Method for determining content of empagliflozin intermediate (S)-3-[4-(5-bromo-2-chlorobenzyl) phenoxy] tetrahydrofuran related substances and enantiomers by reverse phase chromatography
By using a reversed-phase gradient system with a chiral column containing polysaccharide derivative-bonded packing material, combined with a mobile phase of acetonitrile and purified water, the problem of rapid and economical detection of related substances and enantiomers of empagliflozin intermediates was solved, achieving detection results with high specificity, high sensitivity, and high accuracy.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG HONGYUAN PHARMA
- Filing Date
- 2026-01-09
- Publication Date
- 2026-05-12
AI Technical Summary
Existing technologies make it difficult to quickly and economically separate and detect related substances and enantiomers of empagliflozin intermediate (S)-3-[4-(5-bromo-2-chlorobenzyl)phenoxy]tetrahydrofuran, resulting in long testing cycles and high costs for commercial production.
A reverse-phase gradient system using a chiral column with polysaccharide derivative bonded packing material, combined with a mobile phase of acetonitrile and purified water, was employed for high-performance liquid chromatography (HPLC) detection, achieving highly specific, sensitive, and accurate separation and elution of EP4 and EP4B.
It enables rapid, economical, and accurate detection of related substances and enantiomers of empagliflozin intermediates, significantly improving the quality control capabilities of empagliflozin.
Smart Images
Figure CN122017111A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of analytical chemistry and relates to a method for detecting related substances and enantiomers of empagliflozin intermediate (S)-3-[4-(5-bromo-2-chlorobenzyl)phenoxy]tetrahydrofuran. More specifically, this invention relates to a method for detecting related substances (EP4A, EP5, EP4K) and enantiomer (EP4B) of empagliflozin intermediate (S)-3-[4-(5-bromo-2-chlorobenzyl)phenoxy]tetrahydrofuran. Background Technology
[0002] Empagliflozin is a sodium-glucose cotransporter 2 (SGLT2) inhibitor developed by Boehringer Ingelheim and Eli Lilly for the treatment of type 2 diabetes, adult patients with chronic heart failure, and adult patients with chronic kidney disease.
[0003] Eppagliflozin was approved for clinical use by the FDA in 2014. In August 2017, it was first approved in China for the treatment of type 2 diabetes mellitus (T2DM). In June 2022, empagliflozin was approved in China for the treatment of adult patients with heart failure, with or without diabetes and reduced ejection fraction. In May 2023, empagliflozin was approved in China for use in combination with insulin therapy (with or without oral hypoglycemic agents) to improve glycemic control in patients with type 2 diabetes on top of diet and exercise. In November 2023, empagliflozin was approved in China for the treatment of adult chronic kidney disease (CKD). CKD, heart failure, and type 2 diabetes are interconnected diseases affecting more than 1 billion people worldwide.
[0004] Empagliflozin is the only oral hypoglycemic agent to rank among the top ten best-selling drugs in the first half of 2025. According to the website of the National Medical Products Administration, there are currently 89 approved drug registration numbers for empagliflozin (both single-drug and combination formulations) in the Chinese market, with 49 manufacturers.
[0005] PCT patent WO2006120208A[7] reported the mainstream process route of empagliflozin active pharmaceutical ingredient, which includes the key intermediate (S)-3-[4-(5-bromo-2-chlorobenzyl)phenoxy]tetrahydrofuran, as shown in formula EP4. Its synthesis is based on 5-bromo-2-chlorobenzoic acid as the starting material, which is condensed with (S)-3-hydroxytetrahydrofuran after Friedel-Crafts reaction and finally reduced to obtain the product.
[0006] Potential impurities and process impurities in EP4 include: the potential impurity introduced from the feedstock, the enantiomer (R)-3-[4-(5-bromo-2-chlorobenzyl)phenoxy]tetrahydrofuran, as shown in Formula EP4B; the dimer impurity 1,4-bis[4-(5-bromo-2-chlorobenzyl)phenoxy]butane, as shown in Formula EP4K; the process impurity (5-bromo-2-chlorophenyl)[4-[(S)-tetrahydrofuran-3-yl)oxy]phenyl]methanol, as shown in Formula EP4A; and the residual intermediate from the previous step, (S)-(5-bromo-2-chlorophenyl)[4-[(tetrahydrofuran-3-yl)oxy]phenyl]methyl ketone, as shown in Formula EP5.
