Simultaneous analysis of THC and its metabolites in blood using liquid chromatography-tandem mass spectrometry

Cannabis is considered to be the most widely abused illicit drug in Europe. Consequently, sensitive and specific analytical methods are needed for forensic purposes and for cannabinoid pharmacokinetic and pharmacodynamic studies. A simple, rapid and highly sensitive and specific method for the extraction and quantification of Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy- Delta(9)-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy- Delta(9)-tetrahydrocannabinol (THC-COOH) in blood is presented. The method was fully validated according to international guidelines and comprises simultaneous liquid-liquid extraction (LLE) of the three analytes with hexane:ethyl acetate (90:10, v/v) into a single eluant followed by separation and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Chromatographic separation was achieved using a XBridge C(18) column eluted isocratically with methanol:0.1% formic acid (80:20, v/v). Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions. The use of the LLE was demonstrated to be highly effective and led to significant decreases in the interferences present in the matrix. Validation of the method was performed using 250 microL of blood. The method was linear over the range investigated (0.5-40 microg/L for THC, 1-40 microg/L for 11-OH-THC, and 2-160 microg/L for THC-COOH) with excellent intra-assay and inter-assay precision; relative standard deviations (RSDs) were <12% for THC and 11-OH-THC and <8% for THC-COOH for certified quality control samples. The lower limit of quantification was fixed at the lowest calibrator in the linearity experiments. No instability was observed after repeated freezing and thawing or in processed samples. The method was subsequently applied to 63 authentic blood samples obtained from toxicology cases. The validation and actual sample analysis results show that this method is rugged, precise, accurate, and well suited for routine analysis.