Separation of 4 Tetrahydrocannabinol Isomers: Δ6a/10a, Δ8, Δ9, and Δ10 on a Single Quad LCMS

Importance of the Topic

The rapid expansion of the cannabis and hemp industries has heightened the need for precise quantification of tetrahydrocannabinol (THC) isomers. Regulatory definitions, such as the 2018 Farm Bill, classify hemp by a total THC threshold of 0.3 %, driving demand for analytical methods capable of differentiating Δ6a/10a-THC, Δ8-THC, Δ9-THC and Δ10-THC. Accurate isomer separation is critical to verify legal compliance, ensure product safety and support quality control in research and commercial laboratories.

Objectives and Study Overview

This study aimed to develop and validate a rapid, single-run LC-MS method for baseline separation and quantitation of four THC isomers together with cannabidiol (CBD) and tetrahydrocannabinolic acid (THCA). Key goals included:

• Chromatographic resolution of Δ6a/10a-THC, Δ8-THC, Δ9-THC and Δ10-THC in under eight minutes.
• Use of single quadrupole mass detection with selected ion monitoring (SIM) and in-source collision induced dissociation (CID) for structural confirmation.
• Application of the method to real hemp flower samples to verify compliance below 0.3 % THC.

Methodology and Instrumentation

A Shimadzu LCMS-2020 single quadrupole mass spectrometer was coupled to a Nexera 40 UHPLC system equipped with a photodiode array (PDA) detector. Separation employed a Restek Raptor C18 column (150 × 2.1 mm, 2.7 µm) at 40 °C and a gradient of aqueous ammonium formate (5 mM, 0.1 % formic acid) and 50:50 methanol:acetonitrile with 0.1 % formic acid. The flow rate was 0.7 mL/min and injection volume 5 µL.

Mass spectrometry parameters included:

• Drying gas flow 15 L/min, interface temperature 350 °C, DL temperature 250 °C, heat block 400 °C.
• SIM channels at m/z 315.15 (positive) and 357.15 (negative) for target analytes.
• In-source CID at 55 V to generate characteristic fragment ions for each isomer.

Sample preparation involved grinding 100 mg of dry hemp flower, extracting with 10 mL methanol, vortexing and centrifugation. Supernatants were spiked with Δ9-D3-THC internal standard and analyzed against neat calibration curves (0.01–10 ng/µL).

Main Results and Discussion

All four THC isomers, CBD and THCA were baseline separated within eight minutes. Retention times were confirmed with neat standards and PDA detection at 220 nm. In-source CID spectra provided unique fragments: m/z 299 for Δ6a/10a-THC, m/z 247 for Δ8/Δ9-THC, and m/z 217 for Δ10-THC; further differentiation between Δ8 and Δ9 was achieved by monitoring m/z 239.

Calibration curves exhibited excellent linearity (R2 ≥ 0.996) and accuracy between 83.7 % and 117.3 % with RSD ≤ 7.85 %. Application to two hemp samples demonstrated total THC content below the 0.3 % legal limit using both conventional (Δ9-THC + 0.877×THCA) and isomer-inclusive potency calculations.

Benefits and Practical Applications

This method provides a fast, robust approach for simultaneous quantitation and confirmation of multiple THC isomers alongside key cannabinoids. Single-quad instrumentation with SIM and in-source CID offers cost-effective compliance testing for regulatory, quality control and research settings. The short run time and simple sample preparation support high-throughput laboratories.

Future Trends and Application Possibilities

Expanding this approach to additional minor cannabinoids and terpenes could offer broader phytochemical profiling. Integration with high-resolution mass spectrometry may further improve specificity. Automation of sample preparation and data processing will support even higher throughput demands. Emerging regulatory requirements for minor cannabinoid isomers will drive continued method refinement.

Conclusion

A Shimadzu LCMS-2020 single quadrupole system paired with UHPLC-PDA successfully achieved baseline separation and quantitation of four THC isomers in under eight minutes. The method demonstrated strong linearity and accuracy, and effectively measured total THC in hemp samples below the legal threshold. Its speed, simplicity and cost-effectiveness make it a valuable tool for the cannabis and hemp analytical community.

References

None reported in the source document.