[0007] Impurities related to EP4 (EP4A, EP5, EP4K) and enantiomers (EP4B) will be transferred to empagliflozin API through chemical synthesis, generating specific impurities such as empagliflozin enantiomers and empagliflozin dimers. The standard for these specific impurities in the API is ≤0.15%. Therefore, establishing a suitable analytical method for EP4-related impurities and EP4 enantiomers is of great significance for providing a reference for EP4 impurity control and ensuring the quality of empagliflozin API.
[0008]
[0009] (S)-3-[4-(5-bromo-2-chlorobenzyl)phenoxy]tetrahydrofuran (EP4)
[0010]
[0011] (5-Bromo-2-chlorophenyl)[4-[(S)-tetrahydrofuran-3-yl)oxy]phenyl]methanol (EP4A)
[0012]
[0013] (R)-3-[4-(5-bromo-2-chlorobenzyl)phenoxy]tetrahydrofuran (EPB)
[0014]
[0015] (S)-(5-bromo-2-chlorophenyl)[4-[(tetrahydrofuran-3-yl)oxy]phenyl]methyl ketone (EP5)
[0016]
[0017] 1,4-Bis[4-(5-bromo-2-chlorobenzyl)phenoxy]butane (EPK)
[0018] Currently, no literature reports have been found on the detection methods for related substances and enantiomers of EP4. The conventional development scheme uses a non-chiral chromatographic column and reversed-phase elution to separate related impurities; enantiomers are separated by a normal-phase system using a chiral chromatographic column, which requires the development of two analytical methods. The disadvantages are that the commercial production and testing cycle is long and the cost is high.
[0019] This study breaks with conventional method development, employing a reverse-phase gradient elution system on a chiral column with polysaccharide derivative-bonded packing material. This overcomes the challenge of chiral separation of EP4 and EP4B, and also rapidly elutes the dimer impurity EP4K. It enables the simultaneous detection of impurities and enantiomers of EP4 using the same method parameters. The method boasts high specificity, high sensitivity, high accuracy, and simple operation. This research demonstrates outstanding innovation and practicality, significantly surpassing existing technical levels. Summary of the Invention
[0020] The purpose of this invention is to provide a method for detecting related substances and enantiomers of empagliflozin intermediate (S)-3-[4-(5-bromo-2-chlorobenzyl)phenoxy]tetrahydrofuran. This method has advantages such as good specificity, high precision, high accuracy, high sensitivity, and good robustness, and can effectively control the content of specific impurities (EP4A, EP5, EP4K) and enantiomers (EP4B) in ethyl (R)-4-cyano-3-hydroxybutyrate.
[0021] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.
[0022] This invention provides a method for detecting related substances and enantiomers of empagliflozin intermediate (S)-3-[4-(5-bromo-2-chlorobenzyl)phenoxy]tetrahydrofuran, comprising the following steps:
[0023] The EP4 test sample was mixed with an organic solvent to obtain a test sample solution;
[0024] The test solution was detected by high performance liquid chromatography.
[0025] The high-performance liquid chromatography column is a chiral column with polysaccharide derivative bonded packing material; the mobile phase consists of acetonitrile and purified water, and gradient elution is used.
[0026] The detection method of the present invention is characterized in that the mobile phase is a mixture of purified water and acetonitrile.
[0027] The detection method of the present invention is characterized by employing a gradient elution method during the high-performance liquid chromatography (HPLC) detection process. Mobile phase A is purified water, and mobile phase B is acetonitrile. The preferred gradient is: 0–5 min, 55% A; 5–25 min, 55% → 30% A; 25–40 min, 30% → 15% A; 40–52 min, 15% A; 52–52.1 min, 15% → 55% A; 52.1–60 min, 55% A.
[0028] The detection method of the present invention is characterized in that, during the detection process by high performance liquid chromatography, the flow rate is 0.8~1.2 mL / min, preferably 1.0 mL / min.
