Method for determining residual solvent in OAB-14 using HS-GC

Abstract

This invention relates to the field of pharmaceutical analysis technology, specifically a method for determining residual solvents in OAB-14 using HS-GC. A reference solution and a test solution of OAB-14 are prepared. After headspace equilibration of the blank solution, reference solution, and test solution, they are injected into a gas chromatograph via headspace injection. The chromatograms are recorded, and the content of residual solvents in the test solution is calculated based on peak area using the external standard method. The residual solvents are one or more of methanol, ethanol, dichloromethane, triethylamine, pyridine, or xylene. This invention offers high sensitivity, good repeatability, and high accuracy, and can simultaneously detect the content of multiple residual solvents in OAB-14, thereby ensuring the quality of OAB-14 and improving the effectiveness and safety of clinical use.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, specifically a method for determining residual solvents in OAB-14 using HS-GC. Background Technology

[0002] Organic solvents are widely used as synthetic reaction media or extractants in the production of pharmaceutical raw materials and formulations. However, residual solvents that are not completely removed may pose a health hazard. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) classifies solvents into four categories based on their toxicity and potential risks and has established corresponding residue limits. The 2025 edition of the Chinese Pharmacopoeia, Part IV, "Residual Solvents," further clarifies the limits and detection methods for residual solvents in pharmaceuticals, with gas chromatography being a commonly used analytical method.

[0003] OAB-14, chemical name is 4-(3-(2-aminoethyl)ureido)- N -(1,1,4,4,5,5,8,8-octamethyl-1,2,3,4,5,6,7,8-octahydroanthracene-9-yl)benzamide, with the structural formula: The molecular formula is C 32 H 46 N4O2 has a molecular weight of 518.75.

[0004] For the synthesis process of OAB-14, it is crucial to control the residual levels of organic solvents such as methanol, ethanol, dichloromethane, triethylamine, pyridine, and xylene. Specifically, the residual levels of methanol, ethanol, dichloromethane, triethylamine, pyridine, and xylene should not exceed 0.3%, 0.5%, 0.06%, 0.5%, 0.02%, and 0.217%, respectively. These limits aim to ensure drug safety and prevent adverse reactions in patients due to solvent residues.

[0005] According to ICH Q3C(R9) "Impurities: Residual Solvents" and the provisions of General Chapter 0861 of Part IV of the 2025 edition of the Chinese Pharmacopoeia, residual solvents are classified into three categories based on their toxicity and potential risks. Methanol, dichloromethane, pyridine, and xylene belong to Category II solvents (whose use should be restricted) because they have non-genotoxic carcinogenicity in animals or may cause other irreversible toxicities (such as neurotoxicity and teratogenicity). Ethanol and triethylamine belong to Category III solvents (low toxicity), and their use is acceptable without justification at daily doses of 50 mg or less. Category II solvents are the focus of key testing and strict restriction in pharmaceutical quality control.

[0006] The residual limits for the six solvents measured in this invention are based on ICH Q3C and the 2025 edition of the Chinese Pharmacopoeia: methanol ≤3000ppm (0.3%), ethanol ≤5000ppm (0.5%), dichloromethane ≤600ppm (0.06%), triethylamine ≤5000ppm (0.5%), pyridine ≤200ppm (0.02%), and xylene ≤2170ppm (0.217%). Accurate detection of the residual amounts of these solvents in OAB-14 is a necessary prerequisite for ensuring its compliance with drug safety standards and guaranteeing the safety of clinical use.

[0007] There are existing reports on quality control methods for OAB-14. For example, Chinese Patent CN121633362A, published on March 10, 2026, discloses a method for determining the impurity content in OAB-14 using high performance liquid chromatography. This method achieves the separation and quantification of the main component and impurities through high performance liquid chromatography, but it does not involve specific technical solutions for controlling organic solvent residues.

[0008] Therefore, developing effective control technologies for organic solvent residues during the synthesis of OAB-14 is of great significance for improving drug quality. Summary of the Invention

[0009] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for determining residual solvents in OAB-14 using HS-GC. By optimizing specific column stationary phase, headspace conditions and chromatographic temperature program, the method can effectively overcome the defects of the prior art and achieve efficient separation and accurate quantification of multiple residual solvents in OAB-14.

[0010] This invention is achieved using the following technical solution: The method for determining residual solvent in OAB-14 using HS-GC includes the following steps: Prepare a reference solution containing residual solvent and a test solution of OAB-14; After headspace equilibration of the blank solution, reference solution and test solution, respectively, they were injected into the gas chromatograph by headspace injection for chromatographic separation, and the chromatograms were recorded. The content of residual solvent in the test sample solution is calculated based on the peak area using the external standard method. The residual solvent is selected from one or more of methanol, ethanol, dichloromethane, triethylamine, pyridine, and xylene; the gas chromatograph uses a capillary column with cyanopropylphenyl-dimethylpolysiloxane as the stationary phase as the chromatographic column.

[0011] Since the analyte solvents include polar solvents (methanol, ethanol), weakly polar / nonpolar solvents (dichloromethane, xylene), and basic solvents (triethylamine, pyridine), this invention selects a moderately polar stationary phase, cyanopropylphenyl-dimethylpolysiloxane (typically 6% cyanopropylphenyl-94% dimethylpolysiloxane). The cyanopropyl groups in this stationary phase provide dipole-dipole interactions, and the phenyl groups provide π-π interactions, thus exhibiting excellent overall selectivity for mixed solvents with different polarities and boiling points. In particular, it has good inertness towards easily tailing basic components such as triethylamine and pyridine, significantly improving peak shape and ensuring the accuracy of resolution and quantification.

[0012] The blank solution is dimethyl sulfoxide (DMSO); both the reference solution and the test solution use DMSO as the diluent. DMSO has a high boiling point (approximately 189°C) and produces a low vapor pressure at the set headspace heating temperature, thus avoiding large solvent peaks in the chromatogram that could interfere with the determination of the analyte. Simultaneously, DMSO has excellent solubility for OAB-14, facilitating the full release of residual solvent from the matrix into the gas phase in the headspace vial, achieving gas-liquid equilibrium.

[0013] The concentration of OAB-14 in the test solution is 0.01-0.03 g / mL. A suitable test concentration ensures the detection of low levels of residual solvent while avoiding excessively high concentrations that could lead to increased solution viscosity or abnormal changes in the headspace partition coefficient.

[0014] The concentrations of methanol, ethanol, dichloromethane, triethylamine, pyridine, and xylene in the reference solution were 0.6026-120.672 µg / mL, 1.0085-207.152 µg / mL, 0.4057-25.980 µg / mL, 0.01622-203.636 µg / mL, 0.5430-9.404 µg / mL, and 0.1317-88.556 µg / mL, respectively.

[0015] The headspace equilibration conditions include: equilibration temperature of 80-100℃, equilibration time of 25-35 min, and circulation time of 30-40 min.

[0016] The conditions used for the headspace injection method include: a quantitative loop temperature of 90-150℃, a transfer line temperature of 100-160℃, and a headspace injection port temperature of 180-220℃.

[0017] The capillary column is a DB-624 column with the following specifications: 30m length, 0.53mm inner diameter, and 3μm film thickness. The 30m column length provides sufficient theoretical plate number; the wide 0.53mm diameter combined with the 3μm thick liquid film not only increases the phase capacity of the column, allowing low-boiling-point polar solvents (methanol, dichloromethane) to be fully retained and thus avoiding overlap with the solvent front, but also effectively accommodates the large volume of gas brought in by headspace injection, preventing peak broadening.

[0018] The column temperature program of the gas chromatograph is as follows: the initial temperature is 38-42℃, maintained for 3-5 minutes, and then increased to 200-250℃ at a heating rate of 10-20℃ / min, and held for 4-6 minutes.

[0019] The gas chromatograph uses a flame ionization detector with a detector temperature of 245-255℃.

[0020] The gas chromatograph uses nitrogen as the carrier gas with a flow rate of 2.85-3.15 mL / min and a split ratio of 1:1; the detector uses hydrogen with a flow rate of 35-40 mL / min and air with a flow rate of 350-400 mL / min.

[0021] Compared with the prior art, the beneficial effects of the present invention are: (1) The present invention can simultaneously detect the content of six residual solvents, namely methanol, ethanol, dichloromethane, triethylamine, pyridine and xylene, in OAB-14, either qualitatively or quantitatively.

[0022] (2) This invention provides a method for determining residual solvents in OAB-14 using HS-GC. On the one hand, it provides a good reference for controlling residual organic solvents in the OAB-14 production process, ensuring the quality of OAB-14 and thus improving the safety of clinical drug use. On the other hand, this method solves the cumbersome problem of having to use multiple methods to process and detect OAB-14 when multiple residual solvents are present, greatly improving the convenience of operation and saving labor, time, reagent costs, etc.

[0023] (3) The sample pretreatment of the present invention is simple and only requires a single solvent for dissolution; after rigorous methodological verification, the retention time under this condition is stable, and the intra-day and inter-day precision is good. It has strong operability and practicality and is suitable for daily quality control in the industrial production of OAB-14.

