A Sustainable Air-Assisted Liquid–Liquid Microextraction Procedure Coupled to Liquid Chromatography–Tandem Mass Spectrometry for the Determination of 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol and Hexahydrocannabinol Metabolites in Urine

Hexahydrocannabinol (HHC) is a semi-synthetic cannabinoid that has recently emerged in the European market, raising concerns regarding its detection in forensic toxicology. Reliable analytical approaches are required for the determination of HHC metabolites together with conventional cannabis biomarkers in biological samples. This study aimed to develop and validate a sample preparation method based on air-assisted liquid–liquid microextraction (AALLME) coupled to liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the determination of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) and HHC metabolites in urine. Urine samples underwent alkaline hydrolysis followed by AALLME using a cyclohexane/ethyl acetate mixture (9:1) prior to LC–MS/MS analysis. Experimental conditions affecting extraction performance were optimized. Method validation was performed according to international guidelines. The method achieved a limit of detection of 2.5 ng/mL and a lower limit of quantification of 5 ng/mL. Precision and accuracy fulfilled the established acceptance criteria across all concentration levels. The applicability of the method was evaluated using eleven authentic THC-COOH-positive routine casework urine samples. THC-COOH was successfully quantified, whereas no HHC metabolites were detected because no urine samples from confirmed HHC users were available. The proposed workflow provides a reduced-solvent sample preparation approach compared with conventional procedures and demonstrated adequate analytical performance for the simultaneous determination of THC-COOH and HHC metabolites in urine.