Method for detecting and analyzing alpoxivan intermediate 3 and related impurities

Abstract

The invention discloses a detection and analysis method for an alpoxivan intermediate 3 and related impurities, and relates to the field of substance detection methods, the detection and analysis method comprises the following steps: S1, preparation of a test solution: taking a sample, adding a solvent into the sample, dissolving, and diluting for later use; s2, setting chromatographic conditions and a gradient elution program, wherein the chromatographic conditions of the chromatography comprise the use of a reversed-phase C18 chromatographic column, a mobile phase A and a mobile phase B; s3, taking the test solution, injecting the test solution into a liquid chromatograph, recording a chromatogram, and calculating the impurity content; wherein the mobile phase A is prepared from 0.15 to 0.25 weight percent of triethylamine solution, and the pH (Potential of Hydrogen) of the mobile phase A is 2 to 3; the mobile phase B comprises methanol. According to the method disclosed by the invention, the aim of effectively separating and quantifying various related impurities of the alpoxivan intermediate 3 is fulfilled through optimized chromatographic conditions.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of substance detection method, and particularly relates to a detection and analysis method of apresotan intermediate 3 and related impurities. BACKGROUND

[0002] Apresotan, as a new dual endothelin receptor antagonist, its clinical value lies in providing a new treatment option for patients with refractory hypertension. The synthesis path of the drug is long and complex, and the intermediate 3, 5-(4-bromophenyl)pyrimidine-4,6-dichloro, as a key hub for constructing pyrimidine nucleus and introducing halogen functional group, plays a core role in the whole process chain. This intermediate with a molecular weight of about 305 g / mol contains a brominated aromatic ring, an electron-rich pyrimidine heterocycle and two highly reactive chloro substituents, and its chemical properties are quite active. It is extremely easy to undergo hydrolysis substitution, oxidation and dehalogenation under acid, base and humid conditions. In the industrial scale production, the residual starting material p-bromobenzaldehyde, isomers produced by incomplete cyclization reaction, ring-opening by-products, chlorination degree uneven impurities (monochloro product, polychloro product) in chlorination step, and degradation products in storage process, together constitute a complex spectrum containing multiple specific impurities. These impurities have a high degree of similarity with the main component of the skeleton structure, and the polarity difference is small, which brings great challenges to quality control. Once the purity of the intermediate is out of control, it will directly lead to the decrease of selectivity of the subsequent nucleophilic substitution reaction, the increase of by-products, the difficulty of separation and purification of the final product, and ultimately affect the consistency and safety of the bulk drug batches, which becomes an important bottleneck restricting the industrialization of the product.

[0003] However, as a new chemical entity, apresomerant intermediate 3 currently has neither pharmacopoeia quality standards nor literature reported exclusive analysis strategies. Domestic pharmaceutical enterprises can only be forced to refer to the general high performance liquid chromatography conditions of similar structural compounds for non-specific detection in the process of declaration and production, but these conditions generally have problems such as incomplete separation of the main peak and the adjacent impurity peak, insufficient sensitivity due to non-optimized detection wavelength, gradient program unable to consider the separation of multiple components with large polarity difference, poor method robustness, and the like, which are difficult to meet the requirements of strict monitoring of key process parameters in the pharmaceutical production quality management specification. The core problem to be solved in actual production is: how to establish a reliable, stable and repeatable analysis method for the specific impurity spectrum characteristics of the intermediate, which can simultaneously detect and quantify multiple specific impurities, and the method robustness is strong enough to withstand the column batch difference, instrument fluctuation and other common variables without affecting the result determination. This method needs to cover three core use scenarios: first, online quality monitoring at the end of the synthesis reaction, determining whether the reaction is complete by detecting the residual amount of the key impurity, thereby guiding the real-time optimization of process parameters; second, used for commercial production batch release testing, ensuring that each known impurity and unknown impurity in the intermediate product meets the internationally accepted limit standard, as the basis for deciding whether the product can be released; third, supporting accelerated stability study, monitoring the growth trend of degradation impurities under extreme conditions, providing scientific data for formulating reasonable storage conditions and retesting period, and finally filling the technical gap in the quality control field of the new chemical entity, ensuring the controllability of the whole chain quality from the intermediate to the final product. SUMMARY

[0004] The purpose of the present application is to provide a detection and analysis method for apresomerant intermediate 3 and related impurities, which solves the technical problem in the prior art that there is no exclusive detection method and multiple apresomerant intermediate 3 related impurities cannot be effectively separated and quantified at the same time through optimized chromatographic conditions.

