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Accurate quantitation of cannabinoids, particularly Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), is essential for regulatory compliance, forensic investigations, and cannabis product development. Traditional methods, such as liquid chromatography (LC) and gas chromatography (GC) coupled with mass spectrometry, provide reliable results but are time-consuming and resource-intensive. This study introduces a rapid and high-throughput analytical method using zone heat-assisted direct analysis in real time mass spectrometry (DART-MS) combined with in-situ flash derivatization. The method employs trimethylphenylammonium hydroxide (TMPAH) for efficient derivatization, allowing for the differentiation of THC, CBD, and their acidic precursors, Δ9-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA). A custom heated transfer zone was implemented to enhance derivatization efficiency and reduce carryover effects. The method was optimized for reagent concentration and gas stream temperature, achieving high specificity by minimizing interference from isomeric cannabinoids. Validation studies demonstrate good accuracy (relative error within ±15.9 %) and precision (relative standard deviation ≤15 %), with limits of quantitation of 7.5 µg/mL for THC/CBD and 0.5 µg/mL for THCA/CBDA. Comparative analysis of cannabis samples showed a strong correlation with reference LC/MS results, highlighting the reliability of the proposed method. DART-MS offers a significant time advantage, requiring only 10 s per analysis, making it a promising tool for high-throughput screening of cannabis samples in forensic laboratories.
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Chemical identification and optimization of the 4‐aminophenol colorimetric test for the differentiation between hemp‐type and marijuana‐type cannabis plant samples
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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- Award ID(s):
- 2122813
- PAR ID:
- 10510503
- Editor(s):
- Peat, Micahel
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Journal of Forensic Sciences
- Volume:
- 68
- Issue:
- 4
- ISSN:
- 0022-1198
- Page Range / eLocation ID:
- 1162 to 1177
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1111/1556-4029.15309
