The main active ingredients of Cannabis sativa are delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). CBD as the main non-psychoactive constituent has been marketed in a growing number of products for recreational use such as CBD oils and vapes. Pharmacokinetics (PK) studies are needed to determine the absorption, distribution and metabolism of CBD after administration, which is largely affected by the formulation of the product in analogy to nicotine-containing products.

Hence, we developed a quantitative bioanalytical method for the determination of CBD and its three major metabolites – 6-hydroxy-CBD, 7-hydroxy-CBD, and 7-carboxy-CBD – in plasma by an online-LC-MS/MS for this purpose. The method is characterized by an online sample preparation procedure, in order to achieve high sample throughput needed for PK studies. 200 µL of plasma are precipitated with ZnSO4 in methanol and subsequently injected onto the LC-MS/MS system (Shimadzu Nexera X2 uHPLC coupled to Sciex QTrap6500+ MS/MS). Online extraction is achieved by means of a Zorbax C8 column (Agilent) followed by gradient elution on an Amid UHPLC column (Merck).

The method was fully validated according to FDA guidelines for bioanalytical method validation showing excellent results in terms of precision, accuracy and sensitivity.

The LLOQ of 0.39 ng/mL and broad linear range of up to 200 ng/mL for CBD and the three major metabolites in combination with the straightforward sample preparation demonstrate the suitability of this method for its application in CBD PK studies.