Cyclic compound, purification method thereof and application of cyclic compound in quality research

Abstract

The present disclosure provides cyclic compounds and methods of purification and use thereof in quality research. The cyclic compound is clear in structure and reliable in purity, can be used as a reference substance for quality research of a nylon material or a product containing the nylon material, and is expected to improve the reliability of safety evaluation of a medicine packaging material.

Description

Technical Field

[0001] This disclosure belongs to the field of chemical analysis and pharmaceutical safety technology, specifically relating to a cyclic compound extracted from nylon materials or products containing nylon materials, its purification method, and its use in quality research. Background Technology

[0002] Pharmaceutical packaging materials, such as glass, metals, alloys, and polymers, form sealed systems that protect drugs from external contamination and maintain their stability and efficacy during storage and transportation. Multilayer co-extrusion technology combines multiple materials, providing superior chemical stability and barrier properties by effectively blocking oxygen and other gases, thereby extending the shelf life of pharmaceuticals. While multilayer co-extruded infusion bags offer a higher level of protection for drugs, their complex structural design introduces potential risks, particularly regarding additive migration. These additives are incorporated to improve the processing and mechanical properties of the plastics; however, during prolonged contact between the drug and the packaging material, these additives, along with oligomers from the material itself, may migrate into the drug solution, forming leachates that can affect drug safety and efficacy. Therefore, drug regulatory agencies in various countries require systematic packaging material compatibility studies for high-risk formulations to assess their potential risks.

[0003] One of the key aspects of compatibility studies is identifying potentially migratable extractables from packaging materials through extraction experiments, and then accurately qualitatively and quantitatively determining the leachates in the drug. In compatibility studies in this field, when analyzing multilayer co-extruded infusion bags, extractables and leachates frequently detected in these bags contain impurities with high threshold peaks. Although the presence of these impurities in large quantities is known, the lack of high-purity physical reference standards in current technology makes it impossible to establish accurate qualitative and quantitative analytical methods. This results in risk assessments of packaging materials remaining at the stage of "only knowing the presence of impurities, but unable to qualitatively and quantitatively determine them," failing to meet the regulatory requirements for compatibility studies of packaging materials in drug manufacturing. Furthermore, the lack of reference standards makes it impossible to accurately quantify their content, thus hindering the scientific assessment of their migration in the drug solution and their potential toxicological risks.

[0004] To address this, existing technologies have made various attempts, but all have limitations. CN117740974A discloses a method for detecting cyclic oligomers in nylon 66 material, aiming to analyze the total amount or category of known oligomers existing in mixture form; however, it does not involve the process of separating, purifying, structurally identifying, and obtaining high-purity reference standards for any specific, unreported compound. CN119985740A discloses an analytical method using hyphenated techniques to visualize oligomers in nylon polymers to infer the polymerization process. However, this method is essentially an analytical diagnostic tool, not a preparation and purification tool, and cannot provide sufficient quantities of high-purity, single-component compounds as reference standards necessary for routine testing. CN116148361A discloses a method for purifying methylprednisolone impurity B using high-performance liquid chromatography, but its purification target is the degradation impurity of the drug molecule itself, not the separation of cyclic oligomers with similar properties from complex polymer extracts.

[0005] Therefore, it is essential to identify the structure of impurities in the extractables and leachates of nylon products, especially multilayer co-extruded infusion bags, and to accurately determine the content of these impurities. Summary of the Invention

[0006] This disclosure provides a cyclic compound and a purification method thereof, which can obtain a cyclic compound with a well-defined structure and reliable purity. The cyclic compound can be used as a reference standard in quality studies of nylon materials or products containing nylon materials, particularly for accurate qualitative and quantitative analysis of extractables and leachates in compatibility studies of multilayer co-extruded infusion bags, thereby improving the reliability of safety evaluation of pharmaceutical packaging materials.

[0007] A first aspect of this disclosure provides cyclic compounds, their solvates, or salts, said cyclic compounds having the structure shown in Formula I: (I) Where m is selected from integers from 6 to 12; optionally, m is selected from integers from 8 to 10; n is an integer selected from 4 to 10; alternatively, n is an integer selected from 6 to 8.

[0008] In some embodiments, the cyclic compound, its solvate, or salt has a structure as shown in Formula II: (II).

[0009] The cyclic compounds, their solvates, or salts have well-defined structures and are helpful in clarifying the quantitative and qualitative analysis of impurities in the quality study of nylon materials or products containing nylon materials.

[0010] A second aspect of this disclosure provides a method for purifying the cyclic compounds, their solvates, or salts described in the first aspect of this disclosure, the method comprising the following steps: (a) Nylon particles were immersed in a solvent under heating conditions, and then the solvent was removed to obtain a crude extract; (b) The crude extract in step (a) is separated and purified by chromatographic separation, and the corresponding fraction is collected and dried to obtain the cyclic compound, its solvate or salt of the first aspect of this disclosure.

[0011] The purification method can obtain cyclic compounds, their solvates or salts with reliable purity, so that the cyclic compounds, their solvates or salts meet the requirements for use as reference standards and can be used as reference standards in quality research.

[0012] The third aspect of this disclosure provides the use of the aforementioned cyclic compound, its solvate or salt, or the cyclic compound, its solvate or salt obtained by the aforementioned method, in the study of nylon materials or articles containing nylon materials, optionally for use in quality studies.

[0013] The fourth aspect of this disclosure provides a method for the quantitative analysis of the aforementioned cyclic compounds, their solvates, or salts in nylon materials or articles containing nylon materials, comprising the following steps: (I) Establish a test method using the aforementioned cyclic compounds, their solvates, or salts; (II) Extract the nylon material or the product containing the nylon material to obtain the sample to be tested; (III) Use the test method of step (I) to test and analyze the sample to be tested, and calculate the content of the aforementioned cyclic compound, its solvate or salt.

[0014] The quantitative analysis method described above has good reliability and reproducibility, which helps to improve the reliability of safety evaluation of pharmaceutical packaging materials.

[0015] The foregoing and other aspects of this disclosure are described in more detail below. Attached Figure Description

[0016] Figure 1 The extracted ion chromatogram (A), primary mass spectrometry (B), and secondary characteristic spectrum (C) of 1,8-diazacycloeicosane-9,20-dione in Example 1 are shown.

[0017] Figure 2 The 1H NMR spectrum of 1,8-diazacycloeicosane-9,20-dione in Example 1 is shown, with the horizontal axis representing chemical shift (δ, ppm) and the vertical axis representing signal intensity.

[0018] Figure 3The carbon NMR spectrum of 1,8-diazacycloeicosane-9,20-dione in Example 1 is shown, with the horizontal axis representing chemical shift (δ, ppm) and the vertical axis representing signal intensity.

[0019] Figure 4 The thermogravimetric analysis (TGA) results of 1,8-diazacycloeicosane-9,20-dione in Example 1 are shown.

[0020] Figure 5 The typical mass spectrum of the sample solution of the five-layer co-extruded infusion bag containing 1,8-diazacycloeicosane-9,20-dione in Example 7 is shown. A, C, and D correspond to the mass spectra of the blank solution, the control solution, and the sample solution, respectively.

[0021] Figure 6 The linear graph of 1,8-diazacycloeicosane-9,20-dione in Example 7 is shown.

[0022] Figure 7 The chromatogram of the 1,8-diazacycloeicosane-9,20-dione reference standard from Example 7 is shown.

[0023] Figure 8 The chromatogram of the 1,8-diazacycloeicosane-9,20-dione infusion bag sample from Example 7 is shown.

[0024] Figure 9 The dose-response curves (left) of 1,8-diazacycloeicosano-9,20-dione in Example 8 and the cell survival rates of each group (right) are shown.

[0025] Figure 10 The following is an exemplary mass spectrum of impurities in studies of extractables and leachates. Detailed Implementation

[0026] Unless otherwise stated, the terminology used herein has the common meaning understood by one of ordinary skill in the art. It may vary for those skilled in the art depending on the desired properties and effects sought through this application, and each numerical parameter should be interpreted according to the number of significant figures and conventional rounding methods or as understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the experimental procedures described herein are well-known and commonly used in the art. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. Where multiple definitions exist for terms used herein, the definitions in this section shall prevail unless otherwise stated.

[0027] When used in this document, the expression “A and / or B” includes three cases: (1) A; (2) B; and (3) A and B. The expression “A, B and / or C” includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B and C. The meanings of similar expressions can be deduced by analogy.

[0028] As used herein, the terms “include” and “contain” mean that other elements are not excluded in addition to the elements listed.

[0029] Unless otherwise stated, all figures used in this specification and claims to represent content, concentration, proportion, mass, volume, time, temperature, thickness, technical effect, etc., should in any instance be understood to be modified by the terms “about” or “approximately”. Therefore, unless indicated to the contrary, the numerical parameters listed in the following specification and appended claims are approximate values. They can vary for those skilled in the art depending on the desired properties and effects sought through this disclosure, and each numerical parameter should be interpreted according to the number of significant figures and conventional rounding methods or in a manner understood by those skilled in the art.

