Method for determining exo-type norbornimide and related substances thereof

Abstract

This invention relates to the field of pharmaceutical analytical chemistry, and provides a method for the determination of exonorborneilimide and its related substances, wherein the related substances include endonorborneilimide. The determination method is high-performance liquid chromatography (HPLC), and meets the following chromatographic conditions: a chromatographic column packed with octadecylsilane-bonded silica gel is used; the mobile phase consists of an aqueous phase and an organic phase in a volume ratio of (70-90):(30-10), wherein the aqueous phase is an aqueous solution of sodium heptanesulfonate with a pH of 2.8-3.2, and the organic phase is acetonitrile. The determination method provided by this invention has high specificity and accuracy, and can effectively support the quality control of exonorborneilimide.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analytical chemistry, specifically, to a method for determining exomorphic norbornene and its related substances. Background Technology

[0002] Exonorborneneimide (CAS: 14805-29-9), chemically named cis-5-norbornene-exo-2,3-dicarboximide, is a compound with a rigid bridged ring skeleton of norbornene and a highly reactive imide functional group. It has important applications in pharmaceuticals, high-performance polymers, and electronic materials.

[0003] As a key intermediate in the synthesis of many drugs, exonorborneilimide plays an important role in the preparation of the antipsychotic drug lurasidone hydrochloride and the anti-anxiety drug tandospirone. Its quality directly affects the purity of the active pharmaceutical ingredient (API) and is closely related to medication safety. However, during the synthesis and purification of this intermediate, its spatial isomer, endonorborneilimide (CAS: 28871-95-6), is often generated. Both have the same molecular weight and extremely similar physicochemical properties, differing only slightly in the relative position of the longest bridge on the bridged ring, leading to challenges in separation, purification, and quantitative analysis. Under conventional chromatographic and other analytical conditions, baseline separation is often difficult. Furthermore, drug quality studies have shown that even trace amounts of endonorborneilimide impurities can affect the crystallization behavior, stability, and final efficacy of the API, and may even introduce potential toxic side effects.

[0004] Therefore, establishing an analytical method that can efficiently separate and accurately quantify exomorph norbornene imide and its endogenous isomers is crucial for achieving precise quality control of this key intermediate and its downstream drugs. Summary of the Invention

[0005] To address the shortcomings of existing methods in separating exonorbornene imide and its related substances, the present invention aims to provide a novel method for the determination of exonorbornene imide and its related substances. This method effectively separates exonorbornene imide from its endogenous isomers and is suitable for quality monitoring of exonorbornene imide.

[0006] The related substances determined by this invention include endogenous norbornene imide. The determination method provided is high performance liquid chromatography, and the following chromatographic conditions are met: a chromatographic column packed with octadecylsilane-bonded silica gel is used, the mobile phase consists of an aqueous phase and an organic phase in a volume ratio of (70~90):(30~10), wherein the aqueous phase is an aqueous solution of sodium heptanesulfonate with a pH of 2.8~3.2, and the organic phase is acetonitrile.

[0007] The determination method provided by this invention employs a mobile phase system composed of an aqueous solution of sodium heptanesulfonate at a specific pH and acetonitrile, successfully achieving effective separation of exo-norborneimide and its internal isomer. Sodium heptanesulfonate, as an ion-pairing reagent, achieves an optimal balance between hydrophobic interaction and steric hindrance in its C7 alkyl chain length, accurately identifying and distinguishing subtle configurational differences in the bridge rings. Acetonitrile, as the organic phase, contributes to the formation of sharp, symmetrical chromatographic peaks due to its low viscosity, thereby improving resolution and detection sensitivity. Its strong elution capability allows for effective elution of the target analyte even at a low organic phase ratio, helping to maintain the stability of the ion-pairing reagent interaction. This method exhibits excellent specificity. Furthermore, the determination method of this invention uses isocratic elution, which, compared to gradient elution, avoids complex program settings and lengthy column equilibration processes, significantly improving analytical efficiency and reducing baseline drift and retention time fluctuations that may be caused by gradient changes, thus providing superior reproducibility and operational robustness.

[0008] In some embodiments of the present invention, the measured related substances also include other impurities, said other impurities including at least one of impurity a and impurity b, said impurity a being cis-5-norbornane-exo-2,3-dicarboxylic acid having the structure shown in Formula 1; said impurity b being cis-5-norbornane-exo-2-carboxylic acid-3-carboxamide having the structure shown in Formula 2:

[0009]

[0010] In some embodiments of the present invention, the mass concentration of the aqueous solution of sodium heptanesulfonate is 0.05% to 0.15%.

[0011] In some embodiments of the present invention, the volume ratio of the aqueous phase to the organic phase in the mobile phase is (75~85):(25~15).

[0012] In some embodiments of the present invention, the detection wavelength of the high-performance liquid chromatography is 205~215nm, the injection volume is 15~25μL, and the flow rate of the mobile phase is 0.9~1.1mL / min.

[0013] In some embodiments of the present invention, the chromatographic column has an inner diameter of 4.6 mm, a length of 100-250 mm, and a packing particle size of 2.7-5 μm.

[0014] In some embodiments of the present invention, the column temperature used in the high performance liquid chromatography is 35~45°C.

[0015] In some embodiments of the present invention, the determination method includes the following steps:

[0016] (1) Provide the solution to be tested

[0017] The test solution includes a test solution and a control solution; wherein...