[0024] (4) The six residual solvents detected in this invention are all organic solvents actually used or generated in the OAB-14 synthesis process. Their residual levels are key indicators for the stability and quality control of the OAB-14 production process. According to ICH Q3C classification, dichloromethane, pyridine, xylene, and methanol are all Class II restricted solvents, which have potential neurotoxicity or carcinogenic risks; if the residual amount exceeds the limit, it may cause irreversible health damage to patients. Therefore, establishing an accurate detection method for the above solvents is a necessary technical means to ensure the safety of OAB-14 from the source and meet the requirements for drug quality release. After methodological validation and actual sample testing, only methanol was detected in the two batches of OAB-14 raw materials, both of which were below the limit of 3000 ppm specified by ICH Q3C; the other five solvents were not detected. All test results meet the residual solvent limit requirements of the 2025 edition of the Chinese Pharmacopoeia and ICH Q3C. Attached Figure Description

[0025] Figure 1 This is the chromatogram of the blank solution in Example 1.

[0026] Figure 2 This is the chromatogram of the methanol solution in Example 1.

[0027] Figure 3 This is the chromatogram of the ethanol solution in Example 1.

[0028] Figure 4 This is the chromatogram of the dichloromethane solution in Example 1.

[0029] Figure 5 This is the chromatogram of the triethylamine solution in Example 1.

[0030] Figure 6 This is the chromatogram of the pyridine solution in Example 1.

[0031] Figure 7 This is the chromatogram of the xylene solution in Example 1.

[0032] Figure 8 This is the chromatogram of the reference solution in Example 1.

[0033] Figure 9 This is the chromatogram of the test solution in Example 1.

[0034] Figure 10 This is the chromatogram of the spiked solution of the test sample in Example 1.

[0035] Figure 11 This is the chromatogram of the test solution in Comparative Example 1.

[0036] Figure 12 This is the chromatogram of the spiked solution of the test sample in Comparative Example 2.

[0037] Figure 13 This is the hydrogen spectrum of OAB-14 of the present invention.

[0038] Figure 14 This is the carbon spectrum of OAB-14 of the present invention. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the invention.

[0040] Before describing the present invention in conjunction with specific embodiments, in order to ensure that those skilled in the art can fully understand and implement the technical solutions of the present invention, the present invention will now be described in detail: The OAB-14 active pharmaceutical ingredient used in this invention was prepared by Shandong Xinhua Pharmaceutical Co., Ltd. according to the following preparation method, with batch number 2009003 and a content (based on dried product) of 98.0% or more.

[0041] Preparation method of OAB-14: Raw materials and reagents used: 2,5-Dimethyl-2,5-hexanediol (Shanghai Aladdin Biochemical Technology Co., Ltd., purity ≥99%); concentrated hydrochloric acid (Sinopharm Chemical Reagent Co., Ltd., mass fraction 36%-38%); anhydrous aluminum trichloride (Sinopharm Chemical Reagent Co., Ltd., purity ≥99%); benzene (Tianjin Fuyu Fine Chemical Co., Ltd., analytical grade); fuming nitric acid (mass fraction ≥90%); concentrated sulfuric acid (Sinopharm Chemical Reagent Co., Ltd., mass fraction 98%); tin powder (Tianjin Kemeio Chemical Reagent Co., Ltd.); anhydrous ethanol (Sinopharm Chemical Reagent Co., Ltd., analytical grade); dichloromethane, petroleum ether (60-90℃), and ethyl acetate are all commercially available analytical grade reagents and can be used directly; 10% palladium on carbon (palladium content 10%); xylene (analytical grade); pyridine (analytical grade); p-nitrobenzoyl chloride (purity ≥99%); 4-dimethylaminopyridine (DMAP, purity ≥99%); anhydrous potassium carbonate (purity ≥99%); phenyl chloroformate (purity ≥99%); ethylenediamine (purity ≥99%); triethylamine (purity ≥99%); methanol (analytical grade).

[0042] (1) Preparation of 2,5-dichloro-2,5-dimethylhexane In a 1000 mL three-necked flask equipped with a mechanical stirrer, reflux condenser, and thermometer, 100.0 g (0.685 mol) of 2,5-dimethyl-2,5-hexanediol and 500 mL (36%-38% by mass) of concentrated hydrochloric acid were added and stirred at room temperature until partially dissolved. The reaction mixture was heated to 43 °C and stirred for 4 hours. After the reaction was complete, the reaction solution was cooled to room temperature and extracted with dichloromethane (200 mL × 3). The combined organic phases were washed successively with saturated sodium bicarbonate solution (200 mL) and saturated sodium chloride solution (200 mL), and dried over anhydrous sodium sulfate. After filtration, the solvent was removed by concentration under reduced pressure (40 °C, -0.09 MPa) to give a pale yellow oily substance, 2,5-dichloro-2,5-dimethylhexane (118.5 g, 94.2% yield). The product was used directly in the next reaction without further purification.

[0043] (2) Preparation of 1,1,4,4,5,5,8,8-octamethyl-1,2,3,4,5,6,7,8-octahydroanthracene (ZZa) The Friedel-Crafts alkylation method disclosed in CN107304172A was followed. Under nitrogen protection, anhydrous aluminum trichloride (120.0 g, 0.900 mol) and benzene (800 mL) were added to a 2000 mL three-necked flask equipped with a mechanical stirrer, dropping funnel, and reflux condenser, and the flask was cooled to 0 °C in an ice-water bath. Under vigorous stirring, a benzene solution (200 mL) of 2,5-dichloro-2,5-dimethylhexane (118.5 g, 0.645 mol) obtained in step (1) was slowly added dropwise over 1.5 hours, with the reaction temperature controlled not to exceed 10 °C during the addition. After the addition was complete, the ice-water bath was removed, the reaction mixture was slowly heated to room temperature, and the reaction was stirred for 2 hours, followed by heating to 43 °C for 3 hours. After the reaction was completed, the reaction solution was slowly poured into an ice-water mixture (1000 g) and quenched by stirring. The mixture was separated, and the aqueous phase was extracted with benzene (200 mL × 2). The organic phases were combined and washed successively with 5% dilute hydrochloric acid (300 mL), saturated sodium bicarbonate solution (300 mL), and saturated sodium chloride solution (300 mL), and dried over anhydrous sodium sulfate. After filtration, the solvent was removed by concentration under reduced pressure (45 °C, vacuum degree -0.09 MPa) to obtain the crude product. The crude product was recrystallized from petroleum ether (80 °C) to give white needle-like crystals ZZa (89.2 g, yield 82.5%).

[0044] (3) Preparation of 1,1,4,4,5,5,8,8-octamethyl-1,2,3,4,5,6,7,8-octahydro-9-nitroanthracene (ZZb) Under ice-water bath cooling, fuming nitric acid (40 mL, mass fraction ≥90%) was slowly added to a 500 mL three-necked flask equipped with a mechanical stirrer and thermometer. Then, concentrated sulfuric acid (60 mL, mass fraction 98%) was slowly added dropwise at 0-5 °C to prepare a mixed acid, controlling the dropping rate to keep the temperature below 10 °C. The ZZa (50.0 g, 0.155 mol) obtained in step (2) was dissolved in dichloromethane (300 mL) and slowly added dropwise to the above mixed acid at 2 °C, controlling the dropping rate to keep the reaction temperature below 5 °C. After the addition was complete, the reaction was stirred at 0-5 °C for 4 hours, and then slowly raised to room temperature for 1 hour.

[0045] After the reaction was complete, the reaction solution was slowly poured into an ice-water mixture (500 g), and the mixture was stirred to quench the reaction. The aqueous phase was separated and extracted with dichloromethane (150 mL × 2). The combined organic phases were washed successively with saturated sodium bicarbonate solution (200 mL × 2) and saturated sodium chloride solution (200 mL), and dried over anhydrous sodium sulfate. After filtration, the solvent was removed by concentration under reduced pressure (40 °C, vacuum -0.09 MPa) to obtain a pale yellow crude solid. The crude product was recrystallized from ethyl acetate-petroleum ether (1:10, v / v) to give a pale yellow solid ZZb (47.5 g, yield 83.0%).

[0046] (4) Preparation of OAB-14 intermediate (OAB-14-5-aminooctahydroanthracene) In a 1000 mL three-necked flask equipped with a mechanical stirrer and a reflux condenser, add ZZb (40.0 g, 0.109 mol), tin powder (65.0 g, 0.547 mol, 200 mesh), and anhydrous ethanol (400 mL) obtained in step (3), and heat to reflux. Under reflux conditions, slowly add concentrated hydrochloric acid (120 mL, 36%-38% by mass) dropwise over 1 hour, and continue reflux for 5 hours after the addition is complete. After the reaction is complete, cool the reaction solution to room temperature and filter to remove unreacted tin powder. Concentrate the filtrate under reduced pressure (50 °C, vacuum degree -0.09 MPa) to remove most of the ethanol, and dissolve the residue in dichloromethane (500 mL). Adjust the pH to 10 with 20% sodium hydroxide solution, separate the layers, and extract the aqueous phase with dichloromethane (200 mL × 2). The organic phases were combined and washed successively with saturated sodium bicarbonate solution (300 mL) and saturated sodium chloride solution (300 mL), and dried over anhydrous sodium sulfate. After filtration, the solvent was removed by concentration under reduced pressure (45 °C, vacuum degree -0.09 MPa) to obtain the crude product. The crude product was recrystallized from ethyl acetate-petroleum ether (1:3, volume ratio) to give a white solid OAB-14 intermediate (OAB-14-5) (33.8 g, yield 72.5%).