[0030] Although the numerical ranges and parameters described in this disclosure are approximate, the values ​​presented in the specific embodiments are provided as precisely as possible. However, any numerical value will inherently contain some errors, which are necessarily caused by the standard deviation found in its corresponding test measurements. Each numerical range given in this specification will include every narrower numerical range falling within that wider range, as if these narrower numerical ranges were explicitly stated herein.

[0031] In the study of extractables and leachates from multilayer co-extruded infusion bags, the semi-quantitative detection results of non-volatile substances showed that the content of an unknown compound far exceeded the analytical evaluation threshold (exemplary chromatogram shown). Figure 10 The retention time of the unknown compound was 10.442 min. Based on the extraction, separation, and purification of this unknown substance, and the comprehensive use of carbon and hydrogen nuclear magnetic resonance spectroscopy for structural identification, the structure and origin of the compound were finally determined. Based on this, the following aspects are provided in this disclosure: Cyclic compounds, their solvates or salts This disclosure provides cyclic compounds, their solvates or salts, said cyclic compounds having a structure as shown in Formula I: (I) Where m is selected from integers from 6 to 12; n is selected from integers from 4 to 10.

[0032] Salts of the cyclic compounds disclosed herein may be pharmaceutically acceptable salts or chemically formable salts. Examples of salts of cyclic compounds include: hydrochlorides, hydrobromates, hydroiodates, phosphates, phosphonates, sulfates, sulfonates (such as methanesulfonates and p-toluenesulfonates), carboxylates (such as acetates), citrates, malates, tartrates, succinates and salicylates, alkali metal salts (such as sodium and potassium salts), alkali metal salts (such as magnesium and calcium salts), and ammonium salts (such as ammonium salts, alkylammonium salts, dialkylammonium salts, trialkylammonium salts, and tetraalkylammonium salts). These salts are prepared, for example, by contacting the compound with an acid or base that can be used to produce pharmaceuticals.

[0033] In this document, a solvate of a cyclic compound refers to a molecular cluster formed by the compound and a solvent, and when the solvent is water, it is called a hydrate. The solvates of the compounds disclosed herein include not only those formed by a single solvent (such as water, alcohol (e.g., methanol, ethanol, 1...)) propanol or 2 Solvents formed from propanol, acetonitrile, or dimethylformamide, and also include solvates formed from a variety of solvents.

[0034] In some implementations, m is selected from 6-10, 8-10, 7-11, or 8-12. In some implementations, m is selected from an integer between 8 and 10.

[0035] In some implementations, m is selected from 6, 7, 8, 9, 10, 11 or 12, or an integer within the range of any two of the aforementioned values.

[0036] In some implementations, n is selected from 4-10, 5-9, 6-8, or 7-9. In some implementations, n is selected from integers from 6 to 8.

[0037] In some implementations, n is selected from 4, 5, 6, 7, 8, 9, or 10, or is an integer within the range formed by any two of the aforementioned values.

[0038] In some implementations, m is selected from an integer between 8 and 10, and n is selected from an integer between 6 and 8.

[0039] In some embodiments, the cyclic compound, its solvate, or salt has a structure as shown in Formula II: (II).

[0040] In some embodiments, the cyclic compound represented by Formula II, its solvate, or its salt has Figure 2 The hydrogen nuclear magnetic resonance spectrum shown is shown.

[0041] In some embodiments, the 1H NMR spectra of the cyclic compound represented by Formula II, its solvate, or its salt include the following peaks: δ 3.19 (t, J = 6.5 Hz, 4H), 2.17 (t, J = 6.5 Hz, 4H), 1.64-1.60 (m,4H), 1.49-1.46 (m, 4H), 1.36-1.29 (m, 16H).

[0042] In some embodiments, the cyclic compound represented by Formula II, its solvate, or its salt has Figure 3 The carbon NMR spectrum is shown.

[0043] In some embodiments, the carbon NMR spectra of the cyclic compound represented by Formula II, its solvate, or its salt include the following peaks: δ 174.68(2C), 38.69(2C), 35.61(2C), 29.57(2C), 28.86(2C), 28.80(2C), 27.95(2C), 26.54(2C), 25.49(2C).

[0044] In some embodiments, the cyclic compound, its solvate, or salt has a mass content of not less than 95%. In some embodiments, the cyclic compound, its solvate, or salt has a mass content of 95%-100%.

[0045] In some embodiments, the cyclic compound, its solvate, or salt has a mass content of not less than 96%, not less than 97%, not less than 98%, or not less than 99%. In some embodiments, the cyclic compound, its solvate, or salt has a mass content of 96%-100%, 97%-100%, 98%-100%, or 99%-100%.

[0046] The mass content of the cyclic compound, its solvate, or its salt can be determined using any method known in the art. For example, the mass content can be determined by liquid chromatography using the area normalization method, or by the mass balance method.

[0047] The cyclic compound, its solvate, or salt is a non-target polymer in nylon material, specifically a non-target polymer in nylon 612 material. This substance has a low degree of polymerization and small molecular weight, and under conditions of prolonged contact between nylon material or products containing nylon material and liquid, it easily migrates from the nylon material into the liquid it is in contact with. In particular, when products containing nylon material (e.g., multilayer co-extruded infusion bags) are pharmaceutical packaging materials used to contain drug solutions, the cyclic compound, its solvate, or salt migrating into the drug solution can affect the quality of the drug solution, and even more seriously, affect its safety.

[0048] The cyclic compounds, their solvates, or salts represented by Formula I or II possess well-defined structures and excellent purity, which are helpful in clarifying the qualitative and quantitative analysis of impurities in quality studies of nylon materials or products containing nylon materials. Particularly in compatibility studies between pharmaceutical packaging materials and drug solutions, these cyclic compounds, their solvates, or salts can serve as reference standards (or standards) to more accurately perform qualitative and quantitative analysis of impurities in the extractables and leachates of multilayer co-extruded infusion bags. This overcomes the deficiency in existing technologies where the lack of specific impurity compound reference standards (or standards) leads to insufficient risk assessment, thereby improving the reliability of safety evaluation of pharmaceutical packaging materials.

[0049] Extraction and purification methods This disclosure also provides a method for obtaining the cyclic compound, its solvate, or salt from nylon, the method comprising the following steps: (a) Nylon particles were immersed in a solvent under heating conditions, and then the solvent was removed to obtain a crude extract; (b) The crude extract from step (a) is separated and purified by chromatographic separation, and the corresponding fraction is collected and dried to obtain the cyclic compound, its solvate or salt.

[0050] In step (a), nylon particles refer to particulate matter based on nylon (polyamide) material, including but not limited to virgin particles formed directly by granulation after polymerization, or customized particles obtained by secondary processing such as crushing and grinding of block or sheet nylon material. Nylon particles can significantly increase the specific surface area, thereby allowing the solvent to fully contact the nylon material during the extraction process and effectively promoting the dissolution of the target compound.

[0051] Nylon comes in different grades or polymer monomers. Preferably, the grade of the nylon particles is the same as that of the nylon material or articles containing nylon material, thus obtaining compounds that meet the research objectives. In the extraction method provided in this disclosure, the nylon particles can be obtained commercially directly or after appropriate pretreatment to suit the extraction process requirements.

[0052] In some embodiments, in step (a), the nylon particles are selected from one or more of nylon 612, nylon 1010, or nylon 610. In some embodiments, the nylon particles are selected from nylon 612. Therefore, the cyclic compounds, solvates, or salts obtained by the extraction method of this disclosure are particularly suitable for quality studies or quality control of pharmaceutical packaging materials containing nylon 612.

[0053] In some embodiments, step (a) involves Soxhlet extraction, accelerated solvent extraction, or water bath shaking to extract the nylon particles.

[0054] Soxhlet extraction utilizes solvent reflux and siphon principles to continuously extract target compounds from nylon materials. It boasts high extraction efficiency, relatively low solvent consumption, and requires no continuous human monitoring. Accelerated solvent extraction (ASE) can perform rapid extraction under high temperature (50-200℃) and high pressure (1000-3000 psi), offering high automation, speed (typically completed within 15-20 minutes), low solvent consumption, and high extraction efficiency. Water bath shaking extraction, under atmospheric pressure and heating conditions, continuously extracts nylon particles by ensuring sufficient contact between the solvent and the particles through continuous shaking. All these methods can effectively extract target cyclic compounds from nylon particles.

[0055] In some embodiments, step (a) uses a water bath to extract the nylon particles. This extraction method is simple, easy to operate, and allows for controllable time, effectively avoiding degradation of the target compound that may result from prolonged high-temperature heating. Compared to Soxhlet extraction, this method has a shorter extraction time, avoiding the degradation of the target compound caused by the prolonged heating treatment (typically tens of hours) of Soxhlet extraction. Compared to accelerated solvent extraction, it can reduce research costs and also reduce the risk of pyrolysis or structural changes of the target compound under high-pressure testing conditions.

[0056] During compound extraction, temperature can affect the solubility and diffusion rate of the target compound. If the temperature is too low, the extraction efficiency will be insufficient; if the temperature is too high, it may cause a large amount of non-target impurities to dissolve, or even lead to the decomposition of the target compound.

