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

Significance of the Topic

The ability to extract THC and its metabolites from human hair is essential in forensic toxicology, workplace testing, and clinical monitoring. Hair analysis provides a long detection window compared with blood or urine and can reveal cumulative exposure. Reliable and high-throughput extraction methods enhance laboratory efficiency and data quality.

Objectives and Study Overview

This application note presents a workflow to pre-treat and extract tetrahydrocannabinol (THC) and key metabolites from human hair. The aim is to achieve clean extracts with high analyte recovery, low limits of quantitation, and compatibility with both manual column and 96-well plate formats. Performance was assessed using both manual and automated protocols.

Methodology and Instrumentation

Sample Preparation

• Approximately 20 mg of hair was weighed, spiked with deuterated internal standards, and combined with methanol.
• Micropulverization was performed in Biotage Lysera tubes with stainless steel beads (3 cycles at 5.3 m/sec for 3 minutes with 20 sec dwell), followed by centrifugation.
• Supernatant (200 µL) was transferred to supported liquid extraction devices (ISOLUTE SLE+ columns or plates).
• Analytes were eluted with two 600 µL aliquots of MTBE under gravity or positive pressure, evaporated to dryness at 40 °C, and reconstituted in methanol/water (70:30 or 80:20 v/v).

Instrumentation

• Biotage Lysera for sample homogenization
• ISOLUTE SLE+ 400 µL columns and 96-well plates
• Biotage PRESSURE+ 48/96 positive pressure manifolds and Extrahera automation
• TurboVap LV and Biotage SPE Dry 96 evaporators
• Shimadzu Nexera X2 UHPLC system with ACE Excel 2 C18 column and Restek guard column
• Shimadzu 8060 triple quadrupole mass spectrometer with electrospray interface

Main Results and Discussion

The method achieved analyte recoveries greater than 75% for the plate format and over 60% for the column format, with relative standard deviations below 10% for all target compounds. Limits of quantitation ranged from 0.2 pg/mg for THC-COOH to 10 pg/mg for THC and related cannabinoids. Calibration curves across 0.1–200 pg/mg exhibited excellent linearity (r2 ≥ 0.997).

Benefits and Practical Applications

This supported liquid extraction approach eliminates emulsion issues common in liquid–liquid extraction, reduces preparation time, and scales from individual columns to high-throughput 96-well plates. The protocol is well suited for forensic casework, clinical monitoring of cannabis exposure, and workplace drug testing.

Future Trends and Potential Applications

Ongoing developments may include further miniaturization of extraction devices, deeper integration with automated platforms, and expansion to additional drug classes or biomarkers. Coupling with high-resolution mass spectrometry and advanced data processing could further improve sensitivity, selectivity, and interpretative capacity.

Conclusion

The described SLE-based extraction workflow offers a robust, reproducible, and efficient solution for quantifying THC and its metabolites in human hair. It satisfies stringent requirements for recovery, precision, and sensitivity, making it a valuable tool for diverse analytical laboratories.