[0005] The detection and analysis method for apresomerant intermediate 3 and related impurities provided in the embodiments of the present application comprises the following steps: S1: Preparation of test sample solution, including taking a sample, dissolving and diluting the sample with a solvent, and then reserving; S2: Setting chromatographic conditions and gradient elution program, wherein the chromatographic conditions of the chromatography include using a reversed-phase C18 chromatographic column, mobile phase A and mobile phase B; S3: Injecting the test sample solution into a liquid chromatograph, recording the chromatogram, and calculating the impurity content; The mobile phase A comprises 0.15-0.25wt% triethylamine solution, and the pH of the mobile phase A is 2-3; The mobile phase B comprises methanol; The structural formula of the apresomerant intermediate 3 is: .

[0006] The selection principle of the concentration range of the 0.15-0.25 wt% triethylamine solution is based on the dose-effect balance of ion pair action and the critical threshold of the chromatographic system capacity: when the concentration is lower than 0.15 wt%, the concentration of triethylamine cations in the mobile phase is insufficient to form sufficient ion pairs with the weak basic pyrimidine heterocycle and strong polar acidic groups that may exist in the analyte, resulting in a basic compound peak tailing factor exceeding the requirement of 1.5 in the pharmacopoeia, and the retention enhancement effect of the carboxyl-containing impurities is weak, which is easily co-eluted with the main component and fails to separate; when the concentration is higher than 0.25 wt%, the excessive triethylamine cations produce multi-layer adsorption on the surface of the C18 stationary phase, causing column overload phenomenon, which in turn reduces the separation selectivity of structurally similar impurities, and the background absorption of high-concentration amines at a 225 nm ultraviolet detection wavelength significantly increases, which deteriorates the signal-to-noise ratio of trace impurity peaks, and the limit of quantification and the limit of detection cannot meet the limit requirements. The concentration range is verified for the durability of multiple batches of chromatographic columns, ensuring the formation of a monolayer ion pair coverage under the condition of pH 2-3, which fully shields the silanol group activity, enhances the retention of acidic impurities, avoids column overload and baseline noise increase, and cooperates with the methanol gradient to completely separate multiple polar impurities with a large difference in polarity, meeting the stringent standards of system applicability separation degree and impurity peak signal-to-noise ratio not less than 30.

[0007] As an optional implementation, the pH regulator of the mobile phase A is phosphoric acid.

[0008] Phosphoric acid, as a ternary medium-strong acid, provides precise acid-base regulation and buffering capacity in the pH 2-3 range, ensuring stable and reproducible protonation efficiency of triethylamine. Phosphate ions exist in the form of H2PO4⁻ under this pH condition, forming an ion pair system with protonated triethylamine cations, and adjusting the distribution equilibrium of the components to be tested on the surface of the C18 stationary phase through the ion strength effect, inhibiting the retention time drift caused by the fluctuation of the protonation state of the chromatographic column silanol group, and enhancing the tolerance of the method to small fluctuations in column batch differences and instrument parameters. In the ultraviolet detection mode, phosphoric acid has no ultraviolet absorption at 225 nm, avoiding interference with the detection baseline stability caused by the absorption of the regulator itself, and ensuring that the signal-to-noise ratio of low-content impurities meets the quantitative requirements. Compared with other inorganic acids, the buffering capacity of phosphoric acid is significantly higher than that of hydrochloric acid and other monovalent strong acids in the target pH range, which ensures that the pH of the mobile phase remains highly consistent during long-time continuous sampling and transfer between different laboratories.

[0009] As an optional implementation, the chromatographic conditions further include: a detection wavelength of 210-230 nm, a column temperature of 25-35°C, a flow rate of 0.9-1.1 mL / min, and an injection volume of 40-60 μl.

[0010] Specifically, the detection wavelength is strictly set within the range of 210–230 nm, which falls within the characteristic absorption region of the intermediate and its multiple related impurities' conjugate system. If this wavelength is deviated from, the UV response value will decrease significantly, resulting in insufficient sensitivity. The column temperature of 25–35 °C affects column efficiency and peak shape by regulating the viscosity of the mobile phase and the mass transfer rate. When the temperature is below 25 °C, the viscosity of the mobile phase increases, causing peak broadening and prolonged retention time. When the temperature is above 35 °C, the stability of the stationary phase is impaired, and bubbles are easily generated, leading to baseline fluctuations. The flow rate of 0.9–1.1 mL / min directly affects column pressure and separation time. When the flow rate is below 0.9 mL / min, the analytical efficiency decreases, and the increased longitudinal diffusion leads to a decrease in column efficiency. When the flow rate is above 1.1 mL / min, the column pressure exceeds the tolerance of the chromatographic system, and incomplete mass transfer deteriorates the resolution. The injection volume of 40–60 μl ensures that the peak area of ​​trace impurities meets the limit of quantitation requirements while avoiding column overload. When the injection volume is below 40 μl, the impurity peak response is insufficient, making accurate quantification difficult. When the injection volume is above 60 μl, the tailing factor of the main peak increases, and the risk of co-elution of adjacent impurities increases significantly. The parameter range has been verified for system applicability to ensure that the separation degree between the principal component and impurities and the signal-to-noise ratio of the impurity peaks meet the requirements.

