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Pre-analytical challenges for the quantification of endocannabinoids in human serum
D Kratz1, A Sens1, S M G Schäfer2
1Institute of Clinical Pharmacology, pharmazentrum frankfurt/ZAFES, University Hospital of Goethe-University, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany.
Summary
Endocannabinoids (ECs) and related substances are higher in serum than plasma. Prompt processing, ideally on ice, is crucial to minimize ex vivo changes and ensure accurate biomarker analysis.
Area of Science:
- Biochemistry
- Lipidomics
- Clinical Chemistry
Background:
- Endocannabinoids (ECs) are vital lipid mediators implicated in various diseases and metabolic health.
- Accurate EC measurement as biomarkers requires standardized pre-analytical sample handling, which is currently lacking.
- While plasma pre-analytical factors are known, serum handling challenges for ECs remain under-investigated.
Purpose of the Study:
- To systematically investigate the impact of different pre-analytical sample handling protocols on serum concentrations of ECs and EC-like substances.
- To compare EC and EC-like substance concentrations and the 2-AG/1-AG ratio between serum and plasma matrices.
- To provide recommendations for optimal sample handling to ensure data quality in EC biomarker studies.
Main Methods:
- Analysis of anandamide (AEA), 2-arachidonoylglycerol (2-AG), 1-arachidonoylglycerol (1-AG), dehydroepiandrosterone (DHEA), and phenylethanolamine (PEA) using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
- Evaluation of serum samples from nine healthy volunteers subjected to varied pre-analytical conditions: prolonged coagulation, storage at room temperature (RT) or on ice post-centrifugation.
- Simultaneous assessment of analytes in plasma from the same individuals to enable cross-matrix comparison.
Main Results:
- Serum concentrations of ECs and EC-like substances were significantly higher compared to plasma.
- Prolonged coagulation and RT storage of serum samples led to significant ex vivo alterations in analyte levels.
- RT storage increased 1-AG and 2-AG, decreasing the 2-AG/1-AG ratio due to isomerization, highlighting matrix-dependent changes.
Conclusions:
- Serum preparation can induce significant changes in EC and related substance concentrations, impacting their utility as biomarkers.
- Storage on ice post-centrifugation is less critical than RT storage for minimizing ex vivo changes in serum.
- Plasma is the preferred matrix for EC and EC-like substance analysis due to its stability and suitability for rapid, cooled processing, ensuring higher data quality.
