Zhichao ZhangHareem SiddiqiYu-Ping HuangShannon McClorryPeng JiDaniela BarileCarolyn M. Slupsky2025-08-052025-08-052024-01-27https://www.scopus.com/pages/publications/85187260359https://scholars.lib.ntu.edu.tw/handle/123456789/730994Hormones are important signaling molecules controlling physiological homeostasis. ELISA kits are commonly used to measure hormones; however, few ELISA kits are multiplex, not all species-specific ELISA kits are commercially available, and ELISA kits typically require a significant volume of biological fluids. Pigs resemble humans in digestive physiology, making them an excellent model in preclinical research of nutrition and metabolism. In this study, we developed and validated a simple liquid–liquid extraction procedure and LC-MS/MS method for the simultaneous quantification of insulin, cortisol, glucagon-like peptide-1 (GLP-1) (7-37) and (7-36), acyl and des-acyl ghrelin, and carboxylated osteocalcin in pig serum. The proposed method is specific, highly sensitive (LOQ in ng/mL and pg/mL), reasonably accurate (more than 76.2% of all quality control samples within 20% error from nominal values), and precise (intra-day CV ≤ 10% and inter-day CV ≤ 23.1%). The recoveries of all analytes and corresponding internal standards ranged from 83.7 to 116.0%. The method also requires a low serum volume of 50–100 μL, which is invaluable when sample volume is limited. These methods could be easily extended for use in other mammalian species.truecortisolghrelinGLP-1hormone quantitationinsulinLC-MS/MS methodosteocalcinjournal article10.3390/separations110200412-s2.0-85187260359