[0029] The detection method of the present invention is characterized in that the chromatographic column is a polysaccharide derivative-bonded chiral packing material, preferably CHIRAL PAK® IC.
[0030] The detection method of the present invention is characterized in that the high performance liquid chromatography detection column temperature is 25-50℃ and the injection volume is 5-15μL.
[0031] The detection method of the present invention is characterized in that the detection wavelength of the high performance liquid chromatography is 210~230nm.
[0032] This invention provides a method for detecting related substances and enantiomers of empagliflozin intermediate (S)-3-[4-(5-bromo-2-chlorobenzyl)phenoxy]tetrahydrofuran. The detection method provided by this invention has the advantages of good specificity, high precision, high accuracy, high sensitivity, and good robustness. It can effectively control the content of related substances (EP4A, EP5, EP4K) and enantiomer impurities (EP4B) of (S)-3-[4-(5-bromo-2-chlorobenzyl)phenoxy]tetrahydrofuran, which is significantly superior to the existing disclosed technology and is of great significance for improving the quality of empagliflozin. Attached Figure Description
[0033] Figure 1 This is a typical spectrum of a blank solution;
[0034] Figure 2 Location map of EP4A;
[0035] Figure 3 For system applicability map;
[0036] Figure 4 For the chromatogram of the test sample;
[0037] Figure 5 Spectra of solutions at the limit of quantitation;
[0038] Figure 6For the EP4 linear graph;
[0039] Figure 7 The linear graph of EP4A;
[0040] Figure 8 For EP4B linear graph;
[0041] Figure 9 Linear graph of EP5;
[0042] Figure 10 For EP4K linear graphs; Detailed Implementation
[0043] The following specific embodiments will enable those skilled in the art to fully understand the present invention, but do not limit the invention in any way. In the following embodiments, unless otherwise specified, those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention. Reagents and instruments not specifying manufacturers can be commercially available products.
[0044] The EP5, EP4K, and EP4 reference standards used in this invention are self-made by the company, while the EP4A and EP4B reference standards are purchased externally.
[0045] Example 1: Specificity Verification
[0046] Column: CHIRAL PAK ® IC 4.6×250mm, 5μm
[0047] Column temperature: 40℃
[0048] Column flow rate: 1.0 mL / min
[0049] Injection volume: 10µL
[0050] Mobile phase A: purified water
[0051] Mobile phase B: Acetonitrile
[0052] Gradient elution
[0053]
[0054] Solution preparation:
[0055] Test solution: Take an appropriate amount of the test sample, weigh it accurately, and dilute it with a solution containing 0.5 mg of EP4 per mL.
[0056] System suitability solution: Take appropriate amounts of EP4, EP4A, EP4B, EP5, and EP4K reference standards, accurately weigh them, dissolve them in acetonitrile, and dilute them to a mixed solution containing 0.5 mg, 2.5 µg, 1 µg, 1.5 µg, and 2.5 µg per mL, respectively.
[0057] Blank: Acetonitrile.
[0058] EP4 positioning solution: Take an appropriate amount of EP4 reference standard, accurately weigh it, dissolve it in acetonitrile and dilute it to a solution containing 0.5 mg per mL.
[0059] EP4A positioning solution: Take an appropriate amount of EP4A reference standard, accurately weigh it, dissolve it in acetonitrile and dilute it to a solution containing 2.5 µg per mL.
[0060] EP4B positioning solution: Take an appropriate amount of EP4B reference standard, accurately weigh it, dissolve it in acetonitrile and dilute it to a solution containing 1 µg per mL.
[0061] EP5 positioning solution: Take an appropriate amount of EP5 reference standard, accurately weigh it, dissolve it in acetonitrile and dilute it to a solution containing 1.5 µg per mL.
[0062] EP4K positioning solution: Take an appropriate amount of EP4K reference standard, accurately weigh it, dissolve it in acetonitrile and dilute it to a solution containing 2.5 µg per mL.
[0063] Determination: Inject blank solution, each positioning solution, system suitability solution, and test solution into the liquid chromatograph for detection and record the chromatogram.