[0018] The test solution is an exochrome norbornene imide solution with a concentration of C1, and the control solution is an exochrome norbornene imide solution with a concentration of C2, wherein C1 is 3~6 mg / L, C2 = c × C1, and c = 0.1%~1.0%;

[0019] (2) Inject each test solution into a high performance liquid chromatograph for separation and detection, and obtain the corresponding chromatograms;

[0020] (3) Based on the chromatogram obtained in step (2), calculate the content of relevant substances according to Formula 1:

[0021] Formula 1,

[0022] in, X 杂 Indicates the percentage content of a single relevant substance in the test sample;

[0023] A 对 This represents the peak area of ​​the main component in the control solution (i.e., the peak area of ​​the main component).

[0024] A 杂 Indicates the peak area of ​​a single relevant substance in the test solution;

[0025] This method compares the response values ​​of the chromatographic peaks of each related substance in the test sample with the peaks of the main component control solution at a known concentration, and uses a self-comparison method without correction factors to calculate the content of impurities such as endogenous norbornene, thereby avoiding dependence on specific reference standards for each impurity (especially endogenous isomers).

[0026] In some embodiments of the present invention, the concentration C2 of the control solution is 3~6 μg / mL.

[0027] In some embodiments of the present invention, the test solution further includes a system suitability solution containing the following components at known concentrations: exonorborneolimide, endonorborneolimide, impurity a, and impurity b. Analysis of the system suitability solution directly verifies the separation capability of the chromatographic conditions for the target substance, ensuring reliable qualitative identification and accurate quantification of relevant substances (i.e., various impurities) in the test sample, provided that the above components (especially the main component and its endoisomers) are effectively separated.

[0028] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0029] Exemplary embodiments of the present invention will be described in more detail with reference to the accompanying drawings.

[0030] Figure 1 The chromatogram of the test solution in Example 1;

[0031] Figure 2 This is the chromatogram of the control solution in Example 1;

[0032] Figure 3 The chromatogram of the blank solvent in Example 1;

[0033] Figure 4 This is a chromatogram of the system suitability solution in Example 1;

[0034] Figure 5 The chromatogram of the endogenous norbornene reference standard of Example 1 is shown below.

[0035] Figure 6 The chromatogram of impurity a reference standard from Example 1 is shown below.

[0036] Figure 7 This is the chromatogram of impurity b reference standard from Example 1;

[0037] Figure 8 This is a graph showing the limit of quantitation verification of the norbornene reference standard in Example 2;

[0038] Figure 9 This is a graph showing the limit of quantitation verification of the endogenous norbornene reference standard in Example 2;

[0039] Figure 10 This is a verification diagram of the detection limit of the norborneol imide reference standard in Example 2;

[0040] Figure 11 This is a verification graph of the detection limit of the endogenous norbornene reference standard in Example 2;

[0041] Figure 12 The linear relationship curve for the morphological norbornene imide reference standard in Example 2;

[0042] Figure 13 The chromatogram is of the test solution of Comparative Example 1;

[0043] Figure 14 The chromatogram is of the test solution of Comparative Example 2;

[0044] Figure 15 The chromatogram is of the test solution of Comparative Example 3. Detailed Implementation

[0045] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0046] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0047] In the conventional synthetic route of norborneneimide, the formation of the target product, exonorborneneimide, is often accompanied by the generation of its stereoisomer—endonorborneneimide. Because this impurity differs from the main component only in stereoconfiguration, their molecular structures and polarities are highly similar, resulting in extremely similar retention behaviors and severe peak overlap under conventional chromatographic conditions. This makes effective separation and accurate quantification difficult using existing analytical methods. Furthermore, other impurities may exist during the synthesis and purification of norborneneimide, such as residual intermediates or by-reaction products (e.g., cis-5-norbornene-exo-2-carboxylic acid-3-carboxamide, i.e., impurity b), and degradation products from hydrolysis (e.g., cis-5-norbornene-exo-2,3-dicarboxylic acid, i.e., impurity a).

[0048] To address the challenges of effectively separating and determining the endogenous isomers in exoborneolimide, this invention aims to provide a novel method for determining norborneolimide and its related substances.

[0049] The substances determined in this invention include endogenous norborneolimide, and optionally other impurities. As some embodiments, the other impurities include at least one of impurity a and impurity b, wherein...

[0050] The impurity a is cis-5-norbornane-exo-2,3-dicarboxylic acid as shown in structural formula 1:

[0051]

[0052] The impurity b is cis-5-norbornane-exo-2-carboxylic acid-3-carboxamide as shown in structural formula 2:

[0053]

[0054] The determination method of the present invention is high performance liquid chromatography, and at least the following chromatographic conditions are met: a chromatographic column packed with octadecylsilane-bonded silica gel is used, the mobile phase consists of an aqueous phase and an organic phase in a volume ratio of (70~90):(30~10), wherein the aqueous phase is an aqueous solution of sodium heptanesulfonate with a pH of 2.8~3.2, and the organic phase is acetonitrile.

[0055] This invention employs an isocratic elution system constructed using sodium heptanesulfonate as the ion-pairing reagent and acetonitrile as the organic phase, successfully achieving baseline separation of exo- and endo-norbornenimide on a C18 column. In this system, the C7 alkyl chain length of sodium heptanesulfonate achieves a better balance between hydrophobic interaction and stereoselectivity, exhibiting significantly different retention behaviors for the two stereoconfigurations compared to alkyl sulfonates with shorter or longer chains (e.g., sodium pentanesulfonate or sodium octanesulfonate). Simultaneously, acetonitrile is chosen as the organic phase. As a dipolar aprotic solvent, it possesses lower viscosity and stronger elution capacity compared to protic solvents such as methanol. This not only facilitates synergistic effects with sodium heptanesulfonate, enhancing the ability to identify subtle structural differences between the two isomers, but also improves peak shape, increases resolution and detection sensitivity, and achieves effective separation at a lower organic phase ratio, thus contributing to the stability of the entire ion-pairing system.

