Quantifcation of Residual Organic Solvents in Siponimod Using Headspace Capillary Gas Chromatography

Quantifcation of Residual Organic Solvents in Siponimod Using Headspace Capillary Gas Chromatography | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Quantifcation of Residual Organic Solvents in Siponimod Using Headspace Capillary Gas Chromatography Zhao Li, Yi Shi, Jinfeng Zheng, Yanming Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6588784/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective To develop a headspace capillary gas chromatography (HS-GC) method for the determination of residual solvents in Siponimod active pharmaceutical ingredient (API), including methanol, ethanol, acetone, isopropanol, acetonitrile, methylene chloride, ethyl acetate,tetrahydrofuran, n-heptane, toluene, N,N-dimethylformamide, and dimethyl sulfoxide. Methods The analysis was performed using HS-GC with an Agilent DB-624 capillary column (60 m × 0.32 mm, 1.80 µm). The temperature program was set as follows: initial temperature of 45°C held for 17 minutes, then increased to 240°C at a rate of 15°C per minute and held for 10 minutes. Helium was used as the carrier gas at a flow rate of 1.0 mL/min. The injector temperature was set at 210°C, and the FID detector temperature was 260°C. The headspace equilibrium temperature was 105°C, and the equilibrium time was 35 minutes. Results All 12 residual solvents were completely separated. Each solvent showed a good linear relationship between concentration and peak area within the tested range, with correlation coefficients (r) no less than 0.999. The average recovery rates ranged from 95.3% to 100.9%, and the relative standard deviations (RSD) were between 0.8% and 2.7% (n=9). Conclusion This validated analytical approach demonstrates exceptional accuracy, reproducibility, and high sensitivity, enabling robust quantification of 12 residual solvents in siponimod API through a harmonized protocol compliant with ICH Q3C and pharmacopeial standards. Its optimized efficiency and scalability render it a pivotal solution for pharmaceutical quality assurance systems. Siponimod residual solvents gas chromatography headspace method API safety Figures Figure 1 Introduction Siponimod is a next-generation selective sphingosine-1-phosphate (S1P) receptor modulator and immunosuppressant, used for the treatment of relapsing forms of multiple sclerosis (MS) in adults [ 1 - 4 ] . During the synthesis of siponimod active pharmaceutical ingredient (API), 12 organic solvents including methanol, ethanol, acetone, isopropanol, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, n-heptane, toluene, N,N-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO) may be utilized as reaction media or purification agents. According to the International Council for Harmonisation (ICH) Q3C guidelines [ 5 ] , these solvents are classified into different risk categories based on their potential toxicity, and their residual levels must be strictly controlled below the permitted daily exposure (PDE) limits to avoid risks of hepatorenal toxicity or neurotoxicity in patients. Traditional solvent residue detection methods, such as direct-injection gas chromatography (GC), are prone to matrix interference and lack sufficient sensitivity. In contrast, headspace gas chromatography (HS-GC) has emerged as the preferred analytical technique for solvent residue analysis in pharmaceutical quality control due to its efficient enrichment of volatile components, minimal matrix interference, and operational simplicity. Literature detection methods [ 6 - 9 ] generally only detect single or partial solvents. The determination of 12 solvents requires the use of multiple different detection methods and chromatographic conditions, resulting in high analysis costs and a large workload. This study , aligned with the ICH guidelines and Appendix 0861 of Part IV of the Chinese Pharmacopoeia (2020 edition) [ 10 ] , and referencing relevant literature [11-25], systematically optimized chromatographic separation conditions and headspace parameters by integrating the physicochemical properties of siponimod API with the characteristics of the 12 solvents, compatibility of detection technologies, and requirements for sensitivity and accuracy. The aim was to establish a sensitive, accurate, and regulatory-compliant analytical method to ensure that residual organic solvents in siponimod API meet safety standards, thereby providing robust technical support for quality monitoring in its industrial-scale production. Instruments and Reagents Instruments Agilent 8890G3540A Gas Chromatograph equipped with an FID detector and a 7697A headspace autosampler； Mettler XP205 electronic analytical balance with a sensitivity of 0.01 mg. Reagents Siponimod samples from 3 batches (Company A, batch numbers: 0021121, 0031121, 0041121); Methanol, acetonitrile, ethyl acetate, dimethyl sulfoxide, and N-methyl-2-pyrrolidone (diluent) were chromatographic grade reagents; all other reagents were analytical grade. Method and Results Chromatographic Conditions The chromatographic column used was a capillary column with 6% cyanopropylphenyl and 94% dimethylpolysiloxane as the stationary phase (Agilent DB-624 gas chromatography column, 60 m × 0.32 mm, 1.80 µm). The detector was a flame ionization detector (FID). The injector temperature was set at 210°C, and the detector temperature was 260°C. The temperature program was as follows: initial temperature of 45°C held for 17 minutes, then increased to 240°C at a rate of 15°C per minute and held for 10 minutes. The carrier gas was helium with a flow rate of 1.0 mL/min. The injection mode was split injection with a split ratio of 3:1. The headspace vial equilibrium temperature was 105°C, and the equilibrium time was 35 minutes. The injection volume was 1000 µL. Solution Preparation Reference Stock Solution Accurately weighed quantities of 300 mg of methanol, 500 mg each of ethanol, acetone, isopropanol, ethyl acetate, n-heptane, and dimethyl sulfoxide, 41 mg of acetonitrile, 60 mg of methylene chloride, 72 mg of tetrahydrofuran, 88 mg of N,N-dimethylformamide, and 89 mg of toluene, and transferred them into the same 100 mL volumetric flask. The mixture was dissolved and diluted to the mark with N-methyl-2-pyrrolidone, then mixed thoroughly. Reference Solution 2 mL of the reference stock solution was precisely pipetted into a 50 mL volumetric flask, diluted to the mark with N-methyl-2-pyrrolidone, and thoroughly mixed. 1 mL of this solution was precisely pipetted into a headspace vial, and the vial was sealed. Test Solution 40 mg of the sample was accurately weighed into a headspace vial, 1 mL of N-methyl-2-pyrrolidone was precisely added, and the vial was sealed. Spiked Test Solution 40 mg of the sample was accurately weighed into a headspace vial, 1 mL of the reference solution was precisely added, and the vial was sealed. Specificity Test Accurately pipetted appropriate amounts of methanol, ethanol, acetone, isopropanol, acetonitrile, methylene chloride, ethyl acetate, tetrahydrofuran, n-heptane, toluene, N,N-dimethylformamide, dimethyl sulfoxide (12 solvents), and N-methyl-2-pyrrolidone (diluent), and injected following the chromatographic conditions described in Sect.“Chromatographic Conditions” to determine the retention time of each solvent. Then, accurately pipetted the reference solution, test solution, and spiked test solution for injection. The results showed that the blank diluent did not interfere with the detection of the target solvents, and the resolution between the peaks of each solvent in the reference solution was greater than 1.5. The chromatogram of the reference solution is shown in Figure 1, and the retention times and resolutions of the 12 solvents are listed in Table 1. Table. 