[0057] In some embodiments, the heating conditions in step (a) are 40°C-80°C. In some embodiments, the heating conditions in step (a) are 50°C-70°C. In some preferred embodiments, the heating conditions in step (a) are 50°C-65°C. In some preferred embodiments, the heating conditions in step (a) are 50°C-60°C.

[0058] In some embodiments, the heating conditions in step (a) are 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, or a range of any two of the above temperatures or a value within that range.

[0059] The specified temperature range promotes the full dissolution of the target compound within a suitable time, while minimizing the degradation of the target compound and the co-extraction of other impurities.

[0060] In some embodiments, the extraction time in step (a) is 0.1 hours to 20 hours. In some embodiments, the extraction time is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, or 20 hours, or a range of any two of the above times, or a value within such a range.

[0061] In some embodiments, the extraction time in step (a) is 1 hour to 6 hours. In some preferred embodiments, the extraction time in step (a) is 3 hours to 5 hours. In some preferred embodiments, the extraction time in step (a) is 3 hours to 4 hours.

[0062] Extraction time can affect the extraction efficiency of the target compound. If the extraction time is too short, the release or migration of the target compound will not reach equilibrium, resulting in a low recovery rate. If the time is too long, the extraction efficiency will be low, and energy consumption and the risk of impurity dissolution will increase. Extraction time within the above range can shorten the extraction cycle and allow the dissolution of the target compound to reach a basic equilibrium within this time.

[0063] In step (a), the solvent is selected based on the degree of polarity matching with the target compound and the volatility of the solvent. In some embodiments, in step (a), the solvent is selected from one or more of water, methanol, anhydrous ethanol, and acetonitrile.

[0064] In some embodiments, the solvent is selected from one or more of methanol, anhydrous ethanol, and acetonitrile. The solvent exhibits good compatibility with the target compound, enabling thorough extraction of the target compound from the nylon particles and improving the recovery rate of the target compound.

[0065] In some embodiments, the solvent is selected from acetonitrile, which further improves the recovery rate of the target compound.

[0066] In some implementations, step (a) includes: Nylon particles were extracted in one or more organic solvents selected from methanol, anhydrous ethanol and acetonitrile at 50℃-60℃ for 3-4 hours. The solvent was then removed by evaporation to obtain the crude extract.

[0067] In the extraction process, the synergistic optimization of factors such as extraction time, temperature, and solvent results in significant advantages in extraction efficiency, final product recovery rate, and purity, which is helpful for the further purification of the target compound.

[0068] In some embodiments, in step (b), the crude extract is dissolved and then purified by chromatographic separation, wherein the chromatographic separation method is selected from one or more of column chromatography, preparative high performance liquid chromatography, high-speed countercurrent chromatography, and supercritical fluid chromatography.

[0069] Column chromatography typically involves packing a stationary phase (such as silica gel) into a glass column, relying on gravity or low pressure for separation and elution. Column chromatography is extremely inexpensive, requires simple equipment, and is easy to prepare on a large scale.

[0070] Preparative high-performance liquid chromatography (HPLC) uses larger inner diameter columns and higher flow rates, resulting in high resolution and large sample loading capacity. It is a routine and reliable method for obtaining high-purity standards.

[0071] High-speed countercurrent chromatography (HSCLC) is a liquid-liquid extraction partition chromatography technique that does not use any solid support as a stationary phase. It achieves separation by establishing a hydrodynamic equilibrium between two solvent systems with different affinities within a spiral column. It features no irreversible adsorption, high sample recovery, large sample preparation capacity, and moderate cost.

[0072] Supercritical fluid chromatography uses supercritical CO2 as the main mobile phase. It offers fast separation speeds and low solvent consumption (making it environmentally friendly), making it particularly suitable for separating compounds with low to medium polarity.

[0073] In some embodiments, in step (b), the chromatographic separation method is selected from preparative high-performance liquid chromatography (HPLC), and further selected from preparative ultra-high-performance liquid chromatography (UHPLC), which is time-saving and efficient. Compared with column chromatography, it has the advantages of superior resolution and separation efficiency, shorter processing time, and can also separate impurities with high structural similarity. Compared with high-speed countercurrent chromatography, the method development is less difficult and the requirements for solvent system selection are lower. Compared with supercritical fluid chromatography, the equipment cost is lower, but the purification method is more difficult.

[0074] In some embodiments, in step (b), the chromatographic separation employs gradient elution. In some embodiments, the elution procedure includes:

[0075] Phase A is an aqueous solution containing 0.1%-0.5% (v / v) formic acid, and Phase B is an acetonitrile solution containing 0.1%-0.5% (v / v) formic acid.

[0076] In some embodiments, the formic acid concentration in phase A is 0.1% (v / v), 0.2% (v / v), 0.3% (v / v), 0.4% (v / v), 0.5% (v / v), or any range between the aforementioned values, or any value within the range.

[0077] In some embodiments, the formic acid concentration in phase B is 0.1% (v / v), 0.2% (v / v), 0.3% (v / v), 0.4% (v / v), 0.5% (v / v), or any range between the aforementioned values, or any value between the ranges.

[0078] In some embodiments, phase A is an aqueous solution containing 0.1%-0.3% (v / v) formic acid, and phase B is an acetonitrile solution containing 0.1%-0.3% (v / v) formic acid.

[0079] In some embodiments, the detection wavelength in the chromatographic separation process is 210±10 nm, preferably 210±5 nm, and more preferably 210±2 nm. As an example, the detection wavelength can be 208 nm, 209 nm, 210 nm, 211 nm, or 212 nm.

[0080] In some embodiments, the flow rate is 1.5 ± 0.5 mL / min, preferably 1.5 ± 0.3 mL / min, and more preferably 1.5 ± 0.2 mL / min. As an example, the flow rate can be 1.3 mL / min, 1.4 mL / min, 1.5 mL / min, 1.6 mL / min, or 1.7 mL / min.

[0081] In some implementations, the detection parameters for chromatographic separation are: a detection wavelength of 210 nm and a flow rate of 1.5 mL / min.

[0082] In some embodiments, the chromatographic column used for chromatographic separation is a C18 column, particularly a C18 column with an inner diameter of 4.6 mm, a length of 150 mm, and a packing particle size of 5 μm, such as the UltimateXB-C18 column.

[0083] In some embodiments, in step (b), the solvent in the collected fraction is removed by evaporation, and further, the solvent in the fraction is removed by rotary evaporation under reduced pressure. The precipitate is collected and dried under vacuum to obtain the cyclic compound, its solvate, or its salt.

[0084] The extraction method provided in this disclosure has high extraction efficiency, controllable cost, and relatively short cycle time, and can obtain cyclic compounds, their solvates or salts with excellent purity.

[0085] use This disclosure provides for the use of the cyclic compound, its solvates, or salts in the study of nylon materials or articles containing nylon materials, optionally for use in quality studies.

[0086] In this document, "articles containing nylon materials" refers to any finished or semi-finished product composed of nylon (polyamide) material, in forms including but not limited to resin particles, fibers, films, profiles, and components composited with other materials. The nylon component in the article may manifest as a continuous phase, a dispersed phase, or a specific functional layer. These articles particularly include packaging materials used in pharmaceuticals, food, and other applications where the chemical safety of materials is strictly required.

[0087] In this article, "quality study" refers to the process of systematically assessing the quality of materials or products, aiming to determine the extent to which the tested materials conform to predetermined standards, including but not limited to chemical composition analysis, physical performance testing, biosafety verification, and functional stability investigation. Among these, chemical property studies involve aspects such as composition, purity, and impurities that can affect chemical stability and safety.

[0088] In some embodiments, the nylon component in the nylon material or article containing nylon material is selected from one or more of nylon 612, nylon 1010, or nylon 610. In some embodiments, the nylon is selected from nylon 612.

[0089] In some embodiments, the article containing nylon material includes a multilayer co-extruded infusion bag.

[0090] In some embodiments, the quality study includes pharmaceutical-related studies and studies related to the quality control of nylon materials. In some embodiments, the quality study is a compatibility study of pharmaceutical packaging materials.

[0091] In this article, "pharmaceutical-related research" encompasses all scientific research activities related to drug development, production, storage, transportation, and safety of use. "Compatibility studies of pharmaceutical packaging materials" include, but are not limited to, systematically examining whether adverse physical or chemical interactions occur between pharmaceutical packaging materials and pharmaceuticals, and assessing the potential impact of packaging systems on the safety, efficacy, and quality stability of pharmaceuticals.

[0092] In some implementations, the study is a quantitative analysis study.

[0093] In this article, "quantitative analysis" refers to the process of accurately determining the absolute or relative content of a target analyte in a specific matrix using analytical chemistry methods. Specifically, "quantitative analysis research" refers to research activities that use chromatography, spectroscopy, mass spectrometry, or other analytical techniques to determine the content of specific components in a sample.

[0094] In some embodiments, cyclic compounds, their solvates, or salts are used as control substances in the study.