[0047] (5) Preparation of intermediate OAB-14-6 Add xylene (250 mL) to a four-necked flask, then add the intermediate OAB-14 (OAB-14-5) obtained in step (4) (31.4 g, 0.1 mol) and DMAP (1.2 g), and stir for 0.5 hours to dissolve it. Add pyridine (15.8 g, 0.2 mol), followed by p-nitrobenzoyl chloride (22.3 g, 0.12 mol). Under nitrogen protection, heat to reflux and maintain the reaction temperature for 24 hours. Cool to 100 °C, slowly add 10% sodium hydroxide aqueous solution (100 mL) to the reaction mother liquor, and continue stirring for 6 hours. Filter, wash the filter cake with purified water until neutral, then slurry with anhydrous ethanol (150 mL), filter, and dry to obtain a yellow solid intermediate OAB-14-6.

[0048] (6) Preparation of intermediate OAB-14-7 Add 39.3 g of OAB-14-6 and 4.0 g of 10% palladium on carbon to a four-necked flask (or autoclave), then add 200 mL of dichloromethane and 200 mL of anhydrous methanol, and stir until homogeneous. Purge the mixture six times with hydrogen, maintaining an internal temperature of 35°C and a pressure of 0.3 MPa, and continue the reaction for 24 hours. Stop heating and allow the internal temperature to drop to 20°C, then filter to remove the palladium on carbon. Concentrate the filtrate under reduced pressure until no more fractions are distilled off. Add 150 mL of anhydrous ethanol to the residue, slurry, filter, and dry to obtain a white solid intermediate, OAB-14-7.

[0049] (7) Preparation of crude OAB-14 Add 33.1 g of OAB-14-7, 21.1 g of anhydrous potassium carbonate, and 300 mL of dichloromethane to a four-necked flask and stir until homogeneous. Slowly add 13.2 g of phenyl chloroformate dropwise. After the addition is complete, heat to reflux and react for 15-20 hours, monitoring the reaction by TLC until complete. Cool to room temperature and slowly add 23.0 g of ethylenediamine dropwise to the system, reacting at room temperature for 4 hours. Wash with purified water (200 mL), separate the organic phase, concentrate, and remove solvent to obtain crude OAB-14.

[0050] (8) Preparation of OAB-14 refined product Add the crude OAB-14 obtained in step (7) and methanol (150 mL) to a four-necked flask, and add 2 mol / L dilute hydrochloric acid (45 mL) dropwise. Maintain the reaction temperature at 25-30℃ for 15-20 hours. Cool to 5-10℃, allow to stand for crystallization for 2 hours, and filter to collect the hydrochloride solid. Add the filter cake to a flask containing methanol (150 mL), add 10% sodium hydroxide solution (40 mL) dropwise, and react at 25-30℃ for 6 hours to release the target product. Cool to 5-10℃, allow to crystallize for 2 hours, and filter. Add the filter cake to purified water (300 mL) and slurry for 2 hours. Filter and wash until neutral, then dry to obtain the purified OAB-14 product.

[0051] (9) Preparation of finished product Add the purified product (28.0 g), methanol (150 mL), and triethylamine (5 mL) obtained in step (8) to a four-necked flask. Heat to reflux and reflux for 1 hour, then filter while hot. Collect the filter cake, wash with methanol (100 mL) at room temperature for 1 hour, and filter. Place the filter cake in a vacuum drying oven, control the vacuum degree to be not lower than -0.095 MPa, and dry at 60-80℃ to constant weight to obtain the OAB-14 product (approximately 25.5 g, HPLC purity ≥98.0%). The proton NMR spectrum of the obtained OAB-14 is shown below. Figure 13 The carbon spectrum of OAB-14 is shown below. Figure 14 .

[0052] 1. Basic requirements for instruments and reagents The gas chromatograph described in this invention needs to be equipped with a headspace sampler and a flame ionization detector (FID). Among the reagents used, dimethyl sulfoxide (DMSO) should be chromatographically pure to ensure that it does not contain impurities that interfere with the elution of the target analyte; methanol, ethanol, dichloromethane, triethylamine, pyridine, and xylene reference standards should be analytically pure or higher to ensure the accuracy of quantification.

[0053] 2. Basis for Column Selection and Specifications This invention specifies the use of a capillary column (preferably 30m × 0.53mm × 3μm, such as DB-624 or an equivalent column) using cyanopropylphenyl-dimethylpolysiloxane as the stationary phase. This limitation is based on the following technical considerations: First, the six solvents to be tested covered polar (methanol), weakly polar (ethanol), nonpolar (xylene), low-boiling-point (dichloromethane), and basic (triethylamine, pyridine) substances. Conventional 100% dimethylpolysiloxane weakly polar columns cannot effectively separate polar solvents, while polyethylene glycol strongly polar columns exhibit very weak retention of nonpolar solvents and easily lead to severe tailing of basic solvents. 6% cyanopropylphenyl-94% dimethylpolysiloxane, as a moderately polar stationary phase, provides appropriate dipole and π-π interactions through its specific ratio of cyanopropyl and phenyl groups, exhibiting balanced selectivity for the aforementioned complex polar combinations and good deactivation inertness towards basic nitrogen-containing compounds, effectively reducing peak tailing for triethylamine and pyridine.

[0054] Secondly, the 0.53 mm inner diameter and 3 μm thick liquid film design significantly increase the phase capacity of the column (compared to other components). This is particularly important for low-boiling-point methanol and dichloromethane, as the thick liquid film increases the distribution of these low-boiling-point components in the stationary phase, moderately delaying their retention time and thus avoiding overlap with potential air peaks or solvent fronts; at the same time, the large diameter can accommodate the large volume of vapor generated by headspace injection, reducing peak broadening effects.

[0055] 3. Basis for setting headspace injection conditions This method sets the equilibrium temperature at 80-100℃ and the equilibrium time at 25-35 min. The principle is as follows: Xylene, a target analyte, has a high boiling point (approximately 138-144℃), while pyridine has a boiling point of 115℃. If the equilibrium temperature is too low, the vapor pressure of the high-boiling-point solvents distributed to the gas phase will be insufficient, leading to a sharp decrease in detection sensitivity. Meanwhile, the diluent DMSO has a boiling point as high as 189℃. If the equilibrium temperature is above 100℃, a large amount of DMSO will vaporize, not only altering the gas-liquid equilibrium constant in the headspace vial, but also causing column overload due to the large volume of DMSO vapor entering the column, thus masking the target peak. Therefore, an equilibrium temperature of 80-100℃ is a reasonable range that balances sensitivity to high-boiling-point components with avoiding solvent interference.

[0056] Meanwhile, the system piping temperature is strictly set to increase in a gradient: equilibrium temperature (80-100℃) < quantitative loop temperature (90-150℃) < transfer line temperature (100-160℃) < injection port temperature (180-220℃). This "stepped temperature increase" design aims to prevent condensation and accumulation of gaseous samples (especially high-boiling-point vapors containing xylene and pyridine) extracted from the headspace vial in the transfer piping and valve body, which could lead to sample residue, cross-contamination, or inaccurate injection volume.

[0057] 4. Basis for setting temperature programming conditions in gas chromatography The programmed temperature rise of this invention is as follows: initial temperature 3842℃, held for 35 min, then increased at 1020℃ / min to 200-250℃ and held for 46 min. This temperature rise logic is based on the boiling point range of the target analytes: a lower initial temperature (e.g., 40℃) and a holding time can utilize the "solvent focusing effect" to effectively enrich methanol, dichloromethane, and ethanol, which first enter the column, at the column head, thereby obtaining sharp and symmetrical chromatographic peaks and achieving baseline separation of the three; subsequently, a moderate temperature rise rate of 1020℃ / min allows triethylamine, pyridine, and xylene to elute sequentially according to their polarity and boiling point differences, avoiding insufficient separation or excessively long analysis time; finally, the temperature is raised to 200-250℃ and held to promote the rapid elution of trace amounts of DMSO and any high-boiling-point impurities in the matrix, thus providing a clean column environment for the next injection.

[0058] 5. Solution concentration and quantitative calculation method The concentration of OAB-14 in the test solution is set at 0.01~0.03 g / mL. If the concentration is too low, it will not reach the detection limit for trace residual solvents specified by the pharmacopoeia or ICH; if the concentration is too high, the solid OAB-14 may reach saturation in DMSO or make the solution viscosity too high, leading to increased mass transfer resistance between the gas and liquid phases, affecting the constancy of the gas-liquid equilibrium partition coefficient, and causing deviations in the quantitative results. This invention uses the external standard method for quantitative calculation. By comparing the chromatographic peak area (Ax) of each residual solvent in the test solution with the corresponding chromatographic peak area (Ar) in the reference solution, and combining this with the known concentration (Cr) of the reference standard, the content (Cx) of each solvent in the test sample can be accurately calculated. The calculation formula is: Cx=(Ax×Cr) / Ar.

