Impact of buccal cell content and swab retention on tetrahydrocannabinol and cocaine concentrations in oral fluid.

While oral fluid (OF) is increasingly used in forensic and roadside toxicology, measured drug concentrations remain highly dependent on pre-analytical conditions. This study investigated the impact of buccal-cell content and sample fractionation on concentrations of delta 9-tetrahydrocannabinol (THC), cocaine, benzoylecgonine (BZE), and ecgonine methyl ester (EME). Oral-fluid specimens were collected using FLOQSwabs®. Following elution, sonication, and vortex mixing, various fractions (homogenate, pellet and expressed swab) were analyzed via LC-MS/MS. For THC (n = 102), median buccal epithelial cell counts and THC concentrations were significantly higher in the cell-rich pellet than in the homogenate (p < 0.001), with median enrichment ratios of 4.40 and 3.93, respectively. The association between cellular and THC enrichment was strong to very strong (r = 0.77 to 0.88) for concentrations above 5ng/mL. Notably, 4 specimens were below the limit of detection in the homogenate but detectable in the pellet. In contrast, cocaine and its metabolites showed comparable concentrations between the homogenate and pellet, though the expressed swab fraction consistently yielded the highest levels. In 52 specimens, median expressed swab-to-homogenate ratios were 1.16 for cocaine, 1.28 for BZE, and 1.14 for EME. These findings demonstrate that pre-analytical effects are analyte-dependent: buccal cells act as a reservoir for THC, whereas cocaine-related analytes are primarily affected by incomplete release from the swab. These results identify analyte-dependent pre-analytical sources of variability that may affect oral-fluid concentration measurements obtained with this collection device.