Automated Extraction and LC-MS/MS Analysis of 11-Nor-9-carboxy-tetrahydrocannabinol Isomers and Prevalence in Authentic Urine Specimens

Abstract

11-Nor-9-carboxy-Δ9-tetrahydrocannabinol (Δ9-THCCOOH) is the most frequently detected illicit drug metabolite in the military drug testing program. An increasing number of specimens containing unresolved Δ8-THCCOOH prompted the addition of this analyte to the Department of Defense (DoD) drug testing panel. A method was developed and validated for the quantitative confirmation of the carboxylated metabolites of Δ8- and Δ9-THC in urine samples utilizing automated pipette tip dispersive solid phase extraction and analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). Analytes were separated isocratically over an 8.5 min runtime and detected on an MS/MS system equipped with an electrospray ionization source operating in negative mode. A single point calibrator (15 ng/mL) forced through zero demonstrated linearity from 3 to 1,000 ng/mL. Intra- and inter-day precision were ≤9.1% CV, and bias was within ±14.1% for Δ8-THCCOOH and Δ9-THCCOOH. No interferences were found after challenging the method with different over-the-counter drugs, prescription pharmaceuticals, drugs of abuse, and several cannabinoids and cannabinoid metabolites, including Δ1°-THCCOOH. Urine specimens presumptively positive by immunoassay (n=2939; 50 ng/mL Δ9-THCCOOH cutoff) were analyzed with this confirmation method. Specimens that contained Δ8-THCCOOH often had Δ9-THCCOOH above the 15 ng/mL cutoff. However, nearly one-third of the specimens analyzed were positive for Δ8-THCCOOH only. This manuscript describes the first validated automated extraction and confirmation method for Δ8- and Δ9-THCCOOH in urine that provides adequate analyte separation in urine specimens with extreme isomer abundance ratios.