An Accurate Method Based on Ultra‐High‐Performance Liquid Chromatography‐Electrospray Ionization‐Tandem Mass Spectrometry for the Quantification of Five Cannabinoids in Juvenile and Adult Caenorhabditis elegans

Caenorhabditis elegans is an alternative model organism for toxicology research that aligns with the 3Rs principle (Replace, Reduce, Refine) and new approaches to modernize toxicity testing strategies. This study developed and single-laboratory validated a sensitive ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) method for quantifying five cannabinoids- cannabidivarin, cannabigerol, cannabidiol, cannabinol, and cannabichromene- in juvenile and adult C. elegans matrices. Homogenized C. elegans samples were spiked with labeled internal standards and subjected to protein precipitation. The resulting supernatants were injected onto a Waters ACQUITY UPLC BEH C18 column (130 Å, 1.7 µm, 2.1 × 100 mm), coupled to an Agilent 6460 Triple Quadrupole mass spectrometer detector, allowing for the detection and quantification of target analytes in a total run time of 14 min using a 50 µL sample volume. Single-laboratory method validation was performed using spiked quality control samples, consisting of six replicates at four concentrations of all cannabinoids in juvenile and adult C. elegans matrices, analyzed over three consecutive days. The validated method demonstrated linear regression calibration curves with R² ≥ 0.99 across the concentration range of 0.1-7.5 µg/mL for all cannabinoids. The intra-day accuracy in juvenile and adult C. elegans was within 85-108% and 97-109% of the fortified concentration, respectively, with intra-day precision between 1.28-4.62% relative standard deviation (RSD) and 0.64-5.60% RSD. Similarly, the inter-day accuracy was within 86-110% and 97-106% of the fortified concentration, respectively, with the inter-day precision between 2.06-5.62% RSD and 1.42-10.9% RSD. This single laboratory validated UHPLC-ESI-MS/MS method provides accurate and reliable cannabinoid quantification and facilitates the translational utility of cannabinoid toxicity testing in C. elegans. Analytical method development to accurately detect chemicals within diverse tissue matrices supports dosimetry and toxicokinetic analyses, essential steps toward investigating food chemical safety.