[0064] Test results: The blank sample did not interfere with the detection of specific impurities. Figure 1 EP4A reference standard is a racemic mixture, therefore the localization solution spectrum showed two peaks, see [reference needed]. Figure 2 ; See the system suitability solution spectrum. Figure 3 The minimum resolution between two adjacent components (EP4 and EP4B) is 2.1, which is greater than 1.5. See the chromatogram of the test sample solution. Figure 4 The results show that the method has good specificity, and the separation results are shown in Table 1.
[0065] Table 1 Results of the separation test
[0066]
[0067] Example 2: Validation of Limit of Detection and Limit of Quantitation
[0068] The limits of detection (LOD) and limits of quantitation (LOQ) for impurities (EP4A, EP5, EP4B, EP4K, EP4) in EP4 were determined based on the signal-to-noise ratio (SNR). Each component was diluted to different concentrations using a dilution method, and the concentrations were measured. The concentration at which the SNR was greater than or equal to 3 was the limit of detection, and the concentration at which the SNR was greater than or equal to 10 was the limit of quantitation. The results of the limit of quantitation detection have a certain degree of precision and accuracy.
[0069] Solution preparation
[0070] Limit of Quantification Solution: Accurately weigh appropriate amounts of EP4, EP4A, EP4B, EP5, and EP4K reference standards, dissolve and dilute with acetonitrile to a mixed solution containing approximately 0.25 µg, 0.25 µg, 0.25 µg, 0.25 µg, and 0.25 µg per mL, respectively. Prepare 6 aliquots using the same method.
[0071] Detection limit solution: Take appropriate amounts of EP4, EP4A, EP4B, EP5, and EP4K reference standards, accurately weigh them, dissolve and dilute them with acetonitrile to a mixed solution containing approximately 0.15 µg, 0.15 µg, 0.15 µg, 0.15 µg, and 0.15 µg per mL, respectively.
[0072] Determination: Based on the chromatographic parameters of Example 1, blank solution, system suitability solution, solutions at each limit of quantitation, and limit of detection solution were injected into the liquid chromatograph for detection, and the chromatograms were recorded.
[0073] The test results showed that the signal-to-noise ratio of the quantitation limits for each component was greater than 10. Figure 5 In the spectrum of the detection limit solution, the signal-to-noise ratio of each component was greater than 3. In the spectrum of the quantitation limit solution of the 6 needles, the peak area of each component was 0.5%~2.7%, less than 15%, indicating good precision (see Table 2). The quantitation limit concentration of this method is about 0.25µg / mL, which is equivalent to 0.05% of the sample concentration, and the detection limit concentration is 0.15µg / mL, which is equivalent to 0.03%, indicating that this method has high sensitivity.
[0074] Table 2 Results of Precision Validation of Limit of Quantification Concentration
[0075]
[0076]
[0077] Example 3: Linear Correlation Verification
[0078] Solution preparation
[0079] Linear solution L1: Take appropriate amounts of EP4, EP4A, EP4B, EP5, and EP4K reference standards, accurately weigh them, dissolve them in acetonitrile, and dilute them to a mixed solution containing approximately 0.27 µg, 0.25 µg, 0.26 µg, 0.25 µg, and 0.25 µg per mL, respectively.
[0080] Linear solution L2: Take appropriate amounts of EP4, EP4A, EP4B, EP5, and EP4K reference standards, accurately weigh them, dissolve them in acetonitrile, and dilute them to a mixed solution containing approximately 0.50 µg, 1.19 µg, 0.51 µg, 0.76 µg, and 1.20 µg per mL, respectively.
[0081] Linear solution L3: Take appropriate amounts of EP4, EP4A, EP4B, EP5, and EP4K reference standards, accurately weigh them, dissolve them in acetonitrile, and dilute them to a mixed solution containing approximately 0.80 µg, 1.90 µg, 0.82 µg, 1.21 µg, and 1.92 µg per mL, respectively.
[0082] Linear solution L4: Take appropriate amounts of EP4, EP4A, EP4B, EP5, and EP4K reference standards, accurately weigh them, dissolve them in acetonitrile, and dilute them to a mixed solution containing approximately 0.99 µg, 2.38 µg, 1.02 µg, 1.51 µg, and 2.40 µg per mL, respectively.
