Development of a novel Δ⁹-THC-specific color test reagent

Existing on-site color tests for cannabis (e.g., rapid Duquénois-Levine reagent, 4-aminophenol reagent) have insufficient specificity, especially in the point of distinguishing between Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and the other cannabinoids. We developed a novel Δ⁹-THC-specific color test reagent.

Cannabinoid standards were dissolved in 0.5 mL of a 2-hydroxybenzaldehyde solution in acetonitrile (2 g/100 mL) in a glass test tube (solution A). Herbal samples (approximately 10 mg) were extracted using 0.5 mL of the 2-hydroxybenzaldehyde solution in a plastic homogenization vessel. The extract was poured into a glass test tube through a filter cap (solution B). Solutions A and B were mixed with hydrochloric acid (0.5 mL) and allowed to react for 30 s, after which the mixture was extracted with chloroform (1 mL).

When observing the lower layer after the chloroform extraction, Δ⁹-THC and cannabidiol (CBD) exhibited bluish and orangish colors, respectively. In contrast, cannabinol (CBN) did not show any color. The performance of the reagent was evaluated using dried herbal cannabis (n = 58) and other herbal materials (n = 13), including CBD- and CBN-supplemented herbal materials (each n = 2). Only cannabis samples with Δ⁹-THC levels ≥ 0.14% exhibited a bluish or greenish-blue color.

The newly developed reagent shows good specificity for Δ⁹-THC. The new reagent has the potential to replace existing on-site color tests.