Endogenous ethanolamide analysis in human plasma using HPLC tandem MS with electrospray ionization

Area of Science:

• Biochemistry
• Analytical Chemistry
• Pharmacology

Background:

• Endocannabinoids, including anandamide (AEA), are vital signaling lipids.
• Quantification of AEA and related ethanolamides (LEA, OEA, PEA) in human plasma is important for research.
• Existing methods may lack sensitivity or selectivity for simultaneous analysis.

Purpose of the Study:

• To develop and validate a sensitive and selective LC-MS/MS method.
• To enable simultaneous quantification of AEA, LEA, OEA, and PEA in human plasma.
• To establish reliable analytical parameters for these fatty acid ethanolamides.

Main Methods:

• Liquid chromatography tandem mass spectrometry (LC-MS/MS) using positive ion electrospray ionization.
• Protein precipitation of 50 microL human plasma.
• Separation on a Phenomenex Gemini C6-Phenyl HPLC column with gradient elution.
• Quantification using selected reaction monitoring (SRM) and AEA-d8 as internal standard.

Main Results:

• The method achieved lower limits of quantification from 0.05 ng/mL (AEA, LEA) to 1.0 ng/mL (PEA).
• Inter-assay precision and accuracy were within 12% for all analytes.
• Extraction recoveries ranged from 40% to 100%.

Conclusions:

• A robust LC-MS/MS method for simultaneous quantification of four fatty acid ethanolamides in human plasma has been successfully developed.
• The method is sensitive, selective, and suitable for clinical and research applications.
• This analytical tool will aid in understanding the physiological roles of these endocannabinoids.