1 Retention time and separation degree of 12 solvents Solvent RT（min） Resolution Methanol 9.328 / Ethanol 12.226 8.56 Acetone 14.156 5.63 Isopropyl Alcohol 14.870 2.02 Acetonitrile 15.730 2.23 Dichloromethane 16.577 2.14 Ethyl Acetate 21.490 18.97 Tetrahydrofuran 22.068 4.25 n-Heptane 23.828 14.95 Toluene 26.684 31.25 N,N-Dimethylformamide 28.294 14.89 Dimethyl Sulfoxide 29.732 10.72 Linearity, Limit of Detection (LOD), and Limit of Quantitation (LOQ) Accurately measured volumes of 0.2 mL, 0.5 mL, 1 mL, 2 mL, 3 mL, and 4 mL of the reference stock solution were transferred into separate 50 mL volumetric flasks.The solutions were diluted to the mark with N-methyl-2-pyrrolidone (NMP) to prepare a series of linearity solutions with relative concentrations of 10%, 25%, 50%, 100%, 150%, and 200%, respectively.These linearity solutions were injected under the chromatographic conditions described in Sect. “Chromatographic Conditions”. The chromatograms were recorded, and the standard curve was plotted by performing linear regression of peak area (A) versus concentration (C).The limits of detection (LOD) and quantification (LOQ) were determined through serial dilution of the reference solution, with LOD and LOQ defined as the concentrations corresponding to signal-to-noise ratios (S/N) of 3 and 10, respectively.The linear ranges, linear equations, correlation coefficients (r), LOQ, and LOD for the 12 solvents are summarized inTable 2. Tab.2 Linear range， linear equation， correlation coefficient， limit of quantitation and detection limit of 12 solvents Solvent Concentration range (μg/ml) Linearity regression equation r Detection limit (μg/ml) Quantitation limit(μg/ml) Methanol 12.057～241.136 A=3.1972C-5.0508 0.9998 0.46 0.14 Ethanol 20.103～402.056 A=3.6195C-11.3095 0.9998 0.78 0.23 Acetone 20.523～410.464 A=9.7137C-66.0903 0.9997 0.32 0.10 Isopropyl Alcohol 20.138～402.752 A=3.8189C-6.4199 0.9998 0.60 0.19 Acetonitrile 1.650～32.992 A=4.2582C+0.5639 0.9996 0.65 0.20 Dichloromethane 2.612～52.248 A=2.0121C-1.4978 0.9997 0.80 0.27 Ethyl Acetate 20.252～405.040 A=7.0422C-39.2495 0.9997 0.20 0.06 Tetrahydrofuran 2.985～59.696 A=11.3484C-6.2719 0.9997 0.11 0.03 n-Heptane 20.153～403.056 A=39.0246C-345.6378 0.9990 0.006 0.002 Toluene 3.873～77.456 A=7.7571C-4.3504 0.9997 0.05 0.01 N,N-Dimethylformamide 3.818～76.352 A=0.2685C-0.2195 0.9998 6.82 2.04 Dimethyl Sulfoxide 20.452～409.040 A=0.1058C+0.0577 0.9997 8.18 2.45 Repeatability test The reference standard solution and six replicates of the 100% spiked sample solution were prepared according to the "Solution Preparation" protocol. The samples were injected under the chromatographic conditions specified in the "Chromatographic Conditions" section. The measured levels of the 12 solvents (methanol, ethanol, acetone, isopropanol, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, n-heptane, toluene, N,N-dimethylformamide, and dimethyl sulfoxide) were calculated. The relative standard deviations (RSD%) were 1.5%, 1.6%, 1.2%, 1.5%, 0.9%, 0.7%, 1.2%, 1.8%, 0.7%, 0.6%, 0.3%, and 1.0%, respectively, demonstrating the good repeatability of the method. Accuracy test Accurately measured volumes of 1 mL, 2 mL, and 3 mL of the reference standard stock solution were pipetted into separate 50 mL volumetric flasks. The solutions were diluted to the mark with N-methyl-2-pyrrolidone (NMP) to prepare recovery stock solutions at relative concentrations of 50%, 100%, and 150%, respectively. 40 mg of the test substance (accurately weighed) was transferred into headspace vials. 1 mL of the 50%, 100%, and 150% recovery stock solutions was precisely added to each vial. The vials were sealed and prepared as 50%, 100%, and 150% spiked sample solutions (three replicates per concentration). The samples were injected under the chromatographic conditions specified in the "Chromatographic Conditions" section. The recovery rates were calculated, as shown in Table 4. The average recoveries of the 12 solvents (methanol, ethanol, acetone, isopropanol, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, n-heptane, toluene, N,N-dimethylformamide, and dimethyl sulfoxide) ranged from 90% to 110%, demonstrating the method’s satisfactory accuracy. Table. 3 Accuracy test results of 12 solvents Solvent Background Mass (μg) Spiked Mass (μg) Measured Mass (μg) Recovery (%) Average Recovery (%) RSD (%) Methanol 0.00 60.28 58.10，58.62，57.71 96.4，97.2，95.7 99.6 2.7 120.6 123.5，123.3，121.3 102.4，102.2，100.6 180.9 180.2，185.5，179.9 99.6，102.5，99.4 Ethanol 12.22,12.26,12.13 100.5 113.6，112.7，114.0 100.9,99.9,101.4 99.9 1.9 12.26,12.29,11.99 201.0 217.4，213.5，210.6 102.1,100.1,98.8 12.03,12.28,12.26 301.5 306.3，319.4，303.6 97.6,101.9,96.6 Acetone 0.00 102.6 103.4,103.8,102.3 100.8,101.2,99.7 100.2 1.1 205.2 206.7,207.4,205.6 100.7,101.1,100.2 307.8 302.9,309.9,303.9 98.4,100.7,98.7 Isopropyl Alcohol 0.00 100.7 100.5,101.7,100.0 99.8,101.0,99.3 98.7 1.7 201.4 201.2,198.6,197.3 99.9,98.6,98.0 302.1 291.3,298.5,290.8 96.4,98.8,96.3 Acetonitrile 0.00 8.248 8.350,8.393,8.311 101.2,101.8,100.8 100.9 0.8 16.50 16.80,16.75,16.53 101.8,101.5,100.2 24.74 24.68,24.75,25.04 99.8,100.0,101.2 Dichloromethane 0.00 13.06 13.13,13.24,12.89 100.5,101.4,98.7 99.8 0.9 26.12 26.06,26.11,25.86 99.8,100.0,99.0 39.19 38.67,39.43,39.09 98.7,100.6,99.7 Ethyl Acetate 60.78,60.96,60.31 101.3 162.4,162.6,160.6 100.3,100.3,99.0 98.6 1.3 60.98,61.13,59.62 202.5 260.7,260.5,255.8 98.6,98.5,96.9 59.86,61.08,60.99 303.8 355.1,363.4,357.1 97.2,99.5,97.5 Tetrahydrofuran 0.00 14.92 15.01,15.10,15.11 100.6,101.2,101.3 99.5 1.9 29.85 29.69,29.98,29.70 99.5,100.4,99.5 44.77 43.44,44.61,43.01 97.0,99.6,96.1 n-Heptane 0.00 100.8 96.87,97.32,96.85 96.1,96.5,96.1 95.3 1.0 201.5 193.4,191.7,191.4 96.0,95.1,95.0 302.3 283.2,286.9,286.0 93.7,94.9,94.6 Toluene 0.00 19.36 18.60,18.53,18.76 96.1,95.7,96.9 95.5 0.9 38.73 36.98,37.02,37.24 95.5,95.6,96.2 58.09 54.79,55.27,54.85 94.3,95.1,94.4 N,N-Dimethylformamide 0.00 19.09 19.08,19.24,19.19 99.9,100.8,100.5 100.4 0.8 38.18 38.83,38.67,38.15 101.7,101.3,99.9 57.26 56.97,57.56,57.10 99.5,100.5,99.7 Dimethyl Sulfoxide 57.96,58.14,57.52 102.3 159.8,161.5,161.2 99.6,101.0,101.3 100.1 1.2 58.15,58.30,56.86 204.5 263.8,264.3,264.3 100.6,100.7,101.4 57.09,58.25,58.17 306.8 359.4,363.0,361.2 98.5,99.3,98.8 Precision Test The reference standard solution was accurately pipetted, and six consecutive injections were performed under the chromatographic conditions specified in the "Chromatographic Conditions" section. The relative standard deviations (RSD%) of peak areas for the 12 solvents (methanol, ethanol, acetone, isopropanol, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, n-heptane, toluene, N,N-dimethylformamide, and dimethyl sulfoxide) were 0.77%, 0.88%, 0.73%, 0.87%, 0.97%, 0.78%, 0.76%, 0.72%, 0.96%, 0.70%, 2.85%, and 1.66%, respectively, demonstrating good instrumental precision. Robustness Test The chromatographic conditions described in the "Chromatographic Conditions" section were modified through single-factor variations: Initial column temperature: 45°C ± 5°C, Headspace equilibration time: 35 ± 5 min, The blank diluent, reference standard solution, test sample solution, and spiked sample solution (prepared as per the "Solution Preparation" protocol) were injected under these modified conditions. No interference was observed in solvent detection under all modified conditions. The resolution of all solvents in the reference standard solution exceeded 1.5. The detected solvent levels in the spiked sample solution showed no significant differences compared to those obtained under the original chromatographic conditions. Stability Test The reference standard solution from the "Solution Preparation" section was accurately pipetted and injected under the "Chromatographic Conditions" at 0, 6, 12, 24, and 48 h. The relative standard deviations (RSD%) for the 12 solvents (methanol, ethanol, acetone, isopropanol, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, n-heptane, toluene, N,N-dimethylformamide, and dimethyl sulfoxide) were 1.7%, 0.9%, 2.2%, 1.9%, 2.7%, 2.8%, 2.1%, 2.4%, 8.7%, 4.0%, 2.2%, and 2.6%, respectively, demonstrating good method stability. Sample Analysis 3 batches of samples were processed by preparing the reference standard solution and sample solution according to the "Solution Preparation" section. The solutions were injected under the chromatographic conditions specified in the "Chromatographic Conditions" section. The detected levels of the 12 solvents (methanol, ethanol, acetone, isopropanol, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, n-heptane, toluene, N,N-dimethylformamide, and dimethyl sulfoxide) in the 3 batches are summarized in Table 4. Table. 