Method for detecting and analyzing alpoxivan intermediate 6 and related impurities

Abstract

The invention discloses a detection and analysis method for an alpoxivan intermediate 6 and related impurities, and relates to the field of substance detection methods, and the detection and analysis method comprises the following steps: S1, preparation of a test solution: taking an alpoxivan intermediate 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 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 comprises acetonitrile and a 9-11 mmol / L potassium dihydrogen phosphate solution, the pH value of the potassium dihydrogen phosphate solution is 7.4-7.6, and the volume ratio of the potassium dihydrogen phosphate solution to the acetonitrile is (90-100): 5; the mobile phase B comprises acetonitrile. According to the method disclosed by the invention, the aim of effectively separating and quantifying various related impurities of the alpoxivan intermediates 6 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 6 and related impurities. BACKGROUND

[0002] Apresotan, as a new dual endothelin receptor antagonist, provides a new treatment option for patients with refractory hypertension. The synthesis path of the drug is long and complex. The intermediate 6, [5-(4-bromophenyl)-6-[ (2-hydroxyethyl) oxy] pyrimidin-4-yl] azanedisulfonamide, as a key hub connecting the aromatic ring mother nucleus and the sulfonamide side chain, plays a core role in the whole process chain. The intermediate with a molecular weight of about 415 g / mol contains a brominated aromatic ring, an electron-rich pyrimidine heterocycle and a hydrophilic hydroxyethoxy group in its structure, and is quite active in chemical properties. It is extremely easy to hydrolyze, oxidize and debrominate under the conditions of acid, base and high temperature. In the industrial scale production, the residual starting material p-bromobenzaldehyde, the by-products produced by incomplete reaction of fluorinated reagent, the over-chlorinated impurities of chlorination step, and the degradation products in the storage process, together constitute a complex spectrum containing multiple specific impurities. These impurities have a high similarity with the skeleton structure of the main component and a small difference in polarity, 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 the yield of the subsequent coupling reaction, the difficulty of the separation and purification of the final product, and finally affect the consistency and safety of the bulk drug batches, becoming an important bottleneck restricting the industrialization of the product.

[0003] However, as a new chemical entity, apresomerant intermediate 6 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 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 this 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 6 and related impurities, which solves the technical problem in the prior art that there is no exclusive detection method and multiple apresomerant intermediate 6 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 6 and related impurities provided by the embodiments of the present application comprises the following steps: S1: Preparation of test sample solution, including taking apresomerant intermediate sample, dissolving and diluting in the sample with solvent and then reserving; S2: Setting chromatographic conditions and gradient elution program, wherein the chromatographic conditions comprise 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 acetonitrile and 9-11 mmol / L potassium dihydrogen phosphate solution, the pH of the potassium dihydrogen phosphate solution is 7.4-7.6, and the volume ratio of the potassium dihydrogen phosphate solution to acetonitrile is 90-100:5. The mobile phase B comprises acetonitrile. The structural formula of the aprikant intermediate 6 is: .

[0006] As an optional embodiment, the preparation method of the mobile phase A comprises weighing anhydrous potassium dihydrogen phosphate to be dissolved in water to obtain a solution of 1.3-1.4 mg / mL, filtering after adjusting the pH to 7.4-7.6 with a 0.15-0.25 g / mL potassium hydroxide solution, and mixing the filtrate with acetonitrile.

[0007] Specifically, the principle of the preparation method is to ensure the buffer capacity and uniformity of the mobile phase A through step-by-step operation, thereby improving the stability of the chromatographic system. First, the anhydrous potassium dihydrogen phosphate is prepared to a specific concentration of 1.3-1.4 mg / mL, which can provide moderate ionic strength for the mobile phase, maintaining the pH buffering capacity and avoiding excessive salt concentration; then, the pH is adjusted to 7.4-7.6 using a 0.15-0.25 g / mL potassium hydroxide solution, which can quickly and accurately set the pH value with little effect on the overall buffer concentration; the filtration step can remove small particles that may be generated during dissolution or pH adjustment, preventing the chromatographic column from being blocked; finally, the filtrate is mixed with acetonitrile, and the order of "adjusting the pH first and then adding the organic phase" can avoid salting out or pH measurement deviation in the presence of organic solvents. The expected effect is that the pH of the obtained mobile phase A is stable and has good batch consistency, ensuring the retention time stability of intermediate 6 and its impurities in gradient elution, significantly enhancing the tolerance of the method to fluctuations of different chromatographic columns and instruments, thereby realizing the reliability of continuous analysis in industrial production.

