A method for quantifying hepatic and intestinal ceramides on mice by UPLC-MS/MS

Area of Science:

• Biochemistry
• Metabolomics
• Analytical Chemistry

Background:

• Ceramides, a type of sphingolipid, are implicated in metabolic diseases like obesity, diabetes, and cardiovascular disease.
• Dihydroceramides, direct precursors to ceramides, are increasingly recognized for their role in biological processes and metabolic disorders.
• Quantifying ceramide and dihydroceramide levels in the liver and gut is crucial due to their interaction in pathophysiological conditions.

Purpose of the Study:

• To establish a rapid and accurate method for determining ceramide and dihydroceramide levels in mouse liver and small intestinal tissues.
• To facilitate research into the mechanisms underlying ceramide-related metabolic diseases.

Main Methods:

• Developed a method using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for direct quantification.
• Methanol extraction and separation on a Waters BEH C18 column within a 10-minute gradient elution.
• Positive electrospray ionization with multiple reaction monitoring (MRM) for sensitive detection.

Main Results:

• The method demonstrated excellent linearity (R² > 0.997), with limits of detection and quantification for ceramides and dihydroceramides.
• High extraction recoveries (82.32%-118.70%) and good precision (RSD < 15%) were achieved.
• Significant differences in ceramide and dihydroceramide levels were observed between liver and intestinal tissues, identifying major components in each.

Conclusions:

• A simple, rapid, and robust method for quantifying 11 ceramides and 6 dihydroceramides in animal tissues was successfully developed and applied.
• The distinct ceramide compositions in liver and intestinal tissues highlight the importance of considering tissue-specific features when investigating biomarkers and molecular mechanisms of metabolic diseases.