4 Results of 12 solvents in the sample Solvent Batch Batch Batch 0021121 0031121 0041121 Methanol < Not Detected (Less than 0.14 μg/mL) Ethanol 0.03% 0.03% 0.04% Acetone < Not Detected (Less than 0.10 μg/mL) Isopropanol < Not Detected (Less than 0.19 μg/mL) Acetonitrile < Not Detected (Less than 0.20 μg/mL) Dichloromethane < Not Detected (Less than 0.27 μg/mL) Ethyl Acetate 0.15% 0.15% 0.16% Tetrahydrofuran < Not Detected (Less than 0.03 μg/mL) n-Heptane < Not Detected (Less than 0.002 μg/mL) Toluene < Not Detected (Less than 0.01 μg/mL) N,N-Dimethylformamide < Not Detected (Less than 2.04 μg/mL) Dimethyl Sulfoxide 0.15% 0.16% 0.16% Discussion Solvent selection In this study, the solubility of the tested product and the boiling point of each residual solvent were comprehensively considered. N-methyl-2-pyrrolidone with a higher boiling point was used as the solvent. The tested solvent was not only miscible with it, but also did not interfere with the determination of other tested solvents because N-methyl-2-pyrrolidone peaked the latest. In addition, it should be noted that the purity of the solvent N-methyl-2-pyrrolidone reaches the chromatogram pure level and above, because the concentration of N, N-dimethylformamide and dimethyl sulfoxide is low, it is required that the purity of N-methyl-2-pyrrolidone should be as high as possible to avoid interference from impurity peaks near the corresponding component peaks, resulting in deviations in the determination results. Selection of chromatographic conditions The polarity and boiling point of the 12 target solvents to be tested are quite different. Considering the polarity and boiling point of the components to be tested, a column with moderate polarity is selected. In this test, Agilent DB-624 (60m×0.32mm, 1.80µm) capillary column is selected, and the separation degree and column efficiency of each component are ideal, and each substance to be tested can be effectively separated. The interference of solvent is avoided. Due to the large difference in the boiling point of each solvent, programmed heating was adopted, and the heating rate and time were appropriately adjusted to make the 12 residual solvents peak in the appropriate time, and the separation degree of each solvent was greater than 1.5. Selection of headspace balance conditions Due to the large boiling point span of 12 solvents, 105℃ is selected as the headspace equilibrium temperature, which is conducive to saving the gas-liquid equilibrium time. 100℃, 105℃, 110℃ and 115℃ are investigated. With the increase of equilibrium temperature, the chromatographic peak area of each solvent increases to varying degrees. The saturated vapor pressure of the components also gradually increases. At the same time, considering that excessive temperature may cause other impurities in the sample to vaporize or sublimate into the chromatographic system, interfering with the determination of the target object, the equilibrium temperature is selected at 105℃. The change of the peak area of 12 solvents at equilibrium temperature 105℃ was investigated when the equilibrium time was 25, 30, 35 and 40 minutes. The results showed that when the equilibrium time was 35 minutes, the peak area of 12 solvents had reached a stable state, so the equilibrium time was 35 minutes. Conclusion This study successfully established a sensitive, accurate, and efficient headspace gas chromatography (HS-GC) method for the simultaneous determination of 12 residual organic solvents in siponimod active pharmaceutical ingredient (API). By systematically optimizing chromatographic conditions (Agilent DB-624 column, temperature programming) and headspace parameters (105°C equilibrium temperature, 35 min equilibration time), complete separation of all solvents was achieved with resolutions > 1.5 and baseline stability. The method demonstrated excellent linearity (r ≥ 0.999), precision (RSD% 0.3 ~ 2.7%), accuracy (recoveries 90 ~ 110%), and sensitivity (LOD: 0.006 ~ 8.18 µg/mL, LOQ: 0.002 ~ 2.45 µg/mL), fully complying with ICH Q3C and Chinese Pharmacopoeia guidelines. The use of N-methyl-2-pyrrolidone (NMP) as a high-boiling diluent eliminated matrix interference, while robustness testing confirmed method reliability under variable conditions (± 5°C, ± 5 min). Applied to three production batches, the method reliably quantified solvent residues below permitted limits, validating its practicality for industrial quality control. This work addresses a critical gap in multi-solvent analysis, significantly reducing costs and labor compared to traditional fragmented approaches. Its adaptability to other APIs positions it as a universal solution for pharmaceutical safety monitoring, ensuring patient safety and regulatory compliance in drug manufacturing. Declarations Author Contribution Zhao Li wrote the main manuscript text. Xiaoyi Shi prepared Tables 1-4. All authors reviewed and approved the final manuscript. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6588784","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":495023436,"identity":"466b99a8-f14f-4526-9371-c140031390be","order_by":0,"name":"Zhao Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYBACPmYGNhDNw8be2PjwAzFa2KBaZPh4DjcbSxClhQGixUZOIr1NgIcoLezszx78qKjjYZN82MYgwWAnp9tA2GHphj1n2HjYpBPbHhQwJBubHSCs5ZgEbxsPSEu7gQTDgcRthLUwtkn+bZMAOuwgkCROCzObNG+bAQ+bBCPRWtjYpGXOJPCw8SQCA9mACL/w8x9/Jvmmos5evv34w4cfKuzkCGpBAwakKR8Fo2AUjIJRgAMAAKWpMbHHdxFoAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Hunan Institute for Drug Control；","correspondingAuthor":true,"prefix":"","firstName":"Zhao","middleName":"","lastName":"Li","suffix":""},{"id":495023438,"identity":"352c0ea0-ad6c-4d81-ab53-e78ab2e989cb","order_by":1,"name":"Yi Shi","email":"","orcid":"","institution":"Department of Hunan Institute for Drug Control；","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Shi","suffix":""},{"id":495023440,"identity":"a8dc5db6-987e-4733-b203-9bbdb6541e74","order_by":2,"name":"Jinfeng Zheng","email":"","orcid":"","institution":"Department of Hunan Institute for Drug Control；","correspondingAuthor":false,"prefix":"","firstName":"Jinfeng","middleName":"","lastName":"Zheng","suffix":""},{"id":495023441,"identity":"2f1b1277-5695-4227-b8bf-ea92212f0ab0","order_by":3,"name":"Yanming Liu","email":"","orcid":"","institution":"Department of Hunan Institute for Drug Control；","correspondingAuthor":false,"prefix":"","firstName":"Yanming","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-05-04 14:08:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6588784/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6588784/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88300742,"identity":"aafeec33-761c-4c63-ab8b-6cfcefce738e","added_by":"auto","created_at":"2025-08-05 04:36:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":175262,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReference solution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1. methanol, 2. ethanol, 3. acetone, 4. isopropanol, 5. acetonitrile, 6. methylene chloride, 7. ethyl acetate, 8. tetrahydrofuran, 9. n-heptane, 10. toluene, 11. N,N-dimethylformamide, 12. dimethyl sulfoxide.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6588784/v1/6a43362f0eaaefd00020f833.png"},{"id":88301818,"identity":"2799eeea-e513-4488-bf3a-4977f400b94f","added_by":"auto","created_at":"2025-08-05 04:52:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1163741,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6588784/v1/c218fc2a-40ce-4cef-8603-d86f977b682d.pdf"},{"id":88301522,"identity":"017ff7fb-f043-4425-ac44-f080d93d3a29","added_by":"auto","created_at":"2025-08-05 04:44:23","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":261458,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-6588784/v1/4564edd274b611c249b96531.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Quantifcation of Residual Organic Solvents in Siponimod Using Headspace Capillary Gas Chromatography","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cstrong\u003eSiponimod is a next-generation selective sphingosine-1-phosphate (S1P) receptor modulator and immunosuppressant, used for the treatment of relapsing forms of multiple sclerosis (MS) in adults\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e[\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003cstrong\u003e]\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e During the synthesis of siponimod active pharmaceutical ingredient (API), 12 organic solvents\u0026nbsp;including\u0026nbsp;methanol, ethanol, acetone, isopropanol, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, n-heptane, toluene, N,N-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO)\u0026nbsp;may be utilized as reaction media or purification agents. According to the International Council for Harmonisation (ICH) Q3C guidelines\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e[\u003c/strong\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003cstrong\u003e]\u003c/strong\u003e, these solvents are classified into different risk categories based on their potential toxicity, and their residual levels must be strictly controlled below the permitted daily exposure (PDE) limits to avoid risks of hepatorenal toxicity or neurotoxicity in patients. Traditional solvent residue detection methods, such as direct-injection gas chromatography (GC), are prone to matrix interference and lack sufficient sensitivity. In contrast,\u0026nbsp;\u003cstrong\u003eheadspace gas chromatography (HS-GC)\u003c/strong\u003e has emerged as the preferred analytical technique\u0026nbsp;for solvent residue analysis in pharmaceutical quality control due to its efficient enrichment of volatile components, minimal matrix interference, and operational simplicity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLiterature detection methods [\u003c/strong\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cstrong\u003e9\u003c/strong\u003e\u003cstrong\u003e] generally only detect single or partial solvents. The determination of 12 solvents requires the use of multiple different detection methods and chromatographic conditions, resulting in high analysis costs and a large workload.