A method for determining isomers in multi-tiravir sodium by high performance liquid chromatography

Abstract

This invention discloses a method for determining isomers in dolutegravir sodium using high-performance liquid chromatography (HPLC), comprising the following steps: preparing a test solution from a dolutegravir sodium sample, and separating and detecting isomer impurities in dolutegravir sodium by HPLC; the mobile phase A of the HPLC is a phosphoric acid solution with a concentration of 0.08-0.12 wt.%; the mobile phase B is acetonitrile; isocratic elution is performed at a volume ratio of mobile phase A to mobile phase B of 1:16-24, and the sample pan temperature is 2-8℃. This method exhibits strong specificity, short running time, good resolution, and high sensitivity for detecting dolutegravir isomer impurities in dolutegravir sodium. It is also convenient to operate, has a short running time, and high analytical efficiency, significantly improving testing efficiency. Temperature control of the sample pan extends the stability of the reference solution and the sample solution; the external standard method is used for quantification, resulting in more accurate results, thereby effectively controlling the quality of dolutegravir sodium raw material.

Description

Technical Field

[0001] This invention relates to a method for determining isomers in dolutegravir sodium using high performance liquid chromatography, belonging to the field of isomer detection in dolutegravir sodium. Background Technology

[0002] AIDS is a highly dangerous infectious disease caused by infection with the human immunodeficiency virus (HIV). HIV is a virus that attacks the human immune system. It targets the most important CD4+ cytoplasm in the human immune system. ＋ T lymphocytes are the primary target of attack, and the destruction of large numbers of these cells leads to the loss of the body's immune function. As a result, the body becomes susceptible to various diseases and can develop malignant tumors, resulting in a high mortality rate.

[0003] To date, the anti-HIV drugs approved by the U.S. FDA are mainly divided into seven categories: nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), integrase inhibitors (INSTIs), fusion inhibitors (FIs), CCR5 inhibitors, and capsid inhibitors (CAs).

[0004] Dolutegravir Sodium is a highly effective integrase inhibitor that works by binding to the active site of integrase and blocking the key retroviral DNA integration transfer step in the HIV replication cycle, thereby inhibiting HIV integrase, which in turn blocks HIV-1 proviral formation and viral replication.

[0005] Dolutegravir, structural formula is It is a key intermediate in the synthesis of dolutegravir sodium, and it can be converted into dolutegravir sodium through salt formation. Currently, the International Pharmacopoeia stipulates that the content of dolutegravir isomer impurities (including enantiomers and diastereomers) in dolutegravir sodium must not exceed 0.15%. Therefore, developing specific methods to control the content of dolutegravir isomers during production is an important part of ensuring the quality of dolutegravir sodium and its formulations. Although the detection methods for dolutegravir sodium impurities provided in the International Pharmacopoeia include the determination of dolutegravir isomers, these methods suffer from poor specificity, complex analytical procedures, and low detection efficiency in practical applications. Summary of the Invention

[0006] This invention addresses the problems existing in the prior art by providing a method for determining isomers in dolutegravir sodium using high-performance liquid chromatography. This method has strong specificity for detecting dolutegravir isomer impurities in dolutegravir sodium, short running time, good separation, high sensitivity, convenient operation, short running time, and high analytical efficiency, and can effectively control the quality and grade of dolutegravir sodium raw material.

[0007] To achieve the above objectives, the present invention provides a method for determining isomers in dolutegravir sodium using high performance liquid chromatography, the method comprising the following steps: Dolutegravir sodium sample was prepared into a test solution, and isomer impurities in dolutegravir sodium were separated and detected by high performance liquid chromatography. The isomer impurities include at least one of impurity ADC201-020, impurity ADC201-021 and impurity ADC201-022. The detection conditions for the high-performance liquid chromatography include: Mobile phase A: Phosphoric acid solution with a phosphoric acid concentration of 0.08-0.12 wt.%; Mobile phase B: Acetonitrile; Isocratic elution was performed with a mobile phase A to mobile phase B volume ratio of 1:16-24. Sample tray temperature: 2-8℃; Detection wavelength: 250-265nm; The structural formula of the impurity ADC201-020 is as follows: , The structural formula of the impurity ADC201-021 is as follows: , The structural formula of the impurity ADC201-022 is as follows: .

