Isotope-Coded Derivatization Enables Accurate LC–MS/MS Quantification of Urinary THC–COOH without Isotope-Labeled Cannabinoid Standards

Quantitative determination of 11-nor-9-carboxy-Δ 9 -tetrahydrocannabinol (THC–COOH) in urine is the internationally accepted approach for verifying cannabis use in forensic toxicology. Accurate liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis typically relies on stable isotope-labeled internal standards; however, isotope-labeled cannabinoid reference materials are costly, tightly regulated, and not uniformly accessible across laboratories. We report an isotope-coded derivatization (ICD) strategy that enables isotope-dilution–equivalent quantification of urinary THC–COOH without reliance on commercially available isotope-labeled cannabinoid standards. THC–COOH was derivatized with isopropyl-piperidine carboxylic acid hydrazide (IPPAH) and its deuterated analogue (IPPAH- d 6 ), generating a chemically matched analyte/internal standard pair through parallel derivatization. The derivatives exhibited stable chromatographic coelution and closely matched ionization behavior under positive electrospray ionization. Quantitative LC–MS/MS analysis demonstrated linearity over 1–500 ng/mL (r 2 > 0.999), with intra- and interday accuracy of 81.8–108.6% and precision below 10%. Internal standard–normalized matrix factors ranged from 97–109% with minimal variability, indicating effective compensation for matrix effects. This compensation enabled reliable quantification without extensive sample cleanup, allowing a simplified sample preparation workflow compared with conventional methods. Derivatized samples remained stable for at least 72 h in the autosampler without detectable isotopic exchange. This ICD strategy provides a quantitatively reliable alternative to conventional isotope dilution while eliminating dependence on isotope-labeled cannabinoid standards, thereby expanding the accessibility of high-confidence LC–MS/MS quantification and illustrating the broader potential of isotope-coded derivatization in forensic mass spectrometry.