[0011] As an optional implementation, the sample contains at least one of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, and impurity H, and their structural formulas are as follows: , , , , , , and .

[0012] As an optional implementation, the relative retention time of impurity A is 0.29~0.39; the relative retention time of impurity B is 0.70~0.77; the relative retention time of impurity C is 0.73~0.79; the relative retention time of impurity D is 0.89~0.91; the relative retention time of impurity E is 0.91~0.93; the relative retention time of impurity F is 0.95~0.96; the relative retention time of impurity G is 0.97~0.99; and the relative retention time of impurity H is 1.20~1.26.

[0013] As an optional implementation method, the peak area is calculated using the normalized peak area method: The content of impurity A is ≤0.15wt%; the content of impurity B is ≤0.15wt%; the content of impurity C is ≤0.15wt%; the content of impurity D is ≤0.15wt%; the content of impurity E is ≤0.15wt%; the content of impurity F is ≤0.15wt%; the content of impurity G is ≤0.15wt%; and the content of impurity H is ≤0.15wt%.

[0014] As an optional implementation, other impurities are also included, with the content of other impurities ≤0.5wt% and the total amount of impurities ≤2wt%.

[0015] As an optional embodiment, the solvent comprises methanol and water, wherein the volume ratio of methanol to water is (65:35) to (75:25). As an optional implementation, the gradient elution process includes: From 0 to 3 minutes, mobile phase A is 70% and mobile phase B is 30%. Within 3 to 40 minutes, mobile phase A decreased from 70% to 25%, while mobile phase B increased from 30% to 75%. Over 40-45 minutes, mobile phase A decreased from 25% to 10%, while mobile phase B increased from 75% to 90%. For 45-54 minutes, mobile phase A is maintained at 10%, and mobile phase B is maintained at 90%. Within 54-55 minutes, mobile phase A increased from 10% to 70%, while mobile phase B decreased from 90% to 30%. For 55-65 minutes, mobile phase A is maintained at 70% and mobile phase B is maintained at 30%.

[0016] This program, in conjunction with triethylamine concentration and acidic pH, establishes a selectivity range on a reversed-phase C18 column. This allows impurities with similar structures and minor polarity differences to achieve different retention rates due to differences in dipole moment and steric hindrance. The principle is based on precise control of polarity differentiation and dynamic column efficiency balance: an initial 70% A isocratic gradient ensures sufficient retention of polar impurities, preventing co-elution with the solvent front; a slow gradient of A to 25% over 3-40 minutes is crucial for separating positional isomers; an excessively steep gradient will result in insufficient retention differences and a resolution below 1.5; a rapid gradient to 10% A over 40-45 minutes balances the elution efficiency of strongly retained impurities with analysis time; a high organic phase is maintained for 9 minutes to ensure complete elution of hydrophobic byproducts and prevent column memory effect; initial conditions are restored within 1 minute to control the total cycle time to meet online monitoring throughput requirements within 66 minutes; and a 10-minute rebalancing rebuilds the ion-pair adsorption layer to prevent retention drift.

[0017] As an optional implementation, the chromatographic column of the liquid chromatograph uses octadecylsilane-bonded silica gel as the packing material.

[0018] Compared with the prior art, the embodiments of the present invention have the following advantages and beneficial effects: 1. In this embodiment of the invention, the mobile phase A uses a 0.15~0.25wt% triethylamine solution with the pH controlled at 2~3. This allows the triethylamine to be protonated under acidic conditions to form triethylamine cations. These cations interact with the weakly basic pyrimidine heterocycles and strongly polar -COOH easily dissociated groups that may be present in the analyte through ion-pair interactions. This suppresses the secondary adsorption effect of basic compounds on the silanol groups of the C18 column to improve peak tailing, and also enhances the retention capacity of acidic impurities under the reversed-phase chromatography retention mechanism, ensuring that they are fully retained in molecular rather than ionic form, thus producing a sufficient selectivity difference with the main component. The mobile phase B uses pure methanol to provide strong elution power, forming a solvent system with a significant polarity transition with the mobile phase A. The two phases work together in a gradient elution program to achieve sequential elution based on the subtle differences in hydrophobicity between each impurity and the main component. This mobile phase system, through the synergistic effect of precise control of triethylamine concentration and pH value, solves the technical challenge of easy co-elution of apricotitant intermediate 3 and its multiple related impurities due to their highly similar structures and minimal polarity differences. It establishes a specific retention behavior control window, enabling the effective separation of complex impurity profiles that could not be baseline separated under conventional reversed-phase conditions. This fills the technical gap of the lack of pharmacopoeia standards for this new chemical entity and the non-specific methods in the literature, meeting the requirements of method specificity and robustness for online monitoring of the synthesis reaction, batch release inspection, and stability studies.

