LC-MS/MS confirmation of 11-nor-9-carboxy-tetrahydrocannabinol (Δ8, Δ9, Δ1°) and hexahydrocannabinol (HHC) metabolites in authentic urine specimens.

Abstract

Recently, tetrahydrocannabinol (THC) isomers and other semi-synthetic cannabinoids have been introduced into the consumer market as alternatives to botanical cannabis. To assess the prevalence of these potential new analytical targets, a liquid chromatography-tandem mass spectrometry confirmation method was developed for the quantitation of seven cannabinoid metabolites and the qualitative identification of four others in urine. The validated method was applied to authentic urine specimens that screened positive by immunoassay (50 ng/mL cutoff; n=1300). The most commonly observed analytes were 11-nor-9-carboxy-Δ8- and Δ9-THC (Δ8- and Δ9-THCCOOH), with the combination of the two seen as the most prominent analyte combination found. In addition to these metabolites, Δ1°-THCCOOH was observed in 77 specimens. This is the first study to report Δ1°-THCCOOH in authentic urine specimens, with this analyte always appearing in combination with Δ9-THCCOOH. Cross-reactivity studies were performed for (6aR,9R)-Δ1°-THCCOOH using the Beckman Coulter Emit® II Plus Cannabinoid immunoassay and demonstrated cross reactivity equivalent to the Δ9-THCCOOH cutoff, providing added confidence in the reported prevalence and detection patterns. Additionally, 11-nor-9(R)-carboxy-hexahydrocannabinol (9(R)-HHCCOOH) was the most abundant stereoisomer (n=12) in specimens containing HHC metabolites alone (n=14). This is in contrast to 9(S)-HHCCOOH, which was the predominant stereoisomer in specimens containing Δ8- and/or Δ9-THCCOOH. Although HHC and Δ1°-THC metabolites are emerging toxicology findings, based on these specimens collected between April 2022 and May 2024, an analytical panel containing Δ8- and Δ9-THCCOOH appears to be sufficient for revealing cannabinoid exposure within workplace monitoring and deterrence programs.