[0008] It should be noted that the International Pharmacopoeia (IPP) provides a method for detecting impurities in dolutegravir sodium, which can be found in *The International Pharmacopoeia - Twelfth Edition, 2024*, Dolutegravir sodium (Dolutegravirum natricum). This method provides two detection system systems: System 1 is used to detect dolutegravir isomers (impurities A, B, and G), and System 2 is used to detect related substances. The structures of the impurities are shown in Table 1. However, when detecting dolutegravir isomers, System 1 in the IPP has a co-elution problem with isomer impurity G and impurity H under the related substances section. The content of impurity H obtained from System 2 must be subtracted to determine the content of isomer impurity G. Therefore, the method provided by the IPP cannot effectively separate isomer impurity G from related substance impurity H for the detection of dolutegravir isomers, exhibiting poor specificity. The isomer impurities are easily interfered with by other impurities, and the content of isomer impurity G can only be obtained with the help of related substance detection results, making the analytical process complex and inefficient.

[0009] Based on this, the inventors of this invention unexpectedly discovered during the research and development process that by using specific mobile phases A and B in high-performance liquid chromatography (HPLC) detection, along with specific mobile phase ratios and specific sample pan temperatures, it is possible to effectively separate and detect dolutegravir isomer impurities in dolutegravir sodium with good precision and accuracy. This led to the establishment of a detection system for dolutegravir isomer impurities in dolutegravir sodium that is highly specific, has good separation, and high sensitivity. Furthermore, this system is easy to operate, has a short running time, and high analytical efficiency, enabling more accurate detection of isomer content in dolutegravir sodium and better achieving quality control of dolutegravir sodium raw materials and formulations.

[0010] Table 1

[0011] In this invention, isomer impurities in dolutegravir sodium are separated and detected by high-performance liquid chromatography (HPLC), specifically through qualitative or quantitative detection. The qualitative detection process includes: using impurities ADC201-020, ADC201-021, and ADC201-022 as reference standards, determining the corresponding peak times using the HPLC detection conditions provided by this invention (e.g., the peak time for impurity ADC201-020 is approximately 11.067 min, for impurity ADC201-021 it is approximately 20.360 min, and for impurity ADC201-022 it is approximately 9.260 min), and then detecting whether a chromatographic peak appears in the dolutegravir sodium sample at or near the aforementioned peak times under the same HPLC detection conditions, to determine whether the dolutegravir sodium sample contains impurities ADC201-020, ADC201-021, and ADC201-022. Quantitative detection can be performed using the external standard method. The specific process of the external standard method includes: using impurities ADC201-020, ADC201-021, and ADC201-022 as reference standards, preparing reference standard solutions, preparing the dolutegravir sodium sample into a test solution, and performing detection using the high-performance liquid chromatography detection conditions provided by the present invention, calculating the corresponding concentrations for quantification.

[0012] In this invention, the dolutegravir sodium sample can be prepared by referring to a known process (e.g., the method disclosed in CN102933080A), or it can be purchased from an approved dolutegravir sodium active pharmaceutical ingredient manufacturer.

[0013] In a preferred embodiment, the conditions for the high-performance liquid chromatography further include: a total flow rate of mobile phase of 0.9-1.1 ml / min, specifically 0.9 ml / min, 1 ml / min, 1.1 ml / min, or any value between the two aforementioned values, more preferably 1 ml / min; and a column temperature of 28-35℃, specifically 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, or any value between the two aforementioned values, more preferably 28-32℃.

[0014] In a preferred embodiment, the solvent for the test solution is an acetonitrile-water mixture with a volume ratio of 1:1.4-1.6; the volume ratio of acetonitrile to water can be 1:1.4, 1:1.45, 1:1.5, 1:1.55, 1:1.6, or any value between the two aforementioned values.

[0015] In a preferred embodiment, the preparation process of the test solution includes: weighing the dolutegravir sodium sample and placing it in a brown volumetric flask, dissolving and diluting it with solvent; the content of the dolutegravir sodium sample in the test solution is 0.4-0.6 mg / ml. Using a specific ratio of acetonitrile-water solution as a solvent to prepare a dolutegravir sodium sample solution with a specific content can effectively improve the specificity and accuracy of the separation and detection of dolutegravir isomer impurities in dolutegravir sodium.

[0016] In this invention, the chromatographic column can be a DAICL CHIRALCEL OX-3R (3μm), a CHIRALCEL OX-RH column (5μm), or a Phenomenex Lux Cellulose-4 column, etc. As a preferred embodiment, the chromatographic column is a DAICL CHIRALCEL OX-3R, 4.6mm × 150mm, 3μm.