[0056] In this invention, the pH of the aqueous phase is 2.8~3.2, for example, 2.8, 2.9, 3.0, 3.1, 3.2, etc. A pH that is too low (<2.8) may affect the stability of sodium heptanesulfonate reagent and exacerbate potential corrosion of the chromatographic system flow path; while a pH that is too high (>3.2) may decrease the stability of sodium heptanesulfonate and increase the risk of corrosion of the chromatographic system flow path; and when the pH value is higher than 3.2, norbornene imide is difficult to maintain a sufficiently protonated state, resulting in an unstable ion-pair complex formed with sodium heptanesulfonate, weakening the ion-pair retention mechanism, and affecting the separation effect of endo- and exo-isomers.

[0057] In some embodiments, the mass concentration of the aqueous solution of sodium heptanesulfonate is 0.05% to 0.15%, such as 0.05%, 0.07%, 0.08%, 0.10%, 0.12%, 0.15%, etc., preferably 0.10%.

[0058] In some embodiments, the aqueous phase is prepared by weighing 0.5-1.5 g of sodium heptanesulfonate, dissolving it in water to 1000 mL, and adjusting the pH to 2.8-3.2 with phosphoric acid. As a more specific example, the aqueous phase is obtained by dissolving 1.0 g of sodium heptanesulfonate in water to 1000 mL, and then adjusting the pH to 3.0 with phosphoric acid.

[0059] In this invention, pH adjustment of the aqueous phase with phosphoric acid can typically be accomplished in two steps: first, chromatographic grade phosphoric acid (concentration can be 85%) is added to coarsely adjust the pH to near the target range, and then 30% dilute phosphoric acid is used for fine adjustment. As a specific example, when the target pH value of the aqueous phase is 3.0, the pH can be coarsely adjusted to the range of 3.2~3.5 using chromatographic grade phosphoric acid, and then finely adjusted to the target value using dilute phosphoric acid.

[0060] In this invention, the volume ratio of the aqueous phase to the organic phase (acetonitrile) in the mobile phase is (70~90):(30~10), for example, 70:30, 77:23, 75:25, 78:22, 80:20, 85:15, 88:12, 90:10, etc. Preferably, the volume ratio of the aqueous phase to the organic phase in the mobile phase is (75~85):(25~15).

[0061] In some embodiments, the detection wavelength of the high-performance liquid chromatography is 205-215 nm, such as 205 nm, 208 nm, 210 nm, 212 nm, 215 nm, etc. Within this wavelength range, exo-norborneilimide and its endo-isomer have high detection sensitivity.

[0062] In some embodiments, the chromatographic column has an inner diameter of 4.6 mm and a length of 100-250 mm, such as 100 mm, 150 mm, 200 mm, 250 mm, etc., and the packing material particle size is 2.7-5 μm, such as 2.7 μm, 4 μm, 5 μm, etc. Preferably, the chromatographic column has an inner diameter of 4.6 mm, a length of 250 mm, and a packing material particle size of 5 μm.

[0063] In some embodiments, the injection volume of the high-performance liquid chromatography (HPLC) is 15-25 μL, such as 15 μL, 20 μL, or 25 μL, preferably 20 μL; the flow rate of the mobile phase is 0.9-1.1 mL / min, such as 0.9 mL / min, 1.0 mL / min, or 1.1 mL / min, preferably 1.0 mL / min. This injection volume provides sufficient detection response while avoiding column overload; combined with moderate flow rates, it improves analytical efficiency while ensuring separation effectiveness.

[0064] The present invention preferably employs a self-control method without correction factors to determine the content of endogenous norborneneimide. According to some specific embodiments, the determination method includes the following steps:

[0065] (1) Provide the solution to be tested

[0066] The test solution includes the test solution and the control solution;

[0067] (2) Inject each test solution into a high performance liquid chromatograph for separation and detection, and obtain the corresponding chromatograms;

[0068] (3) Based on the chromatogram obtained in step (2), calculate the impurity content according to Formula 1:

[0069] Formula 1,

[0070] in, X 杂 Indicates the percentage content of a single relevant substance in the test sample;

[0071] A 对 This indicates the area of ​​the main peak in the control solution;

[0072] A 杂 It represents the peak area of ​​a single relevant substance in the test solution.

[0073] Formula 1 is a general calculation formula applicable to calculating the content of various related substances. X 杂 The specific determination depends on the target being measured. For example, when testing for the content of endogenous norbornene imide, X 杂 This indicates the percentage content of endogenous norbornene imide. When testing for the content of impurity a, X 杂 This indicates the percentage content of impurity a.

[0074] In step (1), the test solution is an exochrome norbornene solution with a concentration of C1, where C1 is specifically 3~6 mg / L, such as 3.0 mg / L, 3.5 mg / L, 4.0 mg / mL, 4.5 mg / mL, 5.0 mg / mL, 5.2 mg / mL, 5.5 mg / mL, 6.0 mg / mL, etc.

[0075] The control solution is an exoborneol imide solution with a concentration of C2, where C2 = c × C1, and c represents a constant, ranging from 0.1% to 1.0%, such as 0.1%, 0.3%, 0.6%, 0.7%, 0.8%, 1.0%, etc.

[0076] In some embodiments, the concentration C2 of the control solution is 3~6 μg / mL, for example 3.0 μg / mL, 4.0 μg / mL, 5.0 μg / mL, 6.0 μg / mL, etc.

[0077] As some specific examples, the control solution may be prepared by diluting the test solution 1000 times.