[0083] Linear solution L5: Take appropriate amounts of EP4, EP4A, EP4B, EP5, and EP4K reference standards, accurately weigh them, dissolve them in acetonitrile, and dilute them to a mixed solution containing approximately 1.49 µg, 3.57 µg, 1.53 µg, 2.27 µg, and 3.60 µg per mL, respectively.
[0084] Linear solution L6: Take appropriate amounts of EP4, EP4A, EP4B, EP5, and EP4K reference standards, accurately weigh them, dissolve them in acetonitrile, and dilute them to a mixed solution containing approximately 1.99 µg, 4.75 µg, 2.04 µg, 3.03 µg, and 4.79 µg per mL, respectively.
[0085] Linear solution L7: Take appropriate amounts of EP4, EP4A, EP4B, EP5, and EP4K reference standards, accurately weigh them, dissolve them in acetonitrile, and dilute them to a mixed solution containing approximately 2.98 µg, 7.13 µg, 3.06 µg, 4.54 µg, and 7.19 µg per mL, respectively.
[0086] Determination: Based on the chromatographic parameters of Example 1, take each linear solution, inject it into the liquid chromatograph for detection, and record the chromatogram.
[0087] Detection Results: Standard curves were constructed for each component with concentration on the x-axis and peak area on the y-axis. Regression analysis was performed using the least squares multiplication factor (LSM) method. The correlation coefficients for all components were 1.0000, indicating good linearity of the method. The linearity results for each component are shown in Table 3. The linearity plots for EP4, EP4A, EP4B, EP5, and EP4K are shown in Table 3. Figure 6 , Figure 7 , Figure 8 , Figure 9 , Figure 10 .
[0088] Table 3. Linearity verification results and correction factors for each component
[0089]
[0090] The correction factors for each component are calculated using the slopes of the standard curves for each component and the slope of the standard curve for EP4, as shown in Table 3. The correction factors for EP4A, EP4B, and EP4K are in the range of 0.8 to 1.2 and can be calculated using the area normalization method. The correction factor for EP5 is 1.4, and it needs to be multiplied by the correction factor in the result of area normalization.
[0091] Example 4: Precision Validation - Repeatability Validation
[0092] Solution preparation
[0093] Precision solution PR1: Accurately weigh appropriate amounts of EP4 test sample, EP4A, EP4B, EP5, and EP4K reference standards, dissolve and dilute with acetonitrile to a mixed solution containing approximately 0.5 mg, 2.5 µg, 1.0 µg, 1.5 µg, and 2.5 µg per mL, respectively. Prepare 6 aliquots (PR1 to PR6) using the same method.
[0094] Determination: Based on the chromatographic parameters of Example 1, take each precision solution, inject it into the liquid chromatograph for detection, and record the chromatogram.
[0095] Detection results: EP4A, EP4B, EP4K, the largest unknown single impurity, and total impurities were calculated using the area normalization method. EP5 was calculated by multiplying the area normalization result by 1.4. The RSD% of the detection results of each component were between 0.3% and 0.8%, indicating that the method has good repeatability. See Table 4.
[0096] Table 4 Repeatability Validation Results
[0097]
[0098] Example 5: Precision Validation - Intermediate Precision Validation
[0099] Intermediate precision validation was performed on different dates using different instruments and different batches of the same brand of chromatographic columns than the repeatability validation.
[0100] Solution preparation
[0101] Precision solution MP1: Accurately weigh appropriate amounts of EP4 test sample, EP4A, EP4B, EP5, and EP4K reference standards, dissolve and dilute with acetonitrile to a mixed solution containing approximately 0.5 mg, 2.5 µg, 1.0 µg, 1.5 µg, and 2.5 µg per mL, respectively. Prepare 6 aliquots (MP1~MP6) using the same method.
[0102] Determination: Based on the chromatographic parameters of Example 1, take each precision solution, inject it into the liquid chromatograph for detection, and record the chromatogram.
