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This study describes, for the first time, the establishment of an environmentally sustainable reversed-phase HPLC approach for the simultaneous bioanalysis of TP and CIP in human plasma. The developed chromatographic method allowed for rapid separation within 4\u00a0min employing a methanol\u2013water mobile phase, thereby reducing solvent consumption and improving environmental sustainability. The method was validated according to the validation parameters investigated in the present study, with reference to the US FDA Bioanalytical Method Validation Guidance. Excellent linearity was achieved over concentration ranges of 5\u201390\u00a0\u00b5g\/mL (TP) and 5\u201380\u00a0\u00b5g\/mL (CIP) in pure samples, and 0.5\u201330\u00a0\u00b5g\/mL (TP) and 0.5\u201310\u00a0\u00b5g\/mL (CIP) in human plasma. LOD\/LOQ values were 1.60\/4.86\u00a0\u00b5g\/mL for TP and 1.58\/4.78\u00a0\u00b5g\/mL for CIP in pure samples, and 0.16\/0.49\u00a0\u00b5g\/mL for TP and 0.10\/0.31\u00a0\u00b5g\/mL for CIP in spiked human plasma. The proposed method was characterized by adequate sensitivity and demonstrated strong performance in terms of accuracy, precision, specificity, and robustness. Furthermore, it proved effective in assessing the forced degradation of TP and CIP under diverse stress conditions, confirming its capability as a stability-indicating approach. The environmental impact of the method was comprehensively evaluated using multiple established greenness and blueness assessment tools, confirming its favorable environmental profile. Overall, the developed approach presents a rapid, highly accurate, and environmentally sustainable strategy for the simultaneous bioanalysis of TP and CIP in bulk drugs, pharmaceutical formulations, and human plasma.<\/jats:p>","DOI":"10.1038\/s41598-026-65086-3","type":"journal-article","created":{"date-parts":[[2026,8,13]],"date-time":"2026-08-13T02:53:50Z","timestamp":1786589630000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Green stability-indicating RP-HPLC method for simultaneous bioanalysis of tedizolid phosphate and ciprofloxacin in human plasma"],"prefix":"10.1038","volume":"16","author":[{"given":"Gehad Abd","family":"El-Fatah","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amr M.","family":"Mahmoud","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alaa A.","family":"Ahmed-Anwar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ahmed A.","family":"Farghali","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rehab","family":"Mahmoud","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohamed E. 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The study involving participant and human biological material was conducted according to the Declaration of Helsinki and approved by an authorized ethics committee. The named volunteer was fully informed about the aims and risks of the study and subsequently signed a written informed consent form. The protocol, including the consent process, received approval from the institutional ethics committee. Ethical approval for the study was granted by the Institutional Human and Animal Research Ethics Committee (PSAS-BSU-HAREC), Faculty of Postgraduate Studies for Advanced Sciences, Beni-Suef University, Beni Suef 62511, Egypt (Approval No. PSAS-BSU-HAREC.004). Informed consent was obtained from the participant.","order":2,"name":"Ethics","label":"Ethics approval and consent to participate","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"A limitation of the present study is that although extraction recovery and matrix effect were evaluated, these assessments were performed using a single human plasma lot, which does not fully account for inter-individual matrix variability. In addition, IS-normalized matrix factor determination and peak purity assessment during the forced degradation study were not performed, and long-term frozen storage stability was evaluated for only 7 days; therefore, extended frozen storage stability over longer storage periods was not investigated. Future studies will address these limitations to further strengthen the bioanalytical validation and regulatory applicability of the proposed method. Despite these limitations, the developed method demonstrated satisfactory selectivity, linearity, accuracy, precision, sensitivity, extraction recovery, matrix effect, and stability under the investigated conditions.","order":3,"name":"Ethics","label":"Study limitations and future research plans","group":{"name":"EthicsHeading","label":"Declarations"}}],"article-number":"25287"}}