[0059] In summary, this invention achieves accurate determination of six complex residual solvents in OAB-14 through systematic and coordinated matching of dilution solvent, column stationary phase, headspace-liquid equilibrium thermodynamic parameters, and chromatographic separation kinetic parameters.

[0060] The technical solution of the present invention will be further verified and explained below with reference to specific embodiments.

[0061] Example 1 (a) Solution preparation (1) Blank solution: Take 5.0 mL of dimethyl sulfoxide into a 20 mL headspace vial and seal it.

[0062] (2) Methanol solution: Weigh 0.30129 g of methanol by the weight gain method and place it in a 50 mL volumetric flask containing a small amount of dimethyl sulfoxide. Dilute to the mark with dimethyl sulfoxide. Measure 1.0 mL of this solution into a 100 mL volumetric flask containing a small amount of dimethyl sulfoxide. Dilute to the mark with dimethyl sulfoxide and mix well. Take 5.0 mL of this solution into a 20 mL headspace vial and seal it.

[0063] (3) Ethanol solution: Weigh 0.50424 g of anhydrous ethanol by the weight gain method and place it in a 50 mL volumetric flask containing a small amount of dimethyl sulfoxide. Dilute to the mark with dimethyl sulfoxide. Measure 1.0 mL of this solution into a 100 mL volumetric flask containing a small amount of dimethyl sulfoxide and dilute to the mark with dimethyl sulfoxide. Shake well. Take 5.0 mL of this solution into a 20 mL headspace vial and seal it.

[0064] (4) Dichloromethane solution: Weigh 0.06762 g of dichloromethane using the weight gain method and place it in a 50 mL volumetric flask containing a small amount of dimethyl sulfoxide. Dilute to the mark with dimethyl sulfoxide. Measure 1.0 mL of this solution into a 100 mL volumetric flask containing a small amount of dimethyl sulfoxide and dilute to the mark with dimethyl sulfoxide. Shake well. Take 5.0 mL of this solution into a 20 mL headspace vial and seal it.

[0065] (5) Triethylamine solution: Weigh 0.50687 g of triethylamine using the weight gain method, and dilute it to the mark with dimethyl sulfoxide in a 50 mL volumetric flask containing a small amount of dimethyl sulfoxide. Measure 1.0 mL of this solution into a 100 mL volumetric flask containing a small amount of dimethyl sulfoxide, and dilute it to the mark with dimethyl sulfoxide. Shake well. Take 5.0 mL of this solution into a 20 mL headspace vial and seal it.

[0066] (6) Pyridine solution: Weigh 0.02715 g of pyridine using the weight gain method and place it in a 50 mL volumetric flask containing a small amount of dimethyl sulfoxide. Dilute to the mark with dimethyl sulfoxide. Measure 1.0 mL of this solution into a 100 mL volumetric flask containing a small amount of dimethyl sulfoxide and dilute to the mark with dimethyl sulfoxide. Shake well. Take 5.0 mL of this solution into a 20 mL headspace vial and seal it.

[0067] (7) Xylene solution: Weigh 0.21945 g of xylene using the weight gain method and place it in a 50 mL volumetric flask containing a small amount of dimethyl sulfoxide (DMSO). Dilute to the mark with DMSO. Measure 1.0 mL of this solution into a 100 mL volumetric flask containing a small amount of DMSO and dilute to the mark with DMSO. Shake well. Take 5.0 mL of this solution into a 20 mL headspace vial and seal.

[0068] (8) Reference solution: Weigh 0.30168 g of methanol, 0.51788 g of anhydrous ethanol, 0.06495 g of dichloromethane, 0.50909 g of triethylamine, 0.02351 g of pyridine, and 0.22139 g of xylene using the gravimetric method. Dilute to the mark with dimethyl sulfoxide in a 50 mL volumetric flask containing a small amount of dimethyl sulfoxide, and shake well to obtain solution A. Take 1.0 mL of solution A and dilute to the mark with dimethyl sulfoxide in a 100 mL volumetric flask containing a small amount of dimethyl sulfoxide, and shake well to obtain the reference solution. Take 5.0 mL of this solution and seal it in a 20 mL headspace vial.

[0069] (9) Test solution: Weigh 0.10189 g of the sample into a 20 mL headspace vial, add 5.0 mL of dimethyl sulfoxide, and seal.

[0070] (10) Spiked solution for test sample: Weigh 0.10282 g of sample into a 20 mL headspace vial, add 5.0 mL of reference solution, and seal.

[0071] (II) Analysis Conditions The conditions for headspace equilibrium are as follows: Equilibrium temperature: 90℃, equilibrium time: 30min, circulation time: 35min, quantitative loop temperature: 100℃, transfer line temperature: 110℃, headspace inlet temperature: 200℃.

[0072] The chromatographic conditions are as follows: Instruments: Agilent 7890A gas chromatograph; Column: DB-624 (30m × 0.53mm, film thickness 3μm) USR706341H; Column temperature: initial temperature 40℃, hold for 5 min, increase to 200℃ at a rate of 10℃ / min, hold for 5 min; Detector: FID; Detector temperature: 250℃; Carrier gas: nitrogen; Carrier gas flow rate: 3.0 mL / min; Split ratio: 1:1; Hydrogen flow rate: 40 mL / min; Air flow rate: 400 mL / min.

[0073] (III) Determination: After headspace equilibration of the above solutions, they were injected into the gas chromatograph via headspace injection, and the chromatograms were recorded. The results are shown in [Figure number missing]. Figure 1-10 The content of residual solvent in the test sample solution is calculated by peak area using the external standard method.

[0074] Depend on Figure 1-10 It can be seen that, Figure 1 The chromatogram of the blank solution (dimethyl sulfoxide) shows no significant interference at any of the target peaks, indicating that the solvent system has no effect on the determination. Figures 2 to 7 Chromatograms of single solutions of methanol, ethanol, dichloromethane, triethylamine, pyridine, and xylene are shown below. The retention times of each solvent are as follows: methanol approximately 3.613 min, ethanol approximately 4.887 min, dichloromethane approximately 6.471 min, triethylamine approximately 10.148 min, pyridine approximately 12.474 min, and xylene (three isomers) approximately 14.631 min, 14.787 min (maximum peak), and 15.333 min. Figure 8 The chromatogram of the mixed reference solution shows that all target peaks are baseline separated, and the resolution meets the requirements. Figure 9 The chromatogram of the test solution shows that no obvious interfering peaks were observed near the retention time of the target peak, indicating that the sample matrix does not interfere with the determination. Figure 10 The chromatogram of the spiked solution of the test sample shows that the retention times of each target peak are consistent with those in the reference solution, and the peak shapes are symmetrical, further verifying the specificity of the method. In summary, the specificity of this method meets the requirements for residual solvent determination. The retention times of each compound in Example 1 are shown in Table 1.

[0075] Table 1: Retention times of each compound in Example 1

[0076] Method validation (unless otherwise specified, the headspace equilibration and chromatographic conditions used are the same as in Example 1): a. Repeatability (1) Reference solution: Weigh 0.30168g of methanol, 0.51788g of anhydrous ethanol, 0.06495g of dichloromethane, 0.50909g of triethylamine, 0.02351g of pyridine, and 0.22139g of xylene using the gravimetric method. Place them in a 50mL volumetric flask containing a small amount of dimethyl sulfoxide (DMSO). Dilute to the mark with DMSO and shake well to obtain solution A. Take 1.0mL of solution A and place it in a 100mL volumetric flask containing a small amount of DMSO. Dilute to the mark with DMSO and shake well to obtain the reference solution. Take 5.0mL of this solution in a 20mL headspace vial and seal it. (2) Blank solution: Take 5.0mL of DMSO and place it in a 20mL headspace vial and seal it. (3) Repeatability test solution: Weigh 0.1g of the sample and place it in a 20mL headspace vial. Add 5.0mL of the reference solution and seal it. Prepare 6 samples using the same method.

[0077] After headspace equilibration of the above reference solution, blank solution and repeatability test solution, they were injected into the gas chromatograph by headspace injection, and the chromatograms were recorded. The repeatability results are shown in Table 2.

[0078] Table 2: Repeatability Results

[0079] As shown in Table 2, under the reference solution section, the RSD of the peak area of ​​methanol was 0.6%, the RSD of the peak area of ​​ethanol was 0.7%, the RSD of the peak area of ​​dichloromethane was 0.7%, the RSD of the peak area of ​​triethylamine was 0.7%, the RSD of the peak area of ​​pyridine was 0, and the RSD of the peak area of ​​xylene was 0.6, all of which were not greater than 10%. Under the test solution section, the RSD of the six determinations of methanol was 4.7%, the RSD of the six determinations of ethanol was 4.9%, the RSD of the six determinations of dichloromethane was 0.3%, the RSD of the six determinations of triethylamine was 10.0%, the RSD of the six determinations of pyridine was 6.4%, and the RSD of the six determinations of xylene was 4.1%, all of which were not greater than 10% and met the requirements.