[0103] Test results: EP4A, EP4B, EP4K, the largest unknown single impurity, and total impurities were calculated using the area normalization method. EP5 was calculated by multiplying the area normalization result by 1.4. The RSD% of 12 data points, including repeatability test results and intermediate precision results, ranged from 0.8% to 1.9%, less than 15%, indicating good precision of the method (see Table 5).
[0104] Table 5 Precision Validation Results (Repeatability + Intermediate Precision)
[0105]
[0106] Example 6: Accuracy Verification
[0107] Solution preparation
[0108] Test solution: Take an appropriate amount of the test sample, weigh it accurately, and dilute it with a solution containing 0.5 mg of EP4 per mL.
[0109] Accuracy solution AC1: Accurately weigh appropriate amounts of EP4 test sample, EP4A, EP4B, EP5, and EP4K reference standards, dissolve and dilute with acetonitrile to a mixed solution containing approximately 0.5 mg, 0.25 µg, 0.26 µg, 0.25 µg, and 0.25 µg per mL, respectively. Prepare three aliquots, AC1 to AC3, using the same method.
[0110] Accuracy solution AC4: Accurately weigh appropriate amounts of EP4 test sample, EP4A, EP4B, EP5, and EP4K reference standards, dissolve and dilute with acetonitrile to a mixed solution containing approximately 0.5 mg, 1.90 µg, 0.82 µg, 1.21 µg, and 1.92 µg per mL, respectively. Prepare three aliquots (AC4-AC6) using the same method.
[0111] Accuracy solution AC7: Accurately weigh appropriate amounts of EP4 test sample, EP4A, EP4B, EP5, and EP4K reference standards, dissolve and dilute with acetonitrile to a mixed solution containing approximately 0.5 mg, 2.38 µg, 1.02 µg, 1.51 µg, and 2.40 µg per mL, respectively. Prepare three aliquots (AC7-AC9) using the same method.
[0112] Accuracy solution AC10: Accurately weigh appropriate amounts of EP4 test sample, EP4A, EP4B, EP5, and EP4K reference standards, dissolve and dilute with acetonitrile to a mixed solution containing approximately 0.5 mg, 3.57 µg, 1.53 µg, 2.27 µg, and 3.60 µg per mL, respectively. Prepare three aliquots of AC10 to AC12 using the same method.
[0113] Determination: Based on the chromatographic parameters of Example 1, take solutions of each accuracy level, inject them into the liquid chromatograph for detection, and record the chromatogram.
[0114] Results: The recovery rates of each component and the spiked concentrations were measured. The recovery rates of EP4A were 86.5%–99.9%, EP4B 98.8%–104.5%, EP5 99.6%–102.8%, and EP4K 98.3%–103.1%, all within the range of 80%–120%, indicating that the method has high accuracy (see Table 6).
[0115] Table 6 Accuracy Results
[0116]
[0117]
[0118] Example 7: Durability Test
[0119] Adjustments to the detection method (by changing flow rate, column temperature, and column batch) will be made, and the impact on the detection results will be evaluated.
[0120] Solution preparation
[0121] Test solution: Take an appropriate amount of the test sample, weigh it accurately, and dilute it with a solution containing 0.5 mg of EP4 per mL.
[0122] System suitability solution: Take appropriate amounts of EP4, EP4A, EP4B, EP5, and EP4K reference standards, accurately weigh them, dissolve them in acetonitrile, and dilute them to a mixed solution containing 0.5 mg, 2.5 µg, 1 µg, 1.5 µg, and 2.5 µg per mL, respectively.
[0123] Blank: Acetonitrile.
[0124] Detection: By adjusting the methodological parameters, inject blank solution, system suitability solution, and test solution.
[0125] Robustness results: Based on the adjustment parameters listed in Table 7, the minimum resolution of each component in the system suitability spectrum is 2.0, which is greater than 1.5 and comparable to that under normal conditions, indicating that minor changes in chromatographic conditions have no effect on specificity.
[0126] Table 7. Durability Verification System Suitability Verification Results
[0127]
[0128] Adjusting the parameters listed in Table 7 and testing the sample solution, the test results showed no significant changes, indicating that minor changes in chromatographic parameters have no effect on the test results (see Table 8). The overall resolution results show that this method has good robustness.