[0019] 2. In this embodiment of the invention, a specific retention control range is constructed on a reverse-phase C18 column by using a triethylamine concentration of 0.15~0.25wt% and a pH of 2~3 for synergistic ion pairing. This allows for the full separation of multiple components with highly similar structures and minimal polarity differences, such as benzene ring substitution position isomers, open-chain byproducts, and alkoxylated impurities, due to differences in dipole moment and steric hindrance. Combined with a 210nm detection wavelength, this ensures high sensitivity response for each component, with a resolution ≥1.5 and a signal-to-noise ratio ≥30, achieving specific separation of this new chemical entity without pharmacopoeia standards.

[0020] 3. In the gradient program of this invention, the precise balance between the slow descent slope of 3-40 minutes and the high organic phase homogeneity of 9 minutes ensures that polar impurities are not co-eluted and that strongly retained impurities have no column residue. The triethylamine adsorption layer is rebuilt after 10 minutes of rebalancing, so that the retention time RSD is ≤1.0%. The column temperature range of 25-35℃ and the flow rate range of 0.9-1.1 mL / min controls the stability of the mobile phase viscosity and column pressure, avoiding the influence of batch differences and instrument fluctuations on the result judgment, and meeting the stringent reproducibility requirements of online monitoring and release inspection in commercial production.

[0021] 4. The embodiments of this invention take into account both separation efficiency and throughput requirements, completing the separation of the main component and the full impurity spectrum within a total cycle of 66 minutes, supporting real-time determination of the synthesis endpoint and optimization of process parameters; the area normalization method combined with an injection volume range of 40~60μl ensures that the quantitation limit of each known impurity meets the 0.15% limit standard, and unknown impurities are effectively detected, providing reliable data for monitoring degradation trends in stability studies, and filling the technical gap in the quality control of the entire chain from intermediate to active pharmaceutical ingredient of this new chemical entity. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The synthetic route for apxitentan intermediate 3 in this application is as follows; Figure 2 This is a typical diagram of the system applicability solution in an embodiment of the present invention; Figure 3 This is a chromatogram of the interference results in an embodiment of the present invention. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0025] Therefore, the detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0026] During pilot-scale production of apxitentan intermediate 3, abnormal batch stability was observed: some batches passed release but experienced a sharp drop in yield in subsequent steps, while others showed a surge in degradation products in accelerated testing. (See synthetic route). Figure 1 Analysis revealed that each step in the transformation of p-bromobenzaldehyde starting material into a pyrimidine core via cyclization and chlorination may introduce specific impurities—starting material residues, open-chain byproducts from incomplete cyclization, monochlorinated and polychlorinated products with uneven chlorination degrees, and phenolic hydroxyl degradation products generated from pyrimidine ring hydrolysis. These impurities are highly similar in structure to the main component and cannot be separated under conventional HPLC conditions.

[0027] Therefore, the plan is to construct an acidic pH, triethylamine-phosphate ion pair mobile phase system to stabilize and regulate the retention of the pyrimidine ring. Gradient elution starts from 70% aqueous phase, gradually enhancing elution capacity through a combination of linear and plateau elution stages, forcibly separating strongly retained impurities before rapid equilibration. Simultaneously, the plan optimizes the detection of the conjugate absorption wavelength of the main component, and controls the temperature at 25-35℃ to prevent sample transformation. The expectation is that by precisely controlling the three-dimensional parameters of mobile phase composition, gradient time, and detection conditions, easily confused impurities adjacent to the main peak can be baseline separated, while distant impurities can be clearly identified. The method should be able to withstand common fluctuations in column temperature and flow rate, ultimately establishing a dedicated quality control strategy covering the entire process of synthesis monitoring, release inspection, and stability testing, completely resolving the quality and cost risks in industrial production caused by uncontrolled impurities.

[0028] Apoxetine intermediate 3 was prepared via cyclization and chlorination (synthetic route see...). Figure 1 Impurities such as starting material residues, incomplete cyclization products, chlorinated byproducts, and pyrimidine ring degradation products may be generated during the process.