[0078] The high-performance liquid chromatography (HPLC) conditions provided by this invention can effectively separate and quantify the main component (exogenous norbornenimide) and its key stereoisomer impurity (endogenic norbornenimide). Simultaneously, it also exhibits good separation and detection capabilities for other impurities that may exist during synthesis and purification (such as impurity a and impurity b). Under the provided chromatographic conditions, all other impurity peaks that achieve baseline separation from the main peak and the endogenic norbornenimide peak do not require prior knowledge of their specific chemical structures; their relative contents can be calculated using Formula 1 from the same main component reference solution.

[0079] This invention does not impose any particular restriction on the source of the described norbornene imide; it can be a self-made product or a commercially purchased product. As a specific example, the described norbornene imide can be obtained by the following method: First, hexahydro-4,7-methylbenzofuran-1,3-one is mixed with a 15% ammonia solution at a mass ratio of 1:(3.5~4.5). The mixture is first subjected to an ammonolysis ring-opening reaction at 45~50℃ for 30 minutes, and then heated to 98~102℃ for a dehydration cyclization reaction for 1.5 hours. After the reaction is complete, the mixture is cooled and filtered to obtain a crude product, which is then washed with ice water to remove residual ammonia and water-soluble salts. Subsequently, the washed crude product is heated under reflux in ethyl acetate until completely dissolved, decolorized by activated carbon, and filtered while hot. The filtrate is cooled and crystallized to obtain the primary product. The above primary product was added to an ethanol-water mixed solvent with a volume ratio of 4:1, heated to reflux to completely dissolve the solid, then slowly cooled to room temperature and allowed to stand to crystallize. After filtration and vacuum drying, the product was obtained as white crystalline norbornene.

[0080] In some preferred embodiments, to facilitate qualitative and quantitative analysis of the substances including impurities a and b, the test solution may further include a system suitability solution containing the following components at known concentrations: exoborneolimide, endoborneolimide, impurity a, and impurity b. As examples, in the system suitability solution, the concentration of exoborneolimide may be 0.8–1.5 mg / mL, the concentration of endoborneolimide may be 3–100 μg / mL, the concentration of impurity a may be 10–100 μg / mL, and the concentration of impurity b may be 10–100 μg / mL.

[0081] In some embodiments, to perform qualitative and quantitative analysis of impurities a and b in the test sample, the test solution further includes: a reference solution for impurity a and a reference solution for impurity b. By comparing the chromatograms of each reference solution with the test sample solution, the chromatographic peaks corresponding to impurities a and b in the chromatogram of the test sample can be accurately located.

[0082] In some embodiments, each test solution is prepared using a diluent with the same composition as the mobile phase. This eliminates the solvent effect caused by solvent strength mismatch and avoids chromatographic peak distortion and retention time drift.

[0083] The following describes embodiments of the present invention. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0084] Example 1

[0085] 1. Preparation of solution

[0086] 1) Preparation of mobile phase

[0087] Dissolve 1.0 g of sodium heptanesulfonate in water and bring the volume to 1000 mL. Adjust the pH to 3.0 with phosphoric acid to obtain an aqueous phase. Mix the aqueous phase with acetonitrile at a volume ratio of 80:20 to obtain the mobile phase.

[0088] 2) Preparation of the test solution

[0089] Blank solvent: The mobile phase is used as the blank solvent.

[0090] Test solution: Weigh approximately 30 mg of borneolimide (provided by Shenyang Funing Pharmaceutical Co., Ltd., batch number S230701, the same below) accurately, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with the mobile phase, shake well to obtain the test solution (approximately 3.0 mg / mL).

[0091] Control solution: Accurately measure 1 mL of the test solution and place it in a 10 mL volumetric flask. Dilute to the mark with the mobile phase and shake well to obtain the stock solution. Accurately measure 1 mL of the stock solution and place it in a 100 mL volumetric flask. Dilute to the mark with the mobile phase and shake well to obtain the control solution (approximately 3 μg / mL).

[0092] Internal norborneolimide reference stock solution: Weigh approximately 10 mg of internal norborneolimide (provided by Shanghai Jingrong Chemical Technology Co., Ltd., batch number 20120628, the same below) accurately, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with the mobile phase, shake well, and obtain internal norborneolimide reference stock solution (approximately 1.0 mg / mL).

[0093] Endogenous norborneol imide reference solution: Accurately measure 0.3 mL of the above stock solution into a 100 mL volumetric flask, dilute to the mark with the mobile phase, and shake well to obtain (approximately 3 μg / mL).

[0094] Impurity A stock solution: Weigh approximately 10 mg of cis-5-norbornane-exo-2,3-dicarboxylic acid (impurity A, purchased from TLC Pharmaceutical Standards, batch number 3219-066A1, the same below), place it in a 10 mL volumetric flask, dissolve and dilute to the mark with the mobile phase, shake well, and obtain impurity A stock solution (approximately 1 mg / mL).

[0095] Impurity A reference solution: Accurately measure 1 mL of impurity A stock solution into a 10 mL volumetric flask, dilute to the mark with the mobile phase, and shake well to obtain (approximately 100 μg / mL).

[0096] Impurity B stock solution: Weigh approximately 10 mg of cis-5-norbornene-exo-2-carboxylic acid-3-carboxamide (impurity B, purchased from Shenzhen Jianzhu Technology Co., Ltd., batch number 313417, the same below), place it in a 10 mL volumetric flask, dissolve and dilute to the mark with the mobile phase, shake well, and obtain impurity B stock solution (approximately 1 mg / mL).

[0097] Impurity b reference solution: Accurately weigh 1 mL of impurity b stock solution and place it in a 10 mL volumetric flask. Dilute to the mark with the mobile phase and shake well to obtain (approximately 100 μg / mL).