[0017] In this invention, the phosphoric acid concentration of mobile phase A can specifically be 0.08 wt.%, 0.09 wt.%, 0.1 wt.%, 0.11 wt.%, 0.12 wt.%, or any value between the aforementioned two values. As a preferred embodiment, mobile phase A is a phosphoric acid solution with a phosphoric acid concentration of 0.1 wt.%, and mobile phase B is acetonitrile.

[0018] In this invention, the volume ratio of mobile phase A to mobile phase B for isocratic elution can be 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, or any value between the aforementioned two values. As a preferred embodiment, mobile phase A and mobile phase B are eluted isocratically at a volume ratio of 5:95, which can effectively improve the specificity and accuracy of the separation and detection of dolutegravir isomer impurities in dolutegravir sodium.

[0019] In this invention, the temperature of the sample pan can be 2℃, 2.5℃, 3℃, 3.5℃, 4℃, 4.5℃, 5℃, 5.5℃, 6℃, 6.5℃, 7℃, 7.5℃, 8℃, or any value between the two aforementioned values; preferably 4-6℃.

[0020] As a preferred embodiment, the detection conditions of the high performance liquid chromatography further include: the injection volume is 18-22 μl, specifically 18 μl, 19 μl, 20 μl, 21 μl, 22 μl, or any value between the two aforementioned values.

[0021] As a preferred embodiment, the method for determining the isomers in dolutegravir sodium using high performance liquid chromatography includes the following steps: (1) Prepare a test solution from a dolutegravir sodium sample, and prepare a reference solution (preferably with a concentration of 0.6-0.9 µg / ml) from impurities ADC201-020, ADC201-021 and ADC201-022. The test solution and the reference solution shall use the same solvent. (2) Detect each solution in step (1) according to the detection conditions of the above high performance liquid chromatography, and calculate the content of each isomer impurity according to the external standard method.

[0022] As a relatively preferred embodiment of the present invention, the method for determining isomers in dolutegravir sodium using high performance liquid chromatography includes: (1) An acetonitrile-water mixture with a volume ratio of 1:1.4-1.6 was used as the solvent; Reference solution: Take appropriate amounts of ADC201-020 reference standard, ADC201-021 reference standard and ADC201-022 reference standard, accurately weigh them, put them in a brown volumetric flask, add solvent to dissolve and dilute to prepare a solution containing about 0.7-0.8 µg of each of ADC201-020, ADC201-021 and ADC201-022 per ml; Test solution: Accurately weigh dolutegravir sodium raw material, place it in a brown volumetric flask, add solvent to dissolve and dilute to prepare a solution containing approximately 0.4-0.6 mg of dolutegravir sodium sample per 1 ml.

[0023] (2) Detect each solution in step (1) according to the following high performance liquid chromatography detection conditions, record the chromatogram, and calculate the content of each isomer impurity in the sample according to the external standard method; Column: cellulose-tris(4-chloro-3-methylphenylcarbamate) silica gel as packing material (DAICELCHIRALCEL OX-3R, 4.6 mm × 150 mm, 3 μm or equivalent column). Mobile phase: 0.08-0.12 wt.% phosphoric acid solution and acetonitrile at a volume ratio of 1:16-24 for isocratic elution; Column temperature: 28-35℃; Flow rate: 0.9-1.1 ml / min; Detection wavelength: 250-265nm (e.g., 258nm); Injection volume: 18-22 µl; Sample tray temperature: 2-8℃; Running time: Approximately 30 minutes.

[0024] In this invention, the quality control standards for dolutegravir sodium raw material are specifically: ADC201-020 content ≤ 0.15%, ADC201-021 content ≤ 0.15%, and ADC201-022 content ≤ 0.15%.

[0025] Compared with the prior art, the present invention has the following significant advantages: This invention establishes a method for detecting dolutegravir isomers in dolutegravir sodium. This method combines specific parameters such as mobile phase A, mobile phase B, specific mobile phase ratios, and specific sample pan temperature during high-performance liquid chromatography (HPLC). It exhibits strong specificity, short running time, good resolution, and high sensitivity for detecting dolutegravir isomer impurities in dolutegravir sodium. The method is convenient to operate, has a short running time, and high analytical efficiency, significantly improving testing efficiency and saving costs. Furthermore, the quantification method employs an external standard method, resulting in more accurate results and effectively controlling the quality of dolutegravir sodium raw materials or formulations. Attached Figure Description

