Quantitative Analysis of Δ9-tetrahydrocannabinol (Δ9-THC) in Cannabis Plants Using the Fast Blue BB (FBBB) and 4-aminophenol (4-AP) Colorimetric Tests

The Fast Blue BB (FBBB) and 4-aminophenol (4-AP) colorimetric tests have been reportedly used for the qualitative determination of Δ9-THC in plants and for the differentiation between marijuana and hemp-type cannabis. We report the miniaturization of the FBBB colorimetric reaction on a silicone treated filter paper substrate and the analytical figures of merit for a quantitative determination of Δ9-THC for the first time. The reaction between Δ9-THC and FBBB forms a red chromophore that fluoresces when irradiated with visible (480 nm) or UV (365 nm) light, providing a 3-fold increase in sensitivity. Portable instruments are introduced for the objective color determination for both tests and for the fluorescence reading of the THC+FBBB complex. We report a fluorescence signal with Δ9-THC, Δ8-THC, and CBN. The limit of detection (LOD) was determined to be 1.6 ng/μL with precision ~ 12% RSD for standard Δ9-THC solutions ranging between 5-20 ng/μL. The linear dynamic range for this test is reported between 1.6 ng/μL and 20 ng/μL for the portable fluorescence detector. The miniaturization of both colorimetric tests and the increased sensitivity of the FBBB test using fluorescence analysis, coupled to portable instruments allows for limited quantitative analysis of cannabis plants in the field. We also report on the stability of using pre-loaded reagents on a prototype device and color/fluorescence persistence over time. Analytical figures of merit including the linear dynamic range (0.5–10 μg/mL), precision and accuracy for the analysis of plant and oral fluid (OF) samples with known [THC] are also reported. Future studies include an OF extraction method coupled to a robust prototype that could be deployed for field use.