[0095] In this disclosure, "reference standard" or "standard" is synonymous and refers to a chemical reference substance with a defined chemical structure and a purity that meets industry requirements, used for, but not limited to, the following specific purposes: as an external or internal standard in chromatographic analysis for quantitative calculations; as a standard substance in spectral analysis for methodological validation and system suitability testing; and as a comparison standard for qualitative identification in the structural analysis of unknown substances.

[0096] Quantitative analysis methods This disclosure provides a method for the quantitative analysis of the aforementioned cyclic compounds, their solvates, or salts in nylon materials or articles containing nylon materials, comprising the following steps: (I) Establish a test method using the cyclic compound, its solvate or salt; (II) Extract the nylon material or the product containing the nylon material to obtain the sample to be tested; (III) The test sample is tested and analyzed using the test method of step (I) to calculate the content of the cyclic compound, its solvate or salt as described in the first aspect of this disclosure.

[0097] Developing a testing method typically involves a series of systematic methodological validation studies aimed at verifying the method’s suitability for its intended use from different perspectives.

[0098] In some implementations, in step (I), the test method is liquid chromatography-mass spectrometry, which may include one or more of the following studies: specificity, linearity and range, sensitivity, repeatability, accuracy, solution stability and method robustness studies.

[0099] In this article, "specificity" or "specificity" refers to the ability of the method to accurately and uniquely determine the target compound in the presence of other components (such as nylon matrix, other additives, degradation products, or coexisting impurities), and can be used to confirm that analytical signals, such as chromatographic peaks and spectral peaks, originate solely from the target compound and are not affected by other components.

[0100] In this paper, "linearity and range" establishes a mathematical relationship between response values ​​and concentrations using a series of concentration reference solutions to determine the concentration range that the method can accurately measure. "Linearity" refers to the ability of the method to produce a detection response value (e.g., peak area) that is proportional to the concentration of the target analyte. "Range" refers to the concentration range of the analyte in the sample to which the method is applicable, provided that a certain level of accuracy, precision, and linearity is achieved. In some embodiments, the cyclic compound, its solvate, or salt exhibits good linearity in the concentration range of 5–1009 ng / ml.

[0101] In this paper, "sensitivity" refers to the method's ability to detect trace amounts of analytes, expressed as the limit of detection (LOD) and limit of quantitation (LOQ). The LOD and LOQ can be calculated using the signal-to-noise ratio method or based on the standard deviation and slope of the response values. High sensitivity indicates that the method can capture substances in extremely low concentrations in a sample, which is crucial for assessing the safety of extractable / leached substances in packaging materials.

[0102] In this paper, "reproducibility" refers to the degree of consistency between results obtained from multiple measurements of a sample under the same operating conditions. It is typically assessed by preparing at least six sample solutions of the same concentration within the concentration range specified by the method, performing the measurements, and calculating the relative standard deviation of the obtained concentrations. Good repeatability indicates that the method itself has low variability and the operating process is stable and controllable.

[0103] In this paper, "accuracy" refers to the degree of closeness between the analyte concentration determined by this method and a recognized reference value, usually the true value or conventional true value, which is typically demonstrated through recovery experiments. Accuracy determines the reliability of the measurement results; high accuracy means that the method can unbiasedly reflect the actual content of cyclic compounds in the sample.

[0104] In this paper, "solution stability" refers to the degree to which the chemical properties of the analyte remain stable in standard solutions and sample solutions under specific storage conditions and time (such as room temperature, refrigeration, or freezing). Standard solutions and sample solutions are measured simultaneously with freshly prepared solutions after being placed under certain conditions for different times. Changes in peak area and purity, as well as the presence or absence of degradation impurities, are compared.

[0105] In this paper, "method robustness" refers to the ability of a method to maintain consistent analytical results when its parameters are intentionally and reasonably altered, such as by using columns from different brands or batches. During testing, a pre-designed experiment was conducted where a key chromatographic condition was intentionally changed while other conditions remained constant. The same sample was then analyzed to examine changes in key analytical parameters (such as retention time, resolution, and tailing factor). High robustness indicates that the method is insensitive to minor fluctuations in experimental conditions.

[0106] In some embodiments, step (II) includes the extraction parameters in the method provided in the second aspect of this disclosure.

[0107] When used in this document, "liquid chromatography-mass spectrometry" refers to an analytical method that combines liquid chromatography separation technology with mass spectrometry detection technology. This includes, but is not limited to, all equivalent coupling forms such as high-performance liquid chromatography-mass spectrometry (HPLC-MS), ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS / MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), and two-dimensional liquid chromatography-mass spectrometry. It is applicable to the qualitative and quantitative analysis of target compounds and is not limited to specific instrument models, mobile phase compositions, mass spectrometry scanning modes, or data processing software. This term encompasses all liquid chromatography-mass spectrometry combined systems capable of achieving separation and detection functions, and its operating parameters can be adjusted according to actual needs to meet the requirements of different analytical scenarios.

[0108] In some embodiments, liquid chromatography employs gradient elution. In some embodiments, the elution procedure includes:

[0109] Phase A is an aqueous solution containing 0.1%-0.5% (v / v) formic acid, and Phase B is an acetonitrile solution containing 0.1%-0.5% (v / v) formic acid.

[0110] In some embodiments, the formic acid concentration in phase A is 0.1% (v / v), 0.2% (v / v), 0.3% (v / v), 0.4% (v / v), 0.5% (v / v), or any range between the aforementioned values, or any value within the range.

[0111] In some embodiments, the formic acid concentration in phase B is 0.1% (v / v), 0.2% (v / v), 0.3% (v / v), 0.4% (v / v), 0.5% (v / v), or any range between the aforementioned values, or any value between the ranges.

[0112] In some embodiments, phase A is an aqueous solution containing 0.1%-0.3% (v / v) formic acid, and phase B is an acetonitrile solution containing 0.1%-0.3% (v / v) formic acid.

[0113] In some embodiments, the flow rate is 0.3 ± 0.2 mL / min, preferably 0.3 ± 0.1 mL / min. As an example, the flow rate can be 0.2 mL / min, 0.3 mL / min, or 0.4 mL / min.

[0114] In some implementations, the column temperature is 40±2℃, for example, 39℃, 40℃, 41℃.

[0115] In some embodiments, the chromatographic column used for chromatographic separation is a C18 column, particularly a reversed-phase liquid chromatograph with an inner diameter of 2.1 mm, a length of 100 mm, and a packing particle size of 1.8 μm.

[0116] A quantitative analysis method based on the high-purity reference standard prepared in this disclosure is established, which enables accurate and sensitive quantitative analysis of the target compound in the test sample. This successfully solves the problem of "inaccuracy" and lays a solid foundation for accurately quantifying the migration amount of the compound in the drug solution. This, in turn, helps to scientifically assess the toxicological risks and safety evaluation of drug packaging materials.

[0117] Example The following description, in conjunction with the accompanying drawings, illustrates exemplary embodiments of this application, including various details of these embodiments to aid understanding. It should be understood that these are merely exemplary and are in no way intended to limit the scope of protection of this application. The scope of protection of this application is defined only by the claims. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0118] Unless otherwise stated, the experimental methods described in the following examples use commercially available, conventional reagents and instruments. Unless otherwise stated, the procedures were performed according to the techniques or conditions described in the literature or according to the product instructions.

[0119] Example 1: Preparation and structural confirmation of 1,8-diazacycloeicosane-9,20-dione reference standard In this embodiment, the compound 1,8-diazacycloeicosane-9,20-dione of Formula II was isolated and purified from nylon 612 material to obtain a high-purity reference standard of the compound, and its key physicochemical properties were determined.

[0120] 1.1 Experimental Apparatus X500R Q-TOF time-of-flight mass spectrometer (AB SCIEX, USA); ultra-high performance liquid chromatograph (Thermo Fisher Scientific, USA); LC-20 preparative HPLC system (Shimadzu Corporation, Japan); SECURA225D-1CN analytical balance (Sartorius, Germany); MilliQ ultrapure water system (Merck, USA); SY-2230 constant temperature water bath shaker (Hangzhou Jiemei Electronics Co., Ltd., China); thermogravimetric analyzer (Platinum Elmer, USA); N-1300D-W rotary evaporator (EYELA, Japan).

[0121] 1.2 Experimental Materials Acetonitrile, methanol, anhydrous ethanol, and formic acid (all purchased from Sigma Reagents, USA).

[0122] 1.3 Experimental Procedure (1) Extraction Accurately weigh 100.0 g of Nylon 612 particles (DuPont, USA), place them in a 250 mL Erlenmeyer flask, add 100 mL of acetonitrile, and extract by shaking at 150 rpm in a 60°C constant temperature water bath for 4 hours.

[0123] (2) Concentration The extract was transferred to a rotary evaporator and concentrated under reduced pressure at a water bath temperature of 60°C until it was completely evaporated to dryness, yielding a white powdery crude extract with a weight of approximately 85 mg.

