Development of a bioanalytical method for the quantitative analysis of cannabinoids and their metabolites in plasma

MSc (Pharmaceutical), North-West University, Potchefstroom CampusThere is a continuous need to develop sensitive analytical methods for detection of cannabinoids and their metabolites in human plasma for forensic purposes as well as for pharmacokinetics studies. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been the technique of choice due to its sensitivity and rapid sample preparation. High resolution mass spectrometry offers more selectivity due to accurate mass measurement of the targeted compounds and, therefore, better signal-to-noise ratio. The aim of the study was to develop and validate a sensitive liquid chromatography high resolution mass spectrometry (LC-HRMS) method for the quantitative analysis of cannabidiol (CBD), cannabinol (CBN), Δ9-tetrahydrocannabinol (Δ9-THC) and its major metabolites 11-OH-Δ9-THC and 11-Nor-Δ9-THC-9-COOH in human plasma. The method utilized a simple liquid-liquid extraction of the cannabinoids from plasma samples followed by an isocratic chromatographic separation on Zorbax Eclipse reverse phase C18 column (1.8 μm, 50 x 2.1 mm). The aqueous mobile phase (Phase A) consisted of 0.2 % acetic acid in pure HPLC water while the organic mobile phase (Phase B) was acetonitrile. An isocratic program with a composition of 35 % phase A and 65 % phase B at a flow rate of 0.35 mL/min for 10 minutes was used. Detection was performed by electron spray ionization (ESI) HRMS Q-Exactive plus platform in parallel reaction monitoring mode (PRM). One quantitative product ion and one qualitative product ion were monitored for each cannabinoid. Validation was carried out according to FDA guidelines on validation of bioanalytical methods. The method was found to be selective for the target analytes as no interferences were found at the retention times of the cannabinoids in six different blank plasma samples. The specificity was tested by spiking plasma samples with possible concomitant medications, no interferences were found. The method was linear from 0.2 ng/mL to 100.0 ng/mL, having a lower limit of quantitation (LLOQ) of 0.2 ng/mL for the targeted cannabinoids. The average coefficient of determination (r2) was higher than 0.995 for all the analytes. The accuracy was within 15 % at three different concentration levels and within 20 % at LLOQ. The method’s intra-day and inter-day precision were < 11 %. Extraction recovery ranged from 60.4 % to 85.4 % for the target analytes. Matrix effect (ME) was reduced due to high resolution mass separation from backgrounds noise; however, there was still significant ion suppression for some of the analytes originating from the competition of co-eluting compounds for ionization in electrospray ionization source. The lowest ME was observed for 11- OH-THC and ranged from 1.1 % to 7.4 % while for cannabinol, cannabidiol and THC-COOH, ME ranged from 21.3% to 37.5%. THC showed the highest ME of 49.8 % at the low concentration level and 48.9 % at the high concentration level. There was no carry over of the analytes in the blank samples injected after the higher limit of quantitation.Master