[0080] b. Intermediate precision Change the analysts, instruments, and chromatographic columns, and prepare new intermediate precision test solutions for testing on different dates.

[0081] (1) Reference solution: Weigh 0.30152g of methanol, 0.50633g of anhydrous ethanol, 0.06786g of dichloromethane, 0.50916g of triethylamine, 0.02905g of pyridine, and 0.21801g of xylene using the weight gain method. Place them in a 50mL volumetric flask containing a small amount of dimethyl sulfoxide (DMSO). Dilute to the mark with DMSO and shake well to obtain solution A. Take 1.0mL of solution A and place it in a 100mL volumetric flask containing a small amount of DMSO. Dilute to the mark with DMSO and shake well to obtain the reference solution. Take 6 20mL headspace vials and add 5.0mL of the above solution to each, then seal. (2) Blank solution: Place 5.0mL of DMSO in a 20mL headspace vial and seal. (3) Intermediate precision test solution: Weigh 0.1g of sample into a 20mL headspace vial, add 5.0mL of reference solution, and seal; prepare 6 vials in the same way.

[0082] After headspace equilibration of the above reference solution, blank solution and intermediate precision test solution, they were injected into the gas chromatograph by headspace injection, and the chromatograms were recorded. The intermediate precision results are shown in Table 3.

[0083] Table 3: Intermediate Precision Results

[0084] As shown in Table 3, under the reference solution test, the RSD of the peak area of ​​methanol was 2.3%, the RSD of the peak area of ​​ethanol was 2.4%, the RSD of the peak area of ​​dichloromethane was 2.7%, the RSD of the peak area of ​​triethylamine was 2.5%, the RSD of the peak area of ​​pyridine was 2.7%, and the RSD of the peak area of ​​xylene was 2.5%, all not exceeding 10%. Under the test solution test, the RSD of the six determinations of methanol was 2.0%, the RSD of the six determinations of ethanol was 2.1%, the RSD of the six determinations of dichloromethane was 2.4%, the RSD of the six determinations of triethylamine was 2.8%, the RSD of the six determinations of pyridine was 2.5%, and the RSD of the six determinations of xylene was 2.5%, all not exceeding 10%, and all meeting the requirements.

[0085] c. Limit of Detection and Limit of Quantification Limit of Quantitation (LOQ) and Limit of Detection (Detection) Test Solutions: Weigh appropriate amounts of methanol, ethanol, dichloromethane, triethylamine, pyridine, and xylene, and gradually dilute with dimethyl sulfoxide to obtain appropriate concentrations of LOQ and Detection test solutions. Transfer 5.0 mL of each solution to a 20 mL headspace vial and seal.

[0086] Blank solution: Take 5.0 mL of dimethyl sulfoxide and place it in a 20 mL headspace vial. Seal the vial to obtain the blank solution.

[0087] After headspace equilibration of the above solution, it was injected into a gas chromatograph via headspace injection. The chromatogram was recorded, and the signal-to-noise ratio (S / N) of each target peak was measured. When S / N was not less than 2, the concentration of each solvent in the headspace vial was converted to the equivalent content in the test sample, which was the limit of detection (LOD). When S / N was not less than 10, the concentration of each solvent in the headspace vial was converted to the equivalent content in the test sample, which was the limit of quantitation (LOQ). The results of the LOD and LOQ determination in Example 1 are shown in Table 4.

[0088] The calculation formula is: Limit of Detection (LOD) (ppm) = Minimum Detection Concentration (μg / mL) × 50 Limit of Quantification (LOQ) (ppm) = Minimum Quantitative Concentration (μg / mL) × 50.

[0089] Table 4 Results of Limit of Detection and Limit of Quantitation determination

[0090] Table 4 shows that the detection limit for methanol is 6 ppm, with an S / N of 5.0; the quantitation limit is 30 ppm, with an S / N of 12.8; and the RSD of the peak area of ​​the six quantitation limits is 4.4%. The detection limit for ethanol is 13 ppm, with an S / N of 3.3; the quantitation limit is 50 ppm, with an S / N of 13.1; and the RSD of the peak area of ​​the six quantitation limits is 2.6%. The detection limit for dichloromethane is 2 ppm, with an S / N of 5.5; the quantitation limit is 20 ppm, with an S / N of 11.7; and the RSD of the peak area of ​​the six quantitation limits is 4.4%. The detection limit for ethylamine was 0.08 ppm, with an S / N of 8.7; the quantitation limit was 0.8 ppm, with an S / N of 23.4; and the RSD of the peak area of ​​the six quantitation limits was 6.9%. The detection limit for pyridine was 14 ppm, with an S / N of 3.6; the quantitation limit was 27 ppm, with an S / N of 10.5; and the RSD of the peak area of ​​the six quantitation limits was 3.4%. The detection limit for xylene was 1 ppm, with an S / N of 4.0; the quantitation limit was 7 ppm, with an S / N of 10.0; and the RSD of the peak area of ​​the six quantitation limits was 4.4%. All met the requirements.

[0091] d. Linear The extent to which peak area is directly proportional to concentration within the design range was examined.

[0092] (1) Blank solution: Take 5.0 mL of dimethyl sulfoxide and place it in a 20 mL headspace vial and seal it.

[0093] (2) Linear stock solution: Weigh 0.30168g of methanol, 0.51788g of anhydrous ethanol, 0.06495g of dichloromethane, 0.50909g of triethylamine, 0.02351g of pyridine, and 0.22139g of xylene using the weight gain method. Place them in a 50mL volumetric flask containing a small amount of dimethyl sulfoxide (DMSO). Dilute to the mark with DMSO and shake well to obtain solution A. Take 10.0mL of solution A and place it in a 100mL volumetric flask containing a small amount of DMSO. Dilute to the mark with DMSO and shake well. (3) Linearity test solution ①: See the preparation of the quantitation limit test solution under the limit of detection and limit of quantitation section. (4) Linearity test solution ②: Measure 1.0mL of the linear stock solution into the same 50mL volumetric flask. Dilute to the mark with DMSO and shake well. (5) Linearity test solution ③: Measure 2.0 mL of the linearity stock solution into the same 50 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well. (6) Linearity test solution ④: Measure 5.0 mL of the linearity stock solution into the same 50 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well. (7) Linearity test solution ⑤: Measure 6.0 mL of the linearity stock solution into the same 50 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well. (8) Linearity test solution ⑥: Measure 10.0 mL of the linearity stock solution into the same 50 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well.

[0094] Take 5.0 mL each of the blank solution and linear test solutions ①-⑥ and place them in 20 mL headspace vials. Seal the vials, allow them to equilibrate, and then inject them. Record the chromatograms. Plot a linear regression with the peak area of ​​each target peak on the ordinate and the average concentration (µg / mL) of each solvent in the linear test solution on the abscissa. Calculate the linear equation and the correlation coefficient R. 2 The linear measurement data are shown in Table 5.

[0095] Table 5. Results of linearity measurement data

[0096] Table 5 shows that the linear correlation coefficient R is high when the methanol concentration is in the range of 0.6026 μg / mL to 120.672 μg / mL (equivalent to 30 ppm to 6034 ppm of the sample). 2 The linear correlation coefficient R was 0.9997 for ethanol concentrations ranging from 1.0085 μg / mL to 207.152 μg / mL (equivalent to 50 ppm to 10358 ppm of the sample). 2 The linear correlation coefficient R was 0.9998 for dichloromethane concentrations ranging from 0.4057 μg / mL to 25.980 μg / mL (equivalent to 20 ppm to 1299 ppm of the sample). 2The linear correlation coefficient R was 0.9999; for triethylamine concentrations ranging from 0.01622 μg / mL to 203.636 μg / mL (equivalent to 0.8 ppm to 10182 ppm of the sample), the linear correlation coefficient R was 0.9999. 2 The linear correlation coefficient R was 0.9995; for pyridine concentrations in the range of 0.5430 μg / mL to 9.404 μg / mL (equivalent to 27 ppm to 470 ppm of the sample), the linear correlation coefficient R was 0.9995. 2 The linear correlation coefficient R0 was 0.9995; for xylene concentrations ranging from 0.1317 μg / mL to 88.556 μg / mL (equivalent to 7 ppm to 4428 ppm of the sample), the linear correlation coefficient R0 was 0.9995. 2 The value is 0.9998, which meets the requirements.