[0124] (3) Separation and purification Weigh 50.08 mg of the crude extract powder, dissolve it thoroughly in acetonitrile and dilute to 10 mL. Filter the solution through a 0.22 μm microporous membrane to obtain an injection solution with a concentration of approximately 5 mg / mL. Preparative separation was performed using an ultra-high performance liquid chromatography system (Thermo Fisher Scientific, USA).

[0125] Column: Ultimate XB-C18 (4.6 × 100 mm, 5 μm; Agilent Technologies, USA) Column temperature: 30°C Autosampler temperature: 15°C Mobile phase: Phase A: ultrapure water (containing 0.1% formic acid); Phase B: acetonitrile (containing 0.1% formic acid) Elution procedure: Follow the gradient shown in Table 1.

[0126] Flow rate: 1.5 mL / min UV detector wavelength: 210 nm Injection volume: 30 μL (meaning each injection contains approximately 0.15 mg of crude extract. To process 50.08 mg of sample, this process needs to be repeated approximately 333 times).

[0127] Based on the ultraviolet signal, the distillate of the target peak is automatically collected at approximately 2.5 minutes after the peak elution time ends.

[0128] Table 1 Gradient elution program for ultra-high performance liquid chromatography

[0129] (4) Drying treatment All collected target fractions were combined and evaporated under reduced pressure at 60°C until completely dry, yielding a white powdery purified product. The powder was transferred to a weighing bottle and placed in a vacuum drying oven at 105°C for 2 hours until constant weight (mass change Δm < 0.1%), yielding a white powdery pure product with a final accurate weight of 42.12 mg.

[0130] (5) Structural identification The obtained white powder was analyzed by high-resolution time-of-flight mass spectrometry (TOF-MS / MS) using the following method.

[0131] TOF-MS / MS Method: Mass spectrometry was performed using information-dependent acquisition (IDA) mode. Q-TOF parameters were set as follows: ion source gases 1 and 2 50 psi, curtain gas 35 psi, IS 5000 V, DP 80 V, CE 35 V, CES 15 V, ion source temperature 500 °C, scan range 50-1000 Da. TOF-MS / MS mode parameters were set as follows: DP 80 V, CE 35 V, CES 15 V, scan range 50-1000 Da. Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) was used for data acquisition using SCIEX OS Software (version 2.3.6). Total ion current mass spectra were obtained in positive ion mode.

[0132] Under the above test conditions, an ion peak was observed at 2.976 min; a quasi-molecular ion peak [M+H] was observed at m / z 311.2692. + (Calculated value C) 18 H 35 N2O2 + (m / z 311.2698), and the theoretical molecular formula of 1,8-diazacycloeicosane-9,20-dione C 18 H 34The N2O2 concentration showed a high degree of agreement, with an error of less than 0.5 ppm. Its time-of-flight mass spectrometry (TOF-MS) produced several characteristic fragment ion peaks, attributed to the breaking of functional groups such as amide bonds in the structure, further corroborating the molecular structure. The extracted ion chromatogram (A), primary mass spectrum (B), and secondary mass spectrum (C) are shown below. Figure 1 .

[0133] By combining ¹H NMR and ¹³C NMR measurements, the structure of the compound can be accurately identified.

[0134] 1H NMR spectroscopy analysis: The triplet at chemical shift 3.19 of the target compound is the signal of hydrogen atoms on the carbon atom directly bonded to the nitrogen atom of the amide, and the triplet at chemical shift 2.17 is the signal of hydrogen atoms on the carbon atom directly bonded to the carbonyl group of the amide. The other multiplets are the signals of hydrogen atoms on the other methylene groups. Since no amide hydrogen atom signal can be shown in deuterated methanol, the peak area values ​​are consistent with the number of hydrogen atoms, indicating that the measured compound has the structure of compound (II).

[0135] 1 H-NMR (500 MHz, CD3OD): δ 3.19 (t, J = 6.5 Hz, 4H, -NH-CH2-), 2.17(t, J = 6.5 Hz, 4H, -CH2-CO-), 1.64-1.60 (m, 4H), 1.49-1.46 (m, 4H), 1.36-1.29 (m, 16H); ( Figure 2 ).

[0136] Nuclear magnetic resonance carbon spectrum analysis: The peak at chemical shift 174.68 of the target compound is the signal of carbonyl carbon atom, the peak at chemical shift 38.69 is the signal of carbon atom directly bonded to amide nitrogen atom, and the peak at chemical shift 35.61 is the signal of carbon atom directly bonded to carbonyl group. The peak values ​​are consistent with the number of carbon atoms, indicating that the measured compound has the structure of compound (II).

[0137] 13 C-NMR (125 MHz, CD3OD): δ 174.68(2C), 38.69(2C), 35.61(2C), 29.57(2C), 28.86(2C), 28.80(2C), 27.95(2C), 26.54(2C), 25.49(2C);( Figure 3 ).

[0138] Based on the combined analysis results of TOF-MS / MS, ¹H NMR and ¹³C NMR, the white powder was confirmed to be the cyclic monomer 1,8-diazacycloeicosano-9,20-dione in nylon 612, i.e. (II).

[0139] (6) Melting point determination The obtained white powder was thermally analyzed using differential scanning calorimetry (DSC) at a heating rate of 10°C / min. The results showed a sharp endothermic melting peak at 226.7°C. Figure 4 ).

[0140] (7) Purity (content) determination The purity of the prepared 1,8-diazacycloeicosane-9,20-dione reference standard was determined using the internationally accepted mass balance method. This method calculates the absolute content of the main component by deducting the sum of various impurities, including water, residual solvent, inorganic ash, and organic impurities.

[0141] The calculation formula is: Purity (%) = 100% - (Moisture% + Residual Solvent% + Inorganic Ash% + Organic Impurities%) Moisture (Karl Fischer method): Not detected; Residual solvent acetonitrile (headspace-GC / MS method): Not detected; Inorganic ash (residue on ignition method): 0.02%; Organic impurities (refer to the TOF-MS / MS method of “(5) Structure Identification”, calculated by area normalization: the sum of the peak areas of all peaks in the ion chromatogram is regarded as 100%, and the proportion of the peak area of ​​the target peak in the total ion chromatogram (TIC) is compared to evaluate its relative content in different samples): 2.30%. Finally, purity = 100% - (0% + 0% + 0.02% + 2.30%) = 97.68%. The specific determination results are shown in Table 2.

[0142] Table 2 Purity Measurement Results

[0143] 1,8-Diazacycloeicosane-9,20-dione has a purity of over 95%, which meets the requirement of >95% purity for reference standards in quality studies in this field, and can be used as a reference standard for subsequent quality studies.

[0144] Recovery of 1,8-diazacycloeicosane-9,20-dione: 42.12 mg / 50.08 mg = 84.1% The results show that the method for preparing the 1,8-diazacycloeicosane-9,20-dione reference standard provided in this example has excellent purification effect and high recovery rate. The recovery rate of 84.1% and the purity of 97.68% fully verify the excellent performance of this method in separation efficiency, and achieve the goal of efficiently preparing a high-purity reference standard from the crude product.

[0145] Example 2: Investigation of Nylon 612 from Different Sources 2.1 Experimental Materials and Instruments Three types of nylon 612 particles, sourced from a domestic supplier, DuPont (USA), and Evonik (Germany), were selected as raw materials. Acetonitrile (chromatographic grade) and formic acid (chromatographic grade) were used as reagents. The instrumentation included an electronic balance, a constant temperature water bath shaker, and an X500R Q-TOF time-of-flight mass spectrometer coupled with an Exion LC ultra-high performance liquid chromatography system (SCIEX, USA).

[0146] 2.2 Sample Preparation and Analysis Accurately weigh 1.0 g of nylon 612 particles from each of the three suppliers and place them in 20 mL headspace vials, adding 10 mL of acetonitrile to each. Place the vials in a 60°C water bath with a shaker and extract for 4 hours. After extraction, cool the extract to room temperature and filter it through a 0.22 μm microporous membrane. Take 50 μL of the filtrate and dilute it to 1 mL with acetonitrile to prepare the test solution.

[0147] Analysis was performed using ultra-high performance liquid chromatography-time-of-flight mass spectrometry (UPLC-Q-TOF), in which... UPLC Method: The analyte was analyzed using an Exion LC system (SCIEX, Framingham, MA, USA) with an Agilent® SB-Aq column (2.1 × 100 mm, 1.8 μm). Samples were stored at 10°C, and the injection volume was 10 μl. The flow rate was set to 0.3 ml / min, and the column temperature was 40°C. A binary gradient elution system was used, consisting of ultrapure water (containing 0.1% formic acid, phase A) and acetonitrile (containing 0.1% formic acid, phase B). The gradient curves are shown in Table 1.

[0148] TOF-MS / MS method: Refer to the TOF-MS / MS method in the experimental steps “(5) Structure identification” in 1.3.

[0149] Data processing: Referring to the area normalization method in the experimental step “(7) Purity (content) determination” in 1.3, the relative content of the target peak (1,8-diazacycloeicosane-9,20-dione) in different samples was evaluated by comparing the proportion of the peak area in the total ion chromatogram.