[0029] Therefore, embodiments of the present invention provide a method for detecting and analyzing apracitentan intermediate 3 and related impurities, specifically including the following: I. Instruments, Equipment and Main Materials (1) Information on instruments and equipment is shown in Table 1 below: Table 1

[0030] (2) Reagent information is shown in Table 2 below: Table 2

[0031] (3) Information on the reference standard is shown in Table 3 below: Table 3

[0032] (4) Sample information is shown in Table 4 below: Table 4

[0033] (1) Drafting method Instrument: High Performance Liquid Chromatography (HPLC) Chromatographic column: Octadecylsilane-bonded silica gel as the packing material (NapoChrom Core AQ C18, 250mm × 4.6mm, 5μm or equivalent column). Capture Column: Yuexu Ghost-Buster Column Kits, 4.6×50mm or equivalent performance capture column. Mobile phase A: 0.2% triethylamine (adjusted to pH 2.4 with phosphoric acid) Mobile phase B: Methanol.

[0034] The elution procedure is shown in Table 5 below: Table 5

[0035] System suitability solution: Take appropriate amounts of this product and impurity QSR9003-b-4-IMN, add solvent, sonicate to dissolve and dilute to prepare a mixed solution containing approximately 0.1 mg of QSR9003-b-4 and 0.15 μg of QSR9003-b-4-IMN per 1 ml.

[0036] Test solution: Take an appropriate amount of this product, add solvent, sonicate to dissolve and dilute to a solution containing about 0.1 mg per 1 ml.

[0037] Positioning solution: Weigh appropriate amounts of reference standards QSR9003-b-4-IMB, QSR9003-b-4-IME, QSR9003-b-1, QSR9003-b-4-IMM, QSR9003-b-4-IMI, QSR9003-b-4-IMN, QSR9003-b-4-IML and reference standard QSR9003-b-4, accurately weigh them, place them in a volumetric flask, dissolve them in methanol and quantitatively dilute them to prepare a mixed solution containing approximately 0.1 mg of QSR9003-b-4 per 1 ml, with other impurities at 0.15 µg.

[0038] Flow rate: 1.0 ml / min; Column temperature: 30℃; Wavelength: 220 nm; Injection volume: 50 µl System suitability requirements: In the system suitability solution, the separation between the main peak and the impurity QSR9003-b-4-IMN should meet the requirements, and the signal-to-noise ratio of impurity QSR9003-b-4-IMN should not be less than 30. In the positioning solution, impurities QSR9003-b-4-IMA, QSR9003-b-4-IMB, QSR9003-b-4-IME, QSR9003-b-1, QSR9003-b-4-IMM, QSR9003-b-4-IMI, QSR9003-b-4-IMN, QSR9003-b-4, and impurity QSR9003-b-4-IML should elute sequentially.

[0039] Determination method: Accurately measure the test solution and inject it into the liquid chromatograph, and record the chromatogram.

[0040] Acceptance criteria: If the chromatogram of the test solution contains the above-mentioned impurity peaks, the peak area calculated using the peak area normalization method (Note: a correction factor may be added; the accurate correction factor obtained in this validation) shall not exceed the corresponding limit specified in Table 6 below.

[0041] Table 6

[0042] Calculation formula: Where, Aimpurity: the peak area of ​​each impurity in the test solution; Total A: The total peak area of ​​all components in the test solution.

[0043] II. Verification Process and Results (a) System applicability The solution preparation details are shown in Table 7 below: Table 7

[0044] The injection analysis and acceptable criteria are shown in Table 8 below: Table 8

[0045] Verification results refer to Figure 2 And as shown in Table 9 below: Table 9

[0046] Combination Figure 2 As shown in Table 9, in the system suitability solution chromatogram, the resolution of impurity QSR9003-b-4-IMN from the main peak is the lowest at 2.60 (>1.5); the signal-to-noise ratio of impurity QSR9003-b-4-IMN is the lowest at 131.3 (>30).

[0047] Five consecutive injections were performed, and the maximum RSD of the peak area of ​​impurity QSR9003-b-4-IMN relative to the principal component was 0.9% (<2%), and the maximum RSD of the retention time was 0.1% (<1%). The system suitability meets the validation requirements.

[0048] (ii) Exclusivity The solution preparation details are shown in Table 10 below: Table 10

[0049] The injection analysis and acceptance criteria are shown in Table 11 below: Table 11

[0050] Verification results: The blank solvent showed no interference at the positions of the main peak and the elution positions of known impurities. (III) Linearity and Range The solution preparation details are shown in Tables 12, 13, and 14 below: Table 12

[0051] Table 13

[0052] Table 14

[0053] The injection analysis and acceptable criteria are shown in Table 15 below: Table 15

