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

Area of Science:

• Biochemistry
• Analytical Chemistry
• Clinical Diagnostics

Background:

• Urinary sphingolipids show potential as biomarkers for various renal diseases.
• Accurate and efficient quantification methods are needed for clinical application.
• Existing methods may lack the throughput and automation required for large-scale studies.

Purpose of the Study:

• To develop and present a high-throughput, automated protocol for urinary sphingolipid analysis.
• To enable precise quantification of sphingolipids in urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
• To establish a foundation for improved biomarker discovery in kidney diseases.

Main Methods:

• Development of an automated solid-phase extraction protocol for urine samples.
• Implementation of targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) for sphingolipid quantification.
• Detailed description of sample preparation, lipid extraction, and LC-MS/MS analysis procedures.

Main Results:

• A robust and automated protocol for the extraction and quantification of urinary sphingolipids was established.
• The method demonstrates high throughput suitable for clinical and research applications.
• The platform allows for simultaneous metabolomic and lipidomic sample preparation.

Conclusions:

• The presented automated LC-MS/MS protocol provides an efficient means to analyze urinary sphingolipids.
• This method can significantly advance the identification and validation of renal disease biomarkers.
• The protocol is adaptable for other biospecimens and analytical workflows.