[0098] System suitability solution: Accurately weigh approximately 10 mg of the test sample and place it in a 10 mL volumetric flask. Add 1 mL of internal type norbornene reference stock solution, 1 mL of impurity a reference stock solution, and 1 mL of impurity b reference stock solution, respectively. Dissolve and dilute to the mark with the mobile phase, and shake well to obtain the solution.

[0099] 2. Chromatographic analysis

[0100] Chromatographic column: Cosmosil 5C18-AR-Ⅱ, octadecylsilane bonded silica (C18) column (250mm × 4.6mm, 5μm), NacalaiTesque Co., Ltd.

[0101] The detection wavelength is 210nm;

[0102] The injection volume was 20 μL;

[0103] The flow rate was 1.0 mL / min; the column temperature was 40 °C.

[0104] Blank solvent, test solution, control solution, system suitability solution, internal norbornene reference solution, impurity a reference solution, and impurity b reference solution were injected into the high-performance liquid chromatograph (HPLC) and chromatograms were recorded until the main component peak was completely eluted. The test results are as follows: Figures 1-7 As shown.

[0105] pass Figures 1 to 7Analysis shows that the test method in this embodiment has strong specificity and good system applicability. First, in a blank solvent ( Figure 3 No interference was observed at the target analyte peak position, indicating that the solvent does not affect the detection. Additionally, Figure 1 Retention time (t) R The chromatographic peak was at 9.386 min, and... Figure 2 t in R= 9.435min Figure 4 Chinese R The main peak at 9.377 min corresponds to the morphological norbornelimide; Figure 1 Chinese R The chromatographic peak at 7.231 min was observed. Figure 4 Chinese R =7.227min Figure 5 Chinese R The chromatographic peaks corresponding to 7.270 min were all attributed to endorphin imide. Furthermore, Figure 4 Chinese R The chromatographic peak at 5.644 min corresponds to cis-5-norbornene-exo-2-carboxylic acid-3-carboxamide (and... Figure 7 Chinese R =5.678 min peak consistency). Figure 4 Chinese R The chromatographic peak at 12.886 min corresponds to cis-5-norbornene-exo-2,3-dicarboxylic acid (and... Figure 6 Chinese R =13.007min Figure 1 Chinese R =12.944 min peak consistency). It can be seen that each impurity peak is completely separated from the main peak and from each other, and all achieve baseline separation, which meets the system applicability requirements.

[0106] 3. Quantitative analysis

[0107] The content of endo-norborneolimide in the test sample was calculated by substituting the peak area of ​​the endogenous norborneolimide in the test sample solution and the main peak area in the control solution into Formula 1 (where c=1 / 1000), and the content of endogenous norborneolimide in the test sample was found to be 0.227%.

[0108] Example 2: Methodological Validation

[0109] 1. Limit of Quantification (LOQ) and Precision of LOQ

[0110] 1) Limit of quantitation and precision of norbornene imide

[0111] Accurately weigh 10 mg of exonorborneilimide reference standard and place it in a 100 mL volumetric flask. Dissolve and dilute to the mark with the mobile phase, and shake well to obtain an exonorborneilimide stock solution (approximately 0.1 mg / mL). Further dilute the stock solution stepwise with the mobile phase to obtain reference standard solutions of different concentrations. Analyze the reference standard solutions according to the chromatographic method described in Example 1.

[0112] When the concentration of exoborneol imide in the solution is 0.4630 μg / mL, such as Figure 8 As shown, the target chromatographic peak (t) was measured. R The signal-to-noise ratio (S / N = 10.163) of this method was 8.697 min, so the limit of quantitation was determined to be 0.4630 μg / mL, which is equivalent to 9.26 ng based on the injection volume.

[0113] To verify the precision of the limit of quantitation level, six separate solutions of exoborneolimide reference standard at the limit of quantitation concentration were prepared independently according to the above method. Chromatographic analysis was performed on each solution according to the chromatographic analysis method in Example 1, and the peak areas were recorded. The results are shown in Table 1.

[0114] Table 1

[0115]

[0116] 2) Limit of quantitation and precision of endogenous norbornene imide

[0117] Accurately weigh approximately 10 mg of endogenous norbornene imide reference standard and place it in a 100 mL volumetric flask. Dissolve and dilute to the mark with the mobile phase, and mix well to obtain an endogenous norbornene imide stock solution (approximately 0.1 mg / mL). Further dilute the stock solution stepwise with the mobile phase to obtain reference standard solutions of different concentrations. Analyze the reference standard solutions according to the chromatographic method described in Example 1.

[0118] When the concentration of endorphin in the solution is 0.2067 μg / mL, such as Figure 9 As shown, the target chromatographic peak (t) was measured. R The signal-to-noise ratio (S / N = 10.237) of this method was 7.222 min, so the limit of quantitation was determined to be 0.2067 μg / mL; which is equivalent to 4.13 ng based on the injection volume.

[0119] To verify the precision of the limit of quantitation level, six solutions of internal norbornene at the limit of quantitation concentration were prepared independently according to the above method. Chromatographic analysis was performed on each solution under the chromatographic conditions of Example 1, and the peak areas were recorded. The results are shown in Table 2.

[0120] Table 2

[0121]

[0122] As shown in Table 2, the relative standard deviation (RSD) of the peak area measured in the six measurements was 7.52% (<10%), indicating that the method in Example 1 has good precision at the limit of quantitation concentration and can perform reliable and accurate quantitative analysis.

[0123] 2. Limit of Detection (LOD)

[0124] 1) Limit of detection for norborneol imide

[0125] The stock solution of norbornene (approximately 0.1 mg / mL) was gradually diluted with the mobile phase to obtain reference solutions of different concentrations. The reference solutions were analyzed and tested according to the chromatographic method described in Example 1.

