Impurity Profiling and Structural Elucidation of Unknown Degradation Products in Efavirenz Using Integrated HPLC, LC-MS, and NMR Techniques

Impurity Profiling and Structural Elucidation of Unknown Degradation Products in Efavirenz Using Integrated HPLC, LC-MS, and NMR Techniques | 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 Impurity Profiling and Structural Elucidation of Unknown Degradation Products in Efavirenz Using Integrated HPLC, LC-MS, and NMR Techniques Rajesh Meshram, Rajesh Gour This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8688493/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 A comprehensive impurity profiling study was conducted on Efavirenz, an essential antiretroviral drug, using an integrated analytical approach involving HPLC, LC-MS, HRMS, and NMR spectroscopy. A stability-indicating HPLC method was developed and validated as per ICH guidelines, demonstrating excellent linearity (R² = 0.9998), precision (RSD < 2%), accuracy (98–102%), and robustness. Forced degradation studies under acidic, alkaline, oxidative, thermal, and photolytic conditions revealed significant degradation, with alkaline stress being the most severe (33.81% degradation). Nine major degradation products were isolated and characterized using LC-MS and NMR. Structural elucidation confirmed the formation of novel impurities, including cyclized and hydroxylated derivatives. The study provides valuable insights into the degradation pathways of Efavirenz and supports regulatory compliance and quality assurance in pharmaceutical manufacturing. Efavirenz Impurity profiling HPLC LC-MS NMR Forced degradation Degradation products Introduction Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) widely used in the treatment of HIV-1 infection. Ensuring its purity and stability is critical for patient safety and therapeutic efficacy. Impurities in pharmaceuticals can arise from synthesis, storage, or degradation, and their identification is mandated by regulatory bodies such as ICH. This study aims to develop a validated stability-indicating HPLC method, perform forced degradation studies, and characterize unknown degradation products using LC-MS and NMR techniques. Experimental Materials and Methods Efavirenz API was obtained from a certified manufacturer. HPLC-grade acetonitrile, methanol, and formic acid were purchased from Merck. All reagents were of analytical grade. Instrumentation - HPLC: Waters Alliance e2695 with PDA detector - LC-MS: Waters Acquity SQD with ESI source - HRMS: Waters Q-TOF Micro - NMR: Bruker 400 MHz spectrometer - Preparative HPLC: Gilson GX-271 Chromatographic Conditions - Column: C18 (100 × 2.1 mm, 1.7 µm) - Mobile Phase: 0.1% formic acid in water (A) and acetonitrile (B) - Gradient: 0–2 min (60% B), 2–8 min (60–95% B), 8–10 min (95% B) - Flow Rate: 0.4 mL/min - Detection: 247 nm - Column Temperature: 40°C Forced Degradation Studies EFV was subjected to: - Acidic: 0.5 N HCl, 60°C, 12 h - Alkaline: 0.5 N NaOH, 60°C, 16 h - Oxidative: 30% H₂O₂, RT, 24 h - Thermal: 80°C, 24 h - Photolytic: UV 254 nm, 72 h Isolation and Characterization Degradation products were isolated using preparative HPLC and characterized by LC-MS, HRMS, and NMR (¹H, ¹³C, COSY, HSQC, HMBC). Method Validation The HPLC method was validated for linearity, precision, accuracy, LOD, LOQ, and robustness as per ICH Q2(R1). Results and Discussion Method Development and Validation The optimized HPLC method provided baseline separation of EFV and its impurities. System suitability parameters were within acceptable limits (Table 1 ). Table 1 System Suitability Test Results Parameter Acceptance Criteria Observed Value %RSD Retention time – 4.81 min 1.04 Theoretical plates > 3000 5555 1.83 Tailing factor 0.8–1.2 0.98 3.06 Resolution > 1.5 > 1.8 – Table 2 Validation Parameters Parameter Results Acceptance Criteria Linearity (R²) 0.9998 ≥ 0.999 Precision (%RSD) 0.07–0.30% ≤ 2% Accuracy 99.22–101.30% 98–102% LOD 2.5 µg/mL – LOQ 4.0 µg/mL – Forced Degradation Studies EFV was most susceptible to alkaline degradation (33.81%), followed by oxidative (28.37%), photolytic (23.26%), acidic (15.52%), and thermal (12.03%) degradation. Table 3 Degradation Summary Under Stress Conditions Condition % Degradation No. of Degradation Products Acidic 15.52 7 Alkaline 33.81 9 Oxidative 28.37 9 Thermal 12.03 7 Photolytic 23.26 8 LC-MS and HRMS Analysis LC-MS and HRMS provided molecular weights and elemental compositions of degradation products. Key impurities included hydroxylated, cyclized, and chlorinated derivatives. Table 4 HRMS Data of Major Degradation Products Compound [M + H]⁺ (m/z) Molecular Formula Mass Error (ppm) EFV 316.0892 C₁₄H₉ClF₃NO₂ 0 Hydroxydefluoro-EFV 298.0786 C₁₄H₉ClNO₃ 0 Cyclized-EFV 312.0579 C₁₄H₇ClF₃N₂ 0 N-Oxide-EFV 332.0841 C₁₄H₉ClF₃NO₃ 0 NMR Structural Elucidation NMR spectroscopy confirmed the structures of isolated impurities. For example, Alkaline DP-2 showed a new CH₂ signal at 4.18 ppm, indicating cyclization. Oxidative DP-5 displayed hydroxylation signals at 5.51 ppm (OH) and 65.4 ppm (¹³C). Degradation Pathways Acidic: Hydrolysis of cyclopropyl ring and aromatic hydroxylation. Alkaline: Nucleophilic ring opening, cyclization, and amide bond hydrolysis. Oxidative: Hydroxylation of aromatic and aliphatic carbons. Photolytic: Direct photolysis and radical-mediated