[0054] The test results are shown in Tables 16 and 17 below: Table 16

[0055] Table 17

[0056] Combining Tables 16 and 17, we can see that: (1) Test sample concentration level: QSR9003-b-4 showed good linearity in the range of limit of quantitation to 0.45% of the test sample solution concentration, with a correlation coefficient r of 0.9999 (>0.990); the ratio of the Y-axis intercept to the response value of the limit concentration (100%) was 0.95% (within ±10%), the P value was 0.880 (>0.05), and the sum of squared residuals was 0.00004; it also showed good linearity in the range of 50% to 150% of the test sample solution concentration, with a correlation coefficient r of 0.9986 (>0.990); the ratio of the Y-axis intercept to the response value of the limit concentration (100%) was -7.93% (within ±10%), the P value was 0.633 (>0.05), and the sum of squared residuals was 108.2730; (2) Impurity limit levels: impurities QSR9003-b-1, QSR9003-b-4-IMA, QSR9003-b-4-IMB, QSR9003-b-4-IME, QSR9003-b-4-IMI, QSR9003-b-4-IML, QSR9003-b-4-IMM, QSR9003-b-4-IMN and principal component QSR90 03-b-4 exhibits good linearity within the limit of quantitation (LOQ) to 300% limit concentration range, with correlation coefficients (r) ranging from 0.9997 to 1.0000 (>0.990). The ratio of the Y-intercept to the response value at the limit concentration (100%) ranges from -1.30% to 7.87% (within ±10%), with p-values ​​ranging from 0.222 to 0.912 (>0.05) and residual sum of squares ranging from 0.000002 to 0.00024. (3) The correction factors for impurity QSR9003-b-4-IMA are 1.02, QSR9003-b-4-IMB are 1.20, QSR9003-b-4-IME are 1.22, QSR9003-b-1 are 1.12, QSR9003-b-4-IMM are 1.08, QSR9003-b-4-IMI are 0.88, QSR9003-b-4-IMN are 0.92, and QSR9003-b-4-IML are 1.43; the accuracy is calculated by substituting these correction factors.

[0057] In summary, the linearity and range meet the verification requirements.

[0058] (iv) Accuracy Solution preparation: The limits for each impurity (QSR9003-b-4-IMA, QSR9003-b-4-IMB, QSR9003-b-4-IME, QSR9003-b-1, QSR9003-b-4-IMM, QSR9003-b-4-IMI, QSR9003-b-4-IMN, QSR9003-b-4-IML) are all 0.15%; the solutions for each impurity with a proposed LOQ accuracy are prepared at 0.03% of the test sample concentration (equivalent to 20% of the limit), and the preparation process is shown in Table 18 below: Table 18

[0059] Calculation formula: (1) External standard method for impurities: , In the formula: A 对 Peak areas of each impurity in the reference solution; C 对 Concentrations of various impurities in the reference solution. μ g / ml A 准确度 Accuracy: Peak area of ​​each impurity in the solution; S 准确度 : The dilution volume of the accuracy solution; A 本底杂 Peak area of ​​each impurity in the test solution; S 样 : The dilution volume of the test solution; C 加 In the preparation of an accuracy solution, the concentration of each impurity added to the solution is crucial. μ g / ml; V 加 Accuracy: The volume of solution added to the solution, in ml; Measured quantity: Accuracy: The amount of each impurity measured in the solution ( μ g); Background quantity: The amount of various impurities contained in the test sample itself in the accuracy solution ( μ g); Amount added: The amount of each impurity added to the accuracy solution ( μ g).

[0060] (2) Peak area normalization method with correction factor , In the formula: A 杂质 : Peak area of ​​each impurity in the test solution; f： Correction factors for each impurity; W 准供 The sample weight of the test solution for each accuracy level, in mg. W 本底供 Peak area of ​​the main component in each test solution; A 本底杂 The sample weight of the test solution, in mg. 1000: mg and μ The conversion factor of g; C 加 In the preparation of an accuracy solution, the concentration of each impurity added to the solution is crucial. μ g / ml; V 加 Accuracy: The volume of solution added to the solution, in ml; Measured quantity: Accuracy: The amount of each impurity measured in the solution ( μ g); Background quantity: The amount of various impurities contained in the test sample itself in the accuracy solution ( μ g); Amount added: The amount of each impurity added to the accuracy solution ( μ g).

[0061] The injection analysis and acceptable criteria are shown in Table 19 below: Table 19

[0062] The test results are shown in Tables 20 and 21 below: Table 20

[0063] Table 21

[0064] As shown in Tables 20 and 21, the recovery rates of each impurity (QSR9003-b-4-IMA, QSR9003-b-4-IMB, QSR9003-b-4-IME, QSR9003-b-1, QSR9003-b-4-IMM, QSR9003-b-4-IMI, QSR9003-b-4-IMN, QSR9003-b-4-IML) were calculated using the area normalization method with correction factor: 1 sample at the limit of quantitation level: recovery rate between 88.24% and 113.78% (between 80% and 120%); 1 sample each at the 50% and 150% limit concentrations; and 6 samples at the 100% limit concentration: recovery rate between 94.04% and 104.90% (between 90% and 110%). Using the external standard method for impurities: 1 sample at the limit of quantitation level: recovery rate between 91.77% and 111.29% (between 80% and 120%); 1 sample each at the 50% and 150% limit concentrations; and 6 samples at the 100% limit concentration: recovery rate between 94.04% and 104.90% (between 90% and 110%).