[0008] As an optional embodiment, the chromatographic conditions further comprise a detection wavelength of 248-252 nm, a column temperature of 38-42°C, a flow rate of 0.9-1.1 mL / min, an injection volume of 4-6 μl, and an injection tray temperature of 4-6°C.

[0009] As an optional embodiment, 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 the structural formula is as follows in sequence: 、 、 、 、 、 、 and .

[0010] These impurities all retain the conjugated skeleton of bromo-aromatic ring and pyrimidine nucleus of intermediate 6 of alpirohogil, so that they have similar ultraviolet absorption characteristics with the main component at 248~252 nm and can be detected at the same wavelength. Meanwhile, the gradient difference of molecular polarity exists due to the difference of substituent groups, and the hydrophobic retention on C18 stationary phase is different. The potassium dihydrogen phosphate buffer (pH 7.4~7.6)-acetonitrile system in mobile phase A can inhibit the ionization of these nitrogen-containing heterocyclic rings and avoid tailing, and the proportion of acetonitrile increases during gradient elution, so that the fluorinated or debrominated impurities with smaller polarity are eluted first, and the hydroxylated or sulfonylated by-products with smaller polarity are eluted later, so that time separation is realized.

[0011] As an optional implementation, the relative retention time of the impurity A is 1.04~1.14; the relative retention time of the impurity B is 1.36~1.46; the relative retention time of the impurity C is 1.49~1.55; the relative retention time of the impurity D is 1.56~1.65; the relative retention time of the impurity E is 1.66~1.75; the relative retention time of the impurity F is 1.79~1.89; the relative retention time of the impurity G is 2.25~2.79; and the relative retention time of the impurity H is 2.80~2.85.

[0012] The RRT sequence allows each impurity peak to flow out in a predetermined time window during gradient elution, allowing accurate positioning of each impurity through the time window under different brands of chromatographic columns and fluctuations in column temperature of 38~42℃, avoiding false positives or missed detection, and meeting the robustness requirements of impurity tracing during continuous sample analysis in industrial production.

[0013] As an optional implementation, according to the peak area normalization method: The content of impurity A is ≤1.0wt%; the content of impurity B is ≤1.0wt%; the content of impurity C is ≤1.0wt%; the content of impurity D is ≤1.0wt%; the content of impurity E is ≤1.0wt%; 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, it further includes impurity I with a relative retention time of 0.78~0.88 and impurity J with a relative retention time of 1.19~1.29; The content of impurity I is ≤0.5wt%; and the content of impurity J is ≤1.0wt%.

[0015] As an optional implementation, the solvent includes dimethyl sulfoxide and acetonitrile, and the volume ratio of dimethyl sulfoxide and acetonitrile is 50:45~55.

[0016] As an optional implementation, the gradient elution program includes: 0~6 minutes, mobile phase A is 100%, mobile phase B is 0%; 6~20 minutes, mobile phase A decreases from 100% to 85%, mobile phase B increases from 0% to 15%; 20~45 minutes, mobile phase A decreases from 85% to 74%, mobile phase B increases from 15% to 26%; 45~50 minutes, mobile phase A remains 74%, mobile phase B remains 26%; 50~60 minutes, mobile phase A decreases from 74% to 55%, mobile phase B increases from 26% to 45%; 60~70 minutes, mobile phase A decreases from 55% to 10%, mobile phase B increases from 45% to 90%; 70~80 minutes, mobile phase A remains 10%, mobile phase B remains 90%; 80~81 minutes, mobile phase A increases from 10% to 100%, mobile phase B decreases from 90% to 0%; 81~90 minutes, mobile phase A remains 100%, mobile phase B remains 0%.

[0017] As an optional implementation, the chromatographic column of the liquid chromatograph adopts octadecylsilane bonded silica gel as the filler.

