A method for detecting impurities in lebopride starting material LBS5

Abstract

The application provides a method for detecting impurities in leboregen starting material LBS5, and belongs to the field of pharmaceutical analysis chemistry. S ,5 R )‑1‑(3‑fluorophenyl)‑3‑oxabicyclo[3.1.0]hexan‑2‑one. The method adopts high performance liquid chromatography, can simultaneously and accurately quantify one degradation impurity and four process impurities in LBS5, and has strong specificity, high sensitivity, good accuracy, good reproducibility and stable and reliable method. The method can realize the control of the impurities, ensures the safety of LBS5 as a pharmaceutical intermediate, further ensures the safety of a preparation of LBS5, reduces the risk of drug use, simultaneously reduces the difficulty of subsequent synthesis steps, realizes rapid, economic and environmental protection production. The method is suitable for laboratory research and development, and is also suitable for quality control in commercial production.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analytical chemistry, and specifically relates to a method for detecting impurities in LBS5, a starting material for Leborresen. Background Technology

[0002] Leboresen is a dual orexin receptor antagonist that inhibits the wakefulness-inducing effect of orexin by blocking its binding to orexin receptors, thereby inducing physiological sleep. In December 2019, leboresen received FDA approval for the treatment of insomnia characterized by difficulty initiating and / or maintaining sleep in adults. On May 27, 2025, leboresen received marketing approval from the China National Medical Products Administration.

[0003] Leborrheic starting material LBS5 (compound name is (1) S 5 R )-1-(3-fluorophenyl)-3-oxabicyclo[3.1.0]hexane-2-one, with the molecular formula C 11 H9FO2 (molecular weight 192.19) is used as a starting material for the synthesis of leberresen. With increasingly stringent regulatory requirements, strict control of related substances in the leberresen starting material LBS5 is necessary. The literature (Research Progress in the Synthesis of Leberresen, DOI: 10.16522 / j.cnki.cjph.2024.05.003) discloses that LBS1 is a raw material for the synthesis of leberresen starting material LBS5, and LBS1 is highly likely to remain as a residue in the leberresen starting material LBS5.

[0004] Based on the Quality by Design (QBD) principle, effective detection methods and strict control are required for the starting materials of Lebrene. High levels of impurities in the LBS5 of Lebrene starting materials significantly increase the difficulty of impurity removal in subsequent processes, thus affecting the robustness of the production process and adversely impacting the quality of the final product. Therefore, effective monitoring of potential process impurities, intermediate residues, and degradation products in LBS5 is a crucial prerequisite for ensuring the safety of Lebrene. Developing a detection method for impurities in the LBS5 of Lebrene starting materials is of great significance for strictly controlling its quality, ensuring the smooth operation of subsequent production processes, and guaranteeing the safety of the final product.

[0005] Regarding the detection and analysis of impurities related to lemborexant, some existing techniques have been reported. The authoritative literature (OPTIMIZATION OF ROBUST HPLC APPROACH FOR ASSESSING PROCESS RELATEDIMPURITIES OF LEMBOREXANT AND ELUCIDATION OF STRESS INDUCED DEGRADATIONPRODUCTS THROUGH LCMS / MS) discloses an HPLC method for the detection of three process-related impurities (including LBS5) and their forced degradation products in lemborexant active pharmaceutical ingredient (API). This method uses a Kinetex C18 column (150 × 4.6 mm, 5 μm), with isocratic elution using a mobile phase of 10 mM ammonium formate buffer (pH 4.2)-acetonitrile-methanol (65:25:10, v / v / v), a flow rate of 0.7 mL / min, a detection wavelength of 265 nm, a column temperature of 35 °C, and an injection volume of 5 μL. Patent document CN118150745A discloses a method for simultaneously determining five genotoxic impurities, including benzenesulfonates, aldehydes, and nitrobenzenes, in leboresen active pharmaceutical ingredient. The method employs a C18 (3.0 × 150 mm, 3.5 μm) column, using a gradient elution with potassium dihydrogen phosphate aqueous solution as mobile phase A and acetonitrile phosphate solution as mobile phase B. The flow rate is 0.4–0.6 mL / min, the column temperature is 33–43℃, the elution time is 40 min, and the detection wavelength is 220 nm.

[0006] However, the aforementioned existing technologies all target the detection of impurities in the leboresen active pharmaceutical ingredient (API), and their targets are not the related substances in the starting material LBS5. Currently, no relevant patents or published literature have been found to report methods for detecting related substances in the leboresen starting material LBS5 itself. Summary of the Invention

[0007] The purpose of this invention is to provide a method for detecting impurities in LBS5, a starting material of Leborrheic dermatology.

[0008] Based on the synthesis route of the starting material LBS5 from Lebor-Raysen, this invention analyzes the possible impurities (LBS1, LBS5-Z4, LBS5-Z2, LBM1 and LBM7) that may be generated during the synthesis process. The structures and related information are shown in Table 1.

