Development of a colorimetric cannabis test using 4-aminophenol that can discriminate Δ<sup>9</sup>-THC, CBD and Δ<sup>9</sup>-THCA

Colorimetric tests are effective as rapid and simple methods for criminal investigations. Given the recent increase in cannabis-related crime, cannabis detection methods have received significant attention. The widely used Duquenois-Levene (D-L) method, however is known to produce false-positive results, highlighting the need for alternative methods. The 4-aminophenol based method has been proposed as a promising substitute due to its ability to discriminate Δ9-THC and CBD. However, it has a critical limitation: it is inapplicable to mixtures of Δ9-THC and CBD. In this study, we introduced a phase separation technique using an organic and aqueous layer, inspired by the D-L method, into the 4-aminophenol based method. This modification enabled the separation and identification of Δ9-THC and CBD in mixed samples. Furthermore, under the same conditions, the separation of Δ9-THC and Δ9-THCA was also achieved. Based on this newly developed method, we designed the rapid test kit that offers both ease of use and enhanced specificity. By establishing a positive detection criterion that requires the presence of both Δ9-THC and Δ9-THCA, the test kit significantly reduces false-positive results compared to conventional methods. These advantages make it a promising candidate for practical application in forensic field investigations.