Application of experimental design in HPLC method optimisation for the simultaneous determination of multiple bioactive cannabinoids

J Pharm Biomed Anal. 2022 Nov 30;221:115037. doi: 10.1016/j.jpba.2022.115037. Epub 2022 Sep 9.


The scientific interest in Cannabis sativa L. analysis has been rapidly increasing in recent years, especially for what concerns cannabinoids, plant secondary metabolites which are well known for having many biological properties. High-performance liquid chromatography (HPLC) is frequently used for both the qualitative and quantitative analysis of cannabinoids in plant extracts from C. sativa and its derived products. Many studies have been focused on the main cannabinoids, such as ∆9-tetrahydrocannabinolic acid (∆9-THCA), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA) and their decarboxylated derivatives, such as ∆9-tetrahydrocannabinol (∆9-THC), cannabidiol (CBD) and cannabigerol (CBG). In addition to the abovementioned compounds, the plant produces other metabolites of the same chemical class, and some of them have shown interesting biological activities. In the light of this, it is important to have efficient analytical methods for the simultaneous separation of cannabinoids, which is quite complex since they present similar chemical-physical characteristics. The present work is focused on the use of the Design of Experiments technique (DoE) to develop and optimise an HPLC method for the simultaneous separation of 14 cannabinoids. Experimental design optimisation was applied by using a Central Composite Face-Centered design to achieve the best resolution with minimum experimental trials. Five significant variables affecting the chromatographic separation, including ammonium formate concentration, gradient elution, run time and flow rate, were studied. A multivariate strategy, based on Principal Component Analysis (PCA) and Partial Least Squared (PLS) regression, was used to define the best operative conditions. The developed method allowed for the separation of 12 out of 14 cannabinoids. Due to co-elution phenomena, HPLC coupled with a triple quadrupole mass analyser (HPLC-ESI-MS/MS) was applied, monitoring the specific transitions of each compound in the multiple reaction monitoring (MRM) mode. Finally, the optimised method was applied to C. sativa extracts having a different cannabinoid profile to demonstrate its efficiency to real samples. The methodology applied in this study can be useful for the separation of other cannabinoid mixtures, by means of appropriate optimisation of the experimental conditions.

Keywords: Analysis; Cannabinoids; Cannabis sativa L.; Design of experiments; HPLC.

MeSH terms

  • Calcium Gluconate
  • Cannabidiol* / analysis
  • Cannabinoids* / chemistry
  • Cannabis* / chemistry
  • Chromatography, High Pressure Liquid / methods
  • Dronabinol
  • Plant Extracts / chemistry
  • Research Design
  • Tandem Mass Spectrometry / methods


  • Cannabinoids
  • Plant Extracts
  • Cannabidiol
  • Dronabinol
  • Calcium Gluconate