[0150] 2.3. Test Results UPLC-Q-TOF analysis results show that the raw material extracts from the three suppliers differ significantly in terms of chemical complexity, main component content, and impurity types. Specific comparison data are shown in Table 3.

[0151] Table 3 Comparison of analysis results of Nylon 612 particle extracts from different suppliers

[0152] The above results confirm that, although the relative contents of 1,8-diazacycloeicosano-9,20-dione differed significantly, and the types and quantities of impurities also varied, the target compound 1,8-diazacycloeicosano-9,20-dione could be extracted from nylon 612 from different sources. The extraction and analysis methods provided in this disclosure are applicable to the extraction and component evaluation of the target compound in nylon 612 from different sources.

[0153] Example 3: Investigation of different extraction solvents 3.1 Raw materials and solvents Using DuPont Nylon 612 particles as standard raw material, the extraction effects of four different polar solvents—ultrapure water, methanol (chromatographic grade), anhydrous ethanol (chromatographic grade), and acetonitrile (chromatographic grade)—were investigated at the same extraction temperature and time.

[0154] 3.2 Instruments Constant temperature water bath shaker, analytical ultra-high performance liquid chromatography-time-of-flight mass spectrometry.

[0155] 3.3 Experimental Procedure Accurately weigh four 1.0 g portions of DuPont Nylon 612 particles and place them in separate 20 mL headspace vials. Add 10 mL of each of the four different extraction solvents (water, methanol, ethanol, and acetonitrile) to each of the four vials. Place all vials in a 60°C water bath with a shaker and oscillate at 150 rpm for 4 hours. After extraction, cool the samples to room temperature and filter the extract through a 0.22 μm microporous membrane. Take 0.5 mL of the extract from each solvent and dilute it to 10 mL with the corresponding solvent for analysis.

[0156] Analysis was performed using UPLC-Q-TOF, where... UPLC Method: An Exion LC system (SCIEX, Framingham, MA, USA) was used. The column was an Agilent® SB-Aq (2.1 × 100 mm, 1.8 μm). Samples were stored at 10°C, and the injection volume was 10 μl. The flow rate was set to 0.3 mL / min, and the column temperature was 40°C. A binary gradient elution system was used, consisting of ultrapure water (containing 0.1% formic acid, phase A) and acetonitrile (containing 0.1% formic acid, phase B). The elution gradient is shown in Table 4.

[0157] Table 4 Gradient Mode

[0158] TOF-MS / MS Method: Qualitative analysis was performed using Information-Dependent Acquisition (IDA) mode, while quantitative analysis was performed using Multiple Reaction Monitoring High Resolution (MRM-HR) mode. TOF-MS / MS parameters were set as follows: ion source gas 1 and 2 50 psi, curtain gas 35 psi, IS 5000 V, DP 80 V, CE 35 V, CES 15 V, ion source temperature 500 °C, and scan range 50-500 Da. MRM-HR mode parameters were set as follows: precursor ion 311.26 Da; time of flight from initial mass to final mass, ranging from 50.0000 to 315.0000 Da. Other parameters were the same as in Example 2. UPLC-Q-TOF data acquisition was performed using SCIEX OS Software (version 2.3.6).

[0159] The m / z value was extracted by mass spectrometry as 311.27 ± 0.02 ([M+H]). + The ion chromatogram of the target compound (1,8-diazacycloeicosane-9,20-dione) was obtained, and the peak area of ​​the mass spectrum peak was calculated by integration. The size of the peak area was used as an indicator to evaluate the extraction effect of each solvent.

[0160] 3.4 Experimental Results UPLC-Q-TOF analysis showed that the extraction effects of different solvents differed significantly (Table 5).

[0161] Table 5 Comparison of extraction effects with different solvents

[0162] Note: The relative extraction efficiency is calculated with the peak area of ​​acetonitrile as a 100% baseline.

[0163] Data shows that acetonitrile, methanol, and anhydrous ethanol can all extract the target compound with relatively high extraction efficiency. When acetonitrile is used as the extraction solvent, the response value (peak area) of the target compound is much higher than that of other solvents, and its extraction efficiency is at least twice that of ethanol and methanol. Water has the worst extraction effect.

[0164] Experimental results show that acetonitrile extraction is the most effective, followed by methanol and anhydrous ethanol, while water extraction is the least effective.

[0165] Acetonitrile's efficient extraction capability allows it to extract the target compound from the raw material to the maximum extent, laying the foundation for high yield in subsequent purification steps to obtain a higher content of crude extract.

[0166] Example 4: Investigation of extraction temperature and extraction time Using DuPont Nylon 612 particles as the standard raw material, a two-stage investigation was conducted using acetonitrile as the solvent.

[0167] Phase 1 (Temperature Investigation): Under the condition of a fixed extraction time of 4 hours, the extraction effect at four different temperatures (40°C, 50°C, 60°C, 80°C) was investigated.

[0168] The second stage (time-based evaluation): At the determined optimal temperature (60°C), the extraction effects at six different extraction times (1 hour, 2 hours, 3 hours, 4 hours, 8 hours, and 10 hours) were evaluated.

[0169] The content of the target compound and the impurity profile in all extracts were compared by UPLC-Q-TOF analysis.

[0170] 4.1 Experimental Reagents and Instruments Raw material: Nylon 612 particles (DuPont, USA).

[0171] Solvent: Acetonitrile (chromatographic grade).

[0172] Instruments: constant temperature water bath shaker, analytical ultra-high performance liquid chromatography-mass spectrometry system.

[0173] 4.2 Experimental Procedure Extraction temperature study: Accurately weigh four 1.0 g portions of DuPont Nylon 612 particles and place them in separate 20 mL headspace vials. Add 10 mL of acetonitrile to each of the four vials. Place the vials in constant temperature water baths at 40°C, 50°C, 60°C, and 80°C, respectively, and extract at 150 rpm for 4 hours.

[0174] Extraction time study: Six 1.0 g portions of DuPont Nylon 612 particles were accurately weighed and placed in 20 mL headspace vials. 10 mL of acetonitrile was added to each of the six vials. All vials were placed in a 60°C constant temperature water bath shaker and extracted by shaking at 150 rpm. The vials were removed after 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, and 10 hours, respectively.

[0175] After all extractions were completed, the samples were cooled to room temperature. 0.5 mL of the extract under each condition was taken and diluted to 10 mL with acetonitrile. The resulting test solution was then used for UPLC-Q-TOF analysis.

[0176] UPLC-Q-TOF analysis: The method is the same as that in “3.3 Experimental Steps” in Example 3.

[0177] Extraction efficiency evaluation: Extraction m / z was 311.27 ± 0.02 ([M+H]) by mass spectrometry. + The peak area of ​​the target compound's chromatographic peak is calculated by integrating the ion current chromatogram.

[0178] Extraction purity assessment: The relative purity of the extract is assessed by calculating the proportion of the target compound peak area in the total ion current chromatogram (TIC).

[0179] 4.3 Experimental Results (1) Temperature survey results UPLC-Q-TOF analysis results showed that extraction temperature had a significant impact on extraction efficiency and selectivity, as shown in Table 6.

[0180] Table 6 Comparison of extraction effects at different temperatures (extraction time: 4 hours)

[0181] Note: Relative extraction efficiency is calculated using the peak area at 60°C as a 100% baseline.

[0182] (2) Results of time investigation At the optimal temperature of 60°C, extraction time has a significant impact on extraction efficiency and selectivity (Table 7).

[0183] Table 7 Comparison of effects of different extraction times (extraction temperature: 60°C)

[0184] Note: The relative extraction efficiency is calculated using the peak area over 4 hours as a 100% baseline.

[0185] 4.4 Experimental Results (1) Temperature investigation Within the extraction temperature range of 40°C to 80°C, the relative extraction efficiency of the target compound was no less than 50%. Specifically, the extraction yield and peak percentage of the target compound reached their maximum at 60°C (~90%), indicating that the dissolution of other impurities was relatively minimal at this temperature, achieving an optimal balance between extraction efficiency and purity. At 50°C, the relative extraction efficiency of the target compound was 97.3%, with the highest peak percentage (~91%). The relative extraction efficiency was lowest at 40°C, and while the relative extraction yield was slightly higher at 80°C, impurities increased significantly. Therefore, 50°C–60°C is the optimal extraction temperature, followed by 40°C and 80°C.

[0186] (2) Time-based assessment Within a time range of 1 to 10 hours, the relative extraction efficiency of the target compound was no less than 60%. Specifically, at 60°C, the dissolution of the target compound reached near equilibrium within 4 hours. Further extending the extraction time to 8 or 10 hours did not significantly increase the amount of target compound extracted, but the purity of the extract (the percentage of the target compound peak) decreased significantly, and the extract became cloudy, indicating that prolonged heating led to the extraction of a large amount of non-target impurities. The relative extraction efficiency reached 95.2% at 3 hours, with the highest percentage of the target compound peak (~91%). Therefore, an extraction time of 3–4 hours is optimal, maximizing extraction efficiency while maintaining the highest purity.