[0126] When the concentration of exoborneol imide in the solution is 0.1543 μg / mL, such as Figure 10 As shown, the target chromatographic peak (t) was measured. R The signal-to-noise ratio (S / N) of the sample (8.867 min) was 3.786, therefore the limit of detection (LOD) of this method was determined to be 0.1543 μg / mL; which is equivalent to 3.09 ng based on the injection volume.

[0127] 2) Limit of detection for endogenous norborneolimide

[0128] The stock solution of endorphinimide (approximately 0.1 mg / mL) was gradually diluted with the mobile phase to obtain reference solutions of different concentrations. The reference solutions were analyzed and tested according to the chromatographic method described in Example 1.

[0129] When the concentration of endorphin in the solution is 0.0723 μg / mL, such as Figure 11 As shown, the target chromatographic peak (t) was measured. R The signal-to-noise ratio (S / N) of the sample (7.227 min) was 3.694, therefore the limit of detection (LOD) of this method was determined to be 0.0723 μg / mL; which is equivalent to 1.45 ng based on the injection volume.

[0130] 3. Linearity and Range Examination

[0131] Accurately weigh 10.29 mg of exoborneolimide reference standard and place it in a 100 mL volumetric flask. Dissolve and dilute with the mobile phase to prepare a stock solution with an exoborneolimide concentration of 0.1029 mg / mL. Prepare a series of standard solutions of varying concentrations by accurately measuring different volumes of the stock solution and quantitatively diluting them with the mobile phase. Following the chromatographic analysis method of Example 1, accurately measure 20 µL of each standard solution and inject it into the liquid chromatograph, recording the peak area.

[0132] Linear regression analysis was performed with the injection volume W (ng) of the exomorphic norbornene imide as the x-axis and the corresponding peak area A as the y-axis. The results are shown in Table 3, and the linear regression relationship is shown in [Table 3]. Figure 12 .

[0133] Table 3

[0134]

[0135] Figure 12 In the regression equation, A = 6.4772 × 10⁻⁶. 3 W+7.4319×10 4 r=0.9998.

[0136] The results showed that the injection amount of morphoborneolimide in the range of 9.259~1234.596 ng had a good linear relationship with the chromatographic peak area.

[0137] 4. Solution stability

[0138] Accurately weigh 30 mg of the exoborneolimide reference standard and place it in a 10 mL volumetric flask. Dissolve and dilute it with the mobile phase to prepare a solution with an exoborneolimide concentration of approximately 3.0 mg / mL. Place the solution at room temperature and take samples at 0 hours after preparation and at 5, 13, 17, and 27 hours.

[0139] Referring to the chromatographic analysis method in Example 1, 20 μL of each solution at different storage times was accurately measured and injected into the liquid chromatograph for analysis. The peak area was recorded and the relative standard deviation (RSD) was calculated. The results are shown in Table 4.

[0140] Table 4

[0141]

[0142] As shown in Table 4, the peak area RSD at each time point was 3.06%, indicating that the morphological norbornene reference solution had good stability after being placed at room temperature for 27 hours.

[0143] 5. Instrument reproducibility

[0144] Accurately weigh 0.3 mL of exoborneolimide stock solution (approximately 0.1 mg / mL), place it in a 10 mL volumetric flask, dilute with the mobile phase to prepare a volumetric calibrator, and shake well to prepare an exoborneolimide solution (approximately 3.0 μg / mL). Accurately measure 20 μL of this solution and, following the chromatographic analysis method in Example 1, inject it six times consecutively, recording the peak area of ​​each chromatographic peak. The results are shown in Table 5.

[0145] Table 5

[0146]

[0147] As shown in Table 5, the relative standard deviation (RSD) of the peak area measured by 6 consecutive injections was 0.38%, which proves that the instrument has good precision.

[0148] 6. Repeatability

[0149] Following the chromatographic analysis method of Example 1, six test solutions and control solutions were prepared independently in parallel and injected sequentially for analysis. The content (%) of the largest single impurity (impurity a) among endogenous norbornene and other impurities in the test solution was calculated, and the results are shown in Table 6.

[0150] Table 6

[0151]

[0152] The results showed that the content of endogenous norbornene was consistent across six independent determinations, with a relative standard deviation (RSD) of 4.18; the relative standard deviation (RSD) of impurity a was 7.68%. This demonstrates that the test method in Example 1 has good repeatability and the test results are reliable.

[0153] 7. Intermediate precision

[0154] At different times, by different researchers, six test solutions, control solutions, and endogenous norbornene reference solutions were prepared independently and in parallel according to the chromatographic analysis method of Example 1, and were injected and analyzed sequentially. The content (%) of endogenous norbornene in the test solutions was calculated, and the results are shown in Table 7.

[0155] Table 7

[0156]

[0157] The results showed that the content of endogenous norbornene was consistent across six independently determined assays, with a relative standard deviation (RSD) of 3.25. This demonstrates that the test method in Example 1 has good intermediate precision and the test results are reliable.

[0158] 8. Durability

[0159] The chromatographic analysis method of Example 1 was used to determine impurities in exoborneolimide. The difference was that the chromatographic conditions were adjusted. Specifically, the key parameters such as the detection wavelength, mobile phase composition, flow rate, aqueous phase pH, column temperature, and different brands of chromatographic columns were adjusted. The test solution, control solution, and endoborneolimide reference solution were chromatographically detected and quantitatively analyzed. The test results are shown in Tables 8-13. The "standard" in the tables corresponds to the corresponding chromatographic conditions of Example 1.

[0160] Table 8

[0161]

[0162] Table 9

[0163]

[0164] Table 10

[0165]

[0166] Table 11

[0167]

[0168] Table 12

[0169]

[0170] Table 13

[0171]

[0172] Note: The Compass C18 column (250mm×4.6mm, 5μm) was purchased from RPG Electronics Technology Co., Ltd.