[0065] (2) The ratio of the recovery rates of impurities was calculated using the external standard method and the peak area normalization method with correction factor: the ratio of the recovery rates of each impurity at the limit of quantitation concentration was 97.8% to 104.0% (between 90% and 110%); the average recovery rate ratio of each impurity at the limit concentration of 50% to 150% was 98.3% to 105.2% (between 90% and 110%).

[0066] In summary: the accuracy meets the verification requirements.

[0067] (v) Lower limit of the scope Based on the linear range and accuracy test results, the LOQ of the main component (QSR9003-b-4) and each impurity (QSR9003-b-4-IMA, QSR9003-b-4-IMB, QSR9003-b-4-IME, QSR9003-b-1, QSR9003-b-4-IMM, QSR9003-b-4-IMI, QSR9003-b-4-IMN, QSR9003-b-4-IML) is 0.03 μg / ml. This concentration will be used as the LOQ concentration for each component to confirm the lower limit of the range. The solution preparation is shown in Table 22 below. Table 22

[0068] The injection solutions and acceptable standards are shown in Table 23 below: Table 23

[0069] The verification results are shown in Tables 24 and 25 below: Table 24

[0070] Table 25

[0071] Combining Tables 24 and 25, it can be seen that for both injections of the limit of quantitation (LOQ) solution, the S / N ratios of each component peak ranged from 29.4 to 56.2 (all > 10), and the LQ concentrations for both were 0.03% of the sample concentration (all < 0.05% of the sample solution concentration). Two injections were performed for each component's detection limit solution, with S / N ranging from 3.5 to 10.2 (all > 3).

[0072] In summary, the method's sensitivity meets the validation requirements.

[0073] (vi) Precision (repeatability) The solution preparation details are shown in Table 26 below: Table 26

[0074] The injection analysis and acceptable criteria are shown in Table 27 below: Table 27

[0075] The verification results are shown in Table 28 below: Table 28

[0076] As shown in Table 28, for the six spiked solutions (all spiked to the limit), calculated using the peak area normalization method with correction factor, the content of each impurity was less than 0.2%, with a maximum range of 0.02% (<0.05%), and the total impurity content was greater than 1.0%, with a range of 0.1% (<0.2%). The number of impurities greater than 0.05% was the same (8 in total), and the repeatability met the validation requirements.

[0077] (vii) Solution stability The solution preparation details are shown in Table 29 below: Table 29

[0078] The injection analysis and acceptable criteria are shown in Table 30: Table 30

[0079] The verification results are shown in Table 31 below: Table 31

[0080] As shown in Table 31, after the spiked test solution was placed at room temperature for 34.5 hours, the impurity content at each time point was <0.2%, with a range between 0.00% and 0.01% (all <0.05%); the total impurity content was 1.1% to 1.2%, with a range between 0.0% and 0.1% (all <0.2%), and no new impurities greater than 0.05% were generated. Therefore, the spiked test solution was stable at room temperature for 34.5 hours.

[0081] (viii) Durability The durability test conditions are shown in Table 32 below: Table 32

[0082] The solution preparation details are shown in Table 33 below: Table 33

[0083] The injection analysis and acceptable criteria are shown in Table 34. Table 34

[0084] The verification results are shown in Tables 35, 36, and 37 below: Table 35

[0085] Table 36

[0086] Table 37

[0087] Table 37 shows the fine-tuning of chromatographic conditions: detection wavelength (220±2nm), flow rate (1.0±0.2ml / min), column temperature (±5℃), mobile phase pH (2.4±0.2), initial mobile phase ratio (30±5%), and different instruments and columns; (1) Under all durability conditions, the blank solvent does not interfere with the detection of the main peak and any known impurities; (2) In the system suitability solution chromatogram, the minimum resolution between impurity QSR9003-b-4-IMN and the main peak is 2.55 (>1.5); the minimum signal-to-noise ratio of impurity QSR9003-b-4-IMN is 95.7 (>30).

[0088] (3) Compared with the original conditions, the content of each impurity in the spiked solution of the test sample is less than 0.2%, with a range of 0.00%~0.018% (all <0.05%); the total impurity content is greater than 1.0%, with a range of 0.00%~0.045% (all <0.1%), and the number of impurities is 8.