[0009] The literature (Research Progress in the Synthesis of Leborexin, DOI: 10.16522 / j.cnki.cjph.2024.05.003) discloses that LBS1 is a raw material for the synthesis of leborexin starting material LBS5. LBS1 is highly likely to remain as a residue in the leborexin starting material LBS5. Furthermore, the PubChem database explicitly mentions known safety risks of LBS1, including oral, dermal, ocular, and specific target organ polarity toxicity (PubChem Compound Database, CID 68145, Benzeneacetonitrile, 3-fluoro- (CAS No. 501-00-8), National Center for Biotechnology Information (NCBI), National Library of Medicine, https: / / pubchem.ncbi.nlm.nih.gov / compound / 68145, accessed on June 16, 2026.). Therefore, accurate quantitative detection of the LBS1 content in the leborexin starting material LBS5 is of great significance. Furthermore, the verification of this invention revealed that the two batches of commercially available Lebore acetonitrile starting material LBS5 tested in the specific embodiments contained residual impurity LBM1 and degradation impurity LBM7. Acid and alkaline conditions further significantly increased the content of degradation impurity LBM7 in Lebore acetonitrile starting material LBS5. High temperature and light conditions caused the formation of LBS5-Z4 impurity in Lebore acetonitrile starting material LBS5. LBS5-Z4 has been clearly mentioned in the PubChem database as having known safety risks such as oral, skin, eye, and specific target organ polar toxicity (PubChemCompound Database, CID 68145, Benzeneacetonitrile, 3-fluoro- (CAS No. 501-00-8), National Center for Biotechnology Information (NCBI), National Library of Medicine, https: / / pubchem.ncbi.nlm.nih.gov / compound / 68145, accessed on June 16, 2026.). Therefore, accurate quantitative detection of the contents of LBM1, LBM7, and LBS5-Z4 in the starting material LBS5 of Leborrheen is also of great significance.

[0010] Table 1. Impurity Structure and Related Information of Lebor-Raysen Starting Materials Note: "NA" indicates not applicable, and there are no impurity limit requirements for the main component.

[0011] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for detecting impurities in Lebor-Raysen starting material LBS5, wherein the impurities include at least one of LBS1, LBS5-Z4, LBM1, and LBM7; the structural formulas of LBS5, LBS1, LBS5-Z4, LBM1, and LBM7 are as follows: The method includes the following steps: (1) Preparation of test solution: Dissolve the LBS5 starting material to be tested in a solvent to obtain the test solution; (2) Prepare a Leborresen starting material LBS5 reference solution; Dissolve the Leborresen starting material LBS5 reference in a solvent to obtain the Leborresen starting material LBS5 reference solution; (3) Preparation of system suitability solution: Dissolve the LBS5 reference standard and impurity reference standard of Leborrheic starter in solvent to obtain system suitability solution; (4) The content of impurities in the starting material LBS5 of the Lebor-Raysen was calculated by liquid chromatography. The conditions for the liquid chromatography detection include: The chromatographic column was packed with octadecylsilane-bonded silica gel. The mobile phase consists of mobile phase A and mobile phase B. Mobile phase A is a 0.09% to 0.11% aqueous solution of phosphoric acid, one of methanol and acetonitrile, or a mixture of methanol and acetonitrile. In the mixed solution, the ratio of methanol to acetonitrile is 20 to 80:100.

[0012] In addition to residual impurities LBS1 in raw materials, LBM1 in intermediates, LBM7 (a degradation impurity), and LBS5-Z4 (a process-related impurity that also degrades under high temperature and light-induced degradation conditions), LBS5-Z2 (a process-related impurity) may also remain in the LBS5 of Lebor-Raysen starting materials. Therefore, accurate quantitative detection of the LBS5-Z2 content in LBS5 of Lebor-Raysen starting materials is also of great significance.

[0013] To achieve the above-mentioned objectives, the present invention further provides the following technical solutions: Furthermore, the impurity also includes LBS5-Z2; the structural formula of LBS5-Z2 is as follows: .

[0014] Furthermore, the conditions for the liquid chromatography detection include: The chromatographic column used was an Inertsil ODS-3 (250 mm × 4.6 mm 5 μm). Mobile phase B is a mixed solution of methanol and acetonitrile, in which the ratio of methanol to acetonitrile is 40:60.

[0015] The detector is an ultraviolet detector; The detection wavelengths are 220 nm and 266 nm; The column temperature is 35~45℃; The flow rate is 0.9~1.1 mL / min; The injection volume was 5 μL.

[0016] Furthermore, the liquid chromatography detection employs gradient elution, and the gradient elution program is as follows: 0–30 min, mobile phase A decreases from 70% to 10%, and mobile phase B increases from 30% to 90%; 30–31 min, mobile phase A increases from 10% to 70%, and mobile phase B decreases from 30% to 90%; 31–40 min, mobile phase A is maintained at 70%, and mobile phase B is maintained at 30%. Furthermore, the concentration of the phosphoric acid aqueous solution is 0.10%.