[0026] Figure 1 High-performance liquid chromatography (HPLC) chromatogram with solvent as the solvent; Figure 2 High-performance liquid chromatogram of the ADC201-020 localization solution; Figure 3 High-performance liquid chromatogram of the ADC201-021 localization solution; Figure 4 High-performance liquid chromatogram of the ADC201-022 localization solution; Figure 5The high-performance liquid chromatogram of dolutegravir sodium localization solution; Figure 6 This is a high-performance liquid chromatogram of a mixed solution of impurities under the related substances section; Figure 7 High-performance liquid chromatogram of the system suitability solution; Figure 8 This is the high-performance liquid chromatogram of the reference solution; Figure 9 Linear relationship diagram for isomer ADC201-020; Figure 10 Linear relationship diagram for isomer ADC201-021; Figure 11 The linear relationship diagram is for the isomer ADC201-022. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0028] Unless otherwise specified in the examples, standard conditions were followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0029] In this example, the high-performance liquid chromatograph used was a Thermo U3000.

[0030] Dolutegravir sodium raw material was purchased from Yangzhou Aidi Pharmaceutical Technology Co., Ltd., and prepared according to the process in CN102933080A.

[0031] Information on the reference standard is shown in Table 2: Table 2

[0032] Example 1 1. Solution preparation: Solvent: Acetonitrile-water solution (acetonitrile:water = 40:60, v / v).

[0033] ADC201-020 positioning solution: Take an appropriate amount of ADC201-020 reference standard, accurately weigh it, place it in a brown volumetric flask, add solvent to dissolve and dilute it to prepare a solution containing about 0.75µg per ml.

[0034] ADC201-021 positioning solution: Take an appropriate amount of ADC201-021 reference standard, accurately weigh it, place it in a brown volumetric flask, add solvent to dissolve and dilute it to prepare a solution containing 0.75µg per ml.

[0035] ADC201-022 positioning solution: Take an appropriate amount of ADC201-022 reference standard, accurately weigh it, place it in a brown volumetric flask, add solvent to dissolve and dilute it to prepare a solution containing 0.75µg per ml.

[0036] ADC201 localization solution: Weigh 7.5 mg of ADC201 reference standard accurately, place it in a 100 ml brown volumetric flask, dissolve and dilute to the mark with solvent, and shake well to obtain the solution.

[0037] Mixed solution: Accurately weigh appropriate amounts of each reference standard ADC201-019, ADC201-023, ADC201-024, ADC201-025, ADC20-026, ADC201-027, and ADC201-028, place them in a brown volumetric flask, dissolve and dilute with solvent to prepare a solution containing 0.75 µg of each of ADC201-019, ADC201-023, ADC201-024, ADC201-025, ADC20-026, ADC201-027, and ADC201-028 per ml.

[0038] System suitability solution: Take appropriate amounts of dolutegravir sodium, ADC201-020, ADC201-021, and ADC201-022 reference standards, place them in a brown volumetric flask, add solvent to dissolve and dilute to prepare a solution containing 0.5 mg of dolutegravir sodium (ADC201) and 0.75 µg of each of ADC201-020, ADC201-021, and ADC201-022 per 1 ml.

[0039] 2. Chromatographic conditions of high performance liquid chromatography: A cellulose-tris(4-chloro-3-methylphenylcarbamate) silica gel column (DAICEL CHIRALCEL OX-3R, 4.6 mm × 150 mm, 3 μm) was used as the stationary phase, with 0.1% phosphoric acid solution-acetonitrile (5:95, v / v) as the mobile phase, and isocratic elution was performed; the flow rate was 1.0 mL per minute; the column temperature was 30 °C; the sample tray temperature was 5 °C; the detection wavelength was 258 nm; and the injection volume was 20 µL.

[0040] 3. Procedure: Take the solvent, each positioning solution, the mixed solution, and the system suitability solution, and perform high-performance liquid chromatography (HPLC) analysis according to the above conditions. Record the chromatograms. The results are as follows: Figures 1-7 As shown.

[0041] Inject 20 µL of solvent and perform detection under the chromatographic conditions described above using the high-performance liquid chromatograph. The results show ( Figure 1The solvent does not interfere with the determination. Similarly, ADC201-020 positioning solution, ADC201-021 positioning solution, and ADC201-022 positioning solution were taken and detected according to the chromatographic conditions of the high-performance liquid chromatograph described above. The results showed that ( Figures 2-4 The chromatographic peak with a retention time of 11.067 min is the chromatographic peak of ADC201-020, the chromatographic peak with a retention time of 20.360 min is the chromatographic peak of ADC201-021, and the chromatographic peak with a retention time of 9.260 min is the chromatographic peak of ADC201-022.