[0097] e. Accuracy (1) Blank solution: Take 5.0 mL of dimethyl sulfoxide and place it in a 20 mL headspace vial, then seal it. (2) Sample blank solution: Weigh 0.10310 g of the sample into a 20 mL headspace vial, add 5.0 mL of dimethyl sulfoxide, and seal it. (3) Reference solution: Weigh 0.30525 g of methanol, 0.50411 g of anhydrous ethanol, 0.06120 g of dichloromethane, 0.50088 g of triethylamine, 0.02270 g of pyridine, and 0.22683 g of xylene using the weight gain method. Place them in a 50 mL volumetric flask containing a small amount of dimethyl sulfoxide, dilute to the mark with dimethyl sulfoxide, and shake well to obtain solution A. Take 1.0 mL of solution A into a 100 mL volumetric flask containing a small amount of dimethyl sulfoxide, dilute to the mark with dimethyl sulfoxide, and shake well to obtain the reference solution. Take 6 headspace vials of 20 mL each, add 5.0 mL of reference solution to each, and seal. (4) Intermediate stock solution: Take 0.0 mL of solution A1 into a 100 mL volumetric flask containing a small amount of dimethyl sulfoxide, dilute to the mark with dimethyl sulfoxide, and shake well. (5) Accuracy test solution 1: Measure 4.0 mL of intermediate stock solution into a 50 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well to prepare an 80% reference stock solution. Take 3 headspace vials, add 0.1 g of sample to each, and then add 5.0 mL of the above stock solution to each, and seal. (6) Accuracy test solution 2: Measure 5.0 mL of intermediate stock solution into a 50 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well to prepare a 100% reference stock solution. Take 3 headspace vials, add 0.1 g of sample to each, and then add 5.0 mL of the above stock solution to each, and seal. (7) Accuracy test solution 3: Measure 6.0 mL of intermediate stock solution into a 50 mL volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well to prepare a 120% control stock solution. Take 3 headspace vials, add 0.1 g of sample to each vial, and then add 5.0 mL of the above stock solution to each vial. Seal the vials.

[0098] After headspace equilibration of the headspace vials containing the reference solution, blank solution, sample blank solution, and accuracy test solution, the samples were injected, and the chromatograms were recorded. The accuracy test data for methanol, ethanol, dichloromethane, triethylamine, pyridine, and xylene are shown in Table 6-11.

[0099] Calculation formula: Recovery rate (%) = .

[0100] Note: A 测 : The peak area of ​​the solvent in the spectrum of the test solution; A 空 Peak area of ​​the solvent in the blank solution spectrum; A 样空 : Peak area of ​​the solvent in the spectrum of the blank sample solution; A 对 : The average peak area of ​​the solvent in the control solution spectrum; C 测 : The concentration of the solvent in the test solution, in μg / mL; C 对 : The concentration of the solvent in the control solution, in μg / mL; W 测 : The weight of OAB-14 added to the test solution, in g; W 样空 : The weight of OAB-14 in the blank sample solution, in g.

[0101] Table 6. Accuracy test data for methanol

[0102] Table 7 Accuracy test data for ethanol

[0103] Table 8. Accuracy Test Data for Dichloromethane

[0104] Table 9. Accuracy Test Data for Triethylamine

[0105] Table 10 Pyridine Accuracy Test Data

[0106] Table 11 Xylene Accuracy Test Data

[0107] As shown in Table 6-11, the recovery rates of methanol, ethanol, and xylene are all within the range of 96.18%-99.05% with a relative standard deviation of 1.0%; ethanol, ethanol, and dichloromethane are within the range of 96.37%-98.28% with a relative standard deviation of 0.7%; dichloromethane, ethanol, triethylamine, triethylamine, pyridine, and xylene are within the range of 93.21%-96.89% with a relative standard deviation of 1.2%; pyridine, pyridine, and xylene are within the range of 95.59%-102.94% with a relative standard deviation of 3.0%; and xylene, pyridine, and xylene are within the range of 94.85%-96.37% with a relative standard deviation of 0.6%. All of these results meet the requirements.

[0108] f. Sample testing (1) Blank solution: Take 5.0 mL of dimethyl sulfoxide and place it in a 20 mL headspace vial, then seal it. (2) Reference solution: Weigh 0.30525 g of methanol, 0.50411 g of anhydrous ethanol, 0.06120 g of dichloromethane, 0.50088 g of triethylamine, 0.02270 g of pyridine, and 0.22683 g of xylene using the weight gain method. Place them in a 50 mL volumetric flask containing a small amount of dimethyl sulfoxide, dilute to the mark with dimethyl sulfoxide, and shake well to obtain solution A. Take 1.0 mL of solution A and place it in a 100 mL volumetric flask containing a small amount of dimethyl sulfoxide, dilute to the mark with dimethyl sulfoxide, and shake well to obtain the reference solution. Take 6 20 mL headspace vials, add 5.0 mL of the reference solution to each, and seal them. (3) Test solution: Weigh 0.10339g of sample from batch 2009003 and 0.10321g of sample from batch 2009004 into 20mL headspace vials, add 5.0mL of dimethyl sulfoxide, and seal.

[0109] After headspace equilibration of the above-mentioned reference solution, blank solution and test solution, they were injected into the gas chromatograph by headspace injection, and the chromatograms were recorded. The sample detection results are shown in Table 12.

[0110] Table 12: Sample Test Results

[0111] Table 12 shows that only methanol residue was detected in the two batches of OAB-14 samples, with contents of 2123 ppm and 2158 ppm respectively. Both are lower than the methanol limit of 3000 ppm (0.3%) specified by ICH Q3C, with a acceptable margin of approximately 28-29%. Ethanol, dichloromethane, triethylamine, pyridine, and xylene were not detected (below the detection limits of their respective methods) and all met their respective limit requirements. The above results indicate that the method described in this invention can effectively detect residual solvents in OAB-14, and the detection sensitivity (detection limits: methanol 6 ppm, dichloromethane 2 ppm, triethylamine 0.08 ppm, pyridine 14 ppm, xylene 1 ppm) is far lower than the limit requirements of each solvent, which can meet the requirements of drug quality control for the detection of trace residual solvents.

[0112] Example 2 (a) Solution preparation (1) Blank solution: Take 5.0 mL of dimethyl sulfoxide and place it in a 20 mL headspace vial and seal it.

[0113] (2) Reference solution: Weigh 0.30268 g of methanol, 0.50021 g of anhydrous ethanol, 0.06518 g of dichloromethane, 0.51076 g of triethylamine, 0.02095 g of pyridine, and 0.22412 g of xylene using the weight gain method. Place these components in a 50 mL volumetric flask containing a small amount of dimethyl sulfoxide (DMSO). Dilute to the mark with DMSO and shake well to obtain solution A. Take 1.0 mL of solution A and place it in a 100 mL volumetric flask containing a small amount of DMSO. Dilute to the mark with DMSO and shake well to obtain the reference solution. Take a 20 mL headspace vial, add 5.0 mL of the reference solution, and seal.

[0114] (3) Spiked solution for test sample: Weigh 0.1 g of sample into a 20 mL headspace vial, add 5.0 mL of reference solution, and seal.

[0115] (II) Analysis Conditions The headspace equilibration conditions are as follows: equilibration temperature: 90℃, equilibration time: 30min, circulation time: 35min, quantitative loop temperature: 100℃, transfer line temperature: 110℃, headspace inlet temperature: 200℃.

[0116] The chromatographic conditions were as follows: Instrument: Agilent 7890A gas chromatograph; Column: DB-624 (30m × 0.53mm, film thickness 3μm) USR706341H; Column temperature: initial temperature 40℃ held for 5 min, then increased to 200℃ at a rate of 10℃ / min and held for 5 min; Detector: FID; Detector temperature: 250℃; Carrier gas: nitrogen; Carrier gas flow rate: 3.0 mL / min; Split ratio: 1:1; Hydrogen flow rate: 40 mL / min; Air flow rate: 400 mL / min.

[0117] (III) Determination: After headspace equilibration of the above reference solution, blank solution and spiked solution of test sample, the samples were injected into the gas chromatograph by headspace injection, and the chromatograms were recorded. The test results are shown in Table 13-14.

[0118] Table 13 Test results of the reference solution in Example 2

[0119] Table 14 Test results of the spiked solution of the test sample in Example 2

[0120] As shown in Table 13-14, the retention times of each target peak in the chromatogram of the spiked solution of the test sample are basically the same as those in the chromatogram of the reference solution. The resolution between each adjacent target peak in the chromatograms of the spiked solution of the test sample and the reference solution meets the requirements.

[0121] Example 3 (a) The solution preparation is the same as in Example 2.

[0122] (II) Analysis conditions: The headspace equilibration conditions are as follows: equilibration temperature: 90℃, equilibration time: 30min, circulation time: 30min, quantitative loop temperature: 100℃, transfer line temperature: 110℃, headspace inlet temperature: 200℃.

[0123] The chromatographic conditions were as follows: Instrument: Agilent 7890A gas chromatograph; Column: DB-624 (30m × 0.53mm, film thickness 3μm) USR706341H; Column temperature: initial temperature 42℃, hold for 3 min, increase to 200℃ at a rate of 10℃ / min, hold for 4 min; Detector: FID; Detector temperature: 250℃; Carrier gas: nitrogen; Carrier gas flow rate: 3.0 mL / min; Split ratio: 1:1; Hydrogen flow rate: 40 mL / min; Air flow rate: 400 mL / min.

[0124] (III) Determination: After headspace equilibration of the above reference solution, blank solution and spiked solution of test sample, the samples were injected into the gas chromatograph by headspace injection, and the chromatograms were recorded. The test results are shown in Table 15-16.

