{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,19]],"date-time":"2026-10-19T06:39:15Z","timestamp":1792391955293,"version":"4.4.0"},"reference-count":37,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2026,10,18]],"date-time":"2026-10-18T00:00:00Z","timestamp":1792281600000},"content-version":"vor","delay-in-days":17,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T00:00:00Z","timestamp":1790812800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Separation Science Plus"],"published-print":{"date-parts":[[2026,10]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    While developing a topical gel 1% w\/w dosage form, significant unknown impurities were observed above their threshold during shelf\u2010life determination, with relative retention time (RRT) of 0.66, 0.67, and 0.71 in accelerated stability conditions of 40\u00b0C temperature and 75% relative humidity sample. Due to the importance of pharmaceutical quality and purity, impurities during manufacture or storage might cause serious side effects. If an unknown impurity exceeds its threshold limits during product stability testing, its structure must be determined to assess the potential risks. Impurities were separated by gradient HPLC on a Kinetex C18 column with 0.1% formic acid mobile phases, detected at 245\u00a0nm. Structural characterization was done using liquid chromatography with mass spectrometry (LC\u2013MS) and\n                    <jats:sup>1<\/jats:sup>\n                    H NMR (400\u00a0MHz). This study investigated and characterized degradation impurities observed during stability testing of a testosterone topical gel formulation. Three degradation impurities, identified as 6\u03b2\u2010hydroxy\u2010testosterone, 6\u03b2\u2010hydroperoxy\u2010testosterone, and 6\u2010keto\u2010testosterone impurities with molecular masses of 304.42, 320.20, and 302.41, were isolated and characterized using preparative chromatographic, high\u2010resolution mass spectrometric, and nuclear magnetic resonance techniques, followed by in silico (Q)SAR assessment of their potential genotoxicity using Deductive Estimate of Risk from Existing Knowledge (Derek) Nexus and Toxicity Prediction using Komputer Assisted Technology (TOPKAT) silico tools\u2010based knowledge and statistical methods. In silico toxicity evaluation with TOPKAT and Derek Nexus indicated that the testosterone impurities at RRT of 0.66, 0.67, and 0.71 are non\u2010mutagenic, corroborating their designation as ICH M7 Class 5 impurities.\n                  <\/jats:p>","DOI":"10.1002\/sscp.70321","type":"journal-article","created":{"date-parts":[[2026,10,19]],"date-time":"2026-10-19T05:39:51Z","timestamp":1792388391000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Identification and Structural Elucidation of Potential Degradation Impurities in Testosterone Topical Formulation Using LC\u2013MS, NMR and Evaluation of Genotoxicity by Using Knowledge\u2010Based (Derek Nexus) and a Statistical Method\u2010Based (TOPKAT) Silico Tools"],"prefix":"10.1002","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-5447-0698","authenticated-orcid":false,"given":"Venugopalarao","family":"Mukkapati","sequence":"first","affiliation":[{"name":"Department of Chemistry School of Applied Sciences and Humanities (SASH) VIGNAN'S Foundation for Science Technology &amp; Research (VFSTR)  Guntur India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4337-6951","authenticated-orcid":false,"given":"Venkata Kanaka Srivani","family":"Maddala","sequence":"additional","affiliation":[{"name":"Department of Chemistry School of Applied Sciences and Humanities (SASH) VIGNAN'S Foundation for Science Technology &amp; Research (VFSTR)  Guntur India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5852-1656","authenticated-orcid":false,"given":"Hanimi Reddy","family":"Bapatu","sequence":"additional","affiliation":[{"name":"Azurity Pharmaceuticals India LLP  Hyderabad Telangana India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8430-8640","authenticated-orcid":false,"given":"Praveen Kumar","family":"Subbappa","sequence":"additional","affiliation":[{"name":"Azurity Pharmaceuticals  Princeton New Jersey USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,10,18]]},"reference":[{"key":"e_1_2_11_2_1","doi-asserted-by":"publisher","DOI":"10.1210\/jc.2017\u201001658"},{"key":"e_1_2_11_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0140\u20106736(06)68226\u20103"},{"key":"e_1_2_11_4_1","doi-asserted-by":"publisher","DOI":"10.1210\/jc.2009\u20102354"},{"key":"e_1_2_11_5_1","doi-asserted-by":"publisher","DOI":"10.1001\/jama.2017.0129"},{"key":"e_1_2_11_6_1","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1506119"},{"key":"e_1_2_11_7_1","volume-title":"WHO Model List of Essential Medicines","author":"World Health Organization","year":"2023"},{"key":"e_1_2_11_8_1","volume-title":"Testosterone Gel Prescribing Information","author":"US Food and Drug Administration"},{"key":"e_1_2_11_9_1","doi-asserted-by":"publisher","DOI":"10.4236\/ajac.2016.76047"},{"key":"e_1_2_11_10_1","doi-asserted-by":"publisher","DOI":"10.1093\/chromsci\/bmu031"},{"key":"e_1_2_11_11_1","first-page":"671","article-title":"Identification and Determination of Impurities in Drugs","volume":"41","author":"Gorog S.","year":"2006","journal-title":"Journal of Pharmaceutical and Biomedical Analysis"},{"key":"e_1_2_11_12_1","doi-asserted-by":"publisher","DOI":"10.1080\/10826076.2013.836712"},{"key":"e_1_2_11_13_1","doi-asserted-by":"publisher","DOI":"10.1093\/jaoacint\/qsae057"},{"key":"e_1_2_11_14_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jpha.2013.09.003"},{"key":"e_1_2_11_15_1","volume-title":"Impurities in New Drug Substances","author":"ICH Q3A(R2)","year":"2006"},{"key":"e_1_2_11_16_1","volume-title":"Impurities in New Drug Products","author":"ICH Q3B(R2)","year":"2006"},{"key":"e_1_2_11_17_1","doi-asserted-by":"publisher","DOI":"10.1093\/chromsci\/bmv029"},{"key":"e_1_2_11_18_1","doi-asserted-by":"publisher","DOI":"10.1093\/chromsci\/bmad049"},{"key":"e_1_2_11_19_1","doi-asserted-by":"publisher","DOI":"10.1177\/14690667211005335"},{"key":"e_1_2_11_20_1","volume-title":"Stability Testing of New Drug Substances and Products","author":"ICH Q1A(R2)","year":"2003"},{"key":"e_1_2_11_21_1","first-page":"1","article-title":"Stress Testing To Determine Inherent Stability","volume":"24","author":"Singh S.","year":"2000","journal-title":"Pharmaceutical Technology"},{"key":"e_1_2_11_22_1","doi-asserted-by":"crossref","DOI":"10.1201\/9780849359194","volume-title":"Pharmaceutical Stress Testing","author":"Baertschi S. 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