Validation of an LC-MS/MS method for the simultaneous determination of metabolites of cannabis and semi-synthetic cannabinoids in urine
The abuse of cannabis and semi-synthetic cannabinoids has become a public health concern in South Korea. In abusers' urine, tetrahydrocannabinol (THC) and semi-synthetic cannabinoids (Δ8-THC and hexahydrocannabinol (HHC)) are metabolized into carboxylated forms and excreted predominantly as their glucuronide conjugates. To facilitate rapid analysis, a new LC-MS/MS method was developed without the time-consuming hydrolysis. This method enabled the simultaneous quantitation of the free forms (Δ9-carboxy-THC, Δ8-carboxy-THC, 9R-carboxy-HHC, and 9S-carboxy-HHC) and qualitative confirmation of their glucuronide conjugates in human urine. Sample preparation was performed by liquid-liquid extraction (LLE) using ethyl acetate in the presence of 0.1 M acetate buffer (pH 4.0), followed by LC-MS/MS analysis. The method exhibited excellent linearity for all analytes (R² > 0.99) with limits of detection (LOD) of 0.1-1 ng/mL. Minimal carry-over and high reproducibility were observed. The validated method was successfully applied to 18 authentic urine samples from suspected abusers. All 18 samples were positive for Δ9-carboxy-THC (90.4 ± 29.1 ng/mL), 4 for Δ8-carboxy-THC (501.0 ± 334.8 ng/mL) and 2 for carboxy-HHC (47.7 ± 5.5 ng/mL). These results confirm that the proposed method is suitable for authentic urine samples and offers a simpler and faster alternative to conventional methods for routine screening of cannabis and semi-synthetic cannabinoid metabolites in urine.
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