[0018] Compared with the prior art, the embodiment of the present application has the following advantages and beneficial effects: 1、The embodiment of the present application is based on the principle of reversed-phase chromatography, and utilizes the hydrophobic action of a C18 chromatographic column to retain and separate the intermediate 6 of alprostadil and impurities. Mobile phase A is a mixture of 9~11 mmol / L potassium dihydrogen phosphate buffer (pH 7.4~7.6) and acetonitrile at a volume ratio of 90~100:5, wherein the low concentration of acetonitrile forms a weak elution environment, so that the polar starting material residue and early degradation impurities are retained enough and do not flow out with the solvent peak; the neutral pH environment can inhibit the ionization of the sulfonamide group in the molecule, avoid chromatographic peak tailing, and prevent the structural transformation of acid-base sensitive impurities during the analysis process, thereby ensuring the stability of the method. The 9~11 mmol / L phosphate salt concentration provides moderate ionic strength, fine tunes the retention behavior of polar impurities without causing chromatographic column overload or high system back pressure. Mobile phase B is pure acetonitrile, and its proportion is gradually increased through a gradient elution program, and the elution ability is gradually enhanced, so that multiple impurities with large differences in polarity can be considered in a single analysis. This design realizes baseline separation between the main component and eight specific impurities, meets the trace control requirements, and maintains peak symmetry and retention time stability within a reasonable fluctuation range of buffer salt concentration and pH, which is suitable for online monitoring of intermediate synthesis end point, product release inspection and accelerated stability study, and fills the technical gap of the new chemical entity without a dedicated detection method.

[0019] 2, The selection of the detection wavelength range in the embodiment of the application is based on the fact that the aromatic ring and pyrimidine conjugated system common to the intermediate 6 of aprocitentan and its impurities have the maximum absorption in this interval, which can ensure that each component has sufficient response signal, while avoiding end absorption interference; the column temperature is set at 38-42℃, which can reduce the viscosity of the mobile phase, accelerate the mass transfer rate, make the peak shape more sharp and symmetrical, and keep the retention time stable in this temperature range, and can tolerate the temperature fluctuations of the laboratory environment; the flow rate is 0.9-1.1 mL / min, which balances the separation degree and analysis efficiency, and although too slow can achieve better separation, but it takes too long, and too fast can lead to high column pressure and insufficient mass transfer; the injection volume is 4-6 μl, which avoids the column overload and fronting peak phenomenon caused by excessive injection, and at the same time ensures that the signal-to-noise ratio of the trace impurity peak meets the quantitative requirements; the injection tray temperature is controlled at 4-6℃, which prevents the degradation of the test solution due to light or oxidation during the waiting process in the automatic injection queue, and ensures the sample stability and result reproducibility. The above parameters work together to achieve baseline separation between the main peak and the 10 impurities, and small fluctuations in each parameter do not affect the separation effect and method robustness, meeting the stable needs of continuous sample analysis in industrial production process.

[0020] 3, The principle of the solvent system in the embodiment of the application is based on the synergistic solubilization effect of dimethyl sulfoxide (DMSO) and acetonitrile: DMSO, as a high-polarity aprotic solvent, has strong solubilization ability for the bromo-aromatic ring, pyrimidine nucleus and sulfonamide group in the molecule of the intermediate 6 of aprocitentan, which can ensure that the sample does not aggregate or precipitate during the dissolution process; acetonitrile, as a compatible solvent for reverse phase chromatography, can adjust the polarity of the solution, avoid the injection needle blockage or chromatographic peak broadening caused by the high viscosity of pure DMSO, and at the same time ensure that the sample solution is quickly mixed with the initial high water phase system of the mobile phase after injection, reducing the peak shape distortion caused by solvent effect. The volume ratio is fixed at 50:45-55, which can ensure the uniformity of the dissolution behavior of different batches of samples.

[0021] 4, The embodiment of the application follows the principle of gradually increasing elution strength in reverse phase chromatography, and realizes the time-sharing separation of impurity groups with large differences in polarity through the combination of multi-order linear gradient and isocratic equilibrium section. This program realizes the full separation of the main component and eight specific impurities within 90 minutes through the precise control of gradient change in multiple segments, and the method has strong tolerance to column temperature and flow rate fluctuations, meeting the stability and reproducibility requirements of continuous analysis of multiple samples in industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the application, the following will briefly introduce the drawings needed to be used in the embodiments, it should be understood that the following drawings only show some embodiments of the application, therefore should not be regarded as a limitation on the scope, for those skilled in the art, without creative labor, other related drawings can also be obtained from these drawings.

