Quantitative measurement of Δ9-tetrahydrocannabinol and two major metabolites in physiological specimens using capillary column gas chromatography negative ion chemical ionization mass spectrometry

Abstract

delta 9-Tetrahydrocannabinol and two of its metabolites, 11-hydroxy-delta 9-tetrahydrocannabinol and 11-nor-9-carboxy-delta 9-tetrahydrocannabinol, can be measured in a single 1-ml sample of blood, plasma, or urine by a new assay which combines a relatively rapid extraction procedure with capillary column gas chromatography and negative ion chemical ionization mass spectrometry. Deuterium-labeled analogs of each cannabinoid are added to the physiological specimen as internal standards. Two extracts are obtained from each sample: a neutral fraction containing delta 9-tetrahydrocannabinol and 11-hydroxy-delta 9-tetrahydrocannabinol, and an acid fraction containing 11-nor-9-carboxy-delta 9-tetrahydrocannabinol. The neutral fraction is derivatized by treatment with trifluoroacetic anhydride; the acid fraction is first treated with BF3-methanol followed by reaction with trifluoroacetic anhydride. Under electron-capture chemical ionization conditions the derivatized delta 9-tetrahydrocannabinol and 11-nor-9-carboxy-delta 9-tetrahydrocannabinol give abundant molecular anions ideally suited for selected ion monitoring. The negative ion chemical ionization spectrum of the HO-THC-trifluoroacetate shows no molecular anion. Consequently, quantitation of the hydroxy metabolite is achieved by monitoring a fragment ion formed by loss of CF3CO2 from its molecular anion. The limits of reliable measurement are judged to be 0.1 ng ml-1 for 11-nor-9-carboxy-delta 9-tetrahydrocannabinol, 0.2 ng ml-1 for delta 9-tetrahydrocannabinol and 0.5 ng ml-1 for 11-hydroxy-delta 9-tetrahydrocannabinol. Four examples are given of the application of the assay to the analysis of specimens of medico-legal importance.

Determination of ∆-9-Tetrahydrocannabinol (THC), 11-hydroxy-THC, 11-nor-9-carboxy-THC and Cannabidiol in Human Plasma using Gas Chromatography–Tandem Mass Spectrometry

“…The reduced volume supernatants were acidified by the addition of 1 N hydrochloric acid and then extracted with hexane:ethyl acetate (9:1). This course permitted the more neutral cannabinoids (THC, OH-THC, CBD) and the acidic COOH-THC to be extracted in the same fraction, which is a major improvement over previous methods from our laboratory (25,35). The extracts were evaporated to dryness and then reconstituted with a small volume of hexane:ethyl acetate (9:1).…”

“…Freed from protein binding, the analytes became more "available" for partitioning into the organic layer resulting in a more consistent recovery. This step is common to several procedures whether they are followed by LLE (25,26) or SPE (29,34,36,37). The volume of the protein precipitation supernatants was reduced by evaporation.…”

“…Gas chromatography-mass spectrometry (GC-MS) was initially used to measure THC and metabolites in human plasma (14,(25)(26)(27)(28)(29)(30). More recently 2-dimensional GC-MS has been employed to provide more sensitive analysis of plasma cannabinoids (31,32).…”

“…Because cannabinoid compounds are very lipophilic, extraction procedures are often complicated. Most early GC-MS method extraction procedures required a protein precipitation and/or ß-glucuronidase step was used purportedly to disrupt the binding of cannabinoid compounds to plasma proteins (14,25,26,29). Some utilized lengthy liquid-liquid procedures where either separate extracts were obtained for the neutral cannabinoid compounds (THC and OH-THC) and the acidic COOH-THC (25,26), or multiple extractions after derivatization (28).…”

“…Most early GC-MS method extraction procedures required a protein precipitation and/or ß-glucuronidase step was used purportedly to disrupt the binding of cannabinoid compounds to plasma proteins (14,25,26,29). Some utilized lengthy liquid-liquid procedures where either separate extracts were obtained for the neutral cannabinoid compounds (THC and OH-THC) and the acidic COOH-THC (25,26), or multiple extractions after derivatization (28). More recent GC-MS methods use solid phase extraction (SPE) procedures with (29,31,32), or without (27,28) prior protein binding disruption procedure.…”