Using in vivo negligible-depletion SPME in a 6‑OHDA rat model, the study quantified free and total anandamide (AEA) and 2‑AG, finding significantly elevated striatal free AEA in lesioned animals while 2‑AG was undetectable in vivo.

Provides a minimally perturbing in vivo method to measure biologically active endocannabinoids and highlights AEA as a candidate early PD biomarker, supporting translational biomarker development though it offers limited immediate therapeutic mechanistic insight.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder, and diagnosis typically occurs after substantial neuronal loss, underscoring the need for biomarkers capable of detecting early pathological changes. The endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) have been proposed as potential PD biomarkers; however, only their free fractions are biologically active. In this study, an in vivo solid-phase microextraction (SPME) method was developed to quantify free and total concentrations of AEA and 2-AG in rat brain under conditions of negligible depletion. Under these conditions, the SPME probe functions analogously to a sensor, extracting only minute amounts of analyte without perturbing the equilibrium between free and matrix-bound species. Extractions were performed at equilibrium, and experimentally determined distribution constants in PBS were used to calculate free concentrations. Matrix-binding percentages exceeded 99% for both analytes and were used alongside with free concentration measurements to estimate total concentration levels. In vivo analyses in a 6-OHDA rat model of PD revealed significant elevated striatal AEA levels in lesioned animals relative to controls, supporting its potential as a biomarker of early neurochemical alterations. In contrast, ex vivo SPME extractions in dissected brain did not show increased AEA levels in the PD group, indicating loss of physiologically relevant information post-mortem. Although 2-AG was not detected in vivo, it was quantified ex vivo, suggesting limited active release under the examined conditions. Overall, these findings highlight the capability of in vivo SPME to capture changes in heavily bound hydrophobic neurochemicals, thereby supporting its application in studies of endocannabinoid dysregulation.