[0023] Figure 1 Synthetic route of intermediate 6 of alpirohogil in the present application; Figure 2 Typical chromatogram of the sample addition solution in the present application. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme of the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments.

[0025] Therefore, the detailed description of the embodiments of the present application provided below is not intended to limit the scope of the claimed present application, but only represents selected embodiments of the present application. All other embodiments obtained by a person of ordinary skill in the art without creative labor based on the embodiments in the present application belong to the scope of protection of the present application.

[0026] In the pilot scale-up of intermediate 6 of alpirohogil, batch stability abnormalities were observed: some batches were qualified for release but the yield dropped sharply in the subsequent steps, and others had a sharp increase in degradation products in the accelerated test. Combined with the analysis of the synthetic route, it was found that each step of the multi-step conversion (bromination, fluorination, chlorination, cyclization) from the starting material to the final product could introduce specific impurities - residual p-bromobenzaldehyde, incomplete fluorination products, chlorination by-products and pyrimidine ring hydrolysis products. These impurities are highly similar in structure to the main component, and the conventional HPLC conditions cannot separate them at all.

[0027] Therefore, it is envisaged to construct a neutral pH, low salt buffer mobile phase system to stabilize the sulfonamide structure, start with very weak organic phase, gradually strengthen the elution ability through multi-segment linear-platform combination, and force the separation of strong retention impurities and then quickly balance; simultaneously, the main component conjugate absorption wavelength detection is optimized, and low temperature control prevents sample conversion. It is expected that by precisely controlling the three-dimensional parameters of mobile phase composition-gradient time-detection conditions, the closely adjacent main peak of easily confused impurities can be baseline separated, and the far-end impurities can be clearly distinguished. The method can tolerate conventional fluctuations in column temperature, flow rate, etc. Finally, an exclusive quality control strategy covering the whole scene of synthesis monitoring, release inspection and stability investigation is established, and the quality and cost risks of industrial production caused by uncontrolled impurities are completely solved.

[0028] Intermediate 6 of alpirohogil is prepared by bromination, fluorination, chlorination and cyclization reaction (see synthetic route in Figure 1 ), and impurities such as starting material residues, incomplete fluorination products, chlorination by-products and pyrimidine ring degradation products may be produced in the process.

[0029] Therefore, the embodiment of the present application provides a detection and analysis method for an intermediate 6 of alpirohogil and related impurities, and specifically includes the following contents: I. Instruments and main materials (1) The instrument information is shown in Table 1 below: Table 1

[0030] 2) The reagent information is shown in Table 2 below: Table 2

[0031] Table 3

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

[0033] II. Analysis of research results (1) Method development Instrument: high performance liquid chromatograph Chromatographic column: octadecylsilane bonded silica gel as filler (Napsil Chrom Core AQ C18, 5 μm, 4.6×250 mm) Trapping column: Yue Xiu Ghost-Buster Column Kits, 4.6×50 mm or equivalent trapping column.

[0034] Mobile phase A: 10 mmol / L KH2PO4 solution (pH 7.5)-acetonitrile (95:5).

[0035] The preparation method of the mobile phase A includes weighing anhydrous potassium dihydrogen phosphate to obtain a 1.35 mg / mL solution in water, adjusting the pH to 7.5 with a 0.2 g / ml potassium hydroxide solution, filtering, and then mixing the filtrate and acetonitrile to obtain.

[0036] Mobile phase B: acetonitrile.

[0037] Solvent: dimethyl sulfoxide-acetonitrile (50:50).

[0038] Resolution test solution: Take appropriate amounts of this product, QSR9003-b-4 (impurity H) reference standard, QSR9003-b-7 (impurity G) reference standard, QSR9003-b-8 (impurity B) reference standard, QSR9003-b-8-IMH (impurity C) reference standard, QSR9003-b-9-IMA (impurity D) reference standard, QSR9003-b-9-IMD (impurity A) reference standard, QSR9003-b-9-IMG (impurity F) reference standard, and QSR9003-IML (impurity E) reference standard, place them in the same volumetric flask, dissolve and dilute with solvent to prepare a solution containing approximately QSR9003-b-9 per ml. A mixed solution containing 1 mg of QSR9003-b-4, 1.5 μg each of QSR9003-b-7 and QSR9003-b-9-IMG, and 10 μg each of QSR9003-b-8, QSR9003-b-8-IMH, QSR9003-b-8-IMH, QSR9003-b-9-IMD and QSR9003-IML.