[0042] Inject 20 µL of the mixed solution and dolutegravir sodium (ADC201) targeting solution, and perform detection according to the chromatographic conditions of the high-performance liquid chromatograph described above. The results show ( Figures 5-6 The chromatographic peak with a retention time of 15.420 min is the chromatographic peak of dolutegravir sodium (ADC201). The elution time of the impurity mixture under related substances is also different from that of the isomers ADC201-020, ADC201-021, and ADC201-022, indicating that neither the impurity mixture under related substances nor dolutegravir sodium interferes with the detection of the three isomers ADC201-020, ADC201-021, and ADC201-022.

[0043] Inject 20 µL of the system suitability solution and perform detection according to the chromatographic conditions of the high-performance liquid chromatograph described above. The results show ( Figure 7 The resolution of the three isomer chromatographic peaks from their adjacent peaks was greater than 1.5, demonstrating the high specificity of the method provided by this invention. Example 2

[0044] 1. Solution preparation: Solvent: Acetonitrile-water solution (acetonitrile:water = 40:60, v / v).

[0045] Reference solution: Take appropriate amounts of ADC201-020 reference standard, ADC201-021 reference standard and ADC201-022 reference standard, accurately weigh them, place them in a brown volumetric flask, add solvent to dissolve and dilute to prepare a solution containing 0.75µg of each of ADC201-020, ADC201-021 and ADC201-022 per ml.

[0046] 2. Chromatographic conditions of high performance liquid chromatography: A cellulose-tris(4-chloro-3-methylphenylcarbamate) silica gel column (DAICEL CHIRALCEL OX-3R, 4.6 mm × 150 mm, 3 μm) was used as the stationary phase, with 0.1% phosphoric acid solution-acetonitrile (5:95, v / v) as the mobile phase, and isocratic elution was performed; the flow rate was 1.0 mL per minute; the column temperature was 30 °C; the sample tray temperature was 5 °C; the detection wavelength was 258 nm; and the injection volume was 20 µL.

[0047] 3. Procedure: Take the reference solution and perform high-performance liquid chromatography (HPLC) analysis under the conditions described above. Repeat the injection six times, record the chromatograms, and see the results below. Figure 8 The injection precision results are shown in Table 3. The results indicate that the method of the present invention has good injection precision and good system applicability.

[0048] Table 3 Results of the injection precision test Example 3

[0049] 1. Solution preparation: Solvent: Acetonitrile-water solution (acetonitrile:water = 40:60, v / v).

[0050] ADC201-020 reference standard stock solution: Take an appropriate amount of ADC201-020 reference standard, accurately weigh it, place it in a brown volumetric flask, add solvent to dissolve and dilute it to prepare a solution containing about 37.5µg in 1ml.

[0051] ADC201-021 reference standard stock solution: Take an appropriate amount of ADC201-021 reference standard, accurately weigh it, place it in a brown volumetric flask, add solvent to dissolve and dilute it to prepare a solution containing about 37.5µg in 1ml.

[0052] ADC201-022 reference standard stock solution: Take an appropriate amount of ADC201-022 reference standard, accurately weigh it, place it in a brown volumetric flask, add solvent to dissolve and dilute it to prepare a solution containing about 37.5µg in 1ml.

[0053] Limit of Quantitation Solution: Accurately measure 3 ml of ADC201-020 reference stock solution, 6 ml of ADC201-021 reference stock solution, and 2 ml of ADC201-022 reference stock solution, place them in a 50 ml brown volumetric flask, dilute to the mark with solvent, and shake well; then accurately measure 1 ml, place it in a 100 ml brown volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0054] Detection limit solution: Accurately measure 5 ml of the quantitation limit solution and place it in a 10 ml brown volumetric flask. Dilute to the mark with solvent and shake well.

[0055] 2. Chromatographic conditions of high performance liquid chromatography: cellulose-tris(4-chloro-3-methylphenylcarbamate) silica gel was used as the stationary phase (DAICEL CHIRALCEL OX-3R, 4.6 mm × 150 mm, 3 μm), with 0.1% phosphoric acid solution-acetonitrile (5:95, v / v) as the mobile phase, and isocratic elution was performed; the flow rate was 1.0 mL per minute; the column temperature was 30 °C; the sample tray temperature was 5 °C; the detection wavelength was 258 nm; and the injection volume was 20 µL.