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eThis study\u003c/strong\u003e, aligned with the ICH guidelines and Appendix 0861 of Part IV of the Chinese Pharmacopoeia (2020 edition)\u003cstrong\u003e\u0026nbsp;[\u003c/strong\u003e\u003cstrong\u003e10\u003c/strong\u003e\u003cstrong\u003e]\u003c/strong\u003e, and referencing relevant literature [11-25], systematically optimized chromatographic separation conditions and headspace parameters by integrating the physicochemical properties of siponimod API with the characteristics of the 12 solvents, compatibility of detection technologies, and requirements for sensitivity and accuracy. The aim was to establish a \u003cstrong\u003esensitive, accurate, and regulatory-compliant analytical method\u003c/strong\u003e to ensure that residual organic solvents in siponimod API meet safety standards, thereby providing robust technical support for quality monitoring in its industrial-scale production.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstruments and Reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstruments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAgilent 8890G3540A Gas Chromatograph equipped with an FID detector and a 7697A headspace autosampler；\u003c/p\u003e\n\u003cp\u003eMettler XP205 electronic analytical balance with a sensitivity of 0.01 mg.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSiponimod samples from 3 batches (Company A, batch numbers: 0021121, 0031121, 0041121); Methanol, acetonitrile, ethyl acetate, dimethyl sulfoxide, and N-methyl-2-pyrrolidone (diluent) were chromatographic grade reagents; all other reagents were analytical grade.\u003c/p\u003e"},{"header":"Method and Results","content":"\u003cp\u003e\u003cstrong\u003eChromatographic Conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chromatographic column used was a capillary column with 6% cyanopropylphenyl and 94% dimethylpolysiloxane as the stationary phase (Agilent DB-624 gas chromatography column, 60 m \u0026times; 0.32 mm, 1.80 \u0026micro;m). The detector was a flame ionization detector (FID). The injector temperature was set at 210\u0026deg;C, and the detector temperature was 260\u0026deg;C. The temperature program was as follows: initial temperature of 45\u0026deg;C held for 17 minutes, then increased to 240\u0026deg;C at a rate of 15\u0026deg;C per minute and held for 10 minutes. The carrier gas was helium with a flow rate of 1.0 mL/min. The injection mode was split injection with a split ratio of 3:1. The headspace vial equilibrium temperature was 105\u0026deg;C, and the equilibrium time was 35 minutes. The injection volume was 1000 \u0026micro;L.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSolution Preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReference Stock Solution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccurately weighed quantities of 300 mg of methanol, 500 mg each of ethanol, acetone, isopropanol, ethyl acetate, n-heptane, and dimethyl sulfoxide, 41 mg of acetonitrile, 60 mg of methylene chloride, 72 mg of tetrahydrofuran, 88 mg of N,N-dimethylformamide, and 89 mg of toluene, and transferred them into the same 100 mL volumetric flask. The mixture was dissolved and diluted to the mark with N-methyl-2-pyrrolidone, then mixed thoroughly.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReference Solution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2 mL of the reference stock solution was precisely pipetted into a 50 mL volumetric flask, diluted to the mark with N-methyl-2-pyrrolidone, and thoroughly mixed. 1 mL of this solution was precisely pipetted into a headspace vial, and the vial was sealed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTest Solution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e40 mg of the sample was accurately weighed into a headspace vial, 1 mL of N-methyl-2-pyrrolidone was precisely added, and the vial was sealed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpiked Test Solution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e40 mg of the sample was accurately weighed into a headspace vial, 1 mL of the reference solution was precisely added, and the vial was sealed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpecificity Test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccurately pipetted appropriate amounts of methanol, ethanol, acetone, isopropanol, acetonitrile, methylene chloride, ethyl acetate, tetrahydrofuran, n-heptane, toluene, N,N-dimethylformamide, dimethyl sulfoxide (12 solvents), and N-methyl-2-pyrrolidone (diluent), and injected following the chromatographic conditions described in Sect.\u0026ldquo;Chromatographic Conditions\u0026rdquo; to determine the retention time of each solvent. Then, accurately pipetted the reference solution, test solution, and spiked test solution for injection. The results showed that the blank diluent did not interfere with the detection of the target solvents, and the resolution between the peaks of each solvent in the reference solution was greater than 1.5. The chromatogram of the reference solution is shown in Figure 1, and the retention times and resolutions of the 12 solvents are listed in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable. 1\u003c/strong\u003e Retention time and separation degree of 12 solvents\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"85%\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003eSolvent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003eRT（min）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003eResolution\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003eMethanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e9.328\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003eEthanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e12.226\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003e8.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003eAcetone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e14.156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003e5.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003eIsopropyl Alcohol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e14.870\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003e2.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003eAcetonitrile\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e15.730\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003e2.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003eDichloromethane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e16.577\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003e2.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003eEthyl Acetate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e21.490\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003e18.97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003eTetrahydrofuran\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e22.068\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003e4.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003en-Heptane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e23.828\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003e14.95\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003eToluene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e26.684\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003e31.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003eN,N-Dimethylformamide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e28.294\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003e14.89\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 37px;\"\u003e\n \u003cp\u003eDimethyl Sulfoxide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e29.732\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30px;\"\u003e\n \u003cp\u003e10.72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eLinearity, Limit of Detection (LOD), and Limit of Quantitation (LOQ)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccurately measured volumes of 0.2 mL, 0.5 mL, 1 mL, 2 mL, 3 mL, and 4 mL of the reference stock solution were transferred into separate 50 mL volumetric flasks.The solutions were diluted to the mark with N-methyl-2-pyrrolidone (NMP) to prepare a series of linearity solutions with relative concentrations of 10%, 25%, 50%, 100%, 150%, and 200%, respectively.These linearity solutions were injected under the chromatographic conditions described in Sect. \u0026ldquo;Chromatographic Conditions\u0026rdquo;. The chromatograms were recorded, and the standard curve was plotted by performing linear regression of peak area (A) versus concentration (C).The limits of detection (LOD) and quantification (LOQ) were determined through serial dilution of the reference solution, with LOD and LOQ defined as the concentrations corresponding to signal-to-noise