DoE-assisted development and validation of a thin layer chromatography method for optimized separation of major cannabinoids in Cannabis sativa L. samples

Abstract

As interest in the medical use of cannabis and cannabinoids rises, the need to establish appropriate quality specifications for them also becomes evident. Consequently, various methods applied to the analyses of cannabinoids in C. sativa have been reported. Among the chromatographic techniques used for this purpose, TLC stands out as a suitable alternative for fingerprint assessment. Nevertheless, structural similarity between cannabinoids imposes some analytical challenges. In this context, DoE-assisted optimization may favor achieving an adequate resolution between those analytes. Accordingly, this study aimed at developing and validating a TLC method for cannabinoid profile monitoring in C. sativa inflorescences by employing a DoE-driven strategy. A mixture design was applied to estimate the ideal mobile phase composition. Data analyses focused on maximizing resolutions between eight major cannabinoids and improving band distribution throughout the chromatogram. The overall quality of the chromatographic profiles after method optimization was satisfactory. Method validation covered selectivity, precision, and ruggedness assessment. The Youden design was applied for simultaneous analysis of five experimental factors. The proposed method was shown to be fit-for-purpose, allowing chemotype differentiation, in addition to being stability-indicating. Moreover, the results can support the definition of control strategies to ensure consistent achievement of the intended method's performance.

Graphical Abstract

Keywords:

• Cannabinoids
• Cannabis sativa
• design of experiments
• quality control
• thin-layer chromatography

Acknowledgments

The authors would like to thank Prof. Dr. Lilian Auler Mentz for her crucial contribution to the botanical identification of the herbal material and MSc. Mariana Koetz for her assistance regarding the operation of the equipment Automatic TLC Sampler 4 and TLC Scanner 4 during the early study phase.

Disclosure statement

Although one of the authors is a federal employee at the Brazilian National Health Surveillance Agency (Agência Nacional de Vigilância Sanitária, ANVISA), the discussions included in this paper do not necessarily reflect the official position of the Agency nor imply regulatory obligations. That said, the authors report there are no competing interests to declare.

Data availability statement

The data that support the findings of this study are available from the corresponding author, MRS, upon reasonable request.