[0017] Furthermore, the solvent is an organic solvent, which is selected from acetonitrile, methanol, or a mixture of methanol and acetonitrile.

[0018] Furthermore, in the methanol-acetonitrile mixed solution, the ratio of methanol to acetonitrile is 20~80:100.

[0019] Furthermore, the solvent is acetonitrile.

[0020] Furthermore, the concentration of the LBS5 reference solution, the starting material for Leborresen, is 2~20 μg / mL; The concentration of the test solution is 0.2~2 mg / mL; In the system suitability solution, the concentration of the impurity reference standard is 2~20 μg / mL, and the concentration of the Leborresen starting material LBS5 reference standard is 0.2~2 mg / mL.

[0021] Furthermore, the concentration of the LBS5 reference solution, the starting material for Leborresen, is 10 μg / mL; The concentration of the test solution is 1 mg / mL; In the system suitability solution, the concentration of the impurity reference standard is 10 μg / mL, and the concentration of the Leborresen starting material LBS5 reference standard is 1 mg / mL.

[0022] Furthermore, the detection wavelengths of the LBS5 are 220nm and 266nm; The detection wavelength of the LBM7 is 220nm; The detection wavelength of the LBS1 is 266nm; The detection wavelength of the LBS5-Z4 is 266nm; The detection wavelength of the LBS5-Z2 is 220nm; The detection wavelength of the LBM1 is 220 nm.

[0023] Furthermore, the method for calculating the impurity content in the LBS5 starting material of the Lebor-Raysen assay is the external standard method of the main component, and the calculation method is as follows: Impurity content (%) = × ×V× f / / M / 1000×100% in, f Indicates the impurity correction factor; Indicates the peak area of ​​impurities in the test solution; This indicates the peak area of ​​LBS5, the starting material of Leborresen, in the reference solution of LBS5; V represents the concentration of LBS5, the starting material for Leborresen, in the LBS5 reference solution, in μg / mL; V represents the dilution volume of the test solution, in mL; and M represents the sample weight, in mg.

[0024] Furthermore, the correction factor for the LBM7 is 1.62; The correction factor for LBS1 is 0.97; The correction factor for LBS5-Z4 is 1.07; The correction factor for LBS5-Z2 is 1.0; The correction factor for LBM1 is 2.37.

[0025] The present invention has achieved the following beneficial effects: This invention reveals that existing methods for detecting impurities in leboresen active pharmaceutical ingredient (LPI) LBS5 (e.g., CN118150745A and literature (doi.org / 10.32737 / 0005-2531-2024-4-27-40)) suffer from poor separation and ineffective separation of some impurities. This invention, through the selection of chromatographic conditions, provides a liquid chromatography method capable of simultaneously and accurately quantifying five impurities (LBS1, LBS5-Z4, LBS5-Z2, LBM1, and LBM7) in LIBresen LBS5. This method exhibits high specificity, high sensitivity, good accuracy, good reproducibility, and stability. The detection method of this invention can accurately detect the content of the aforementioned five impurities in LIBresen LBS5, allowing for control of these impurities and ensuring the safety of LIBresen LBS5 as a drug starting material, thereby further guaranteeing the safety of its formulation and reducing medication risks. This method is applicable to both laboratory research and development and quality control in commercial production.

[0026] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0027] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0028] Figure 1 This is a chromatogram of the blank solution in Example 1 of the present invention.

[0029] Figure 2 This is a chromatogram of the system suitability solution ① of Example 1 of the present invention.

[0030] Figure 3 This is a chromatogram of the system suitability solution ② of Example 1 of the present invention.

[0031] Figure 4 This is a chromatogram of test solution 1 in Example 1 of the present invention.

[0032] Figure 5 This is the chromatogram of test solution 2 in Example 1 of the present invention.

[0033] Figure 6 This is a superimposed chromatogram of the forced degradation test in Example 1 of the present invention.

[0034] Figure 7The chromatograms are of the system suitability solution and the LBS1 positioning solution of Comparative Example 1 of this invention.

[0035] Figure 8 The chromatograms are of the system suitability solution and the LBM7 positioning solution in Comparative Example 2 of this invention. Detailed Implementation

[0036] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0037] This invention discloses a liquid phase detection method for multiple impurities in Lebor-Raysen starting material LBS5. Those skilled in the art can, based on the content of this document, appropriately modify the parameters to implement this method. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of this invention. The method and application of this invention have been described through embodiments. Those skilled in the art can make modifications or appropriate alterations and combinations to the method and application without departing from the content, spirit, and scope of this invention to achieve and apply the technology of this invention.

[0038] In this invention, "room temperature" means 25±10 ℃.

[0039] The reagents or instruments used in the liquid phase detection method for multiple impurities in the Lebor-Raysen starting material LBS5 provided by this invention are all commercially available.