ratios (S/N) of 3 and 10, respectively.The linear ranges, linear equations, correlation coefficients (r), LOQ, and LOD for the 12 solvents are summarized inTable 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTab.2\u003c/strong\u003e Linear range， linear equation， correlation coefficient， limit of quantitation and detection limit of 12 solvents\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"642\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 131px;\"\u003e\n \u003cp\u003eSolvent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003eConcentration range\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(\u0026mu;g/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eLinearity regression equation\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003er\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eDetection limit\u003c/p\u003e\n \u003cp\u003e(\u0026mu;g/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003eQuantitation limit(\u0026mu;g/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 131px;\"\u003e\n \u003cp\u003eMethanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e12.057～241.136\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eA=3.1972C-5.0508\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e0.9998\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 131px;\"\u003e\n \u003cp\u003eEthanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e20.103～402.056\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eA=3.6195C-11.3095\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e0.9998\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 131px;\"\u003e\n \u003cp\u003eAcetone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e20.523～410.464\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eA=9.7137C-66.0903\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e0.9997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 131px;\"\u003e\n \u003cp\u003eIsopropyl Alcohol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e20.138～402.752\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eA=3.8189C-6.4199\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e0.9998\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 131px;\"\u003e\n \u003cp\u003eAcetonitrile\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e1.650～32.992\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eA=4.2582C+0.5639\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e0.9996\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 131px;\"\u003e\n \u003cp\u003eDichloromethane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e2.612～52.248\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eA=2.0121C-1.4978\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e0.9997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 131px;\"\u003e\n \u003cp\u003eEthyl Acetate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e20.252～405.040\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eA=7.0422C-39.2495\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e0.9997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 131px;\"\u003e\n \u003cp\u003eTetrahydrofuran\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e2.985～59.696\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eA=11.3484C-6.2719\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e0.9997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 131px;\"\u003e\n \u003cp\u003en-Heptane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e20.153～403.056\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eA=39.0246C-345.6378\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e0.9990\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 131px;\"\u003e\n \u003cp\u003eToluene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e3.873～77.456\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eA=7.7571C-4.3504\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e0.9997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 131px;\"\u003e\n \u003cp\u003eN,N-Dimethylformamide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e3.818～76.352\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eA=0.2685C-0.2195\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e0.9998\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e6.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e2.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 131px;\"\u003e\n \u003cp\u003eDimethyl Sulfoxide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 133px;\"\u003e\n \u003cp\u003e20.452～409.040\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eA=0.1058C+0.0577\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e0.9997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e8.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e2.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eRepeatability test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe reference standard solution and six replicates of the 100% spiked sample solution were prepared according to the \u0026quot;Solution Preparation\u0026quot; protocol. The samples were injected under the chromatographic conditions specified in the \u0026quot;Chromatographic Conditions\u0026quot; section. The measured levels of the 12 solvents (methanol, ethanol, acetone, isopropanol, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, n-heptane, toluene, N,N-dimethylformamide, and dimethyl sulfoxide) were calculated. The relative standard deviations (RSD%) were 1.5%, 1.6%, 1.2%, 1.5%, 0.9%, 0.7%, 1.2%, 1.8%, 0.7%, 0.6%, 0.3%, and 1.0%, respectively, demonstrating the good repeatability of the method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccuracy test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccurately measured volumes of 1 mL, 2 mL, and 3 mL of the reference standard stock solution were pipetted into separate 50 mL volumetric flasks. The solutions were diluted to the mark with N-methyl-2-pyrrolidone (NMP) to prepare recovery stock solutions at relative concentrations of 50%, 100%, and 150%, respectively. 40 mg of the test substance (accurately weighed) was transferred into headspace vials. 1 mL of the 50%, 100%, and 150% recovery stock solutions was precisely added to each vial. The vials were sealed and prepared as 50%, 100%, and 150% spiked sample solutions (three replicates per concentration). The samples were injected under the chromatographic conditions specified in the \u0026quot;Chromatographic Conditions\u0026quot; section. The recovery rates were calculated, as shown in Table 4. The average recoveries of the 12 solvents (methanol, ethanol, acetone, isopropanol, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, n-heptane, toluene, N,N-dimethylformamide, and dimethyl sulfoxide) ranged from 90% to 110%, demonstrating the method\u0026rsquo;s satisfactory accuracy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable. 3\u003c/strong\u003e\u0026nbsp; Accuracy test results of 12 solvents\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"580\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003eSolvent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eBackground Mass (\u0026mu;g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003eSpiked Mass (\u0026mu;g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003eMeasured Mass (\u0026mu;g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003eRecovery (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003eAverage Recovery (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003eRSD (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 65px;\"\u003e\n \u003cp\u003eMethanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e60.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e58.10，58.62，57.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e96.4，97.2，95.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 74px;\"\u003e\n \u003cp\u003e99.6 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 47px;\"\u003e\n \u003cp\u003e2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e120.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e123.5，123.3，121.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e102.4，102.2，100.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e180.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e180.2，185.5，179.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e99.6，102.5，99.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 65px;\"\u003e\n \u003cp\u003eEthanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e12.22,12.26,12.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e100.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e113.6，112.7，114.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e100.9,99.9,101.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 74px;\"\u003e\n \u003cp\u003e99.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e12.26,12.29,11.