The emerging disciplines of lipidomics and metabolomics show great potential for the discovery of diagnostic biomarkers, but appropriate pre-analytical sample-handling procedures are critical because several analytes are prone to ex vivo distortions during sample collection. To test how the intermediate storage temperature and storage period of plasma samples from K3EDTA whole-blood collection tubes affect analyte concentrations, we assessed samples from non-fasting healthy volunteers (n = 9) for a broad spectrum of metabolites, including lipids and lipid mediators, using a well-established LC-MS-based platform. We used a fold change-based approach as a relative measure of analyte stability to evaluate 489 analytes, employing a combination of targeted LC-MS/MS and LC-HRMS screening. The concentrations of many analytes were found to be reliable, often justifying less strict sample handling; however, certain analytes were unstable, supporting the need for meticulous processing. We make four data-driven recommendations for sample-handling protocols with varying degrees of stringency, based on the maximum number of analytes and the feasibility of routine clinical implementation. These protocols also enable the simple evaluation of biomarker candidates based on their analyte-specific vulnerability to ex vivo distortions. In summary, pre-analytical sample handling has a major effect on the suitability of certain metabolites as biomarkers, including several lipids and lipid mediators. Our sample-handling recommendations will increase the reliability and quality of samples when such metabolites are necessary for routine clinical diagnosis.

BACKGROUND: Arachidonoyl ethanolamide (AEA) and 2-arachidonoyl glycerol (2-AG) are central lipid mediators of the endocannabinoid system. They are highly relevant due to their involvement in a wide variety of inflammatory, metabolic or malign diseases. Further elucidation of their modes of action and use as biomarkers in an easily accessible matrix, like blood, is restricted by their susceptibility to deviations during blood sampling and physiological co-dependences, which results in high variability of reported concentrations in low ng/mL ranges.
OBJECTIVE: The objective of this review is the identification of critical parameters during the pre-analytical phase and proposal of minimum requirements for reliable determination of endocannabinoids (ECs) in blood samples.
METHODS: Reported physiological processes influencing the EC concentrations were put into context with published pre-analytical research and stability data from bioanalytical method validation.
RESULTS: The cause for variability in EC concentrations is versatile. In part, they are caused by inter-individual factors like sex, metabolic status and/or diurnal changes. Nevertheless, enzymatic activity in freshly drawn blood samples is the main reason for changing concentrations of AEA and 2-AG, besides additional non-enzymatic isomerization of the latter.
CONCLUSIONS: Blood samples for EC analyses require immediate processing at low temperatures (>0 °C) to maintain sample integrity. Standardization of the respective blood tube or anti-coagulant, sampling time point, applied centrifugal force and complete processing time can further decrease variability caused by sample handling. Nevertheless, extensive characterization of study participants is needed to reduce distortion of clinical data caused by co-variables and facilitate research on the endocannabinoid system.