[0125] Table 15 Test results of the reference solution in Example 3

[0126] Table 16 Test results of the spiked solution of the test sample in Example 3

[0127] As shown in Tables 15-16, the retention times of each target peak in the chromatogram of the spiked solution of the test sample are basically consistent with those in the chromatogram of the reference solution, and the resolution between each adjacent target peak in the chromatograms of the spiked solution of the test sample and the reference solution meets the requirements.

[0128] Example 4 (a) The solution preparation is the same as in Example 2.

[0129] (II) Analysis conditions: The headspace equilibration conditions are as follows: equilibration temperature: 90℃, equilibration time: 30 min, circulation time: 40 min, quantitative loop temperature: 100℃, transfer line temperature: 110℃, headspace inlet temperature: 200℃.

[0130] The chromatographic conditions were as follows: Instrument: Agilent 7890A gas chromatograph; Column: DB-624 (30m × 0.53mm, film thickness 3μm) USR706341H; Column temperature: initial temperature 38℃, hold for 5 min, increase to 250℃ at a rate of 20℃ / min, hold for 5 min; Detector: FID; Detector temperature: 250℃; Carrier gas: nitrogen; Carrier gas flow rate: 3.0 mL / min; Split ratio: 1:1; Hydrogen flow rate: 35 mL / min; Air flow rate: 350 mL / min.

[0131] (III) Determination: After headspace equilibration of the above reference solution, blank solution and spiked solution of test sample, the samples were injected into the gas chromatograph by headspace injection, and the chromatograms were recorded. The test results are shown in Table 17-18.

[0132] Table 17 Test results of the reference solution in Example 4

[0133] Table 18 Test results of the spiked solution of the test sample in Example 4

[0134] As shown in Tables 17-18, the retention times of each target peak in the chromatogram of the spiked solution of the test sample are basically consistent with those in the chromatogram of the reference solution, and the resolution between each adjacent target peak in the chromatograms of the spiked solution of the test sample and the reference solution meets the requirements.

[0135] Example 5 (a) The solution preparation is the same as in Example 2.

[0136] (II) Analysis conditions: The headspace equilibration conditions are as follows: equilibration temperature: 90℃, equilibration time: 30min, circulation time: 35min, quantitative loop temperature: 100℃, transfer line temperature: 110℃, headspace inlet temperature: 200℃.

[0137] The chromatographic conditions were as follows: Instrument: Agilent 7890A gas chromatograph; Column: DB-624 (30m × 0.53mm, film thickness 3μm) USR706341H; Column temperature: initial temperature 40℃ held for 4 min, then increased to 200℃ at a rate of 10℃ / min and held for 6 min; Detector: FID; Detector temperature: 250℃; Carrier gas: nitrogen; Carrier gas flow rate: 3.15mL / min; Split ratio: 1:1; Hydrogen flow rate: 40mL / min; Air flow rate: 400mL / min.

[0138] (III) Determination: After headspace equilibration of the above reference solution, blank solution and spiked solution of test sample, the samples were injected into the gas chromatograph by headspace injection, and the chromatograms were recorded. The test results are shown in Tables 19-20.

[0139] Table 19 Test results of the reference solution in Example 5

[0140] Table 20 Test results of the spiked solution of the test sample in Example 5

[0141] As shown in Tables 19-20, the retention times of each target peak in the chromatogram of the spiked solution of the test sample are basically consistent with those in the chromatogram of the reference solution, and the resolution between each adjacent target peak in the chromatograms of the spiked solution of the test sample and the reference solution meets the requirements.

[0142] Example 6 (a) The solution preparation is the same as in Example 2.

[0143] (II) Analysis conditions: The conditions for headspace equilibrium are as follows: Equilibrium temperature: 90℃, equilibrium time: 30min, circulation time: 35min, quantitative loop temperature: 100℃, transfer line temperature: 110℃, headspace inlet temperature: 200℃.

[0144] The chromatographic conditions are as follows: Instruments: Agilent 7890A gas chromatograph; Column: DB-624 (30m × 0.53mm, film thickness 3μm) USR706341H; Column temperature: initial temperature 40℃, hold for 5 min, increase to 200℃ at a rate of 10℃ / min, hold for 5 min; Detector: FID; Detector temperature: 250℃; Carrier gas: nitrogen; Carrier gas flow rate: 2.85mL / min; Split ratio: 1:1; Hydrogen flow rate: 40mL / min; Air flow rate: 400mL / min.

[0145] (III) Determination: After headspace equilibration of the above reference solution, blank solution and spiked solution of test sample, the samples were injected into the gas chromatograph by headspace injection, and the chromatograms were recorded. The test results are shown in Tables 21-22.

[0146] Table 21 Test results of the reference solution in Example 6

[0147] Table 22 Test results of the spiked solution of the test sample in Example 6

[0148] As shown in Tables 21-22, the retention times of each target peak in the chromatogram of the spiked solution of the test sample are basically consistent with those in the chromatogram of the reference solution, and the resolution between each adjacent target peak in the chromatograms of the spiked solution of the test sample and the reference solution meets the requirements.

[0149] Example 7 (a) The solution preparation is the same as in Example 2.

[0150] (II) Analysis conditions: The headspace equilibration conditions are as follows: equilibration temperature: 100℃, equilibration time: 25 min, circulation time: 35 min, quantitative loop temperature: 150℃, transfer line temperature: 160℃, headspace inlet temperature: 220℃.

[0151] The chromatographic conditions are as follows: Instruments: Agilent 7890A gas chromatograph; Column: DB-624 (30m × 0.53mm, film thickness 3μm) USR706341H; Column temperature: initial temperature 40℃, hold for 5 min, increase to 200℃ at a rate of 10℃ / min, hold for 5 min; Detector: FID; Detector temperature: 255℃; Carrier gas: nitrogen; Carrier gas flow rate: 3.0 mL / min; Split ratio: 1:1; Hydrogen flow rate: 40 mL / min; Air flow rate: 400 mL / min.

[0152] (III) Determination: After headspace equilibration of the above reference solution, blank solution and spiked solution of test sample, the samples were injected into the gas chromatograph by headspace injection, and the chromatograms were recorded. The test results are shown in Tables 23-24.

[0153] Table 23 Test results of the reference solution in Example 7

[0154] Table 24 Test results of the spiked solution of the test sample in Example 7

[0155] As shown in Tables 23-24, the retention times of each target peak in the chromatogram of the spiked solution of the test sample are basically consistent with those in the chromatogram of the reference solution, and the resolution between each adjacent target peak in the chromatograms of the spiked solution of the test sample and the reference solution meets the requirements.

[0156] Example 8 (a) The solution preparation is the same as in Example 2.

[0157] (II) Analysis conditions: The conditions for headspace equilibrium are as follows: Equilibrium temperature: 80℃, equilibrium time: 35min, circulation time: 35min, quantitative loop temperature: 90℃, transfer line temperature: 100℃, headspace inlet temperature: 180℃.

[0158] The chromatographic conditions are as follows: Instruments: Agilent 7890A gas chromatograph; Column: DB-624 (30m × 0.53mm, film thickness 3μm) USR706341H; Column temperature: initial temperature 40℃, hold for 5 min, increase to 200℃ at a rate of 10℃ / min, hold for 5 min; Detector: FID; Detector temperature: 245℃; Carrier gas: nitrogen; Carrier gas flow rate: 3.0 mL / min; Split ratio: 1:1; Hydrogen flow rate: 40 mL / min; Air flow rate: 400 mL / min.

[0159] (III) Determination: After headspace equilibration of the above reference solution, blank solution and spiked solution of test sample, the samples were injected into the gas chromatograph by headspace injection, and the chromatograms were recorded. The test results are shown in Tables 25-26.

[0160] Table 25 Test results of the reference solution in Example 8

[0161] Table 26 Test results of the spiked solution of the test sample in Example 8

[0162] As shown in Tables 25-26, the retention times of each target peak in the chromatogram of the spiked solution of the test sample are basically consistent with those in the chromatogram of the reference solution, and the resolution between each adjacent target peak in the chromatograms of the spiked solution of the test sample and the reference solution meets the requirements.

[0163] Example 9 (a) The solution preparation is the same as in Example 2.

[0164] (II) Analysis conditions: The conditions for headspace equilibrium are as follows: Equilibrium temperature: 90℃, equilibrium time: 35min, circulation time: 35min, quantitative loop temperature: 100℃, transfer line temperature: 110℃, headspace inlet temperature: 200℃.

[0165] The chromatographic conditions are as follows: Instruments: Agilent 7890A gas chromatograph; Column: DB-624 (30m × 0.53mm, film thickness 3μm) USR706341H; Column temperature: initial temperature 40℃, hold for 5 min, increase to 200℃ at a rate of 10℃ / min, hold for 5 min; Detector: FID; Detector temperature: 250℃; Carrier gas: nitrogen; Carrier gas flow rate: 3.0 mL / min; Split ratio: 1:1; Hydrogen flow rate: 40 mL / min; Air flow rate: 400 mL / min.

[0166] (III) Determination: After headspace equilibration of the above reference solution, blank solution and spiked solution of test sample, the samples were injected into the gas chromatograph by headspace injection, and the chromatograms were recorded. The test results are shown in Tables 27-28.