[0056] 3. Procedure: Take the limit of detection (LOD) solution and the limit of quantitation (LOQ) solution, and perform high-performance liquid chromatography (HPLC) analysis according to the above conditions. Inject the LOD solution once and the LOQ solution six times. Record the chromatograms. The results of the LOD and LOQ are shown in Table 4.

[0057] Table 4 Results of Limit of Detection and Limit of Quantitation Tests

[0058] The results of Example 3 show that the method of the present invention has good detection sensitivity. Example 4

[0059] 1. Solution preparation: Solvent: Acetonitrile-water solution (acetonitrile:water = 40:60, v / v).

[0060] ADC201-020 reference standard stock solution: Take an appropriate amount of ADC201-020 reference standard, accurately weigh it, place it in a brown volumetric flask, add solvent to dissolve and dilute it to prepare a solution containing about 37.5µg per ml.

[0061] ADC201-021 reference stock solution: Take an appropriate amount of ADC201-021 reference standard, accurately weigh it, place it in a brown volumetric flask, add solvent to dissolve and dilute it to prepare a solution containing about 37.5µg per ml.

[0062] ADC201-022 reference standard stock solution: Take an appropriate amount of ADC201-022 reference standard, accurately weigh it, place it in a brown volumetric flask, add solvent to dissolve and dilute it to prepare a solution containing about 37.5µg per ml.

[0063] Linear stock solution: Take 20 ml of each of the reference stock solutions of ADC201-020, ADC201-021 and ADC201-020, place them in a 100 ml brown volumetric flask, dilute to the mark with solvent, and shake well to obtain the linear stock solution.

[0064] Linear solutions: Dilute and bring to volume the linear stock solution with solvent according to Table 5, shake well, and obtain linear solutions L1 to L5. Among them, linear solution L0 is the same as the limit of quantitation solution in Example 3.

[0065] Table 5. Preparation of linear solutions

[0066] 2. Chromatographic conditions of high performance liquid chromatography: cellulose-tris(4-chloro-3-methylphenylcarbamate) silica gel was used as the stationary phase (DAICEL CHIRALCEL OX-3R, 4.6 mm × 150 mm, 3 μm), with 0.1% phosphoric acid solution-acetonitrile (5:95, v / v) as the mobile phase, and isocratic elution was performed; the flow rate was 1.0 mL per minute; the column temperature was 30 °C; the sample tray temperature was 5 °C; the detection wavelength was 258 nm; and the injection volume was 20 µL.

[0067] 3. Procedure: Take the linear solution and perform high-performance liquid chromatography (HPLC) analysis under the conditions described above. Record the chromatograms. The linearity results are shown in Tables 6-8, and the linearity curve is shown in Table 6. Figures 9-11 .

[0068] Table 6. Linearity and Range Results of ADC201-020

[0069] Table 7 Linearity and Range Results for ADC201-021

[0070] Table 8 Linearity and Range Results for ADC201-022

[0071] The results showed that isomers ADC201-020 exhibited good linearity in the concentration range of 0.01881–2.2442 µg / ml; ADC201-021 exhibited good linearity in the concentration range of 0.04367–2.2198 µg / ml; and ADC201-022 exhibited good linearity in the concentration range of 0.01825–2.2409 µg / ml. Example 5

[0072] 1. Solution preparation: Solvent: Acetonitrile-water solution (acetonitrile:water = 40:60, v / v).

[0073] Reference solution: Accurately weigh appropriate amounts of ADC201-020, ADC201-021, and ADC201-022 reference standards, place them in the same brown volumetric flask, dissolve and dilute with solvent to prepare a solution containing 0.75 µg of each of ADC201-020, ADC201-021, and ADC201-022 per ml. Prepare two parallel solutions.

[0074] Reference stock solution: Take appropriate amounts of ADC201-020 reference standard, ADC201-021 reference standard and ADC201-022 reference standard, accurately weigh them, place them in the same brown volumetric flask, add solvent to dissolve and dilute to prepare a solution containing 37.5µg of each of ADC201-020, ADC201-021 and ADC201-022 per ml.

[0075] Spiked solution for test sample: Accurately weigh approximately 25 mg of dolutegravir sodium raw material (purchased from Yangzhou Aidi Pharmaceutical Technology Co., Ltd.), place it in a 50 ml brown volumetric flask, accurately measure 1 ml of the reference stock solution into the above volumetric flask, dissolve and dilute to the mark with solvent, and shake well to obtain the test sample. Prepare 6 parallel solutions.

