Determination of Cannabinoids (THC) in Biological Samples

Importance of the Topic

The accurate measurement of Delta-9-THC and its primary metabolites in biological fluids is essential for forensic investigations, clinical monitoring, and interpreting patterns of cannabis use. By distinguishing between the parent compound and its hydroxyl and carboxy metabolites, laboratories can infer time since use, differentiate recent versus past exposure, and support legal and medical decision-making.

Objectives and Study Overview

This study aimed to develop and validate a robust GC/EI-MS/MS method using a quadrupole ion trap to simultaneously identify and quantify Delta-9-THC, 11-hydroxy-THC (11-OH-THC), and 11-nor-Delta-9-THC-COOH (THC-COOH) in various biological matrices. Linearity, sensitivity, selectivity, and reproducibility were assessed over relevant concentration ranges.

Methodology and Instrumentation

Sample preparation involved the following steps:

• Enzymatic hydrolysis of urine with beta-glucuronidase.
• Protein precipitation of blood or serum with acetonitrile.
• Liquid–liquid extraction using hexane:ethyl acetate (7:1).
• Concentration and derivatization of extracts with BSTFA + 1% TMS.

Analytes were separated by GC on a DB-5ms column and detected by EI-MS/MS in multiple reaction monitoring (MRM) mode. Deuterated internal standards ensured accurate quantitation and correction for matrix effects.

Instrumentation

• Gas chromatograph: Agilent GC with DB-5ms Ultra Inert column (25 m × 0.20 mm, 0.33 µm).
• Mass spectrometer: Agilent 220 Quadrupole Ion Trap, EI source, tuned automatically.
• Carrier gas: Helium at 1.3 mL/min; splitless injection at 250 °C.
• Oven program: 160 °C (1 min), ramp 25 °C/min to 260 °C (2 min), then 5 °C/min to 300 °C (1 min).

Results and Discussion

The method exhibited excellent linearity over 2.5–25.0 ng/mL for THC and 11-OH-THC, and 5.0–150 ng/mL for THC-COOH (R2 > 0.99). Limits of detection were 1.0 ng/mL for THC and 11-OH-THC, and 5.0 ng/mL for THC-COOH. Limits of quantitation were 2.5 ng/mL and 10.0 ng/mL, respectively. Ion ratios, retention times, and chromatographic resolution met forensic acceptance criteria. No carry-over or significant interferences were observed.

Benefits and Practical Applications

This GC/MS/MS approach offers high selectivity and sensitivity while minimizing matrix interferences. Its ability to resolve parent drug and metabolites supports accurate interpretation of cannabis use timing and assists forensic toxicology laboratories in routine casework.

Future Trends and Potential Applications

Emerging advances may include coupling high-resolution mass spectrometry for even greater specificity, miniaturized sample preparation formats for high-throughput workflows, and automation of derivatization steps. Expansion to novel cannabinoids and implementation in portable forensic instrumentation are anticipated developments.

Conclusion

A sensitive, selective, and reproducible GC/EI-MS/MS method for simultaneous quantification of Delta-9-THC and its key metabolites in biological specimens was established and validated. With robust performance and low detection limits, this protocol is well-suited for forensic and clinical toxicology applications.

References

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