Extraction of THC and its Metabolites from Human Nail Samples Using ISOLUTE® SLE+ Prior to UPLC-MS/MS Analysis

Importance of the Topic

Human nails are emerging as a minimally invasive and stable matrix for detecting long-term exposure to pharmacological agents, including cannabinoids. Unlike blood or urine, nails can accumulate analytes over weeks to months, offering an extended detection window. This capability is valuable in forensic and clinical toxicology to assess patterns of cannabis use or abstinence reliably.

Objectives and Overview of the Study

This work describes an optimized protocol for extracting Δ9-tetrahydrocannabinol (THC) and its main metabolites from human nail samples, using bead-based pulverisation followed by supported liquid extraction (SLE) with ISOLUTE SLE+ formats. The workflow is tailored for both individual 400 µL columns and high-throughput 96-well plates. Key performance targets include high analyte recovery, low variability, and sub-picogram per milligram sensitivity, compatible with UPLC-MS/MS analysis.

Methodology and Instrumentation

The sample preparation comprises the following steps:

• Sample pulverisation: 10 mg of nail is weighed into reinforced tubes with stainless steel beads and ground to a fine powder using the Biotage Lysera system under controlled speed and duration.
• Extraction: Pulverised nails are extracted with 0.1% formic acid in methanol, spiked with deuterated internal standards, and centrifuged. An aliquot of the supernatant is evaporated and reconstituted in aqueous-organic solvent.
• Supported Liquid Extraction: The reconstituted extract (up to 250 µL) is loaded onto ISOLUTE SLE+ 400 µL columns or 96-well plate. Elution is performed with two sequential 600 µL aliquots of methyl tert-butyl ether under gravity, followed by positive pressure to complete recovery. Eluates are evaporated and reconstituted for analysis.
• Chromatographic separation and detection: UPLC separation is achieved on a biphenyl column with a gradient of 0.01% formic acid in water and acetonitrile. Detection uses a triple-quadrupole mass spectrometer in positive electrospray mode with multiple reaction monitoring transitions specific to each analyte.

Used Instrumentation

• Biotage Lysera bead homogeniser
• ISOLUTE SLE+ supported liquid extraction columns and 96-well plate
• Biotage PRESSURE+ positive pressure manifolds
• TurboVap LV solvent evaporator and SPE Dry-96 concentrator
• Shimadzu Nexera X2 UHPLC system with biphenyl column
• Shimadzu 8060 Triple Quadrupole mass spectrometer

Main Results and Discussion

The procedure yielded clean extracts with analyte recoveries exceeding 75% for column format and 70% for plate format, with relative standard deviations below 10%. Limits of quantification ranged from 1 pg/mg for THC-COOH, CBN, and CBD to 0.02 pg/mg for THCAA in column format. Calibration curves exhibited excellent linearity (r2>0.999) over three orders of magnitude.

Benefits and Practical Applications

• High sensitivity and robust quantification of cannabis biomarkers in nails
• Avoidance of emulsion issues common to liquid-liquid extraction
• Compatibility with both manual and high-throughput formats
• Reduced sample preparation time and improved reproducibility

Future Trends and Opportunities

• Expansion to other drug classes and metabolites in nail and alternative matrices
• Integration with high-resolution mass spectrometry for untargeted screening
• Automation and miniaturisation for clinical and forensic laboratories
• Potential application in workplace testing, doping control, and longitudinal exposure studies

Conclusion

The described SLE+ workflow coupled with UPLC-MS/MS provides a reliable, sensitive, and streamlined method for detecting THC and its metabolites in human nails. Its adaptability to column and plate formats supports diverse throughput requirements, delivering reproducible data for forensic and clinical toxicology.

Reference

Biotage Application Note AN929 (2020) Extraction of THC and its Metabolites from Human Nails Using ISOLUTE SLE+