[0089] (4) The RRT range of impurity QSR9003-b-4-IMA is 0.29~0.39, the RRT range of impurity QSR9003-b-4-IMB is 0.70~0.77, the RRT range of impurity QSR9003-b-4-IME is 0.73~0.79, the RRT range of impurity QSR9003-b-1 is 0.89~0.91, the RRT range of impurity QSR9003-b-4-IMM is 0.91~0.93, the RRT range of impurity QSR9003-b-4-IMI is 0.95~0.96, the RRT of impurity QSR9003-b-4-IMN is 0.98, and the RRT range of impurity QSR9003-b-4-IML is 1.20~1.26. The relative retention times of each impurity are relatively stable.

[0090] Conclusion: Durability meets verification requirements.

[0091] In summary, the validation results of the above-mentioned analytical method for related substances of apracitentan intermediate 3 (QSR9003-b-4) show that the method has good system suitability, specificity, accuracy, linearity and range, lower limit of range, precision (repeatability), solution stability and robustness, and is suitable for the determination of related substances of apracitentan intermediate 3 (QSR9003-b-4).

[0092] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting and analyzing an intermediate 3 of Alpirozan and related impurities, characterized by, The method comprises the following steps: S1: preparing a sample solution, including taking a sample, dissolving and diluting the sample with a solvent, and then preparing the sample solution; S2: setting chromatographic conditions and a gradient elution program, wherein the chromatographic conditions of the chromatography comprise using a reversed-phase C18 chromatographic column, mobile phase A, and mobile phase B; S3: injecting the sample solution into a liquid chromatograph, recording a chromatogram, and calculating the content of impurities; The mobile phase A comprises 0.15-0.25 wt% triethylamine solution, and the pH of the mobile phase A is 2-3. The mobile phase B comprises methanol. The structural formula of the alpiroxentan intermediate 3 is: .

2. A method for detecting and analyzing aliskiren intermediate 3 and related impurities according to claim 1, characterized in that, The pH regulator of the mobile phase A is phosphoric acid.

3. A method for detecting and analyzing one of the intermediate 3 of Alpirotenan and related impurities according to claim 2, characterized in that, The chromatographic conditions further comprise a detection wavelength of 210-230 nm, a column temperature of 25-35 °C, a flow rate of 0.9-1.1 mL / min, and an injection volume of 40-60 μl.

4. The method for detecting and analyzing aliskiren intermediate 3 and related impurities according to claim 1, wherein, The sample contains at least one of impurities A, B, C, D, E, F, G, and H, and the structural formulas are as follows in sequence: , , , , , , and .

5. The method for detecting and analyzing one intermediate 3 of Alpiroxentan and related impurities according to claim 4, characterized in that, The relative retention time of the impurity A is 0.29-0.39; the relative retention time of the impurity B is 0.70-0.77; the relative retention time of the impurity C is 0.73-0.79; the relative retention time of the impurity D is 0.89-0.91; the relative retention time of the impurity E is 0.91-0.93; the relative retention time of the impurity F is 0.95-0.96; the relative retention time of the impurity G is 0.97-0.99; and the relative retention time of the impurity H is 1.20-1.

26.

6. The method for detecting and analyzing one intermediate 3 of Alpiroxentan and related impurities according to claim 4, characterized in that, The content of the impurity A is ≤0.15 wt%; the content of the impurity B is ≤0.15 wt%; the content of the impurity C is ≤0.15 wt%; the content of the impurity D is ≤0.15 wt%; the content of the impurity E is ≤0.15 wt%; the content of the impurity F is ≤0.15 wt%; the content of the impurity G is ≤0.15 wt%; and the content of the impurity H is ≤0.15 wt%. The content of other impurities is ≤0.5 wt%, and the total content of impurities is ≤2 wt%.

7. A method for detecting and analyzing an intermediate 3 of Alpirozan and related impurities according to claim 6, characterized in that, The solvent comprises methanol and water, and the volume ratio of the methanol to the water is (65:35)-(75:25).

8. The method for detecting and analyzing one intermediate 3 of Alpiroxentan and related impurities according to claim 1, wherein, The gradient elution program comprises:

9. The method for detecting and analyzing an intermediate 3 of Alpiroxentan and related impurities according to claim 1, characterized in that, 0-3 minutes, 70% of the mobile phase A and 30% of the mobile phase B; 3-40 minutes, the mobile phase A decreases from 70% to 25%, and the mobile phase B increases from 30% to 75%; 40-45 minutes, the mobile phase A decreases from 25% to 10%, and the mobile phase B increases from 75% to 90%; 45-54 minutes, the mobile phase A remains 10%, and the mobile phase B remains 90%; 54-55 minutes, the mobile phase A increases from 10% to 70%, and the mobile phase B decreases from 90% to 30%; 55-65 minutes, the mobile phase A remains 70%, and the mobile phase B remains 30%. The chromatographic column of the liquid chromatograph uses octadecylsilane-bonded silica gel as a filler.

10. The method for detecting and analyzing one intermediate 3 of Alpiroxentan and related impurities according to claim 1, wherein, ​

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