[0039] Test solution: Accurately weigh an appropriate amount of this product, dissolve and dilute it with solvent to prepare a solution containing approximately 1 mg per ml, and shake well. The gradient elution program is shown in Table 5 below: Table 5

[0040] The flow rate was 1.0 ml / min; the column temperature was 40 ℃; the detection wavelength was 250 nm; and the injection volume was 5 μl.

[0041] Determination method: Accurately measure the test solution, inject it into the liquid chromatograph, record the chromatogram, and the results are as follows. Figure 2 As shown.

[0042] Limits: If any impurity peaks are present in the chromatogram of the test solution, the peak area, calculated using the normalization method, shall not exceed the corresponding limits specified in Table 6.

[0043] Table 6

[0044] The calculation formula is: , Among them, A 杂 : Peak area of ​​each impurity in the test solution; A 主 The sum of the peak areas of all components in the test solution.

[0045] III. Data Analysis (1) System applicability A. The solution preparation details are shown in Table 7 below: Table 7

[0046] B, Injection analysis and acceptable standards are shown in Table 8 below: Table 8

[0047] C, Analysis Figure 2 System suitability results - retention time (min) are shown in Table 9 below, system suitability results - peak area are shown in Table 10 below, system suitability results - peak area (%) are shown in Table 11 below, and system suitability results - resolution are shown in Table 12 below: Table 9

[0048] Table 10

[0049] Table 11

[0050] Table 12

[0051] In combination with Tables 9-12, the peak area RSD of the test sample spiked solution was between 0.1-3% (≤5%), the peak area percentage RSD was ≤5% between 0.00-2% (≤5%), and the retention time RSD was between 0.04-0.2% (≤1%) for 5 consecutive injections.

[0052] In the test sample spiked solution, the resolution between the main peak and the adjacent impurity peak was between 9.99-12.53 (≥1.5), and the resolution between each impurity peak and the adjacent peak was between 5.49-47.81 (≥1.5).

[0053] (2) Specificity A, The solution configuration is shown in Table 13 below: Table 13

[0054] B, Injection analysis and acceptable standards are shown in Table 14 below: Table 14

[0055] C, The verification results and conclusions are shown in Table 15 below: Table 15

[0056] As shown in Table 15, the blank solution and the impurity QSR9003-A0-3 did not interfere with the detection of the main peak (QSR9003-b-9) and other impurities.

[0057] The minimum separation between the main peak and the adjacent impurity peak in the test sample spiked solution is 10.02 (>1.5); the minimum separation between each impurity peak and the adjacent peak is 5.53 (>1.5).

[0058] (3) Linearity and range A. Acceptable criteria Test sample concentration level: For QSR9003-b-9, in the concentration range of 25% to 200% of the test sample solution, r≥0.990 (n≥5), the ratio of the Y-axis intercept to the response value of the limit concentration (100%) should not exceed ±10%, and the P value should be >0.05. Report the linear equation, linear graph, residual sum of squares, and residual normal probability graph. Impurity limit level: For each known impurity, in the concentration range of the quantitative limit to 300% of the limit concentration, r≥0.990 (n≥5), the ratio of the Y-axis intercept to the response value of the limit concentration (100%) should not exceed ±10%, and the P value should be >0.05. Report the linear equation, linear graph, residual sum of squares, and residual normal probability graph. Report the correction factor of each known impurity.

[0059] B. Test sample concentration level of the embodiments of the present application is shown in Table 16 below: Table 16

[0060] C. Impurity limit level is shown in Table 17 and Table 18 below: Table 17

[0061] Table 18

[0062] From Table 16, Table 17, and Table 18, it can be known that: Test sample concentration level: For QSR9003-b-9, in the concentration range of 25% to 200% of the test sample solution, the correlation coefficient r is 0.994 (>0.990), the ratio of the Y-axis intercept to the response value of the limit concentration (100%) is -4.2% (within ±10%), and the P value is 0.584 (>0.05).

