Determination of the Cross-Reactivity of the Biological Metabolite (–)-trans-Δ9-Tetrahydrocannabinol-Carboxylic Acid-Glucuronide (THC-COOH-Gluc) for Cannabinoid Immunoassays

Abstract

Abstract The highest concentrated metabolite of (−)-trans-Δ9-tetrahydrocannabinol (THC) in urine, the main psychoactive constituent of Cannabis sativa, is 11-nor-9-carboxy-(−)-trans-Δ9-tetrahydrocannabinol-β-D-glucuronide [(−)-trans-THC-COOH-Gluc]. Even though reference standards for THC, 11-hydroxy-THC (11-OH-THC) and THC-COOH are commercially available as the biological (−)-trans-stereoisomers, the reference standard of THC-COOH-Gluc is only available as the racemic 11-nor-9-carboxy-(±)-cis-Δ9-tetrahydrocannabinol-β-D-glucuronide. This poses the problem for immunoassays, because different stereoisomers may have different cross-reactivity (CR). The aim of the current study was to extract the biological stereoisomer (−)-trans-THC-COOH-Gluc from a urine sample of two marihuana consumers by solid-phase extraction with a Chromabond® C18 cartridge. The cannabinoids in the obtained extract were quantified by Liquid-chromatography coupled to tandem mass spectrometry (LC–MS-MS) and used after dilution for further testing of the CR of (−)-trans-THC-COOH-Gluc with a homogenous enzyme immunoassay assay (hEIA) (Urine HEIA® Cannabinoids (THC), Immunalysis™, Pomona, CA, USA). The CR was determined as the measured HEIA® signal (ng/mL) per THC-COOH-Gluc concentration (ng/mL) in percentage. Results showed that the CR (determined in concentration ratios) is concentration dependent and is 72–87% in the calibration range (20–50 ng/mL). At the cut-off of the hEIA (40 ng/mL), the CR was determined to be 75%. With a molecular weight quotient of 1.51 (MWTHC-COOH-Gluc/MWTHC-COOH = 520.568 g/mol/344.451 g/mol), this means that CR (in molar ratios) is 106–131%. This finding is important, since the major metabolite of THC in urine is (−)-trans-THC-COOH-Gluc and not (−)-trans-THC-COOH, which is used for calibration and no hydrolysis is performed during the determination by hEIA.