oxidation. Conclusion A stability-indicating HPLC method was successfully developed and validated for impurity profiling of Efavirenz. Forced degradation studies revealed significant degradation under alkaline and oxidative conditions. Nine degradation products were isolated and characterized using LC-MS and NMR, providing insights into degradation mechanisms. The study supports quality control and regulatory compliance for Efavirenz formulations. Declarations Acknowledgments The authors thank for LNCT University for instrumental support and guidance. Funding Declaration This research received no external funding. Author Contribution Declaration Rajesh Meshram and Rajesh Gour contributed equally to the conceptualization, design, and execution of the study. Rajesh Meshram was responsible for the synthesis and characterization of the prodrug, while Rajesh Gour conducted the biological evaluation studies. Both authors contributed to the writing and editing of the manuscript. Clinical Trial Registration Not Applicable Consent to Publish Declaration Not Applicable Ethics and Consent to Participate Declarations Not Applicable Data Availability: The datasets generated and/or analysed during the current study are not publicly available due to patentability of data; but are available from the corresponding author on reasonable request. Conflict of Interest: The authors declare that they have no conflict of interest. References ICH Q3A(R2). Impurities in New Drug Substances. 2006. ICH Q3B(R2). Impurities in New Drug Products. 2006. Sudhakar, et al. J Pharm Biomed Anal. 2011;54:667. Reddy, et al. J Pharm Biomed Anal. 2007;43:1262. Vasanthakumar, et al. J Pharm Biomed Anal. 2010;52:579. Kumar A, et al. J Pharm Biomed Anal. 2014;96:15. Raman, et al. J Pharm Biomed Anal. 2011;55:1081. Talluri, et al. J Pharm Biomed Anal. 2015;104:10. Chowdary, et al. J Pharm Biomed Anal. 2016;117:434. Bhimavarapu, et al. J Pharm Biomed Anal. 2013;84:12. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8688493","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":595577846,"identity":"83afa9d4-462c-4813-ab96-2d8415b25233","order_by":0,"name":"Rajesh Meshram","email":"data:image/png;base64,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","orcid":"","institution":"LNCT University","correspondingAuthor":true,"prefix":"","firstName":"Rajesh","middleName":"","lastName":"Meshram","suffix":""},{"id":595577847,"identity":"2fa442df-2bdf-4f1f-b513-95c98db53c83","order_by":1,"name":"Rajesh Gour","email":"","orcid":"","institution":"LNCT University","correspondingAuthor":false,"prefix":"","firstName":"Rajesh","middleName":"","lastName":"Gour","suffix":""}],"badges":[],"createdAt":"2026-01-24 17:53:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8688493/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8688493/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106021904,"identity":"2cb394d0-aade-41d9-9d4c-ed0c861af208","added_by":"auto","created_at":"2026-04-02 13:57:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":623132,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8688493/v1/2250071d-1ac6-4698-9353-30b446bb30e1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eImpurity Profiling and Structural Elucidation of Unknown Degradation Products in Efavirenz Using Integrated HPLC, LC-MS, and NMR Techniques\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEfavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) widely used in the treatment of HIV-1 infection. Ensuring its purity and stability is critical for patient safety and therapeutic efficacy. Impurities in pharmaceuticals can arise from synthesis, storage, or degradation, and their identification is mandated by regulatory bodies such as ICH. This study aims to develop a validated stability-indicating HPLC method, perform forced degradation studies, and characterize unknown degradation products using LC-MS and NMR techniques.\u003c/p\u003e"},{"header":"Experimental","content":"\u003cp\u003e\u003cstrong\u003eMaterials and Methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEfavirenz API was obtained from a certified manufacturer. HPLC-grade acetonitrile, methanol, and formic acid were purchased from Merck. All reagents were of analytical grade.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstrumentation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e- HPLC: Waters Alliance e2695 with PDA detector\u003c/p\u003e\n\u003cp\u003e- LC-MS: Waters Acquity SQD with ESI source\u003c/p\u003e\n\u003cp\u003e- HRMS: Waters Q-TOF Micro\u003c/p\u003e\n\u003cp\u003e- NMR: Bruker 400 MHz spectrometer\u003c/p\u003e\n\u003cp\u003e- Preparative HPLC: Gilson GX-271\u003c/p\u003e\n\u003cp\u003eChromatographic Conditions\u003c/p\u003e\n\u003cp\u003e- Column: C18 (100 \u0026times; 2.1 mm, 1.7 \u0026micro;m)\u003c/p\u003e\n\u003cp\u003e- Mobile Phase: 0.1% formic acid in water (A) and acetonitrile (B)\u003c/p\u003e\n\u003cp\u003e- Gradient: 0\u0026ndash;2 min (60% B), 2\u0026ndash;8 min (60\u0026ndash;95% B), 8\u0026ndash;10 min (95% B)\u003c/p\u003e\n\u003cp\u003e- Flow Rate: 0.4 mL/min\u003c/p\u003e\n\u003cp\u003e- Detection: 247 nm\u003c/p\u003e\n\u003cp\u003e- Column Temperature: 40\u0026deg;C\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eForced Degradation Studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEFV