographic column uses octadecylsilane bonded silica as the filler, pH 2.8-3.0 phosphate buffer as the mobile phase A, acetonitrile as the mobile phase B, and gradient elution; the detection wavelength is 258-262 nm; the column temperature is 25-35°C; the flow rate is 0.9-1.1 mL / min; the injection volume is 20 μL; and the gradient elution program is:

6. The method for determining potential genotoxic impurities in esomeprazole sodium by HPLC according to claim 5, characterized in that: The chromatographic column is selected from Kromasil-C18 chromatographic column (150×4.6 mm, 5 μm) and YMC-Triart C18 chromatographic column (4.6 mm×150 mm, 5 μm).

7. The method for determining potential genotoxic impurities in esomeprazole sodium by HPLC according to claim 5, characterized in that: The pH 2.8-3.0 phosphate buffer solution is prepared by dissolving 2.72 g of potassium dihydrogen phosphate in 900 mL of water, adding 1 mL of triethylamine, and then adding water to 1000 mL. The pH value is adjusted to 2.8-3.0 with phosphoric acid.

8. The method for determining potential genotoxic impurities in esomeprazole sodium by HPLC according to claim 1 or 5, characterized in that: The HPLC detection conditions are as follows: Kromasil-C18 column (4.6 mm × 150 mm, 5 μm), pH 3.0 phosphate buffer as mobile phase A, acetonitrile as mobile phase B, gradient elution; detection wavelength, 260 nm; column temperature, 30° C.; flow rate, 1.0 mL / min; injection volume, 20 μL; gradient elution program:

9. The method for determining potential genotoxic impurities in esomeprazole sodium by HPLC according to claim 8, characterized in that: The pH 3.0 phosphate buffer solution was prepared by dissolving 2.72 g of potassium dihydrogen phosphate in 900 mL of water, adding 1 mL of triethylamine, and then adding water to 1000 mL. The pH value was adjusted to 3.0 with phosphoric acid.

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