The Separation of ∆8-THC, ∆9-THC, and Their Enantiomers by UPC2 Using Trefoil Chiral Columns

Significance of the Topic

Chiral resolution of cannabis-derived cannabinoids, particularly Δ⁸-THC and Δ⁹-THC enantiomers, is critical for pharmaceutical quality control, forensic profiling, and consumer safety. The stereochemistry of THC influences its pharmacological and toxicological properties, mandating precise separation and quantification under regulatory guidelines.

Objectives and Study Overview

This application study aimed to develop and validate a rapid, high-resolution method for separating Δ⁸-THC, Δ⁹-THC, and their respective (+)/(−) enantiomers using supercritical fluid chromatography (SFC) on Waters ACQUITY UPC2 with Trefoil chiral columns. Key objectives included:

• Screening three Trefoil chiral stationary phases (AMY1, CEL1, CEL2) for selectivity and resolution.
• Optimizing isocratic conditions for baseline separation in <3 minutes.
• Establishing calibration curves and assessing method repeatability.
• Applying the method to a commercial CBD oil sample before and after heat and acid conversion to THC.

Methodology and Instrumentation

Sample Preparation:

• Standards of (±)Δ⁸-THC and (±)Δ⁹-THC diluted in ethanol or heptane for screening and calibration.
• Cannabidiol oil samples treated under three conditions: untreated, heated at 55 °C, and heated with 0.1 M HCl at 55 °C.

Chromatographic Conditions:

• System: Waters ACQUITY UPC2 with PDA detector set at 228 nm.
• Columns: Trefoil AMY1, CEL1, CEL2 (2.5 µm, 3 mm × 150 mm).
• Mobile Phases: CO₂ (A) and ethanol (B).
• Flow Rate: 2 mL/min; ABPR: 2000 psi; Column Temp: 50 °C; Injection: 1 µL.
• Optimized Isocratic: 15 % ethanol in CO₂ for complete enantiomeric separation in under 3 min.

Main Results and Discussion

Column Screening:

• All three Trefoil phases achieved full resolution of four THC stereoisomers under a 2–20 % ethanol gradient.
• AMY1 exhibited greatest retention and resolution, selected for further optimization.

Isocratic Optimization:

• Calculated elution solvent strength (≈15.7 % ethanol) led to successful isocratic separation.
• 15 % ethanol delivered baseline resolution of Δ⁸-THC and Δ⁹-THC enantiomers in <3 min.

Calibration and Repeatability:

• Linear calibration (0.003125–0.05 mg/mL) for each enantiomer (R² > 0.998).
• Repeatability RSD < 2 % (n = 7) at mid-range concentration.

Application to CBD Oil:

• Untreated oil showed trace (−)Δ⁹-THC, confirming “THC-free” labeling.
• Heat alone did not alter THC content significantly.
• Acidic heating converted CBD to (−)Δ⁹-THC (~7.6 % of sample) with minor (−)Δ⁸-THC formation.

Benefits and Practical Applications of the Method

• Rapid analysis (<3 min) increases throughput and reduces solvent use.
• Orthogonal selectivity of AMY1, CEL1, CEL2 columns offers flexibility in complex matrices.
• High sensitivity and repeatability support both qualitative profiling and quantitative QC.
• Applicable to pharmaceutical enantiomeric purity testing, forensic drug analysis, and stability monitoring of infused products.

Future Trends and Potential Applications

Advances in SFC and chiral stationary phases will broaden enantiomeric separations for emerging cannabinoids and other chiral natural products. Integration with high-resolution mass spectrometry and greener co-solvents will further enhance selectivity, sensitivity, and sustainability. Automated method scouting and AI-driven optimization may accelerate development workflows and regulatory compliance for novel therapeutics.

Conclusion

The developed UPC2 method on a Trefoil AMY1 column provides a robust, rapid, and reproducible approach for baseline separation and quantification of Δ⁸-THC, Δ⁹-THC, and their enantiomers. This technique meets stringent analytical requirements for pharmaceutical, forensic, and consumer product testing while offering high throughput and minimal solvent consumption.

Reference

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