Citation: Abdelshakour, M.A.; Mostafa, A.E.; Hadad, G.M.; Hamed, D.M.; El-Darder, O.M.; El-Gindy, A.; Khinkar, R.M.; Aldurdunji, M.M.; Murshid, S.S.; Abdel Salam, R.A. Experimental Design Assisted HPLC/UV and LC-MS/MS for Simultaneous Determination of Selected Veterinary Antibiotics in Broiler Chicken. Separations 2022, 9, 427. https://doi.org/10.3390/ separations9120427 Academic Editors: Yiyang Dong and Yuwei Yuan Received: 16 November 2022 Accepted: 8 December 2022 Published: 10 December 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). separations Article Experimental Design Assisted HPLC/UV and LC-MS/MS for Simultaneous Determination of Selected Veterinary Antibiotics in Broiler Chicken Mohamed A. Abdelshakour 1, *, Aziza E. Mostafa 2 , Ghada M. Hadad 2 , Dalia M. Hamed 3 , Omayma M. El-Darder 4 , Alaa El-Gindy 2 , Roaa M. Khinkar 5 , Mohammed M. Aldurdunji 6 , Samar S. Murshid 7 and Randa A. Abdel Salam 2 1 Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Sohag University, Sohag 82524, Egypt 2 Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt 3 Department of Avian and Rabbit Medicine, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522, Egypt 4 Department of Home Economics, Faculty of Education, Suez Canal University, Ismailia 41522, Egypt 5 Department of Pharmacy Practice, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia 6 Department of Clinical Pharmacy, College of Pharmacy, Umm Al-Qura University, P.O. Box 13578, Makkah 21955, Saudi Arabia 7 Department of Natural Products, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia * Correspondence: mohamed.abdelshakour@pharm.sohag.edu.eg Abstract: Antibiotics are used in the poultry industry to treat and prevent diseases. Their frequent use resulted in the appearance of antibiotic residuals in poultry meat, which is considered a serious public health issue. Among frequently used antibiotics are cefotaxime (CTX), ciprofloxacin (CIP), colistin (CST), doxycycline (DOX), flumequine (FLU), sulfamethoxazole (SMZ), trimethoprim (TMP) and tylosin (TYL). This study aimed to develop an optimized and validated method for concurrent estimation of the eight antibiotics in broiler chicken samples based on an easy extraction method fol- lowed by HPLC-UV and LC/MS/MS analysis. An experimental design was used for the optimization of the extraction procedure. Optimal conditions for separation were determined by using a central composite design after studying (1) mobile phase initial concentration, (2) column temperature, and (3) flow rate. The method was validated on the bases of ICH guidelines. The detection limits ranged from 3 to 5 μg kg -1 for HPLC- UV and ranged from 0.01 to 0.05 μg kg -1 for LC/MS/MS, while quan- tification limits ranged from 10 to 16 μg kg -1 for HPLC- UV and ranged from 0.01 to 0.11 μg kg -1 for LC/MS/MS. The chromatographic techniques were utilized for the analysis of spiked broiler chicken samples at a concentration range from 30 to 300 μg kg -1 ) for HPLC-UV and 0.01–20 μg kg -1 for LC/MS/MS. The proposed methods were used for quantification of the residues of the studied antibiotics in real broiler samples obtained from local supermarkets in Ismailia governorate, Egypt. The detected levels of residual antibiotics were within the permissible limits. Keywords: antibiotics; food analysis; liquid chromatography; sustainability of natural resources 1. Introduction Antibacterial medicines are used in poultry for treatment and protection purposes as well as increasing efficiency and growth factors [1]. Several groups of antibacterial agents, including cephalosporins, tetracyclines, beta-lactams, sulfonamides, lincosamides, fluoro- quinolones, aminoglycosides, macrolides, and others, are widely used in the treatment of food-producing animals [2]. Macrolides are an important group of antibiotics extensively Separations 2022, 9, 427. https://doi.org/10.3390/separations9120427 https://www.mdpi.com/journal/separations