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The anti-HIV drug emtricitabine (FTC) is co-administered with two direct acting anti-HCV drugs; daclatasvir (DAC) and ledipasvir (LDV). The three drugs are simultaneously determined in human urine for the first time by a validated, simple and sensitive RP-HPLC with programmed fluorescence detection. The column used is Exsil 100 ODS C18 column (250\u2009\u00d7\u20094.6\u00a0mm, 5\u00a0\u03bcm). The used mobile phase is acetonitrile: methanol: 0.01\u00a0M ammonium acetate buffer, pH 4.5 in ratio (20: 60: 20) in isocratic mode pumped at flow rate 1 mL\/min. The proposed method is successfully validated according to FDA bioanalytical validation guidelines. The calibration curves are linear over the ranges (500-15000, 1\u201350 and 10\u2013100 ng\/mL) with average recoveries (97.9-99.54%, 98.78-104.17% and 98.49\u2013100.96%) for FTC, DAC and LDV, respectively. The intraday and inter-day accuracy and precision results are within the acceptable limits. Stability assays reveal that the three studied drugs were stable during preparation, injection and storage. The method can be applied for the quantification of the three drugs co-administered to HIV\/HCV co-infected patients\u2019 urine which aids in therapeutic drug monitoring and dosage adjustment for chronic patients.<\/jats:p>","DOI":"10.1038\/s41598-025-07401-y","type":"journal-article","created":{"date-parts":[[2025,7,1]],"date-time":"2025-07-01T22:14:49Z","timestamp":1751408089000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Sensitive RP-HPLC method with fluorimetric detection for concurrent quantification of emtricitabine, Daclatasvir and Ledipasvir in human urine"],"prefix":"10.1038","volume":"15","author":[{"given":"Aya A.","family":"Youssef","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"N.","family":"Magdy","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lobna A.","family":"Hussein","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"A. 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