[0187] (3) Determine the optimal process Combining the results of the two experiments, the combination of an extraction temperature of 50-60°C, an extraction time of 3-4 hours, and acetonitrile solvent can maximize the recovery rate of the target compound and minimize the co-extraction of impurities. This makes the extraction method highly efficient and selective, enabling near-quantitative extraction of the target cyclic compound in a short time, while maximally suppressing the dissolution of impurities.

[0188] Example 5: Investigation of Soxhlet Extraction Method This embodiment examines the effects of conventional Soxhlet extraction and the extraction method optimized by this disclosure. This embodiment uses the industry-standard Soxhlet extraction method, and the subsequent concentration, purification, drying, and identification steps are exactly the same as in Example 1, to compare the impact of different extraction methods on the recovery rate and purity of the final product.

[0189] 5.1 Sample preparation method Extraction: Accurately weigh 100.0 g of Nylon 612 particles (DuPont, USA) and place them in the filter paper tube of a Soxhlet extractor. Extract by continuous reflux in 100 mL of acetonitrile for 24 hours (60°C).

[0190] The concentration, separation, purification, and drying steps were the same as in Example 1, resulting in a white powder with a final weight of 35.8 mg.

[0191] 5.2 Experimental Results The extract was tested using the same method as in Example 1.

[0192] Recovery: 50.12 mg of crude extract was fed to produce 35.8 mg of pure product. The recovery rate of the target compound using this method was 71.4%.

[0193] Purity: The area normalization method showed that the purity of the product was 90.47%.

[0194] 5.3 Results Analysis The results show that Soxhlet extraction can also effectively extract the target compound from nylon particles. However, compared with the extraction method (60°C extraction for 4 hours) in Example 1.3 of this disclosure, Soxhlet extraction has the following disadvantages: The recovery rate was lower: 71.4% was significantly lower than the 84.1% in Example 1. The extraction time was as long as 24 hours, and the long heating time resulted in more impurity peaks in the crude extract in liquid chromatography, indicating that side reactions or more impurities may have been extracted; this increased the difficulty of subsequent purification, resulting in more loss of the target product in the multi-step processing.

[0195] The product purity was poor: the purity of 90.47% failed to meet the requirements of the chromatographic reference standard (usually >95%), and was significantly lower than the 97.68% of Example 1. This indicates that the crude extract obtained by Soxhlet extraction has a more complex composition and a higher content of impurities.

[0196] Inefficient and risky: The 24-hour extraction time is extremely inefficient, and prolonged heating may cause other components in the nylon particles to degrade or the target monomer itself to change, introducing new impurities, which increases the burden of subsequent purification and affects the quality of the final product.

[0197] A comparison of Examples 1 and 5 demonstrates that even using the same advanced purification technology (UPLC), the inefficient and complex traditional Soxhlet extraction method becomes a bottleneck in the entire process. In contrast, the shaking extraction method under heating conditions exhibits significant advantages in extraction efficiency, final product recovery rate, and purity, making it a gentler, more efficient, and more reliable extraction method.

[0198] Example 6: Preparative HPLC Purification Investigation This embodiment examines the effectiveness of conventional preparative HPLC and the UPLC preparation method disclosed herein. In this embodiment, a conventional preparative HPLC system (LC-20, Shimadzu, Japan) was used with a larger particle size column containing the same packing material to purify the same batch of crude extract obtained in Example 1, in order to compare separation efficiency.

[0199] 6.1 Sample preparation method Extraction and concentration: Same as in Example 1.

[0200] Separation and purification: A standard Prep-HPLC system was used, with a Waters XBridge Prep C18 column (250 × 19 mm, 10 μm). Mobile phase: Phase A: ultrapure water (containing 0.1% formic acid); Phase B: acetonitrile (containing 0.1% formic acid). The gradient program is shown in Table 8. The UV detector wavelength was 210 nm, the flow rate was 10.0 mL / min, and the run time was extended to 60 minutes to complete the separation. The injection volume was 0.5 mL (i.e., each injection contained approximately 2 mg of crude extract. Note: To process 50.15 mg of sample, this process needs to be repeated approximately 25 times). Based on the UV signal, the fraction collected at 11–13 min elution time (approximately 12.5 min) was collected. Fraction collection was performed manually.

[0201] Table 8 Gradient elution program for conventional Prep-HPLC system

[0202] 6.2 Experimental Results Only 20.15 mg of pure product was obtained from 50.15 mg of crude extract, with a product recovery rate of only 40.1%. The solvent consumption was twice that of the UPLC method, and the product purity was comparable to that of the product prepared in Example 1 (97.5%).

[0203] 6.3 Results Analysis (1) Reasons for the significant difference in recovery rate Conventional Prep-HPLC systems operate at lower pressures and typically use larger particle sizes of packing material and thicker columns, resulting in severe peak broadening. When manually collecting fractions, to avoid interference from impurities before and after the fraction, some purity must be sacrificed to ensure the "purity" of the target analyte. This usually involves using a wider collection window ("cutting off the head and tail"), which is the main reason for the significant loss of the target product and low recovery rate.

[0204] (2) Disadvantages in efficiency and cost The purification cycle is long, requiring multiple injections to obtain sufficient product, and Prep-HPLC involves manual fraction collection, significantly increasing time and labor costs. Furthermore, the solvent consumption per unit output is far higher than that of the method disclosed herein, making it neither economical nor environmentally friendly.

[0205] Experimental results demonstrate that although conventional Prep-HPLC may achieve similar levels of final product purity, its process efficiency is low, solvent waste is severe, and recovery rate is too low. The UPLC preparation method in Example 1, with its high column efficiency and narrow peak width resulting from ultra-high pressure and small particle size packing material (5 μm), combined with a fully automated fraction identification and collection system, offers significant advantages in terms of high recovery rate, high speed, and low consumption.

[0206] Example 7: Establishment and application of a quantitative analysis method based on reference standards 7.1 Experimental Methods Analysis was performed using UPLC-Q-TOF, where... (1) UPLC method An Exion LC system (SCIEX, Framingham, MA, USA) was used with an Agilent® SB-Aq column (2.1 × 100 mm, 1.8 μm). Samples were stored at 10°C, and the injection volume was 10 μl. The flow rate was set to 0.3 ml / min, and the column temperature was 40°C. A binary gradient elution system was used, consisting of ultrapure water (containing 0.1% formic acid, phase A) and acetonitrile (containing 0.1% formic acid, phase B). The gradient is shown in Table 4.

[0207] (2) TOF-MS / MS method Qualitative analysis was performed using Information-Dependent Acquisition (IDA) mode, while quantitative analysis was performed using Multiple Reaction Monitoring High Resolution (MRM-HR) mode. TOF-MS / MS parameters were set as follows: ion source gases 1 and 2 50 psi, curtain gas 35 psi, IS 5000 V, DP 80 V, CE 35 V, CES 15 V, ion source temperature 500 °C, and scan range 50-500 Da. MRM-HR mode parameters were set as follows: precursor ion 311.26 Da; time of flight from initial mass to final mass, ranging from 50.0000 to 315.0000 Da. Other parameters were the same as described above. Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) was used for data acquisition using SCIEX OS Software (version 2.3.6).

[0208] (3) Preparation of reference solution Using the reference standard prepared in Example 1, an appropriate amount was accurately weighed to prepare a series of standard solutions of different concentrations (10, 30, 50, 100, 1000 ng / mL) for linearity studies.

[0209] (4) Sample solution preparation The five-layer co-extruded infusion bag was cut into (3 cm × 1 cm) fragments, immersed in 10 mL of 50% ethanol solution, and then extracted by heating at 40°C for 1 hour. The resulting solution was used as the test sample solution.

[0210] (5) Methodological validation Refer to the ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) Guidelines for Analytical Method Validation. The suitability of the proposed method is evaluated through specificity, limit of detection (LOD) and limit of quantitation (LOQ), linearity, precision, recovery, stability, and robustness.

[0211] 7.2 Experimental Results (1) Exclusivity Figure 5 The image shows a typical mass spectrum of a five-layer co-extruded infusion bag sample solution containing 1,8-diazacycloeicosano-9,20-dione. A, B, and C represent the mass spectra of the blank solution, reference solution, and sample solution, respectively. As shown in the figure, the blank solution shows no obvious chromatographic peaks, only baseline fluctuations or noise signals. Compared to the blank solution, 1,8-diazacycloeicosano-9,20-dione in the sample and reference solutions eluted at the same retention time (approximately 2.9 min), with no interfering signals observed, indicating good specificity.

[0212] (2) Linearity and range 1,8-diazacycloeicosano-9,20-dione showed good linearity in the concentration range of 5.04–1008.06 ng / ml, with the correlation coefficient and linear equation being: Y = 56318.1X + 4019.51692, r = 0.99958. Figure 6 ).

[0213] (3) Sensitivity The limit of detection (LOD) and limit of quantitation (LOQ) of this method were 2.02 ng / mL and 5.04 ng / mL, respectively.