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e201.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e217.4，213.5，210.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e102.1,100.1,98.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e12.03,12.28,12.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e301.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e306.3，319.4，303.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e97.6,101.9,96.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 65px;\"\u003e\n \u003cp\u003eAcetone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e102.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e103.4,103.8,102.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e100.8,101.2,99.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 74px;\"\u003e\n \u003cp\u003e100.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e205.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e206.7,207.4,205.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e100.7,101.1,100.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e307.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e302.9,309.9,303.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e98.4,100.7,98.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 65px;\"\u003e\n \u003cp\u003eIsopropyl Alcohol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e100.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e100.5,101.7,100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e99.8,101.0,99.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 74px;\"\u003e\n \u003cp\u003e98.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e201.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e201.2,198.6,197.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e99.9,98.6,98.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e302.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e291.3,298.5,290.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e96.4,98.8,96.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 65px;\"\u003e\n \u003cp\u003eAcetonitrile\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e8.248\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e8.350,8.393,8.311\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e101.2,101.8,100.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 74px;\"\u003e\n \u003cp\u003e100.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e16.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e16.80,16.75,16.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e101.8,101.5,100.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e24.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e24.68,24.75,25.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e99.8,100.0,101.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 65px;\"\u003e\n \u003cp\u003eDichloromethane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e13.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e13.13,13.24,12.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e100.5,101.4,98.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 74px;\"\u003e\n \u003cp\u003e99.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e26.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e26.06,26.11,25.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e99.8,100.0,99.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e39.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e38.67,39.43,39.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e98.7,100.6,99.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 65px;\"\u003e\n \u003cp\u003eEthyl Acetate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e60.78,60.96,60.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e101.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e162.4,162.6,160.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e100.3,100.3,99.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 74px;\"\u003e\n \u003cp\u003e98.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e60.98,61.13,59.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e202.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e260.7,260.5,255.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e98.6,98.5,96.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e59.86,61.08,60.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e303.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e355.1,363.4,357.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e97.2,99.5,97.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 65px;\"\u003e\n \u003cp\u003eTetrahydrofuran\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e14.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e15.01,15.10,15.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e100.6,101.2,101.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 74px;\"\u003e\n \u003cp\u003e99.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e29.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e29.69,29.98,29.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e99.5,100.4,99.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e44.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e43.44,44.61,43.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e97.0,99.6,96.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 65px;\"\u003e\n \u003cp\u003en-Heptane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e100.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e96.87,97.32,96.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e96.1,96.5,96.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 74px;\"\u003e\n \u003cp\u003e95.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e201.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e193.4,191.7,191.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e96.0,95.1,95.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e302.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e283.2,286.9,286.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e93.7,94.9,94.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 65px;\"\u003e\n \u003cp\u003eToluene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e19.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e18.60,18.53,18.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e96.1,95.7,96.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 74px;\"\u003e\n \u003cp\u003e95.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e38.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e36.98,37.02,37.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e95.5,95.6,96.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e58.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e54.79,55.27,54.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e94.3,95.1,94.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 65px;\"\u003e\n \u003cp\u003eN,N-Dimethylformamide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e19.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e19.08,19.24,19.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e99.9,100.8,100.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 74px;\"\u003e\n \u003cp\u003e100.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e38.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e38.83,38.67,38.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e101.7,101.3,99.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e57.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e56.97,57.56,57.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e99.5,100.5,99.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 65px;\"\u003e\n \u003cp\u003eDimethyl Sulfoxide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e57.96,58.14,57.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e102.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e159.8,161.5,161.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e99.6,101.0,101.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 74px;\"\u003e\n \u003cp\u003e100.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e58.15,58.30,56.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e204.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e263.8,264.3,264.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e100.6,100.7,101.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e57.09,58.25,58.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e306.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 114px;\"\u003e\n \u003cp\u003e359.4,363.0,361.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e98.5,99.3,98.