Highly sensitive and specific derivatization strategy to profile and quantitate eicosanoids by UPLC-MS/MS

Area of Science:

• Biochemistry
• Analytical Chemistry
• Physiology

Background:

• Eicosanoids, derived from fatty acids like ARA, EPA, and DHA, are vital signaling molecules.
• Quantifying eicosanoids is challenging due to their low abundance, structural similarity, instability, and poor ionization.
• Existing methods struggle with comprehensive and sensitive eicosanoid profiling.

Purpose of the Study:

• To develop a novel ultra-high performance liquid chromatograph coupled with tandem mass spectrometry (UPLC-MS/MS) strategy.
• To enable comprehensive profiling and quantification of over 60 eicosanoids.
• To overcome the limitations of existing methods in eicosanoid analysis.

Main Methods:

• Utilized an efficient derivatization reagent, 2,4-bis(diethylamino)-6-hydrazino-1,3,5-triazine (T3), to modify eicosanoid carboxylic acids.
• Employed general multiple reaction monitoring (MRM) parameters for broad eicosanoid detection.
• Validated the method for bio-sample analysis using isotopic internal standards within 19 minutes.

Main Results:

• Achieved high derivatization yield (>99%) in 30 minutes at 4°C.
• Enhanced sensitivity by 10- to 5000-fold, with limits of quantitation (LOQs) from 0.05 to 50 pg.
• Demonstrated improved stability of derivatized eicosanoids compared to free forms.
• Successfully profiled and quantified 46 and 50 eicosanoids in rat plasma and heart tissue, respectively.
• Identified significant changes in 24/46 plasma and 25/50 heart tissue eicosanoids in an acute myocardial ischemia (AMI) model.

Conclusions:

• The developed UPLC-MS/MS method with T3 derivatization offers a sensitive, stable, and reliable approach for comprehensive eicosanoid profiling.
• This method facilitates the study of eicosanoid alterations in disease states, such as AMI.
• The findings provide insights into eicosanoid metabolism pathways relevant to disease pathogenesis.