Separation and sensitive assay of THC in biological fluids by HPLC and GLC

HPLC systems were developed to permit quantitative separation of delta9-tetrahydrocannabinol from many of the heptane extractable lipoidal and other endogenous substances in biological fluids. These substances interfered with the quantification by flame ionization GLC of unmodified compound and by electron capture GLC of pentafluorobenzoylated compound. Reverse phase HPLC elution, with 47% acetonitrile in water, and normal phase HPLC with 25% chloroform in heptane, separated tetrahydrocannabinol from 11-hydroxy-delta9-tetrahydrocannabinol and other monohydroxylated tetrahydrocannabinols. These systems also purified stock solutions of tetrahydrocannabinol from accompanying contaminants. The various monohydroxylated tetrahydrocannabinols were resolved from each other in normal phase, 80% chloroform in heptane. The delta8 and delta9-tetrahydrocannabinols were separable in normal phase with 5% tetrahydrofuran in hexane. The GLC analysis of pentafluorobenzoylated tetrahydrocannabinol had a sensitivity of 1 ng/ml of plasma with an estimated 5% standard error of an assay with the extraction and GLC procedures given herein. Radiochemical analysis of the HPLC separated fraction had s sensitivity of 0.2 ng/ml of plasma with an estimated 2% standard error of an assay. There was no significant difference between the liquid scintillation and electron capture GLC assays of the HPLC separated delta9-tetrahydrocannabinol obtained from the plasma of dogs administered the drug. Radiolabelled compounds can be added to plasma samples as internal standards to determine the recovery efficiencies of the several procedures in the analysis of unlabelled tetrahydrocannabinol.