[0040] The leboretin starting material LBS5 used in this invention was purchased from Nanjing Weinuode Pharmaceutical Technology Co., Ltd., batch number: 25W0503-a-250823, purity 99.9%, batch number: 25W0503-LBLS-IM3-251026, purity 99.7%; the impurity reference standards LBS1, LBS5-Z2, LBS5-Z4, LBM1, and LBM7 were purchased from PLC Company (purity 99.96%), Sichuan Aobang Gude Pharmaceutical Co., Ltd. (purity 99.59%), PLC Company (purity 90.05%), Shenzhen Bolte Company (purity 95.88%), and Sichuan Aobang Gude Pharmaceutical Co., Ltd. (purity 94.00%), respectively.

[0041] Example 1: Liquid phase detection method for multiple impurities in Lebor-Raysen starting material LBS5 (1) Instrument: Shimadzu 2030 liquid chromatograph.

[0042] (2) Chromatographic conditions: 0.1% phosphoric acid water was used as mobile phase A, and methanol-acetonitrile (40:60) was used as mobile phase B. Gradient elution was performed according to the table below.

[0043] Column: Inertsil ODS-3 250×4.6mm, 5μm.

[0044] Detector: Ultraviolet detector, detection wavelengths 220 nm and 266 nm.

[0045] Column temperature: 40 ℃.

[0046] Flow rate: 1.0 mL / min.

[0047] Injection volume: 5 μL.

[0048] (3) Preparation of blank solution: Acetonitrile was used as blank solution (referring to "solvent" in the following examples).

[0049] (4) Preparation of reference solution: Weigh 10 mg of LBS5 reference standard accurately, place it in a 10 mL volumetric flask, add solvent to dissolve and dilute to the mark, and shake well; accurately measure 1 mL, place it in a 100 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0050] (5) Preparation of test solution 1: Take 10 mg of LBS5 test sample (batch number: 25W0503-a-250823), accurately weigh it, put it in a 10 mL volumetric flask, add solvent to dissolve and dilute to the mark, shake well to obtain a test solution with a concentration of 1 mg / mL.

[0051] (6) Preparation of test solution 2: Take 10 mg of LBS5 test sample (batch number: 25W0503-LBLS-IM3-251026), accurately weigh it, put it in a 10 mL volumetric flask, add solvent to dissolve and dilute to the mark, shake well to obtain a test solution with a concentration of 1 mg / mL.

[0052] (7) Preparation of system suitability solution: ① Take the impurity reference standards (LBS1, LBS5-Z4, LBM1 and LBM7) and the main component LBS5 reference standard, respectively, accurately weigh them, dissolve them in solvent and quantitatively dilute them to prepare a mixed solution containing 10 μg of each impurity and 1 mg of LBS5 per 1 mL, as the system suitability solution ①.

[0053] ② Take the impurity LBS5-Z2 reference standard and the main component LBS5 reference standard, accurately weigh them, dissolve them in solvent and quantitatively dilute them to prepare a mixed solution containing 10 μg of each impurity and 1 mg of LBS5 per 1 mL, as the system suitability solution ②.

[0054] (8) Impurity determination: Inject the blank solution, reference solution, test solution, and system suitability solution into the liquid chromatograph using direct injection, record the chromatogram, and calculate the content based on peak area using the external standard method for principal components. The calculation formula is: Impurity content (%) = × ×V× f / / M / 1000×100% In the formula: Peak area of ​​impurities in the test solution; : Peak area of ​​LBS5 in the reference solution; : The concentration of LBS5 in the reference solution, in μg / mL; V: Dilution volume of the test solution, mL; M: Sample weight, mg; Among them, LBM7 f The value is 1.62, for LBS1. f The value is 0.97, for LBS5-Z4. f The value is 1.07, for LBS5-Z2. f For 1.0, LBM1 f It is 2.37.

[0055] (8) Experimental results: Experimental results ( Figures 1-3 The results showed that the blank solution had no interference, and the peaks of each component in the system suitability solution were well separated. The content was calculated by peak area using the external standard method for the principal component. The results showed that the test solution of commercially available Leborrheic starting material LBS5 (…) Figure 4 , Figure 5 In the test sample solution 1, LBM7 was detected with a content of 0.018%, and LBM1 was detected with a content of 0.033%. The test results meet the following standards: LBM7, LBS1, LBS5-Z2, and LBM1 should not exceed 1.0%, LBS5-Z4 should not exceed 0.1%, unknown impurities should not exceed 1.0%, and total impurities should not exceed 2.0%. This indicates that the impurity control of the two batches of test samples meets the requirements.

[0056] The following comparative method was used to detect various impurities in the Lebor-Raysen starting material LBS5.

[0057] Comparative Example 1: Detection Method for Multiple Impurities in Lebor-Raysen Starting Material LBS5 The chromatographic conditions described in the patent document "A method for simultaneous determination of multiple genotoxic impurities in leboresen active pharmaceutical ingredient" (application publication number: CN118150745A) were used to detect multiple impurities in the leboresen starting material LBS5 of this invention.

