The Study of Three HPLC Column Chemistries for Optimal Separation of THC Isomers

Significance of the Topic

The emergence of isomeric forms of THC metabolites presents significant challenges in forensic, clinical and regulatory analyses. Accurate separation of Δ8-THC-COOH and Δ9-THC-COOH is critical to ensure reliable quantitation in biological samples and to prevent misinterpretation of exposure or impairment.

Objectives and Study Overview

This work aimed to evaluate three HPLC column chemistries for resolving Δ8-THC-COOH and Δ9-THC-COOH efficiently. The goal was to develop a rapid, robust and reproducible method suitable for high-throughput and complex matrices such as urine.

Materials and Methods

• Columns evaluated
  • FluoroPhenyl (100 × 2.1 mm, 2.7 µm)
  • C18 (100 × 2.1 mm, 2.7 µm)
  • Biphenyl (100 × 2.1 mm, 2.7 µm)
• Mobile phases
  • Solvent A: 0.1% formic acid in water
  • Solvent B: 0.1% formic acid in methanol

Used Instrumentation

A Shimadzu LCMS-8045 system with electrospray ionization (positive mode) was used. The column compartment was set to 30 °C, and all methods were constrained to a maximum pressure of 8700 psi.

Main Results and Discussion

• FluoroPhenyl column achieved baseline separation in a 7-minute run, reducible to 5.25 minutes on a 50 × 2.1 mm, 2.7 µm format.
• C18 phase provided partial resolution but required ~16 minutes per analysis.
• Biphenyl column failed to resolve the isomers under tested gradients.
• Method robustness confirmed by analyzing extreme isomer ratios (1000 ng/mL to 10 ng/mL) with no peak interference.

Benefits and Practical Applications of the Method

• High throughput with rapid cycle times.
• Accurate quantitation of isomeric metabolites in urine and other biological matrices.
• Flexible method adaptation to different column formats and laboratory needs.
• Reproducibility supports compliance testing and routine quality control.

Future Trends and Potential Applications

• Integration with automated sample preparation to further increase workflow efficiency.
• Application to other isomeric analytes in complex matrices.
• Development of miniaturized HPLC or column-switching systems for field analysis.
• Coupling with high-resolution mass spectrometry for enhanced selectivity.

Conclusion

The Raptor FluoroPhenyl phase outperformed C18 and Biphenyl chemistries in separating Δ8-THC-COOH and Δ9-THC-COOH. The resulting method is fast, efficient and reproducible, resolving key analytical challenges and adaptable to varied laboratory workflows.

References

• Lupo S. 2023 The Big Pain Development of Pain Free Methods for Analyzing 231 Multiclass Drugs and Metabolites by LC MS MS Restek technical literature library
• Schwartz N. 2017 Fluorophenyl LC Phases What You Should Know Restek chromablography
• York J. 2021 THC COOH Detected Δ-8 or Δ-9 Restek chromablography