[0076] 2. Chromatographic conditions of high performance liquid chromatography: cellulose-tris(4-chloro-3-methylphenylcarbamate) silica gel was used as the stationary phase (DAICEL CHIRALCEL OX-3R, 4.6 mm × 150 mm, 3 μm), with 0.1% phosphoric acid solution-acetonitrile (5:95, v / v) as the mobile phase, and isocratic elution was performed; the flow rate was 1.0 mL per minute; the column temperature was 30 °C; the sample tray temperature was 5 °C; the detection wavelength was 258 nm; and the injection volume was 20 µL.

[0077] 3. Procedure: Take the spiked solution of the test sample and the reference solution, perform high performance liquid chromatography analysis under the above conditions, record the chromatograms, and the repeatability test results are shown in Table 9.

[0078] Table 9 Results of Repeatability Tests

[0079] The results show that the method of the present invention has good repeatability. Example 6

[0080] The reference solution and the spiked solution of the test sample were prepared using the same method as in Example 5, and the detection was performed under the same chromatographic conditions. The difference was that the solutions were prepared by different analysts on different dates and the tests were performed using another high-performance liquid chromatograph of the same model as an intermediate precision test. The results are shown in Table 10.

[0081] Table 10 Results of intermediate precision test

[0082] The results show that the method of the present invention has good intermediate precision. Example 7

[0083] 1. Solution preparation: Solvent: Acetonitrile aqueous solution (acetonitrile:water = 40:60, v / v).

[0084] ADC201-020 reference standard stock solution: Take an appropriate amount of ADC201-020 reference standard, accurately weigh it, place it in a brown volumetric flask, add solvent to dissolve and dilute it to prepare a solution containing 37.5µg per ml.

[0085] ADC201-021 reference stock solution: Take an appropriate amount of ADC201-021 reference standard, accurately weigh it, place it in a brown volumetric flask, add solvent to dissolve and dilute it to prepare a solution containing 37.5µg per ml.

[0086] ADC201-022 reference standard stock solution: Take an appropriate amount of ADC201-022 reference standard, accurately weigh it, place it in a brown volumetric flask, add solvent to dissolve and dilute it to prepare a solution containing 37.5µg per ml.

[0087] Impurity stock solution: Take 20 ml of each of the reference stock solutions of ADC201-020, ADC201-021 and ADC201-022, place them in a 100 ml brown volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0088] 50% Accuracy Solution: Accurately weigh approximately 25 mg of dolutegravir sodium raw material and place it in a 50 ml brown volumetric flask. Transfer 2.5 ml of the impurity stock solution, dissolve and dilute to the mark with solvent, and shake well. Prepare three parallel portions.

[0089] 100% Accuracy Solution: Accurately weigh approximately 25 mg of dolutegravir sodium raw material and place it in a 50 ml brown volumetric flask. Transfer 5 ml of the impurity stock solution, dissolve and dilute to the mark with solvent, and shake well. Prepare three parallel solutions.

[0090] 150% accuracy solution: Accurately weigh approximately 25 mg of dolutegravir sodium raw material and place it in a 50 ml brown volumetric flask. Transfer 7.5 ml of the impurity stock solution, dissolve and dilute to the mark with solvent, and shake well. Prepare three parallel solutions.

[0091] Test solution: Take an appropriate amount of dolutegravir sodium raw material, accurately weigh it, place it in a brown volumetric flask, add solvent to dissolve and dilute it to prepare a solution containing about 0.5 mg per 1 ml.

[0092] 2. Chromatographic conditions of high performance liquid chromatography: cellulose-tris(4-chloro-3-methylphenylcarbamate) silica gel was used as the stationary phase (DAICEL CHIRALCEL OX-3R, 4.6 mm × 150 mm, 3 μm), with 0.1% phosphoric acid solution-acetonitrile (5:95, v / v) as the mobile phase, and isocratic elution was performed; the flow rate was 1.0 mL per minute; the column temperature was 30 °C; the sample tray temperature was 5 °C; the detection wavelength was 258 nm; and the injection volume was 20 µL.

[0093] 3. Procedure: Take the accuracy solution, test solution, and reference solution, and perform high-performance liquid chromatography (HPLC) analysis according to the above chromatographic conditions. Record the chromatograms. The linearity results are shown in Table 11. The results show that the recoveries of isomers ADC201-020, ADC201-021, and ADC201-022 are all between 80.0% and 120.0%, and the RSD of the recoveries is less than 10.0%. The method of this invention has good accuracy.