[0058] (1) Instrument: Agilent 1260Ⅱ liquid chromatograph.

[0059] (2) Chromatographic conditions: A mobile phase A was 0.01 mol / L potassium dihydrogen phosphate aqueous solution, and B was 0.1% acetonitrile phosphate solution. Gradient elution was performed according to the table below.

[0060] Column: ZORBAX SB-AQ 150 4.6mm, 3.5μm.

[0061] Detector: Ultraviolet detector, detection wavelength 220 nm.

[0062] Column temperature: 38℃.

[0063] Flow rate: 0.5 mL / min.

[0064] Injection volume: 5 μL.

[0065] (3) Preparation of blank solution: Acetonitrile-water (50:50) was used as blank solution (referring to the "solvent" in this comparative example).

[0066] (4) Preparation of test solution: Take 10 mg of LBS5 test sample (batch number: 25W0503-a-250823), accurately weigh it, put it in a 10 mL volumetric flask, add solvent to dissolve and dilute to the mark, shake well to obtain a test solution with a concentration of 1 mg / mL.

[0067] (5) Preparation of system suitability solution: Take the impurity reference standards (LBS1, LBS5-Z2, LBS5-Z4, LBM1 and LBM7) and the main component LBS5, respectively, accurately weigh them, dissolve them in solvent and quantitatively dilute them to prepare a mixed solution containing 10 μg of each impurity and 1 mg of LBS5 per 1 mL, which is used as the system suitability solution.

[0068] (6) Impurity localization solution: Weigh approximately 5 mg of each of the impurity reference standards LBS1, LBS5-Z2, LBS5-Z4, LBM1, and LBM7 accurately, dissolve them in solvent, and dilute quantitatively to prepare a solution containing 10 μg of impurity per mL. This solution is used as the impurity localization solution. (7) Impurity determination: Inject 5 μL each of the above blank solution, localization solution, test solution, and system suitability solution into the liquid chromatograph and record the chromatogram.

[0069] (8) Results: Experimental results ( Figure 7 The results show that, under the chromatographic conditions of the patented method (CN118150745A), the LBS1 peak overlaps with the main peak, and the resolution does not meet the requirements.

[0070] Comparative Example 2: Detection Method for Multiple Impurities in Lebor-Raysen Starting Material LBS5 The optimized and robust HPLC method described in references D. Amrutha Varshini1, M. Radha Madhavi2, etc., was used to evaluate process-related impurities of Lemborexant and Elucidation of Stres Induced Degradation Products through LC-MS / MS to elucidate the chromatographic conditions in the forced degradation products (OPTIMIZATION OF ROBUST HPLC APPROACH FOR ASSESSING PROCESS RELATED IMPURITIES OF LEMBOREXANT AND ELUCIDATION OF STRESS INDUCED DEGRADATION PRODUCTS THROUGH LCMS / MS) for the detection of various impurities in the Lemborexant starting material LBS5 of this invention.

[0071] (1) Instrument: Agilent 1260Ⅱ liquid chromatograph.

[0072] (2) Chromatographic conditions: Mobile phase: 0.01 mol / L ammonium formate (pH 4.2) - acetonitrile - methanol (65:25:10).

[0073] Column: ZORBAX SB-AQ 150 4.6mm, 3.5μm.

[0074] Detector: Ultraviolet detector, detection wavelength 265 nm.

[0075] Column temperature: 35℃.

[0076] Flow rate: 0.7 mL / min.

[0077] Injection volume: 10 μL.

[0078] (3) Preparation of blank solution: Methanol was used as blank solution (referring to the "solvent" in this comparative example).

[0079] (4) Preparation of test solution: Take 10 mg of LBS5 test sample (batch number: 25W0503-a-250823), accurately weigh it, put it in a 10 mL volumetric flask, add solvent to dissolve and dilute to the mark, shake well to obtain a test solution with a concentration of 1 mg / mL.

[0080] (5) Preparation of system suitability solution: Take the impurity reference standards (LBS1, LBS5-Z2, LBS5-Z4, LBM1 and LBM7) and the main component LBS5, respectively, accurately weigh them, dissolve them in solvent and quantitatively dilute them to prepare a mixed solution containing 10 μg of each impurity and 1 mg of LBS5 per 1 mL, which is used as the system suitability solution.

[0081] (6) Impurity localization solution: Take about 5 mg of impurity reference standards LBS1, LBS5-Z2, LBS5-Z4, LBM1 and LBM7 respectively, accurately weigh them, dissolve them in solvent and dilute them quantitatively to prepare a solution containing 10 μg of impurity per 1 mL, which is used as the impurity localization solution.

[0082] (7) Impurity determination: Inject 10 μL each of the above blank solution, positioning solution, test solution and system suitability solution into the liquid chromatograph and record the chromatogram.