was subjected to:\u003c/p\u003e\n\u003cp\u003e- Acidic: 0.5 N HCl, 60\u0026deg;C, 12 h\u003c/p\u003e\n\u003cp\u003e- Alkaline: 0.5 N NaOH, 60\u0026deg;C, 16 h\u003c/p\u003e\n\u003cp\u003e- Oxidative: 30% H₂O₂, RT, 24 h\u003c/p\u003e\n\u003cp\u003e- Thermal: 80\u0026deg;C, 24 h\u003c/p\u003e\n\u003cp\u003e- Photolytic: UV 254 nm, 72 h\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation and Characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDegradation products were isolated using preparative HPLC and characterized by LC-MS, HRMS, and NMR (\u0026sup1;H, \u0026sup1;\u0026sup3;C, COSY, HSQC, HMBC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod Validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe HPLC method was validated for linearity, precision, accuracy, LOD, LOQ, and robustness as per ICH Q2(R1).\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMethod Development and Validation\u003c/h2\u003e \u003cp\u003eThe optimized HPLC method provided baseline separation of EFV and its impurities. System suitability parameters were within acceptable limits (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSystem Suitability Test Results\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcceptance Criteria\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eObserved Value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e%RSD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRetention time\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.81 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTheoretical plates\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;3000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5555\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTailing factor\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8\u0026ndash;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eResolution\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eValidation Parameters\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResults\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAcceptance Criteria\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLinearity (R\u0026sup2;)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.9998\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;0.999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePrecision (%RSD)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.07\u0026ndash;0.30%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;2%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAccuracy\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.22\u0026ndash;101.30%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98\u0026ndash;102%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLOD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.5 \u0026micro;g/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLOQ\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.0 \u0026micro;g/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eForced Degradation Studies\u003c/h3\u003e\n\u003cp\u003eEFV was most susceptible to alkaline degradation (33.81%), followed by oxidative (28.37%), photolytic (23.26%), acidic (15.52%), and thermal (12.03%) degradation.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDegradation Summary Under Stress Conditions\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCondition\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e% Degradation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo. of Degradation Products\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAcidic\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAlkaline\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e33.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOxidative\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e28.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eThermal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePhotolytic\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e23.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eLC-MS and HRMS Analysis\u003c/h3\u003e\n\u003cp\u003eLC-MS and HRMS provided molecular weights and elemental compositions of degradation products. Key impurities included hydroxylated, cyclized, and chlorinated derivatives.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHRMS Data of Major Degradation Products\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[M\u0026thinsp;+\u0026thinsp;H]⁺ (m/z)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMolecular Formula\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMass Error (ppm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEFV\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e316.0892\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC₁₄H₉ClF₃NO₂\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHydroxydefluoro-EFV\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e298.0786\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC₁₄H₉ClNO₃\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCyclized-EFV\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e312.0579\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC₁₄H₇ClF₃N₂\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eN-Oxide-EFV\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e332.0841\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC₁₄H₉ClF₃NO₃\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eNMR Structural Elucidation\u003c/h2\u003e \u003cp\u003eNMR spectroscopy confirmed the structures of isolated impurities. For example, Alkaline DP-2 showed a new CH₂ signal at 4.18 ppm, indicating cyclization. Oxidative DP-5 displayed hydroxylation signals at 5.51 ppm (OH) and 65.4 ppm (\u0026sup1;\u0026sup3;C).