Retention behavior of 39 cannabinoids in reverse-phase liquid chromatography using C₁₈ or phenyl hexyl columns

Reverse-phase liquid chromatography with ultraviolet detection (LC-UV) is the primary analytical technique for quantifying Δ⁹-THC in Cannabis plant materials since the passage of the Agriculture Improvement Act of 2018. This legislation established a Δ⁹-THC threshold of 0.3 % (by "dry weight") to distinguish hemp from marijuana, effectively removing hemp from the United States Drug Enforcement Administration's list of controlled substances. In 2022, the Chemical Sciences Division (CSD) at the National Institute of Standards and Technology (NIST) demonstrated accurate and precise analytical measurements of Δ⁹-THC, Δ⁸-THC, and nine other cannabinoids in Cannabis plant and oil samples using LC-UV absorbance detection. This method used a C₁₈ column to resolve cannabinoids using an ACN/H₂O mobile phase. Recently, the commercial landscape has shifted as manufacturers utilize synthetic processes to convert cannabidiol (CBD) from hemp extracts into finished products containing various isomers or analogs of Δ⁹-THC. This practice has introduced chromatographic interferences from synthetic by-products, which can compromise the accuracy of Δ⁹-THC measurements. In response, NIST CSD has modified the original LC-UV method to develop two refined methods designed to minimize these interferences. The LC-UV screening method currently employed by NIST CSD was evaluated for its ability to resolve 39 cannabinoids and compared with a novel LC-UV method using a Phenyl Hexyl (pH) column. This study evaluates trends in cannabinoid retention for Δ⁹-THC, its isomers, and analogs on both column types. While both stationary phases exhibited predictable retention trends for THC homologs based on alkyl side-chain length, the pH column provided a unique correlation between THC isomer elution and the specific position of the double bond within the cyclohexene ring.