[0083] (8) Results: Experimental results ( Figure 8 This indicates that, under the chromatographic conditions described in the reference method, the LBS5-Z4 peak overlapped with the LBM7 peak, and the LBS1 peak was not baseline separated from the main peak, resulting in a resolution that did not meet the requirements.

[0084] The following experimental examples demonstrate the methodological verification of this invention.

[0085] Experimental Example 1: Methodological Validation The "solvent" mentioned later in this experimental example is acetonitrile; the "diluent" mentioned later in this experimental example is acetonitrile.

[0086] (1) Specificity and forced degradation test Under the chromatographic conditions of Example 1, system suitability solution, blank solution, and test sample (batch number: 25W0503-a-250823) solution were prepared according to the method of Example 1. 10 μL of each of the system suitability solution, blank solution, and test sample solution were injected into the liquid chromatograph for detection. The liquid chromatogram is shown below. Figures 1-4 As shown, the blank solution does not interfere with the determination of each impurity, and the separation degree between each component in the system suitability solution is not less than 1.5, indicating good separation and meeting the requirements.

[0087] Preparation of high temperature forced degradation test solution: Take 10.588 mg of LBS5 test sample (batch number: 25W0503-a-250823), place it under high temperature 608 for about 2 months, take it out, and prepare the high temperature forced degradation test solution according to the test sample solution preparation method in Example 1 (4).

[0088] Preparation of forced degradation test solution under strong light: Take about 10 mg of LBS5 test sample (batch number: 25W0503-a-250823) and place it under high temperature of 60°C and strong light irradiation conditions for about 2 months respectively. Take it out and prepare the forced degradation test solution under strong light according to the test sample solution preparation method in Example 1 (4).

[0089] Acid degradation of the test solution: Accurately weigh 10.623 mg of this product, place it in a 10 ml volumetric flask, add 2 mL of solvent to dissolve it, then add 1 ml of 0.5 M hydrochloric acid solution, place at 80 °C for 30 min, cool immediately, add 1 mL of 0.5 M sodium hydroxide solution, mix well, dilute to the mark with solvent, and shake well.

[0090] Alkali-induced degradation of the test solution: Accurately weigh 10.360 mg of this product and place it in a 10 mL volumetric flask. Add 2 mL of solvent to dissolve the product, then add 1 mL of 1 M sodium hydroxide solution. Let it stand at room temperature for 4 h, then add 1 mL of 1 M hydrochloric acid solution, mix well, dilute to the mark with solvent, and shake well.

[0091] Oxidative destruction of the test solution: Accurately weigh 10.591 mg of this product, place it in a 10 mL volumetric flask, add 1 mL of solvent to dissolve it, then add 1 mL of 3% hydrogen peroxide, let it stand at room temperature for 47 h, dilute to the mark with solvent, and shake well.

[0092] Take 10 μL of each of the above forced degradation test solutions and inject them into the liquid chromatograph for detection. The liquid chromatogram is shown below. Figure 6 As shown in Table 2, the results of the forced degradation test are detailed. It can be seen that high temperature and light degradation produce 0.02%-0.4% LBS5-Z4 impurities, while acid and alkali degradation increase the LBM7 impurity from 0.018% to 3.4%-14%. The separation degree between each component is not less than 1.5, which is good and meets the requirements.

[0093] The above results indicate that: (1) High temperature and light conditions will cause LBS5-Z4 impurities to be generated in the leboresen starting material LBS5. LBS5-Z4 impurities have been clearly mentioned in the PubChem database as having known safety risks such as oral, skin, eye, and specific target organ polar toxicity. Therefore, accurate quantitative detection of LBS5-Z4 content in leboresen starting material LBS5 is of great significance; (2) Commercially available leboresen starting material LBS5 contains degradation impurity LBM7. Acid and alkaline conditions will further significantly increase the content of degradation impurity LBM7 in leboresen starting material LBS5. Therefore, accurate quantitative detection of LBM7 content in leboresen starting material LBS5 is also of great significance.

[0094] Table 2 Results of Forced Degradation Test (2) Limit of quantitation and limit of detection Stock solution of impurity LBS5-Z4 reference standard: Accurately weigh 1.010 mg of impurity LBS5-Z4 reference standard, place it in a 10 mL volumetric flask, add diluent to dissolve and dilute to the mark, and shake well.

[0095] Impurity LBS5-Z2 reference standard stock solution: Accurately weigh 5.307 mg of LBS5-Z2 reference standard, place it in a 10 mL volumetric flask, add diluent to dissolve and dilute to the mark, and shake well.

[0096] Stock solution of impurity LBS1 reference standard: Accurately weigh 1.218 mg of impurity LBS1 reference standard, place it in a 10 mL volumetric flask, add diluent to dissolve and dilute to the mark, and shake well.

[0097] Impurity LBM1 reference standard stock solution: Accurately weigh 1.114 mg of impurity LBM1 reference standard, place it in a 10 mL volumetric flask, add diluent to dissolve and dilute to the mark, and shake well.

