Combined Lipidomic and Metabolomic Analyses on Cardiac Organoids.

Cardiac organoids are increasingly used to model human cardiac development and disease, but their small size often limits molecular characterization, especially if different approaches and protocols are necessary to extract and quantify metabolites and lipids. Here, we present a multistep workflow for combined targeted free amino-acid (FAA) based metabolomic and lipidomic profiling from a single pooled cardiac organoid sample. The protocol covers organoid harvesting, detergent-assisted lysis, and a modified Folch extraction that generates an organic phase for lipid analysis and an aqueous phase for FAA metabolite analysis. Lipids are quantified by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS) in Multiple Reaction Monitoring (MRM) mode using class-matched external standards and an internal standard to support calibration and reduce technical variability. Free amino acids and derivatives are analyzed from the same sample after filtration, drying, and AccQ-Tag derivatization, with norvaline as an internal standard. Together, this approach maximizes information yield from limited material and enables integrated analysis of metabolic and lipid pathways within the same biological specimen, facilitating organoid-based studies of cardiac maturation, disease modeling, and pharmacological responses.