Protocol for high-throughput, automated, solid-phase extraction of sphingolipids from human urine

Urinary sphingolipids may serve as biomarkers of renal diseases. Here, we present a high-throughput, automated protocol for solid-phase extraction and tandem mass spectrometry (LC-MS/MS) quantification of lipids from urine. We describe steps for sample preparation and lipid extraction, and then detail procedures for targeted LC-MS/MS analysis of urinary sphingolipids. In this protocol, we focus on the analysis of urine samples; however, this platform can be optimized for alternative biospecimens. Additionally, this method allows for tandem preparation of metabolomic and lipidomic samples. For complete details on the use and execution of this protocol, please refer to Nicholson et al.¹.