Multiple peptide hormones are secreted from islets of Langerhans to maintain blood glucose homeostasis. Defects in the amount and patterns of hormone secretion can lead to metabolic disorders, such as diabetes. To understand the relationships between the peptides, analytical methods that can quantify multiple hormones in short time increments are required. To this end, an automated and online system was developed for sampling perfusate from ∼30 human islets held on a glass microfluidic device with sequential liquid chromatography (LC)-MS/MS runs every 2 min to resolve secretion dynamics. Islet perfusate was mixed with an isotopically labeled internal standard and loaded into a 2 μL sample loop which was injected for 0.1 min every 1.9 min onto a 2.1 mm × 30 mm (I.D. × length) C18 column held at 70 °C. Online detection of insulin, C-peptide, glucagon, and somatostatin levels was performed using a triple quadrupole mass spectrometer. Optimization of separation conditions using linear solvent strength theory enabled rapid separation of the four peptides. Calibration curves were linear from 0.5 to 50 nM with an RSD for all analytes between 3-15% and <
3% RSD for all retention times. Results showed secretion dynamics such as first-phase insulin release and negatively correlated release of glucagon and insulin. This simple LC-MS method that used a single 6-port valve with a single sample loop is expected to be useful for examining secretion of other biologically relevant molecules from islets and could be applied to other biological systems for rapid and automated sampling to investigate cellular communication.