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDegradation Pathways\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eAcidic: Hydrolysis of cyclopropyl ring and aromatic hydroxylation.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eAlkaline: Nucleophilic ring opening, cyclization, and amide bond hydrolysis.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eOxidative: Hydroxylation of aromatic and aliphatic carbons.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003ePhotolytic: Direct photolysis and radical-mediated oxidation.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eA stability-indicating HPLC method was successfully developed and validated for impurity profiling of Efavirenz. Forced degradation studies revealed significant degradation under alkaline and oxidative conditions. Nine degradation products were isolated and characterized using LC-MS and NMR, providing insights into degradation mechanisms. The study supports quality control and regulatory compliance for Efavirenz formulations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank for \u0026nbsp;LNCT University \u0026nbsp;for instrumental support and guidance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no external funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRajesh Meshram and Rajesh Gour contributed equally to the conceptualization, design, and execution of the study. Rajesh Meshram was responsible for the synthesis and characterization of the prodrug, while Rajesh Gour conducted the biological evaluation studies. Both authors contributed to the writing and editing of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trial Registration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics and Consent to Participate Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are not publicly available due to patentability of data; but are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eICH Q3A(R2). Impurities in New Drug Substances. 2006.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eICH Q3B(R2). Impurities in New Drug Products. 2006.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSudhakar, et al. J Pharm Biomed Anal. 2011;54:667.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReddy, et al. J Pharm Biomed Anal. 2007;43:1262.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVasanthakumar, et al. J Pharm Biomed Anal. 2010;52:579.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar A, et al. J Pharm Biomed Anal. 2014;96:15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaman, et al. J Pharm Biomed Anal. 2011;55:1081.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTalluri, et al. J Pharm Biomed Anal. 2015;104:10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChowdary, et al. J Pharm Biomed Anal. 2016;117:434.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhimavarapu, et al. J Pharm Biomed Anal. 2013;84:12.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Efavirenz, Impurity profiling, HPLC, LC-MS, NMR, Forced degradation, Degradation products","lastPublishedDoi":"10.21203/rs.3.rs-8688493/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8688493/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA comprehensive impurity profiling study was conducted on Efavirenz, an essential antiretroviral drug, using an integrated analytical approach involving HPLC, LC-MS, HRMS, and NMR spectroscopy. A stability-indicating HPLC method was developed and validated as per ICH guidelines, demonstrating excellent linearity (R\u0026sup2; = 0.9998), precision (RSD\u0026thinsp;\u0026lt;\u0026thinsp;2%), accuracy (98\u0026ndash;102%), and robustness. Forced degradation studies under acidic, alkaline, oxidative, thermal, and photolytic conditions revealed significant degradation, with alkaline stress being the most severe (33.81% degradation). Nine major degradation products were isolated and characterized using LC-MS and NMR. Structural elucidation confirmed the formation of novel impurities, including cyclized and hydroxylated derivatives. The study provides valuable insights into the degradation pathways of Efavirenz and supports regulatory compliance and quality assurance in pharmaceutical manufacturing.\u003c/p\u003e","manuscriptTitle":"Impurity Profiling and Structural Elucidation of Unknown Degradation Products in Efavirenz Using Integrated HPLC, LC-MS, and NMR Techniques","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-24 11:18:03","doi":"10.21203/rs.3.rs-8688493/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":"5455d811-50f3-4ba7-b903-392fd4f3e61a","owner":[],"postedDate":"February 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-02T13:56:17+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-24 11:18:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8688493","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8688493","identity":"rs-8688493","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}