Comprehensive Drug Analysis in Hair: Extraction and UPLC-MS/MS Quantification

Importance of the Topic

Hair is an increasingly preferred matrix in forensic toxicology due to its noninvasive collection, ease of storage at room temperature, and the ability to detect drug exposure over extended periods ranging from months to years. This long detection window supports investigations where other biological specimens may be unavailable or inappropriate, such as post-mortem cases, drug-facilitated sexual assault, and long-term compliance or abuse monitoring.

Objectives and Study Overview

This work set out to develop, optimize, and fully validate a comprehensive method for extracting and quantifying an extensive panel of illicit and pharmaceutical drugs in hair. The method was designed to meet confirmation cut-off criteria recommended by the Society of Hair Testing (SoHT) and to provide reliable, high-throughput analysis suitable for routine forensic laboratories.

Used Instrumentation

• Precellys Tissue Homogenizer with CKMix Lysing Kits for sample pulverization
• Waters Oasis MCX 30 mg plates for solid phase extraction
• Waters ACQUITY UPLC I-Class FTN System coupled to a Xevo TQ Absolute Tandem Quadrupole MS detector
• Waters UPLC BEH C18 column (1.7 µm, 2.1 × 100 mm)

Methodology and Instrumentation

Samples were sequentially washed with aqueous buffer and organic solvents, then pulverized to a fine powder. Analytes were extracted via mixed-mode cation exchange SPE, eluted, and concentrated prior to injection. UHPLC separation employed a gradient from 2% to 90% acetonitrile (both phases containing 0.1% formic acid) over 4 minutes. MS parameters were optimized for source temperature, gas flows, and cone and capillary voltages to maximize sensitivity and selectivity.

Main Results and Discussion

The method demonstrated excellent linearity across relevant ranges (R2 ≥ 0.994 for all analytes). Recoveries spanned 6–79%, with 84% of compounds exceeding 40% recovery and all relative standard deviations under 17%. Matrix effects were consistent and well controlled (only six compounds showed >40% ion suppression). Intra-batch and inter-batch precision and accuracy met acceptance criteria (most CVs <10%), and all analytes satisfied SoHT confirmation cut-off levels. External quality control samples correlated closely with nominal values (overall slope ~1.05, R2 ~0.79), confirming method reliability in an independent assessment.

Benefits and Practical Applications

• Long-term detection of drug use for forensic and clinical investigations
• Reliable confirmation of substances in cases of drug-facilitated sexual assault and post-mortem toxicology
• High-throughput workflow allowing same-day batch analysis
• Compliance monitoring in pain management and workplace testing

Future Trends and Applications

Advancements may include integration of high-resolution MS for untargeted screening, automation of sample preparation to further increase throughput, development of additional biomarkers for novel psychoactive substances, and combined analysis of hair with other keratinous matrices such as nails.

Conclusion

The optimized UHPLC-MS/MS workflow provides a rapid, sensitive, and robust platform for comprehensive drug analysis in hair. It fulfills SoHT confirmation criteria and offers forensic laboratories a validated solution for routine, high-confidence testing.

References

• Cooper G.A.A., Kristofics N. & Kronstrand R. 2012. Forensic Science International 281:20–24.