[0063] Impurity limit level: QSR9003-b-9, QSR9003-b-4, QSR9003-b-7, QSR9003-b-8, QSR9003-b-9-IMG, QSR9003-b-8-IMH, QSR9003-b-9-IMA, QSR9003-b-9-IMD, QSR9003-IML were in the range of 300% limit concentration, the correlation coefficient r was 0.9994-1.0000 (all >0.990), the ratio of Y-axis intercept to limit concentration (100%) response value was-4.2-2.2% (within ±10%), and the P value was 0.142-0.789 (all >0.05).

[0064] QSR9003-b-4 correction factor 0.93, QSR9003-b-7 correction factor 0.56, QSR9003-b-8 correction factor 0.73, QSR9003-b-9-IMG correction factor 0.79, QSR9003-b-8-IMH correction factor 0.84, QSR9003-b-9-IMA correction factor 1.01, QSR9003-b-9-IMD correction factor 1.12, QSR9003-IML correction factor 1.01. In summary, the linearity and range meet the verification requirements.

[0065] (4) Accuracy A, the solution preparation is shown in Table 19 as follows: Table 19

[0066] The calculation formula is: , .

[0067] Among them, A 对 : peak area of each impurity peak of the reference solution; C 对 : concentration of each impurity of the reference solution, μg / ml A 准确度 : peak area of each impurity peak in the accuracy solution; A 本底杂 : peak area of each impurity peak in the test solution; C 加 : concentration of each impurity in the solution added in the preparation of the accuracy solution, μg / ml; M 准 : sample weight of the test sample in the accuracy solution, mg; M 供 : sample weight of the test sample in the test solution, mg; Measured amount: total amount of each component measured in the accuracy solution (μg / ml); Background: Total amount of each component contained in the test sample itself in the accuracy solution (μg / ml); Added amount: Total amount of each component added in the accuracy solution (μg / ml).

[0068] B. Injection analysis and acceptable standards are shown in Table 20 below: Table 20

[0069] C. Verification results and conclusions are shown in Tables 21-28: Table 21

[0070] Table 22

[0071] Table 23

[0072] Table 24

[0073] Table 25

[0074] Table 26

[0075] Table 27

[0076] Table 28

[0077] From Tables 21-28, we can see that: Quantitative limit - 1 part of the accuracy solution, the recovery rate is between 92.2% and 115.7% (between 80% and 120%).

[0078] 50%, 100%, 200% limit concentration accuracy solution, the recovery rate is between 97.4% and 102.3% (between 90% and 110%), the recovery rate RSD (%) is between 0.2 and 1.9% (all <10%).

[0079] In summary: the accuracy meets the verification requirements.

[0080] (5) Lower limit of the range A. Acceptable standards Main components and each known impurity: The quantitative limit solution is injected twice, and the S / N should be ≥10; the quantitative limit concentration should be ≤0.05% of the test sample concentration.

[0081] Limit of detection solution continuous injection 2 needle, S / N≥3.

[0082] B, the quantitative limit and the detection limit of the embodiment of the present application are shown in Table 29 and Table 30 respectively as follows: Table 29

[0083] Table 30

[0084] From Table 29 and Table 30, it can be seen that: The limit of quantification solution continuous injection 2 needle, the minimum S / N of each known impurity peak is 43.6 (>10), and the maximum limit of quantification is 0.04% (<0.05%) of the concentration of the test sample; The limit of detection solution continuous injection 2 needle, the minimum S / N of each known impurity peak is 15.0 (>3).

[0085] Therefore, the sensitivity meets the requirements.

[0086] (6) Precision (repeatability) A, the solution preparation is shown in Table 31 as follows: Table 31

[0087] B, the injection analysis and acceptable standards are shown in Table 32 as follows: Table 32

[0088] C, the verification results and conclusions are shown in Table 33 as follows: Table 33

[0089] From Table 33, among the 6 test sample solutions with standard addition (all with standard addition to the limit), the maximum range of impurities less than 0.2% is 0.01 (<0.05%); the maximum range of impurities between 0.2% and 1.0% is 0.01 (<0.1%); the maximum range of impurities greater than 1.0% and total impurities is 0.11 (<0.2%), and the impurities greater than 0.05% are all 12. In summary, the repeatability meets the requirements.

