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Abstract 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 withm/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.
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This content will become publicly available on August 1, 2025
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 and 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.
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- Award ID(s):
- 2122813
- PAR ID:
- 10510501
- Editor(s):
- Banks, Craig
- Publisher / Repository:
- Elsevier
- Date Published:
- Journal Name:
- Talanta Open
- Volume:
- 9
- Issue:
- C
- ISSN:
- 2666-8319
- Page Range / eLocation ID:
- 100287
- Subject(s) / Keyword(s):
- Δ9-THC Fast Blue BB 4-AP Marijuana Quantitative analysis
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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