UC Davis

Hormones are chemical messengers that travel through blood and tissue to regulate millions of metabolic pathways. Accurate measurement of hormones is important for reliable diagnostics and health research. Current methods for hormone quantification rely on antibodies to capture and detect hormones. These forms of assays suffer from poor specificity and reproducibility and can involve large money and time commitments. Liquid chromatography (LC) coupled tandem mass spectrometry (MS/MS) is an alternative quantitation method already widely used for the quantification of small metabolites. Although advances in mass spectrometry have allowed for the quantification of larger peptides and steroid hormones, there exist no methods specific to pig hormones. Pigs are considered an excellent model system for the study of human metabolism as the two species share many similarities in digestive anatomy and physiology. Additionally, despite the great potential of LC-MS/MS for simultaneous quantitation, multiplex methods are scarce. In this study, we developed and validated an LC-MS/MS-based method to simultaneously quantify several post-prandial hormones including cortisol, GLP-1 (7-37), GLP-1 (7-36), acyl and desacyl ghrelin, and carboxylated osteocalcin. Post-prandial hormones are those that respond to feeding and are essential in understanding the impact of diet on appetite regulation, glycemic control, and body composition. Although not a post-prandial hormone, cortisol was included since stress is known to affect post-prandial hormonal response. Hormones were isolated from 100 uL of pig serum using an optimized acetonitrile-based protein precipitation extraction. The extracted sample was analyzed by LC-MS/MS. The final method had excellent recovery and reproducibility. The validated detection range encompasses the typical ranges for the target hormones in adult pigs and piglets. Although calibration curves for all analytes demonstrated R2 > 0.9, additional work should be done to further improve linearity and sensitivity for more precise and accurate measurement. Overall, our method meets the threshold as a bioanalytical method for the measurement of seven hormones from a small sample size.