[0167] Table 27 Test results of the reference solution in Example 9

[0168] Table 28 Test results of the spiked solution of the test sample in Example 9

[0169] As shown in Tables 27-28, the retention times of each target peak in the chromatogram of the spiked solution of the test sample are basically consistent with those in the chromatogram of the reference solution, and the resolution between each adjacent target peak in the chromatograms of the spiked solution of the test sample and the reference solution meets the requirements.

[0170] Example 10 (a) The solution preparation is the same as in Example 2.

[0171] (II) Analysis conditions: The conditions for headspace equilibrium are as follows: Equilibrium temperature: 90℃, equilibrium time: 25min, circulation time: 35min, quantitative loop temperature: 100℃, transfer line temperature: 110℃, headspace injection port temperature: 200℃.

[0172] The chromatographic conditions are as follows: Instruments: Agilent 7890A gas chromatograph; Column: DB-624 (30m × 0.53mm, film thickness 3μm) USR706341H; Column temperature: initial temperature 40℃, hold for 5 min, increase to 200℃ at a rate of 10℃ / min, hold for 5 min; Detector: FID; Detector temperature: 250℃; Carrier gas: nitrogen; Carrier gas flow rate: 3.0 mL / min; Split ratio: 1:1; Hydrogen flow rate: 40 mL / min; Air flow rate: 400 mL / min.

[0173] (III) Determination: After headspace equilibration of the above reference solution, blank solution and spiked solution of test sample, the samples were injected into the gas chromatograph by headspace injection, and the chromatograms were recorded. The test results are shown in Tables 29-30.

[0174] Table 29 Test results of the reference solution in Example 10

[0175] Table 30 Test results of the spiked solution of the test sample in Example 10

[0176] As shown in Tables 29-30, the retention times of each target peak in the chromatogram of the spiked solution of the test sample are basically consistent with those in the chromatogram of the reference solution, and the resolution between each adjacent target peak in the chromatograms of the spiked solution of the test sample and the reference solution meets the requirements.

[0177] Example 11 (a) Solution preparation is the same as in Example 2. (II) Analysis conditions: The conditions for headspace equilibrium are as follows: Equilibrium temperature: 90℃, equilibrium time: 30min, circulation time: 35min, quantitative loop temperature: 100℃, transfer line temperature: 110℃, headspace inlet temperature: 200℃.

[0178] The chromatographic conditions are as follows: Instruments: Agilent 7890A gas chromatograph; Column: DB-624 (30m × 0.53mm, film thickness 3μm) UST287311H; Column temperature: initial temperature 40℃, hold for 5 min, increase to 200℃ at a rate of 10℃ / min, hold for 5 min; Detector: FID; Detector temperature: 250℃; Carrier gas: nitrogen; Carrier gas flow rate: 3.0 mL / min; Split ratio: 1:1; Hydrogen flow rate: 40 mL / min; Air flow rate: 400 mL / min.

[0179] (III) Determination: After headspace equilibration of the above reference solution, blank solution and test solution, respectively, they were injected into the gas chromatograph by headspace injection, and the chromatograms were recorded. The test results are shown in Tables 31-32.

[0180] Table 31 Test results of the reference solution in Example 11

[0181] Table 32 Test results of the spiked solution of the test sample in Example 11

[0182] As shown in Tables 31-32, the retention times of each target peak in the chromatogram of the spiked solution of the test sample are basically consistent with those in the chromatogram of the reference solution, and the resolution between each adjacent target peak in the chromatograms of the spiked solution of the test sample and the reference solution meets the requirements.

[0183] Comparative Example 1 (a) Solution preparation: (1) Blank solution: Take 5.0 mL of N,N-dimethylformamide into a 20 mL headspace vial and seal it. (2) Test solution: Weigh 0.10 g of the sample into a 20 mL headspace vial, add 5.0 mL of N,N-dimethylformamide, and seal it.

[0184] (ii) Analysis conditions: Same as in Example 1.

[0185] (III) Determination: After headspace equilibration of the above solutions, inject them into the gas chromatograph using headspace injection and record the chromatograms. The chromatogram of the test solution is shown in [reference needed]. Figure 11 .

[0186] During the experiment, it was found that the test sample was not easily soluble in N,N-dimethylformamide. The experimental results showed that there was an obvious interfering chromatographic peak at the methanol elution point in the chromatogram of the test sample solution, indicating that if N,N-dimethylformamide is used as a blank solvent, the methanol content in the sample cannot be accurately quantified. N,N-dimethylformamide is not suitable as a blank solvent for this test method.

[0187] Comparative Example 2 (a) Solution preparation: (1) Blank solution: Take 5.0 mL of dimethyl sulfoxide into a 20 mL headspace vial and seal it. (2) Reference solution: Weigh 0.3 g of methanol, 0.5 g of anhydrous ethanol, 0.06 g of dichloromethane, 0.5 g of triethylamine, 0.02 g of pyridine, and 0.217 g of xylene using the weight gain method. Add dimethyl sulfoxide to a 50 mL volumetric flask containing a small amount of dimethyl sulfoxide, dilute to the mark, and shake well to obtain solution A. Take 1.0 mL of solution A into a 100 mL volumetric flask containing a small amount of dimethyl sulfoxide, dilute to the mark, and shake well to obtain the reference solution. (3) Spiked solution for test sample: Weigh 0.10 g of the sample into a 20 mL headspace vial, add 5.0 mL of the reference solution, and seal it.

[0188] (ii) Analysis conditions: split ratio: 1:1, other analysis conditions are the same as in Example 1.

[0189] (III) Determination: After headspace equilibration of the blank solution and the spiked solution of the test sample, inject them into the gas chromatograph using headspace injection and record the chromatograms. The chromatogram of the spiked solution of the test sample is shown in [reference needed]. Figure 12 .

[0190] The results showed that the detection sensitivity of dichloromethane and pyridine in the chromatogram of the spiked solution of the test sample was poor. This indicates that if the sample is analyzed with a split ratio of 1:1, the response signal is weak if there is residual dichloromethane and pyridine in the sample. The response is easily affected by the sample matrix and baseline noise is formed. Trace residues may result in false negatives and poor stability of parallel tests.

Claims

1. A method for determining residual solvent in OAB-14 using HS-GC, characterized in that, Includes the following steps: Prepare a reference solution containing residual solvent and a test solution of OAB-14; After headspace equilibration of the blank solution, reference solution and test solution, respectively, they were injected into the gas chromatograph by headspace injection for chromatographic separation, and the chromatograms were recorded. The content of residual solvent in the test sample solution is calculated based on the peak area using the external standard method. The residual solvent is selected from one or more of methanol, ethanol, dichloromethane, triethylamine, pyridine, and xylene; the gas chromatograph uses a capillary column with cyanopropylphenyl-dimethylpolysiloxane as the stationary phase as the chromatographic column.

2. The method for determining residual solvent in OAB-14 using HS-GC according to claim 1, characterized in that, The blank solution is dimethyl sulfoxide; both the reference solution and the test solution use dimethyl sulfoxide as the diluent.

3. The method for determining residual solvent in OAB-14 using HS-GC according to claim 2, characterized in that, The concentration of OAB-14 in the test solution is 0.01-0.03 g / mL.

4. The method for determining residual solvent in OAB-14 using HS-GC according to claim 2, characterized in that, The concentrations of methanol, ethanol, dichloromethane, triethylamine, pyridine, and xylene in the reference solution were 0.6026-120.672 µg / mL, 1.0085-207.152 µg / mL, 0.4057-25.980 µg / mL, 0.01622-203.636 µg / mL, 0.5430-9.404 µg / mL, and 0.1317-88.556 µg / mL, respectively.

5. The method for determining residual solvent in OAB-14 using HS-GC according to claim 1, characterized in that, The headspace equilibration conditions include: equilibration temperature of 80-100℃, equilibration time of 25-35 min, and circulation time of 30-40 min.

6. The method for determining residual solvent in OAB-14 using HS-GC according to claim 5, characterized in that, The conditions used for the headspace injection method include: a quantitative loop temperature of 90-150℃, a transfer line temperature of 100-160℃, and a headspace injection port temperature of 180-220℃.

7. The method for determining residual solvent in OAB-14 using HS-GC according to claim 1, characterized in that, The capillary column is a DB-624 chromatographic column with the following specifications: column length 30m, inner diameter 0.53mm, and film thickness 3μm.

8. The method for determining residual solvent in OAB-14 using HS-GC according to claim 1, characterized in that, The column temperature program of the gas chromatograph is as follows: the initial temperature is 38-42℃, maintained for 3-5 minutes, and then increased to 200-250℃ at a heating rate of 10-20℃ / min, and maintained for 4-6 minutes.

9. The method for determining residual solvent in OAB-14 using HS-GC according to claim 1, characterized in that, The gas chromatograph uses a flame ionization detector with a detector temperature of 245-255℃.

10. The method for determining residual solvent in OAB-14 using HS-GC according to claim 1, characterized in that, The gas chromatograph uses nitrogen as the carrier gas with a flow rate of 2.85-3.15 mL / min and a split ratio of 1:1; the detector uses hydrogen with a flow rate of 35-40 mL / min and air with a flow rate of 350-400 mL / min.

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