Fast LC/MS/MS Analytical Method for the Separation of Isobaric Isomers of Delta 8, 9 and 10 THC and Carboxy THC metabolites

Importance of the Topic

Accurate separation of delta-8, delta-9, and delta-10 tetrahydrocannabinol isomers and their carboxy metabolites is critical for forensic, clinical, and quality control laboratories. Isobaric forms share identical molecular weights and similar fragmentation patterns, making chromatographic resolution indispensable for reliable identification and quantitation of these compounds.

Study Objectives and Overview

This study aimed to develop a fast and robust LC-MS/MS method capable of baseline chromatographic separation between delta-8, delta-9, and delta-10 THC isomers and their corresponding carboxy metabolites, 11-nor-9-carboxy-delta-8-THC and 11-nor-9-carboxy-delta-9-THC. The work focused on optimizing column selection, mobile phases, and detection modes to ensure specificity and sensitivity within a 10-minute run time.

Methodology and Instrumentation Used

Sample Preparation:

• Standards prepared by serial dilution of a stock solution to produce calibration levels from 1 ng/mL to 1000 ng/mL
• Direct injection of 20 μL onto the LC column without further cleanup

Instrumentation Used:

• Agilent 1290 Infinity II UHPLC with binary pump, thermostatted autosampler at 5 °C, and column compartment at 55 °C
• Agilent 6495C triple quadrupole mass spectrometer with JetStream electrospray ionization in positive mode
• Poroshell CS-C18 column, 2.1 x 100 mm, 2.7 μm particle size

LC Conditions:

• Mobile phase A: water with 5 mM ammonium formate and 0.1% formic acid
• Mobile phase B: methanol
• Flow rate: 0.3 mL/min; gradient from 75% B to 98% B over 6.5 min, total run time 8.5 min plus 1.5 min re-equilibration

MS Conditions:

• Gas temperature 300 °C, sheath gas 350 °C, nebulizer 50 psi
• Capillary voltage 4000 V, nozzle voltage 1500 V
• Dynamic MRM mode for individual transitions of each analyte

Main Results and Discussion

Column Selection:
The charged surface CS-C18 column delivered superior resolution, achieving baseline separation of all target isomers, compared to EC-C18, phenyl-hexyl, and SB-AQ phases.

Dynamic MRM vs. Regular MRM:
Dynamic MRM labeling of individual analytes minimized matrix interferences and prevented peak overlap, enhancing selectivity.

Calibration Performance:
Calibration curves in the 1–1000 ng/mL range showed excellent linearity with correlation coefficients (R2) above 0.998 for all analytes, demonstrating the method’s quantitative reliability.

Benefits and Practical Applications

This method offers rapid analysis with high specificity for distinguishing structurally similar THC isomers and metabolites. Its straightforward sample preparation and compatibility with common triple quadrupole platforms make it suitable for forensic drug screening, clinical toxicology, doping control, and quality control in the cannabis industry.

Future Trends and Potential Applications

Advances may include integration with high-resolution mass spectrometry for wider compound coverage, microflow LC for reduced solvent consumption, and automation combined with artificial intelligence-driven data analysis to increase throughput and analytical confidence.

Conclusion

The developed LC-MS/MS method achieves robust baseline separation and accurate quantitation of delta-8, delta-9, and delta-10 THC isomers and their carboxy metabolites within a 10-minute assay. The approach is easily adopted by any laboratory equipped with LC/MS triple quadrupole systems and supports demanding analytical applications.

References

• Szczesniewski A. Fast LC/MS/MS Analytical Method for the Separation of Isobaric Isomers of Delta 8, 9 and 10 THC and Carboxy THC Metabolites. ASMS 2023, Poster WP675. Agilent Technologies.