[0173] The results showed that endogenous norbornene imide could be stably detected under varying chromatographic parameters. Although the measured values ​​fluctuated, the absolute content changes were minimal, and all endogenous norbornene imide results were below the quality control standard limit (<0.8%). This indicates that the error introduced by the variations in the above parameters is acceptable in rigorous pharmaceutical quality control practices, fully demonstrating the robustness of the method.

[0174] Example 3

[0175] The impurities in exoborneolimide were determined using the chromatographic analysis method described in Example 1, except that the test samples were replaced with Sample I and Sample II. The test solution and control solution were prepared according to the method in Example 1, and then injected into a high-performance liquid chromatograph for chromatographic analysis. The results are shown in Table 14.

[0176] Table 14

[0177]

[0178] Note: Sample I was provided by Beijing Kefurui Technology Development Co., Ltd., and Sample II was provided by Shenyang Funing Pharmaceutical Co., Ltd.

[0179] As shown in Table 14, the endo-norborneolimide in both batches of samples could be accurately determined. It is evident that the method of Example 1 can effectively determine the endo-isomer in exo-norborneolimide samples from different sources.

[0180] Comparative Example 1

[0181] Chromatographic column: Compass C18 column (250mm × 4.6mm, 5μm) from R&D Technology Co., Ltd.

[0182] The detection wavelength is 210nm;

[0183] The injection volume was 20 μL;

[0184] The flow rate was 1.0 mL / min;

[0185] The column temperature is 40℃.

[0186] Mobile phase: Dissolve 1.0 g of sodium heptanesulfonate in water and bring the volume to 1000 mL. Adjust the pH to 3.0 with phosphoric acid to obtain an aqueous phase. Use this aqueous phase as mobile phase A and acetonitrile as mobile phase B, and perform gradient elution according to the procedure shown in Table 15.

[0187] Table 15

[0188]

[0189] Diluent: It is prepared by mixing mobile phase A and mobile phase B at a volume ratio of 90:10.

[0190] Test solution: Weigh approximately 30 mg of borneolimide (batch number S230701) accurately, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with diluent, shake well to obtain the test solution (approximately 3.0 mg / mL).

[0191] Impurity reference solution: Weigh 10.0 mg of intra-type norborneneimide accurately, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with diluent, and shake well to obtain the reference stock solution; accurately measure 3 mL of the stock solution and place it in a 100 mL volumetric flask, dissolve and dilute to the mark with diluent, and shake well to obtain the impurity reference solution (concentration approximately 30 μg / mL).

[0192] The test solution and impurity reference solution were injected into the high-performance liquid chromatograph for analysis.

[0193] The test results of the test sample solution are as follows Figure 13 As shown in the figure, because the exogenous norborneneimide uses a terminal absorption wavelength of 210 nm, the gradient baseline at this wavelength is not stable, with many ghost peaks (non-impurity peaks) and significant background interference. Furthermore, comparison with the endogenous isomer reference standard revealed that the main peak (t) in the test sample... RThe peak (6.716 min) and stereoisomeric impurities (endotype) could not be effectively separated. Furthermore, unknown impurities co-eluted after the main peak, interfering with its determination. Therefore, this gradient flow elution method is unsuitable for determining the intermediate norbornene.

[0194] Comparative Example 2

[0195] Chromatographic column: Compass C18 column (250mm × 4.6mm, 5μm) from R&D Technology Co., Ltd.

[0196] The detection wavelength is 210nm;

[0197] The injection volume was 20 μL;

[0198] The flow rate was 1.0 mL / min;

[0199] The column temperature is 40℃.

[0200] Mobile phase: Dissolve 1.0 g sodium pentanesulfonate in water and bring the volume to 1000 mL. Adjust the pH to 3.0 with phosphoric acid to obtain an aqueous phase. Mix the aqueous phase with acetonitrile at a volume ratio of 80:20 to obtain the mobile phase.

[0201] Test solution: Weigh approximately 30 mg of borneolimide (batch number S230701) accurately, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with the mobile phase, shake well to obtain the test solution (approximately 3.0 mg / mL).

[0202] Impurity reference solution: First, accurately weigh about 10 mg of endogenous norborneneimide and place it in a 10 mL volumetric flask. Dissolve and dilute to the mark with the mobile phase, and shake well to obtain a stock solution (about 1.0 mg / mL). Then, accurately measure 3 mL of the stock solution and place it in a 100 mL volumetric flask. Dilute to the mark with the mobile phase, and shake well to obtain an impurity (endogenous norborneneimide) reference solution (about 30 μg / mL).

[0203] The test solution and impurity reference solution were injected into the high-performance liquid chromatograph for analysis.

[0204] The test results of the test sample solution are as follows: Figure 14 As shown. Localization analysis using the spectrum of the impurity reference solution (endo-norborneolimide) revealed that the retention time t of the main peak of exo-norborneolimide was... R =6.700 min, retention time t of endogenous isomer R =5.119min, the separation between the two was poor, failing to achieve baseline separation. Meanwhile, near the main peak (t RAn unknown impurity peak, which was difficult to completely separate from the main peak, appeared at 6.289 min, and the main peak exhibited significant tailing. These phenomena indicate that under these chromatographic conditions, the resolution of the key substance pair was poor, and the peak shape was unsatisfactory. Therefore, the ion-pair chromatographic conditions constructed with sodium pentanesulfonate cannot reliably determine the related substances of exoborneolimide.

[0205] Comparative Example 3

[0206] Chromatographic column: Compass C18 column (250mm × 4.6mm, 5μm) from R&D Technology Co., Ltd.

