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The Iranian surfactant, Beraksurf, was used as the sample. A simple extraction procedure involving methanol dissolution and protein precipitation was employed, followed by HPLC analysis. The method underwent comprehensive validation for various parameters, including system suitability, repeatability (intra\u2010assay precision), intermediate precision, linearity, accuracy, limit of detection (LOD), limit of quantification (LOQ), and robustness. The results demonstrated excellent system suitability. Repeatability and intermediate precision were confirmed with low %RSD values, while linearity was established with correlation coefficients (<jats:italic>R<\/jats:italic><jats:sup>2<\/jats:sup>) exceeding 0.99 for all analytes. The method exhibited high accuracy, with recoveries ranging from 97% to 100%, and low LOD and LOQ values, indicating its sensitivity. Robustness testing revealed that minor variations in method parameters did not significantly impact results. This validated RP\u2010HPLC\u2010RID method provides a reliable and efficient approach for the analysis of key surfactant components, contributing to improved quality control in surfactant preparations and enhancing therapeutic strategies for managing RDS in infants.<\/jats:p>","DOI":"10.1002\/bmc.70151","type":"journal-article","created":{"date-parts":[[2025,6,20]],"date-time":"2025-06-20T00:07:43Z","timestamp":1750378063000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Development and Validation of an HPLC\u2010RI Method for the Simultaneous Determination of DPPC, Palmitic Acid, and Cholesterol in Bovine Pulmonary Surfactant"],"prefix":"10.1002","volume":"39","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1248-8029","authenticated-orcid":false,"given":"Mahsa","family":"Aghaei","sequence":"first","affiliation":[{"name":"ARC Bioassay (Iran Food and Drug Administration Accredited QC Laboratory of 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