Matrix-Tolerant Quantification of THC and THCA in Complex Cannabis Products Using In-Sample Calibration with Multiple Isotopologue Reaction Monitoring

Accurate quantification of Δ9-tetrahydrocannabinol (THC) and Δ9-tetrahydrocannabinolic acid (THCA) across diverse cannabis- and hemp-derived products remains challenging due to severe matrix effects, wide concentration variability, and the need for matrix-matched calibration in traditional LC–MS workflows. Here, we develop an in-sample calibration curve (ISCC) method based on multiple isotopologue reaction monitoring (MIRM) from stable-isotope-labeled (SIL) analytes to enable robust quantification of THC and THCA without external calibration curves. The approach leverages the theoretical relative isotopic abundances of SIL calibrators to generate multiple internal calibration points within each injection. By incorporating two SIL calibrators for THC (THC-D3 and THC-D9), applying a response-correction factor to harmonize labeled and unlabeled analytes, and utilizing native-analyte isotopologue transitions at high abundance, the method achieves a >600-fold dynamic range. The ISCC method demonstrated excellent linearity (R 2 > 0.999), precision (<10% RSD), and accuracy (±10%) in commercial CBD oils, gummies, creams, waxes, dietary supplements, and plant materials. Comparison with external calibration showed strong agreement across all matrices. Collectively, this work develops the ISCC–MIRM framework for heterogeneous consumer and forensic samples and establishes a practical, matrix-tolerant calibration strategy for routine cannabinoid analysis.