[0098] Impurity LBM7 reference standard stock solution: Accurately weigh 1.041 mg of impurity LBM7 reference standard, place it in a 10 mL volumetric flask, add diluent to dissolve and dilute to the mark, and shake well.

[0099] LBS5 reference standard stock solution: Accurately weigh 10.372 mg of LBS5 reference standard, place it in a 10 mL volumetric flask, add diluent to dissolve and dilute to the mark, and shake well.

[0100] Each reference standard stock solution was serially diluted and analyzed under the chromatographic conditions of Example 1. Chromatograms were recorded. The limit of quantitation was set at a signal-to-noise ratio of approximately 10, and the limit of detection was set at a signal-to-noise ratio of approximately 3. The results of the limits of detection and quantitation for each component are shown in Table 3.

[0101] Table 3 Results of Limit of Quantitation and Limit of Detection Experimental results show that the ratio of the quantitation limit solution concentration to the limit concentration for each analyte is no greater than 5.0%, and the signal-to-noise ratio is no less than 10; the ratio of the detection limit solution concentration to the limit concentration for each analyte is no greater than 2.5%, and the signal-to-noise ratio is no less than 3. The sensitivity meets the requirements.

[0102] (3) Linearity and Range Take appropriate amounts of LBS5-Z4, LBS1, LBM1, LBM7 and LBS5 reference stock solutions from the sensitivity test, dilute with solvent to prepare a series of linear solutions, inject and analyze according to the chromatographic conditions of Example 1, record the chromatograms, and perform linear regression with the injection concentration (μg / mL) as the abscissa (x) and the peak area as the ordinate (y). The results are shown in Table 4.

[0103] Table 4. Results of Linear Relationships The results showed that the main component and each impurity exhibited good linearity within the linear range of the limit of quantitation to 200% limit.

[0104] (4) Repeatability Reference solution: Accurately weigh the LBS5 reference standard from Leborresen, dissolve it in solvent and dilute it quantitatively to prepare a solution containing approximately 10 μg per mL.

[0105] Test solution: Take LBS5 test sample (batch number: 25W0503-a-250823), accurately weigh it, dissolve it in solvent and dilute it quantitatively to prepare a solution containing about 1 mg per 1 mL. Prepare 6 parallel solutions.

[0106] Take the above solution and analyze it according to the chromatographic conditions of Example 1. Record the chromatogram and calculate the content of each impurity by peak area according to the external standard method of principal component. The results are shown in Table 5.

[0107] Table 5 Repeatability Results Experimental results show that when six test solutions were measured in parallel, the content of each impurity was calculated using the principal component external standard method with correction factor. The detection amount of each impurity met the standard described in Example 1, and the repeatability requirements were met.

[0108] (5) Accuracy Test solution (baseline value): Referenced "repeatability" data, LBS5 test sample (lot number: 25W0503-a-250823).

[0109] Three 20%, 100%, and 150% spiked recovery solutions were prepared from the stock solutions of each reference standard under the sensitivity test. The solutions were injected and analyzed under the chromatographic conditions of Example 1. The chromatograms were recorded, and the recovery rate of the analyte in each recovery solution was calculated using the external standard method. The results are shown in Table 6.

[0110] Table 6 Accuracy Results Experimental results show that, within the concentration range of 20% to 150% of the limit, the recovery rates of each impurity are between 91.5% and 104.0%, and the RSDs are all no greater than 1.7%, meeting the accuracy requirements.

[0111] (6) Solution stability Take the reference solution and the test solution (batch number: 25W0503-a-250823) and place them at room temperature. Inject and analyze them at different time periods, record the chromatograms, and calculate the relative standard deviation of the peak area or content of each impurity in each solution. The test results are shown in Table 7.

[0112] Table 7 Solution stability results The experimental results showed that the reference solution and the test solution were stable at room temperature for 73 hours, while the reference solutions for each impurity were stable for 142 hours.

[0113] (7) Durability Diluent, system suitability solution, reference solution and test solution: Same as under “Repeatability”, LBS5 test sample (batch number: 25W0503-a-250823).

[0114] Adjust the chromatographic method parameters according to Table 8, inject the above solutions for analysis, record the chromatograms, calculate the content of each impurity according to the external standard method of principal component, and the results are shown in Table 9.

[0115] Table 8. Robustness Chromatographic Parameters Table 9 Summary of Durability Results Experimental results show that after minor changes to various chromatographic parameters (column temperature ±5 ℃, flow rate ±0.1 mL / min, mobile phase A concentration ±10%, column and instrument), the solvent blank does not interfere with the detection of each analyte. The system suitability is good, with the resolution between impurity peaks in the solution not less than 3.0, the resolution between the main component peak and adjacent impurities not less than 2.4, the detection amount of each analyte in the test solution meets the requirements, and the range is not greater than 0.013%. The method robustness meets the requirements.

