# A Minimally Invasive LC–MS/MS Approach for Assessing Endocannabinoids in Saliva and Capillary Blood Microsamples

## TL;DR

This study introduces a minimally invasive method using saliva and finger-prick blood samples to measure endocannabinoids, enabling easier and scalable research into the endocannabinoid system.

## Contribution

The novel contribution is a validated LC–MS/MS method for quantifying endocannabinoids in minimally invasive biological matrices.

## Key findings

- The method successfully quantifies AEA and 2-AG in saliva and finger-prick blood microsamples with acceptable analytical performance.
- Blood microsample concentrations do not directly match plasma concentrations, suggesting their use for relative rather than absolute measurements.
- Venipuncture does not significantly affect salivary AEA or 2-AG concentrations.

## Abstract

N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) are lipid signalling molecules within the endocannabinoid system, which regulates numerous physiological processes and is implicated in diverse pathological conditions. Given the limited feasibility of obtaining human tissue samples, quantifying AEA and 2-AG in biological matrices is essential for understanding the endocannabinoid system in humans. While many studies have used blood samples for this purpose, the collection of this matrix typically requires invasive venipuncture, which limits the scalability and practicality of endocannabinoid research. This study validated extraction and LC–MS/MS methods for quantifying AEA and 2-AG (co-quantified with its isomer 1-AG) in minimally invasive matrices, including saliva and finger-prick blood microsamples, with acceptable linearity, recovery, reproducibility, and matrix effects. The assay additionally enabled exploratory quantification of arachidonic acid, oleoylethanolamide (OEA), palmitoylethanolamide (PEA), and selected steroid hormones, supporting multiplexed assessment from a single sample. Analyte concentrations measured in blood microsamples did not directly correspond to plasma concentrations, indicating that microsampling is suited for assessing relative within-study changes rather than absolute plasma equivalence. Application of the method demonstrated that venipuncture did not significantly alter salivary AEA or 2-AG concentrations. Overall, this method provides a minimally invasive and accessible approach for investigating endocannabinoid dynamics alongside other physiological biomarkers.