Chemical identification and optimization of the 4-aminophenol colorimetric test for the differentiation between hemp-type and marijuana-type cannabis plant samples

The 4- Aminophenol (4-AP) colorimetric test is a fast, easy-to-use, and cost-effective presumptive assay of cannabis plant material producing different chromophores with THC-rich cannabis (blue color) and with CBD-rich cannabis (pink color). The main drawback of the 4-AP test is a brief observation window where the color rapidly changes to black, limiting the utility of the test. We now report for the first time, the identification of the product chromophores between 4-AP and CBD/THC as well as propose an explanation and a solution for the color degradation of the chromophores. The identification of the chromophores is provided by spectroscopic (UV-Vis), chromatography, and mass spectrometry (TLC and LC-QToF-MS). Oxidation of excess 4-AP (Reagent A) in the presence of NaOH (Reagent B) produces the black color observed for the previously reported 4-AP tests and reported in the literature. The adjustment of reactants concentrations and volumes of 4-AP:THC/CBD to a 1:1 ratio significantly reduces the black oxidation by-product and increases the observation window up to 2 h instead of the previously reported 5-10 min. For the first time, mass spectrometry and chromatography confirmed that the reaction of THC and CBD with 4-AP produced chromophores with m/z (M + H) = 420, consistent with proposed indophenol structures. The TLC method developed confirmed the separation between CBD and THC chromophores. The specificity of the test is also reported, showing false positive results for the presence of THC (blue color) for samples of thyme and oregano. LDA and SIMCA models showed that the optimized 4-AP procedure performs better than the previously reported 4-AP color test.