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003ePrecision Test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe reference standard solution was accurately pipetted, and six consecutive injections were performed under the chromatographic conditions specified in the \u0026quot;Chromatographic Conditions\u0026quot; section. The relative standard deviations (RSD%) of peak areas for the 12 solvents (methanol, ethanol, acetone, isopropanol, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, n-heptane, toluene, N,N-dimethylformamide, and dimethyl sulfoxide) were 0.77%, 0.88%, 0.73%, 0.87%, 0.97%, 0.78%, 0.76%, 0.72%, 0.96%, 0.70%, 2.85%, and 1.66%, respectively, demonstrating good instrumental precision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRobustness Test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chromatographic conditions described in the \u0026quot;Chromatographic Conditions\u0026quot; section were modified through single-factor variations: Initial column temperature: 45\u0026deg;C \u0026plusmn; 5\u0026deg;C, Headspace equilibration time: 35 \u0026plusmn; 5 min, The blank diluent, reference standard solution, test sample solution, and spiked sample solution (prepared as per the \u0026quot;Solution Preparation\u0026quot; protocol) were injected under these modified conditions. No interference was observed in solvent detection under all modified conditions. The resolution of all solvents in the reference standard solution exceeded 1.5. The detected solvent levels in the spiked sample solution showed no significant differences compared to those obtained under the original chromatographic conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStability Test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe reference standard solution from the \u0026quot;Solution Preparation\u0026quot; section was accurately pipetted and injected under the \u0026quot;Chromatographic Conditions\u0026quot; at 0, 6, 12, 24, and 48 h. The relative standard deviations (RSD%) for the 12 solvents (methanol, ethanol, acetone, isopropanol, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, n-heptane, toluene, N,N-dimethylformamide, and dimethyl sulfoxide) were 1.7%, 0.9%, 2.2%, 1.9%, 2.7%, 2.8%, 2.1%, 2.4%, 8.7%, 4.0%, 2.2%, and 2.6%, respectively, demonstrating good method stability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e3 batches of samples were processed by preparing the reference standard solution and sample solution according to the \u0026quot;Solution Preparation\u0026quot; section. The solutions were injected under the chromatographic conditions specified in the \u0026quot;Chromatographic Conditions\u0026quot; section. The detected levels of the 12 solvents (methanol, ethanol, acetone, isopropanol, acetonitrile, dichloromethane, ethyl acetate, tetrahydrofuran, n-heptane, toluene, N,N-dimethylformamide, and dimethyl sulfoxide) in the 3 batches are summarized in Table 4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable. 4\u003c/strong\u003e Results of 12 solvents in the sample\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"90%\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 29px;\"\u003e\n \u003cp\u003eSolvent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003eBatch\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 23px;\"\u003e\n \u003cp\u003eBatch\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 23px;\"\u003e\n \u003cp\u003eBatch\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e0021121\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 23px;\"\u003e\n \u003cp\u003e0031121\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 23px;\"\u003e\n \u003cp\u003e0041121\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29px;\"\u003e\n \u003cp\u003eMethanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026lt; Not Detected (Less than 0.14 \u0026mu;g/mL)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29px;\"\u003e\n \u003cp\u003eEthanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e0.03%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 23px;\"\u003e\n \u003cp\u003e0.03%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 23px;\"\u003e\n \u003cp\u003e0.04%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29px;\"\u003e\n \u003cp\u003eAcetone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026lt; Not Detected (Less than 0.10 \u0026mu;g/mL)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29px;\"\u003e\n \u003cp\u003eIsopropanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026lt; Not Detected (Less than 0.19 \u0026mu;g/mL)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29px;\"\u003e\n \u003cp\u003eAcetonitrile\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026lt; Not Detected (Less than 0.20 \u0026mu;g/mL)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29px;\"\u003e\n \u003cp\u003eDichloromethane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026lt; Not Detected (Less than 0.27 \u0026mu;g/mL)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29px;\"\u003e\n \u003cp\u003eEthyl Acetate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e0.15%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 23px;\"\u003e\n \u003cp\u003e0.15%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 23px;\"\u003e\n \u003cp\u003e0.16%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29px;\"\u003e\n \u003cp\u003eTetrahydrofuran\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026lt; Not Detected (Less than 0.03 \u0026mu;g/mL)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29px;\"\u003e\n \u003cp\u003en-Heptane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026lt; Not Detected (Less than 0.002 \u0026mu;g/mL)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29px;\"\u003e\n \u003cp\u003eToluene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026lt; Not Detected (Less than 0.01 \u0026mu;g/mL)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29px;\"\u003e\n \u003cp\u003eN,N-Dimethylformamide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026lt; Not Detected (Less than 2.04 \u0026mu;g/mL)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29px;\"\u003e\n \u003cp\u003eDimethyl Sulfoxide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.15%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 24px;\"\u003e\n \u003cp\u003e0.16%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20px;\"\u003e\n \u003cp\u003e0.16%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eSolvent selection\u003c/h2\u003e\u003cp\u003eIn this study, the solubility of the tested product and the boiling point of each residual solvent were comprehensively considered. N-methyl-2-pyrrolidone with a higher boiling point was used as the solvent. The tested solvent was not only miscible with it, but also did not interfere with the determination of other tested solvents because N-methyl-2-pyrrolidone peaked the latest. In addition, it should be noted that the purity of the solvent N-methyl-2-pyrrolidone reaches the chromatogram pure level and above, because the concentration of N, N-dimethylformamide and dimethyl sulfoxide is low, it is required that the purity of N-methyl-2-pyrrolidone should be as high as possible to avoid interference from impurity peaks near the corresponding component peaks, resulting in deviations in the determination results.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eSelection of chromatographic conditions\u003c/h2\u003e\u003cp\u003eThe polarity and boiling point of the 12 target solvents to be tested are quite different. Considering the polarity and boiling point of the components to be tested, a column with moderate polarity is selected. In this test, Agilent DB-624 (60m\u0026times;0.32mm, 1.80\u0026micro;m) capillary column is selected, and the separation degree and column efficiency of each component are ideal, and each substance to be tested can be effectively separated. The interference of solvent is avoided. Due to the large difference in the boiling point of each solvent, programmed heating was adopted, and the heating rate and time were appropriately adjusted to make the 12 residual solvents peak in the appropriate time, and the separation degree of each solvent was greater than 1.5.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eSelection of headspace balance conditions\u003c/h2\u003e\u003cp\u003eDue to the large boiling point span of 12 solvents, 105℃ is selected as the headspace equilibrium temperature, which is conducive to saving the gas-liquid equilibrium time. 100℃, 105℃, 110℃ and 115℃ are investigated. With the increase of equilibrium temperature, the chromatographic peak area of each solvent increases to varying degrees. The saturated vapor pressure of the components also gradually increases. At the same time, considering that excessive temperature may cause other impurities in the sample to vaporize or sublimate into the chromatographic system, interfering with the determination of the target object, the equilibrium temperature is selected at 105℃. The change of the peak area of 12 solvents at equilibrium temperature 105℃ was investigated when the equilibrium time was 25, 30, 35 and 40 minutes. The results showed that when the equilibrium time was 35 minutes, the peak area of 12 solvents had reached a stable state, so the equilibrium time was 35 minutes.