[0207] The detection wavelength is 210nm;

[0208] The injection volume was 20 μL;

[0209] The flow rate was 1.0 mL / min;

[0210] The column temperature is 40℃.

[0211] Mobile phase: Dissolve 1.0 g sodium pentanesulfonate in water and bring the volume to 1000 mL. Adjust the pH to 3.0 with phosphoric acid to obtain an aqueous phase. Mix the aqueous phase with methanol at a volume ratio of 80:20 to obtain the mobile phase.

[0212] Test solution: Weigh approximately 30 mg of borneolimide accurately, place it in a 10 mL volumetric flask, dissolve and dilute to the mark with the mobile phase, and shake well to obtain the test solution (approximately 3.0 mg / mL).

[0213] Impurity reference solution: First, accurately weigh about 10 mg of endogenous norborneneimide and place it in a 10 mL volumetric flask. Dissolve and dilute to the mark with the mobile phase, and shake well to obtain a stock solution (about 1.0 mg / mL). Then, accurately measure 3 mL of the stock solution and place it in a 100 mL volumetric flask. Dilute to the mark with the mobile phase, and shake well to obtain an impurity (endogenous norborneneimide) reference solution (about 30 μg / mL).

[0214] The test solution and impurity reference solution were injected into the high-performance liquid chromatograph for analysis.

[0215] The test results of the test sample solution are as follows: Figure 15As shown in the figure, localization analysis of the spectrum of the impurity reference solution revealed that the internal norbornene imide in the test sample eluted at a retention time of approximately 10.207 min. However, this peak exhibited severe peak-fronting distortion. Such asymmetric peak shapes significantly affect integration accuracy, leading to a decrease in the precision and accuracy of the quantitative results for this impurity. This indicates that under these chromatographic conditions, methanol, as an organic phase, exhibits a mismatch in its solubility-mass transfer behavior with this key impurity, causing band broadening within the column. Therefore, a mobile phase system using methanol is not suitable for the reliable detection of internal norbornene imide.

[0216] In summary, the determination method provided by this invention is highly specific and accurate, effectively supporting the quality control of exoborneolimide. In the system suitability test, this method can achieve baseline separation between the main peak of exoborneolimide and its internal isomers, impurity a, and impurity b, with no interference from the blank solvent, indicating good method specificity. Verification showed that the method has a low limit of quantitation, meeting the accurate quantification requirements for trace impurities; good linearity (correlation coefficient r > 0.999); the relative standard deviation (RSD) of repeatability and intermediate precision meets the requirements; the test solution exhibits good stability within the specified time; and robustness test results show that fluctuations in key chromatographic parameters within a certain range do not affect the separation and quantification results. In conclusion, this method is suitable for process monitoring and finished product release inspection of exoborneolimide raw materials and their key intermediates, providing a reliable analytical tool for their quality control.

[0217] The various embodiments of the present invention have been described above. These embodiments are merely illustrative of the technical solutions of the present invention and are not intended to limit it, nor are they exhaustive, nor are they limited to the disclosed embodiments. Those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention. In particular, as long as there is no structural conflict, the various technical features mentioned in the various embodiments can be combined in any way. The present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A method for determining morphological norbornene and its related substances, characterized in that, The related substances include endorphinimide, and the determination method is high-performance liquid chromatography (HPLC), and the following chromatographic conditions are met: A chromatographic column packed with octadecylsilane-bonded silica gel was used. The mobile phase consisted of an aqueous phase and an organic phase in a volume ratio of (70~90):(30~10), wherein the aqueous phase was an aqueous solution of sodium heptanesulfonate with a pH of 2.8~3.2, and the organic phase was acetonitrile. The aqueous solution of sodium heptanesulfonate has a mass concentration of 0.05% to 0.15%. The detection wavelength of the high-performance liquid chromatography is 205~215nm, the injection volume is 15~25μL, and the flow rate of the mobile phase is 0.9~1.1mL / min.

2. The determination method according to claim 1, characterized in that, In the mobile phase, the volume ratio of the aqueous phase to the organic phase is (75~85):(25~15).

3. The determination method according to claim 1, characterized in that, The chromatographic column has an inner diameter of 4.6 mm, a length of 100~250 mm, and a packing particle size of 2.7~5 μm.

4. The determination method according to claim 1, characterized in that, The column temperature used in the high-performance liquid chromatography is 35~45℃.

5. The determination method according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Provide the solution to be tested; The test solution includes a test solution and a control solution; wherein... The test solution is an exonorborneolimide solution with a concentration of C1, and the control solution is an exonorborneolimide solution with a concentration of C2. C1 is 3~6 mg / L, C2 = c × C1, c = 0.1%~1.0%; (2) Inject each test solution into a high performance liquid chromatograph for separation and detection, and obtain the corresponding chromatograms; (3) Based on the chromatogram obtained in step (2), calculate the content of relevant substances according to Formula 1: Formula 1, in, X 杂 Indicates the percentage content of a single relevant substance in the test sample; A 对 This indicates the area of ​​the main peak in the control solution; A 杂 It represents the peak area of ​​a single relevant substance in the test solution.

6. The determination method according to claim 5, characterized in that, The concentration C2 of the control solution was 3~6 μg / mL.

7. The determination method according to claim 5, characterized in that, The related substances also include other impurities, which include at least one of impurity a and impurity b, wherein impurity a is cis-5-norbornane-exo-2,3-dicarboxylic acid as shown in Formula 1, and impurity b is cis-5-norbornane-exo-2-carboxylic acid-3-carboxamide as shown in Formula 2. 。 8. The determination method according to claim 7, characterized in that, The test solution also includes a system suitability solution containing the following components at known concentrations: exoborneolimide, endoborneolimide, impurity a, and impurity b.

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