[0129] Table 8. Test results of the test specimens for durability verification
[0130]
[0131] Example 8: Solution Stability Test
[0132] Solution stability study: The test solution and system suitability solution were placed at room temperature for 1 day, 2 days and 3 days to observe the change of impurities and to assess the shelf life of the solution.
[0133] Solution preparation
[0134] Test solution: Take an appropriate amount of the test sample, weigh it accurately, and dilute it with a solution containing 0.5 mg of EP4 per mL.
[0135] System suitability solution: Take appropriate amounts of EP4, EP4A, EP4B, EP5, and EP4K reference standards, accurately weigh them, dissolve them in acetonitrile, and dilute them to a mixed solution containing 0.5 mg, 2.5 µg, 1 µg, 1.5 µg, and 2.5 µg per mL, respectively.
[0136] Blank: Acetonitrile.
[0137] Detection: Following the method parameters of Example 1, take the blank solution, test solution, and system suitability solution that have been placed at room temperature, inject them into the liquid chromatography system, and record the chromatogram.
[0138] The solution stability results showed that after 3 days at room temperature, both the test solution and the system suitability solution maintained the required separation, with no significant changes in any component and no new unknown impurities detected. This indicates that the solution was stable after 3 days at room temperature. The solution stability results are shown in Table 9.
[0139] Table 9. Test Results of Test Specimens for Durability Verification
[0140]
[0141] Example 9: Method Application and Sample Detection
[0142] Test samples, batches: Z1212-250725, Z1212-250726, Z1212-250727
[0143] Solution preparation
[0144] Test solution: Take an appropriate amount of the test sample, weigh it accurately, and dilute it with a solution containing 0.5 mg of EP4 per mL.
[0145] System suitability solution: Take appropriate amounts of EP4, EP4A, EP4B, EP5, and EP4K reference standards, accurately weigh them, dissolve them in acetonitrile, and dilute them to a mixed solution containing 0.5 mg, 2.5 µg, 1 µg, 1.5 µg, and 2.5 µg per mL, respectively.
[0146] Blank: Acetonitrile.
[0147] Detection: Inject blank solution, system suitability solution, and test solution according to the chromatographic parameters of Example 1, and record the chromatogram.
[0148] The test results for the three batches of test samples are shown in Table 10.
[0149] Table 10 Test results of three batches of samples
[0150]
Claims
1. A method for determining the content of related substances and enantiomers of empagliflozin intermediate (S)-3-[4-(5-bromo-2-chlorobenzyl)phenoxy]tetrahydrofuran by reversed-phase chromatography, characterized in that, include: The EP4 test sample was mixed with an organic solvent to obtain a test sample solution; The test solution was analyzed by high performance liquid chromatography (HPLC); wherein the HPLC column was a chiral column with polysaccharide derivative bonded packing material; the mobile phase consisted of acetonitrile and purified water, and gradient elution was used.
2. The detection method as described in claim 1, characterized in that, In the high-performance liquid chromatography (HPLC) detection process, a gradient elution method is used. Mobile phase A is purified water, and mobile phase B is acetonitrile. The preferred gradient is: 0~5 min, 55% A; 5~25 min, 55%→30% A; 25~40 min, 30%→15% A; 40~52 min, 15% A; 52~52.1 min, 15%→55% A; 52.1~60 min, 55% A.
3. The detection method as described in claim 1, characterized in that, During high-performance liquid chromatography (HPLC) detection, the flow rate is 0.8–1.2 mL / min. -1 Preferably 1.0 mL·min -1 .
4. The detection method as described in claim 1, characterized in that, The chromatographic column is a chiral packing material bonded with polysaccharide derivatives, preferably CHIRAL PAK. ® IC.
5. The detection method as described in claim 1, characterized in that, The high-performance liquid chromatography (HPLC) method uses a column temperature of 25-35℃ and an injection volume of 5-15 μL.
6. The detection method as described in claim 1, characterized in that, The high-performance liquid chromatography method is used for detection at wavelengths of 210-230 nm.