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study successfully established a sensitive, accurate, and efficient headspace gas chromatography (HS-GC) method for the simultaneous determination of 12 residual organic solvents in siponimod active pharmaceutical ingredient (API). By systematically optimizing chromatographic conditions (Agilent DB-624 column, temperature programming) and headspace parameters (105\u0026deg;C equilibrium temperature, 35 min equilibration time), complete separation of all solvents was achieved with resolutions\u0026thinsp;\u0026gt;\u0026thinsp;1.5 and baseline stability. The method demonstrated excellent linearity (r\u0026thinsp;\u0026ge;\u0026thinsp;0.999), precision (RSD% 0.3\u0026thinsp;~\u0026thinsp;2.7%), accuracy (recoveries 90\u0026thinsp;~\u0026thinsp;110%), and sensitivity (LOD: 0.006\u0026thinsp;~\u0026thinsp;8.18 \u0026micro;g/mL, LOQ: 0.002\u0026thinsp;~\u0026thinsp;2.45 \u0026micro;g/mL), fully complying with ICH Q3C and Chinese Pharmacopoeia guidelines.\u003c/p\u003e\u003cp\u003eThe use of N-methyl-2-pyrrolidone (NMP) as a high-boiling diluent eliminated matrix interference, while robustness testing confirmed method reliability under variable conditions (\u0026plusmn;\u0026thinsp;5\u0026deg;C, \u0026plusmn;\u0026thinsp;5 min). Applied to three production batches, the method reliably quantified solvent residues below permitted limits, validating its practicality for industrial quality control.\u003c/p\u003e\u003cp\u003eThis work addresses a critical gap in multi-solvent analysis, significantly reducing costs and labor compared to traditional fragmented approaches. Its adaptability to other APIs positions it as a universal solution for pharmaceutical safety monitoring, ensuring patient safety and regulatory compliance in drug manufacturing.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eZhao Li wrote the main manuscript text. Xiaoyi Shi prepared Tables 1-4. All authors reviewed and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eGiancarlo C ,Hans-Peter H ,Rajesh B , et al.Benefit-Risk Profile of Sphingosine-1-Phosphate Receptor Modulators in Relapsing and Secondary Progressive Multiple Sclerosis.[J].Drugs,2017,77(16):1755-1768. https://doi.org/10.1007/s40265-017-0814-1\u003c/li\u003e\n \u003cli\u003eMartin V ,Anat A ,Peter H H , et al.The Benefits and Risks of Switching from Fingolimod to Siponimod for the Treatment of Relapsing-Remitting and Secondary Progressive Multiple Sclerosis.[J].Drugs in R\u0026amp;D,2023,23(4):331-338. https://doi.org/10.1007/S40268-023-00434-6\u003c/li\u003e\n \u003cli\u003eCHEN Benchuan. New drug for multiple sclerosis - siponimod[J]．Herald of Medicine，2019 ,38 (09): 1243-1253. https://doi.org/10.3870/j.issn.1004-0781.2019.09.030\u003c/li\u003e\n \u003cli\u003eMAO Xiaomeng，ZHANG Jing，YANG Haijing，et al．Considerations for cardiac safety risk management in phase I clinical trials of sphingosine phosphate receptor 1 modulators．Chin J Clin Pharmacol，2024 ,40 (11): 1646-1649. https://doi.org/10.13699/j.cnki.1001-6821.2024.11.022\u003c/li\u003e\n \u003cli\u003eCouncil of Europe，European Pharmacopoeia. Eighth Edition Volumel[M]．Europe: EDQM，2013: 583-584．\u003c/li\u003e\n \u003cli\u003eZhou Haijun. International Technical requirements for drug registration: Quality part [M]. Beijing: People\u0026apos;s Medical Publishing House, 2006:112-151.\u003c/li\u003e\n \u003cli\u003eKuss HJ, Kromidas S. Guidelines for quantitative analysis by liquid and gas chromatography [M]. Chen Xiaoming, Tang Yayan, trans. 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Beijing: China Medical Science and Technology Press, 2020:116-120.\u003c/li\u003e\n \u003cli\u003eXiaoyi S ,Shuai L ,Zhao L , et al.Quantification of Residual Organic Solvents in Clobetasol Propionate using Headspace Capillary Gas Chromatography[J].Chromatographia,2024,87(4):195-202. https://doi.org/10.1007/S10337-024-04313-3\u003c/li\u003e\n \u003cli\u003eLI Suhua, WU Shaofeng, Determination of residual solvent in Clopidogrel bisulfate by gas chromatography [J]. Shanxi Chemical Industry, 2023,43 (08): 41-43. https://doi.org/10.16525/j.cnki.cn14-1109/tq.2023.08.015\u003c/li\u003e\n \u003cli\u003eSHEN Xin, ZHOU Zhenyu, Determination of residual solvent in Meropenem for injection by headspace gas chromatography [J]. Chinese Journal of Pharmaceutical Evaluation, 2023,40 (04): 305-308.\u003c/li\u003e\n \u003cli\u003eGU Xiaofeng, TANG QianQian, Determination of five organic solvents in pirampanel by headspace gas chromatography [J]. 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Polymers 14(15):3002. https:// doi.org/10.3390/polym14153002.\u003c/li\u003e\n \u003cli\u003eZou L, Guo X, McElderry JD (2023) Platform headspace gas chromatography method for high-throughput determination of residual solvents in pharmaceutical materials. J Pharm Biomed Anal 229:115349. https://doi.org/10.1016/j.jpba.2023.115349\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eNacham O, Ho TD, Anderson JL, Webster GK (2017) Use of ionic liquids as headspace gas chromatography diluents for the analysis of residual solvents in pharmaceuticals. J Pharm Biomed Anal 145:879\u0026ndash;886. https://doi.org/10.1016/j.jpba.2017.05.033\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSalatti-Dorado J\u0026Aacute;, Gonz\u0026aacute;lez-Rubio S, Garc\u0026iacute;a-G\u0026oacute;mez D, Lucena R, C\u0026aacute;rdenas S, Rubio S (2019) A high thermally stable oligomerbased supramolecular solvent for universal headspace gas chromatography: proof-of-principle determination of residual solvents in drugs. Anal Chim Acta 1046:132\u0026ndash;139. https://doi.org/10.1016/j. aca.2018.09.023\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLin L, Su LL, Li HH, Mao CQ, Ji D, Xie H et al (2022) Study on quality markers and action mechanisms of inulae fos on antihepatitis through network pharmacology and high-performance liquid chromatography fngerprints. World J Tradit Chin Med 8:426\u0026ndash;435. https://doi.org/10.4103/wjtcm.wjtcm_1_22\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Siponimod, residual solvents, gas chromatography, headspace method, API safety","lastPublishedDoi":"10.21203/rs.3.rs-6588784/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6588784/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective \u003c/strong\u003eTo develop a headspace capillary gas chromatography (HS-GC) method for the determination of residual solvents in Siponimod active pharmaceutical ingredient (API), including methanol, ethanol, acetone, isopropanol, acetonitrile, methylene chloride, ethyl acetate,tetrahydrofuran, n-heptane, toluene, N,N-dimethylformamide, and dimethyl sulfoxide.\u003cbr\u003e\n\u003cstrong\u003eMethods \u003c/strong\u003eThe analysis was performed using HS-GC with an Agilent DB-624 capillary column (60 m × 0.32 mm, 1.80 µm). The temperature program was set as follows: initial temperature of 45°C held for 17 minutes, then increased to 240°C at a rate of 15°C per minute and held for 10 minutes. Helium was used as the carrier gas at a flow rate of 1.0 mL/min. The injector temperature was set at 210°C, and the FID detector temperature was 260°C. The headspace equilibrium temperature was 105°C, and the equilibrium time was 35 minutes.\u003cbr\u003e\n\u003cstrong\u003eResults \u003c/strong\u003eAll 12 residual solvents were completely separated. Each solvent showed a good linear relationship between concentration and peak area within the tested range, with correlation coefficients (r) no less than 0.999. The average recovery rates ranged from 95.3% to 100.9%, and the relative standard deviations (RSD) were between 0.8% and 2.7% (n=9).\u003cbr\u003e\n\u003cstrong\u003eConclusion \u003c/strong\u003eThis validated analytical approach demonstrates exceptional accuracy, reproducibility, and high sensitivity, enabling robust quantification of 12 residual solvents in siponimod API through a harmonized protocol compliant with ICH Q3C and pharmacopeial standards. Its optimized efficiency and scalability render it a pivotal solution for pharmaceutical quality assurance systems.\u003c/p\u003e","manuscriptTitle":"Quantifcation of Residual Organic Solvents in Siponimod Using Headspace Capillary Gas Chromatography","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-05 04:36:18","doi":"10.21203/rs.3.rs-6588784/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"415ee325-30ea-480b-a530-4ed4c46ce12a","owner":[],"postedDate":"August 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-05T04:36:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-05 04:36:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6588784","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6588784","identity":"rs-6588784","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}