[0214] (4) Instrument precision The LOQ was used as the test sample for precision. The results showed that the relative standard deviation (RSD) of the instrument precision was 1.41%, and the intermediate precision of 1,8-diazacycloeicosane-9,20-dione was 1.06%.

[0215] (5) Repeatability As shown in Table 9, the average content of 1,8-diazacycloeicosane-9,20-dione body was 98.91 ng / cm³. 2 The RSD% is 0.23%.

[0216] Table 9. Reproducibility of Nylon 1,8-diazacycloeicosane-9,20-dione in five-layer co-extruded infusion bags

[0217] (6) Accuracy As shown in Table 10, the method has good accuracy, with a recovery rate of 97.65%–101.53% and an RSD of 1.33%.

[0218] Table 10 Recovery rate of 1,8-diazacycloeicosane-9,20-dione in five-layer co-extruded infusion bags (n=9)

[0219] (7) Solution stability The RSD% of the five-layer co-extruded infusion bag test solution and the 1,8-diazacycloeicosano-9,20-dione reference solution were 0.128% and 0.412% at room temperature (25±2℃) for 0h, 12h, 24h, 48h and 72h, respectively, indicating that the reference and test solutions remained stable within 72 hours (Table 11).

[0220] Table 11 Solution stability test results

[0221] (8) Method durability To evaluate the reliability of this method, sample solutions were analyzed under adjusted parameters including column temperature (±2 ℃), mobile phase composition (±0.2%), flow rate (±0.02 mL / min), and different chromatographic columns (Column 1: Agilent® SB-Aq 2.1×100 mm, 1.8 μm; Column 2: Waters® HSS T3 2.1 mm×100 mm, 1.8 µm). The changes in recovery rate under different conditions were systematically evaluated.

[0222] Robustness data are detailed in Table 12. Experimental results show that the method has good robustness, with recoveries of 1,8-diazacycloeicosano-9,20-dione remaining between 98.17% and 100.2%. Under different detection conditions, the results all meet the acceptable standards specified in the ICH guidelines.

[0223] Table 12. Durability test results of 1,8-diazacycloeicosane-9,20-dione (expressed as percentage of recovery under different conditions).

[0224] The above method was used to test different batches of five-layer co-extruded infusion bags, and the test results are shown in Table 13. Table 13 Test results of five-layer co-extruded infusion bags from different batches

[0225] Figures 7-8 The chromatograms are for a 1,8-diazacycloeicosane-9,20-dione reference standard and an infusion bag sample, respectively. Chromatographic peak information is as follows:

[0226] The chromatographic peaks appearing in the sample had the same retention time as the reference standard under the same conditions, and the mass-to-charge ratio (m / z) of the chromatographic peaks in the sample was consistent with that of the reference standard. Verification by retention time and high-resolution mass spectrometry data indicated that the component detected in the five-layer co-extruded infusion bag sample was the same substance as the high-purity reference standard prepared in Example 1, namely 1,8-diazacycloeicosano-9,20-dione.

[0227] The contents of 1,8-diazacycloeicosano-9,20-dione in the three batches of samples were 98.81, 98.83, and 99.11 ng / cm³, respectively. 2 The results were highly similar and reproducible, indicating that the method provided in this embodiment has high durability.

[0228] In summary, the high-purity reference standard prepared in this disclosure can be used for the qualitative and quantitative analysis of the target compound (1,8-diazacycloeicosane-9,20-dione) in pharmaceutical packaging materials, providing a reliable technical basis for accurately quantifying its migration level in formulations, assessing batch-to-batch consistency of materials, and conducting quality and compatibility studies on pharmaceutical packaging materials.

[0229] Example 8: Cytotoxicity study of 1,8-diazacycloeicosane-9,20-dione 8.1 Experimental Methods Using the CCK-8 assay, human hepatocytes (L02 cell line, Yimo Biotechnology) were used as a model. Cells were treated with different concentrations (0, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, 51.2 mM) of 1,8-diazacycloeicosano-9,20-dione (prepared in Example 1) for 24 hours, and cell viability was calculated.

[0230] 8.2 Experimental Results according to Figure 9 The dose-response curves (left) and cell viability (right) results show that compound 1,8-diazacycloeicosano-9,20-dione exhibits cytotoxicity against L02 cells. Its IC50 was calculated. 50 The value was 4.011 mM, which provides a crucial basis for the subsequent toxicological safety evaluation of this compound.

Claims

1. A cyclic compound, its solvate, or its salt, said cyclic compound having the structure shown in Formula I: （I） in, m is selected from integers from 6 to 12; alternatively, m is selected from integers from 8 to 10. n is an integer selected from 4 to 10; alternatively, n is an integer selected from 6 to 8. or, The cyclic compound, its solvate, or its salt has the structure shown in Formula II: (II)。 2. The cyclic compound, its solvate, or its salt according to claim 1, wherein, The cyclic compound, its solvate or salt has Figure 2 shows the 1H NMR spectrum; or, the 1H NMR spectrum includes the following peaks: δ 3.19 (t, J = 6.5 Hz, 4H), 2.17 (t, J = 6.5 Hz, 4H), 1.64-1.60 (m, 4H), 1.49-1.46 (m, 4H), 1.36-1.29 (m, 16H); And / or, Figure 3 shows a carbon NMR spectrum; or, the carbon NMR spectrum includes the following peaks: δ 174.68(2C), 38.69(2C), 35.61(2C), 29.57(2C), 28.86(2C), 28.80(2C),27.95(2C), 26.54(2C), 25.49(2C); And / or, The mass content shall not be less than 95%.

3. A method for purifying the cyclic compound, its solvate, or its salt according to claim 1 or 2, the method comprising the following steps: (a) Nylon particles were immersed in a solvent under heating conditions, and then the solvent was removed to obtain a crude extract; (b) The crude extract in step (a) is separated and purified by chromatographic separation, and the corresponding fraction is collected and dried to obtain the cyclic compound, its solvate or salt as described in claim 1 or 2.

4. The method according to claim 3, wherein, At least one of the following conditions must be met: In step (a), the nylon particles are selected from one or more of nylon 612, nylon 1010 or nylon 610, and optionally, the nylon is selected from nylon 612; In step (a), Soxhlet extraction, accelerated solvent extraction, or water bath shaking extraction is used; optionally, water bath shaking extraction is used. In step (a), the heating conditions are 40℃-80℃; or, the heating conditions are 50℃-70℃; or, the heating conditions are 50℃-60℃. In step (a), the extraction time is 0.1 hours to 24 hours; or, the extraction time is 1 hour to 6 hours; or, the extraction time is 3 hours to 4 hours. In step (a), the solvent is selected from one or more of methanol, anhydrous ethanol, and acetonitrile; optionally, the solvent is selected from acetonitrile; and / or, In step (b), the solvent of the corresponding fraction is removed by rotary evaporation under reduced pressure, followed by vacuum drying.

5. The method according to claim 4, wherein the chromatographic separation method in step (b) is selected from one or more of column chromatography, preparative high-performance liquid chromatography, high-speed countercurrent chromatography, and supercritical fluid chromatography; optionally, it is selected from preparative high-performance liquid chromatography, for example, preparative ultra-high-performance liquid chromatography.

6. The method according to claim 5, wherein, The chromatographic separation method is gradient elution, and its parameters include: The detection wavelength is 210±10 nm; and / or, The flow rate is 1.5 ± 0.5 mL / min; and / or, The elution process includes: Phase A is an aqueous solution containing 0.1%-0.5% (v / v) formic acid; Phase B is an acetonitrile solution containing 0.1%-0.5% (v / v) formic acid.

7. Use of the cyclic compound, its solvate or salt as described in claim 1 or 2, or the cyclic compound, its solvate or salt obtained by the method of any one of claims 3-6, in the study of nylon materials or articles containing nylon materials, optionally for use in quality studies.

8. The use according to claim 7, wherein, The nylon component in the nylon material or article containing nylon material is selected from one or more of nylon 612, nylon 1010, or nylon 610; optionally, the nylon is selected from nylon 612; and / or, The products containing nylon material include multilayer co-extruded infusion bags; and / or, The studies include pharmaceutical-related research, which may include compatibility studies of pharmaceutical packaging materials; and / or, The study is a quantitative analysis study; and / or, Cyclic compounds, their solvates, or salts were used as controls in the study.

9. A method for quantitative analysis of the cyclic compound, its solvate, or salt described in claim 1 or 2 in nylon materials or articles containing nylon materials, comprising the following steps: (I) Establish a test method using the cyclic compound of claim 1 or 2, its solvate or salt; (II) Extract the nylon material or the product containing the nylon material to obtain the sample to be tested; (III) The test sample is tested and analyzed using the test method of step (I), and the content of the cyclic compound, its solvate or salt as described in claim 1 or 2 is calculated.

10. The quantitative analysis method according to claim 9, wherein, In step (I), the test method is liquid chromatography-mass spectrometry, which may include one or more of the following studies: specificity, linearity and range, sensitivity, repeatability, accuracy, solution stability, and method robustness studies; and / or, In step (II), the extraction treatment includes the extraction parameters of the method according to any one of claims 3-6.

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