[0094] Table 11 Accuracy Test Results

[0095] In summary, the method for determining isomers in dolutegravir sodium of the present invention facilitates the separation, elution, and detection of isomers in dolutegravir sodium, and fully demonstrates that the method is scientific, reliable, and can control the isomers in dolutegravir sodium. Example 8

[0096] Dolutegravir sodium raw material was purchased from Yangzhou Aidi Pharmaceutical Technology Co., Ltd., with batch numbers APH201-1-230501, APH201-1-230502, and APH201-1-230503. Test solutions for the three batches of dolutegravir sodium raw material samples were prepared using the method in Example 7, and reference solutions were prepared using the method in Example 2. The chromatographic conditions of the high performance liquid chromatograph in Example 7 were used for detection. The peak areas were obtained, and the content of each isomer impurity in the raw material was calculated using the external standard method. The results are shown in Table 12.

[0097] Table 12. Isomer Detection Results of Multiple Batches of Dolutegravir Sodium Raw Material Comparative Example 1

[0098] The same solution preparation process and detection method as in Example 1 were used, except that the elution ratio of mobile phase A and mobile phase B was replaced with 0.1% phosphoric acid solution: acetonitrile = 1:30 (v / v). The results showed that the system suitability solution required 60 minutes to elute completely, which resulted in a long running time and low efficiency. Comparative Example 2

[0099] Using the same solution preparation process and HPLC conditions as in Example 1, the sample pan temperature was set to room temperature (approximately 25°C), 10°C, and 5°C to investigate the stability of the system suitability solution in Example 1. The results showed that at room temperature, the baseline near the main peak in the chromatogram of the system suitability solution was significantly elevated; at 10°C, the baseline near the main peak was slightly elevated; and at 5°C, the baseline near the main peak was more stable.

Claims

1. A method for determining isomers in dolutegravir sodium using high performance liquid chromatography, characterized in that, The method includes the following steps: Dolutegravir sodium sample was prepared into a test solution, and isomer impurities in dolutegravir sodium were separated and detected by high performance liquid chromatography. The isomer impurities include at least one of impurity ADC201-020, impurity ADC201-021 and impurity ADC201-022. The detection conditions for the high-performance liquid chromatography include: Column: cellulose-tris(4-chloro-3-methylphenylcarbamate) silica gel as the packing material; Mobile phase A: Phosphoric acid solution with a phosphoric acid concentration of 0.08-0.12 wt.%; Mobile phase B: Acetonitrile; Isocratic elution was performed with a mobile phase A to mobile phase B volume ratio of 1:16-24. Sample tray temperature: 2-8℃; Detection wavelength: 250-265nm; The structural formula of the impurity ADC201-020 is as follows: , The structural formula of the impurity ADC201-021 is as follows: , The structural formula of the impurity ADC201-022 is as follows: .

2. The method according to claim 1, characterized in that, The conditions for the high performance liquid chromatography also include: a total flow rate of 0.9-1.1 ml / min, preferably 1 ml / min; and a column temperature of 28-35℃, preferably 28-32℃.

3. The method according to claim 1, characterized in that, The solvent for the test sample solution is an acetonitrile-water mixture with a volume ratio of 1:1.4-1.

6.

4. The method according to claim 1, characterized in that, The preparation process of the test solution includes: weighing the dolutegravir sodium sample and placing it in a brown volumetric flask, adding solvent to dissolve and dilute it; Preferably, the content of dolutegravir sodium sample in the test solution is 0.4-0.6 mg / ml.

5. The method according to claim 1, characterized in that, The chromatographic column used was a DAICL CHIRALCEL OX-3R, 4.6 mm × 150 mm, 3 μm.

6. The method according to claim 1, characterized in that, The mobile phase A is a phosphoric acid solution with a phosphoric acid concentration of 0.1 wt.%, and the mobile phase B is acetonitrile.

7. The method according to claim 1, characterized in that, The mobile phase A and mobile phase B are eluted isocratically at a volume ratio of 5:

95.

8. The method according to claim 1, characterized in that, The temperature of the sample pan is 4-6℃.

9. The method according to claim 1, characterized in that, The detection conditions for the high performance liquid chromatography also include an injection volume of 18-22 μl.

10. The method according to any one of claims 1-9, characterized in that, The method includes the following steps: (1) Prepare a test solution from a dolutegravir sodium sample and prepare a reference solution from impurities ADC201-020, ADC201-021 and ADC201-022. The test solution and the reference solution use the same solvent. (2) Detect each solution in step (1) according to the detection conditions of the high performance liquid chromatography, and calculate the content of each isomer impurity by external standard method.

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