[0116] In summary, the present invention provides a method for determining impurities in LBS5, a starting material of Lebor-Raysen, which is highly specific, sensitive, linear, repeatable, accurate, has good solution stability and robustness.

[0117] In summary, this invention provides a method for detecting impurities in Lebor-Raysen starting material LBS5. This method can simultaneously and accurately quantify one degradation impurity and four process impurities in Lebor-Raysen starting material LBS5, and exhibits strong specificity, high sensitivity, good accuracy, good reproducibility, and stable reliability. This method is suitable for laboratory research and development, as well as for quality control in commercial production.

[0118] Obviously, the above description is merely a preferred embodiment of the present invention, and is only an example to clearly illustrate the invention, and is not intended to limit the embodiments of the present invention. It should be noted that those skilled in the art can make various other improvements and modifications without departing from the principles of the present invention, and it is neither necessary nor possible to exhaustively list all embodiments here. Any modifications, equivalent improvements, and substitutions made within the spirit and principles of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. A method for detecting impurities in Lebor-Raysen starting material LBS5, characterized in that: The impurities include at least one of LBS1, LBS5-Z4, LBM1, and LBM7; the structural formulas of LBS5, LBS1, LBS5-Z4, LBM1, and LBM7 are as follows: The method includes the following steps: (1) Preparation of test solution: Dissolve the LBS5 starting material to be tested in a solvent to obtain the test solution; (2) Prepare a Leborresen starting material LBS5 reference solution; Dissolve the Leborresen starting material LBS5 reference in a solvent to obtain the Leborresen starting material LBS5 reference solution; (3) Preparation of system suitability solution: Dissolve the LBS5 reference standard and impurity reference standard of Leborrheic starter in solvent to obtain system suitability solution; (4) The content of impurities in the starting material LBS5 of the Lebor-Raysen was calculated by liquid chromatography. The conditions for the liquid chromatography detection include: The chromatographic column was packed with octadecylsilane-bonded silica gel. The mobile phase consists of mobile phase A and mobile phase B. Mobile phase A is a 0.09% to 0.11% aqueous solution of phosphoric acid, and mobile phase B is a solution of methanol, acetonitrile, or a mixture of methanol and acetonitrile. In the mixed solution, the ratio of methanol to acetonitrile is 20 to 80:

100.

2. The method according to claim 1, characterized in that: The impurities also include LBS5-Z2; the structural formula of LBS5-Z2 is as follows: 。 3. The method according to claim 1 or 2, characterized in that: The conditions for the liquid chromatography detection include: The chromatographic column used was Inertsil ODS-3; Mobile phase B is a mixed solution of methanol and acetonitrile, in which the ratio of methanol to acetonitrile is 40:60; The detector is an ultraviolet detector; The detection wavelengths are 220 nm and 266 nm.

4. The method according to claim 1 or 2, characterized in that: The concentration of the phosphoric acid aqueous solution is 0.10%.

5. The method according to claim 1 or 2, characterized in that: The solvent is an organic solvent, selected from acetonitrile, methanol, or a mixture of methanol and acetonitrile, wherein the ratio of methanol to acetonitrile in the mixture is 20-80:

100.

6. The method according to claim 5, characterized in that: The solvent is acetonitrile.

7. The method according to claim 1 or 2, characterized in that: The concentration of the LBS5 reference solution, the starting material for Leborresen, is 2-20 μg / mL; The concentration of the test solution is 0.2~2 mg / mL; In the system suitability solution, the concentration of the impurity reference standard is 2~20 μg / mL, and the concentration of the Leborresen starting material LBS5 reference standard is 0.2~2 mg / mL.

8. The method according to claim 2, characterized in that: The detection wavelengths of the LBS5 are 220nm and 266nm; The detection wavelength of the LBM7 is 220nm; The detection wavelength of the LBS1 is 266nm; The detection wavelength of the LBS5-Z4 is 266nm; The detection wavelength of the LBS5-Z2 is 220nm; The detection wavelength of the LBM1 is 220 nm.

9. The method according to claim 2, characterized in that: The method for calculating the impurity content in the Lebor-Raysen starting material LBS5 is the external standard method, and the calculation method is as follows: Impurity content (%) = × ×V× f / / M / 1000×100% in, f Indicates the impurity correction factor; Indicates the peak area of ​​impurities in the test solution; This indicates the peak area of ​​LBS5, the starting material of Leborresen, in the reference solution of LBS5; V represents the concentration of LBS5, the starting material of Leborresen, in the LBS5 reference solution, in μg / mL; V represents the dilution volume of the test solution, in mL; and M represents the sample weight of the test sample, in mg.

10. The method according to claim 9, characterized in that, The correction factor for the LBM7 is 1.62; The correction factor for LBS1 is 0.97; The correction factor for LBS5-Z4 is 1.07; The correction factor for LBS5-Z2 is 1.0; The correction factor for LBM1 is 2.37.

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