[0090] (7) Solution stability A, acceptable standards Under the condition of 5°C, at least 24h was observed: At each observation time point: The reference solution: the area ratio of each impurity peak to 0h is within the range of 90% to 110%. The impurity of the QSR9003-b-9 test sample spiked solution is less than 0.2%, and the maximum difference of the impurity is 0.002% (<0.05%), the impurity of the QSR9003-b-9 test sample spiked solution is between 0.2% and 1.0%, and the maximum difference of the impurity is 0.02% (<0.1%), the impurity of the QSR9003-b-9 test sample spiked solution is greater than 1.0%, and the maximum difference of the impurity is 0.2% (≤0.2%), and no new impurity greater than 0.05% is generated.

[0091] B, the verification result of the embodiment of the application: The ratio of each impurity peak in the reference solution to that at 0h is in the range of 97.0% to 101.1% (95% to 105%) within 44.5h at 5°C.

[0092] The maximum difference of the impurity of the QSR9003-b-9 test sample spiked solution is 0.002% (<0.05%) within 123h at 5°C, the maximum difference of the impurity of the QSR9003-b-9 test sample spiked solution is 0.02% (<0.1%) within 123h at 5°C, the maximum difference of the impurity of the QSR9003-b-9 test sample spiked solution is 0.2% (≤0.2%) within 123h at 5°C, and no new impurity greater than 0.05% is generated.

[0093] Conclusion: The reference solution is stable within 44.5h at 5°C, and the test sample spiked solution is stable within 123h at 5°C.

[0094] (7) Durability A, acceptable standard Investigation conditions: column temperature (40±2°C), flow rate (1.0±0.1ml / min), wavelength (250±2nm), mobile phase A pH (7.5±0.1), mobile phase A salt concentration (10mmol / L±10%), mobile phase A proportion (95:5±1%), instrument (Thermo, Shimadzu) Under each durability condition, the blank solvent does not interfere with the detection of the main peak and each known impurity; The separation degree between the main peak and the adjacent impurity peak in the chromatogram of the test sample spiked solution (QSR9003-b-9, QSR9003-b-4, QSR9003-b-7, QSR9003-b-8, QSR9003-b-9-IMG, QSR9003-b-8-IMH, QSR9003-b-9-IMA, QSR9003-b-9-IMD, QSR9003-IML spiked to the limit) under each durability condition is ≥1.5, and the separation degree between each known impurity and the adjacent peak is ≥1.5; Compared with the original condition: the individual impurity with a content less than 0.2% has a range of ≤0.05%, the individual impurity with a content of 0.2% to 1.0% has a range of ≤0.1%, the individual impurity with a content greater than 1.0% has a range of ≤0.2%, the total impurity content has a range of ≤1.0%, and the number of impurities greater than 0.05% is the same; The RRT of each impurity is reported.

[0095] B, the verification results of the embodiments of the application: The blank solvent does not interfere with the detection of the main component and each known impurity; Under each durability condition, the minimum separation degree between the main peak and the adjacent peak in the test sample spiked solution (QSR9003-b-9, QSR9003-b-4, QSR9003-b-7, QSR9003-b-8, QSR9003-b-9-IMG, QSR9003-b-8-IMH, QSR9003-b-9-IMA, QSR9003-b-9-IMD, QSR9003-IML spiked to the limit) is 8.4 (>1.5), and the minimum separation degree between the known impurity and the adjacent peak is 3.6 (>1.5); Compared with the original condition: the individual impurity with a content less than 0.2% has a range of 0.05% (≤0.05%), the individual impurity with a content of 0.2% to 1.0% has a range of 0.06% (<0.1%), the individual impurity with a content greater than 1.0% has a range of 0.2% (≤0.2%), the total impurity content has a range of 0.6% (<1.0%), and the number of impurities greater than 0.05% is 13.

[0096] The RRT of each impurity under each durability condition is relatively stable, and the RRT can be directly used for positioning each known impurity and specific unknown impurity in the future, and the RRT is the average value under each durability condition, as shown in the following Table 34: Table 34

[0097] From Table 34, it can be seen that the column temperature (40±2℃), flow rate (1.0±0.1ml / min), wavelength (250±2nm), pH of mobile phase A (7.5±0.1), salt concentration of mobile phase A (10mmol / L±10%), proportion of mobile phase A (95:5±1%), and instrument (Thermo, Shimadzu) meet the verification requirements.

