Multi-panel detection of drugs and drug metabolites in hair samples using a comprehensive extraction method

Importance of the Topic

The accurate detection of drugs, novel psychoactive substances (NPS) and endogenous metabolites in biological specimens is essential for forensic, clinical and toxicological investigations. Hair analysis offers a non-invasive sampling approach with extended detection windows (months to years), minimal risk of sample alteration, simplified transport and storage, and the ability to reconstruct long-term exposure through segmental analysis.

Objectives and Study Overview

This study aimed to develop and validate a comprehensive workflow for the simultaneous detection and quantification of a broad panel of analytes in human hair. Three distinct panels were targeted: (1) 77 NPS covering synthetic cannabinoids, opioids, phenethylamines and tryptamines; (2) 23 classical drugs of abuse (DOA) including opioids, stimulants, antidepressants, cannabinoids and benzodiazepines; and (3) ethyl glucuronide (EtG) as a biomarker of alcohol consumption. The method integrates a streamlined 14-step extraction with the sensitivity and selectivity of the SCIEX QTRAP® 6500+ LC-MS/MS system using Scheduled MRM™ acquisition.

Methodology

Hair samples (25–50 mg) were decontaminated by washing with methanol and diethyl ether, dried under nitrogen, and spiked with calibration standards and internal standards. Aqueous extraction was performed at 100 °C for 60 min, followed by centrifugation and organic phase transfer.

Instrumentation

• UHPLC: ExionLC™ AC system with Phenomenex Synergi Hydro-RP column (50 × 3 mm, 2.5 µm).
• MS/MS: SCIEX QTRAP® 6500+ with IonDrive™ Turbo V ESI source, Scheduled MRM acquisition.
• Software: Analyst® 1.7.1 for acquisition; SCIEX OS™ 1.5 for data processing and quantitation.

Main Results and Discussion

NPS panel achieved near-baseline separation in an 18 min gradient, with R² > 0.98 across 10–200 pg/mg. Case samples confirmed JWH-122 and AM-2201 at 111.8 and 11.7 pg/mg.

DOA panel separated 23 analytes in an 8 min run, linear across 0.05–2 ng/mg (R² > 0.98). Real hair samples revealed ketamine, oxycodone, tramadol and zolpidem (sample 2) and THC, cocaine, benzoylecgonine and cocaethylene (sample 3).

EtG panel used a 4 min gradient, linear from 20–300 pg/mg (R² > 0.99). Three case samples from social and heavy drinkers were distinguished accurately, demonstrating sensitivity below 30 pg/mg cutoff.

Benefits and Practical Applications

• Comprehensive multi-analyte coverage in a single workflow.
• High sensitivity (high pg/mg to low ng/mg) with robust linearity.
• Non-invasive sampling and long detection window suitable for forensic, clinical and compliance testing.
• Efficient data processing with Scheduled MRM and automated peak integration.

Future Trends and Potential Applications

Expansion of targeted panels to emerging NPS, incorporation of high-resolution MS for untargeted screening, automation of sample preparation, integration of segmental hair analysis for temporal exposure profiling, and development of data analytics platforms for rapid case interpretation.

Conclusion

The presented workflow combining a robust extraction protocol with the sensitivity of the SCIEX QTRAP 6500+ LC-MS/MS system enables accurate, high-throughput detection and quantification of a diverse range of drugs, metabolites and biomarkers in hair samples. Its demonstrated performance in real case samples underscores its applicability for forensic, clinical and regulatory toxicology.

References

1. P. Kintz, Consensus of the Society of Hair Testing on hair testing for chronic excessive alcohol consumption (2011), Forensic Sci. Int. 218, 2.