[0098] The above description is merely a specific implementation of the present application. It should be understood that the above description is only a specific implementation of the present application and is not intended to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for detecting and analyzing an intermediate 6 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 the chromatographic conditions and 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 acetonitrile and a 9-11 mmol / L potassium dihydrogen phosphate solution, the pH of the potassium dihydrogen phosphate solution is 7.4-7.6, and the volume ratio of the potassium dihydrogen phosphate solution to acetonitrile is 90-100:5; The mobile phase B comprises acetonitrile; The structural formula of the alpiroxentan intermediate 6 is: .

2. A method for detecting and analyzing aliskiren intermediate 6 and related impurities according to claim 1, characterized in that, The preparation method of the mobile phase A comprises weighing anhydrous potassium dihydrogen phosphate, dissolving the anhydrous potassium dihydrogen phosphate in water to obtain a 1.2-1.5 g / L solution, adjusting the pH to 7.4-7.6 by using a 0.15-0.25 g / ml potassium hydroxide solution, and then filtering, and then mixing the filtrate and acetonitrile to obtain the mobile phase A.

3. A method for detecting and analyzing one of the intermediate 6 of Alpirotenan and related impurities according to claim 2, characterized in that, The chromatographic conditions further comprise that the detection wavelength is 248-252 nm, the column temperature is 38-42 ℃, the flow rate is 0.9-1.1 mL / min, the injection amount is 4-6 μl, and the injection tray temperature is 4-6 ℃.

4. The method for detecting and analyzing aliskiren intermediate 6 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 6 of Alpiroxetant and related impurities according to claim 4, characterized in that, The relative retention time of the impurity A is 1.04-1.14, the relative retention time of the impurity B is 1.36-1.46, the relative retention time of the impurity C is 1.49-1.55, the relative retention time of the impurity D is 1.56-1.65, the relative retention time of the impurity E is 1.66-1.75, the relative retention time of the impurity F is 1.79-1.89, the relative retention time of the impurity G is 2.25-2.88, and the relative retention time of the impurity H is 2.80-2.

96.

6. The method for detecting and analyzing one intermediate 6 of Alpiroxetant and related impurities according to claim 4, wherein, The content of the impurity A is ≤1.0 wt%, the content of the impurity B is ≤1.0 wt%, the content of the impurity C is ≤1.0 wt%, the content of the impurity D is ≤1.0 wt%, the content of the impurity E is ≤1.0 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%. Further comprising impurity I with a relative retention time of 0.78-0.88 and impurity J with a relative retention time of 1.19-1.29; 7. A method for detecting and analyzing one of the intermediate 6 of Alpirotenan and related impurities according to claim 6, characterized in that, The content of the impurity I is ≤0.5 wt%, and the content of the impurity J is ≤1.0 wt%. The solvent comprises dimethyl sulfoxide and acetonitrile, and the volume ratio of the dimethyl sulfoxide to acetonitrile is 50:45-55.

8. The method for detecting and analyzing aliskiren intermediate 6 and related impurities according to claim 1, wherein, The gradient elution program comprises:

9. The method for detecting and analyzing aliskiren intermediate 6 and related impurities according to claim 1, wherein, 0-6 minutes, the mobile phase A is 100%, and the mobile phase B is 0%; 6-20 minutes, the mobile phase A decreases from 100% to 85%, and the mobile phase B increases from 0% to 15%; 20-45 minutes, the mobile phase A decreases from 85% to 74%, and the mobile phase B increases from 15% to 26%; 45-50 minutes, the mobile phase A remains 74%, and the mobile phase B remains 26%. ​ 50~60 minutes, mobile phase A decreased from 74% to 55%, mobile phase B increased from 26% to 45%; 60~70 minutes, mobile phase A decreased from 55% to 10%, mobile phase B increased from 45% to 90%; 70~80 minutes, mobile phase A kept 10%, mobile phase B kept 90%; 80~81 minutes, mobile phase A increased from 10% to 100%, mobile phase B decreased from 90% to 0%; 81~90 minutes, mobile phase A kept 100%, mobile phase B kept 0%.

10. A method for detecting and analyzing an intermediate 6 of Alpiroxetant and related impurities according to claim 1, characterized in that, The chromatographic column of the liquid chromatograph adopts octadecylsilane bonded silica gel as the filler.

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