Streamlined Sample Preparation of a Drugs of Abuse Panel in Human Nails Using ISOLUTE® SLE+ Prior to UPLC-MS/MS Analysis

Significance of the Topic

Forensic and clinical toxicology increasingly adopt alternative matrices such as human nails for long-term drug monitoring due to their non-invasive collection and extended detection windows. A robust, high-throughput sample preparation method is essential to deliver reliable, sensitive multi-analyte quantitation in these matrices.

Objectives and Study Overview

This study presents a streamlined workflow combining mechanical micropulverization and supported liquid extraction (ISOLUTE SLE+) for the simultaneous extraction of 49 drugs of abuse from human nail samples. The protocol was optimized for both manual (pressure-driven) and automated (Extrahera) formats, using 400 µL column and 96-well plate configurations, prior to UPLC-MS/MS analysis.

Methodology and Instrumentation

• Sample Preparation:
   – Weigh 10 mg of nail clippings and grind to powder using Biotage Lysera (8 × 6.95 m/sec, 45 s dwell).
   – Add 1 mL methanolic 0.1% NH₄OH and 10 µL of 100 pg/mL deuterated internal standard mix.
• Supported Liquid Extraction (ISOLUTE SLE+ 400 µL):
   – Load 400 µL supernatant, allow absorption (5 min).
   – Elute analytes with DCM/IPA (95:5, 600 µL) and MTBE (600 µL) under gravity, finalize with positive pressure.
• Evaporation and Reconstitution:
   – Evaporate eluates at 40 °C with 50 mM HCl in MeOH, reconstitute in 200 µL mobile phase A/B (80:20).
• UPLC-MS/MS Analysis:
   – UPLC: Shimadzu Nexera X2, Restek Raptor Biphenyl column, gradient from 80:20 to 0:100 (A: aqueous 2 mM ammonium formate/0.1% formic acid; B: methanolic 2 mM ammonium formate/0.1% formic acid), 0.4 mL/min, 30 °C.
   – MS/MS: Shimadzu 8060 triple quadrupole, ESI+, optimized MRM transitions and source parameters.

Key Results and Discussion

• Recoveries exceeded 80% for most compounds, with RSDs below 10% across manual and automated formats.
• Linearity demonstrated over 1–1000 pg/mg with r²>0.99 for nearly all analytes.
• Limits of quantitation ranged from 1 to 10 pg/mg, depending on analyte.
• Comparable performance achieved using both column and plate SLE+ formats and different processing platforms.

Benefits and Practical Applications

• High-throughput capability suitable for forensic, clinical and QA/QC laboratories.
• Efficient solvent use, elimination of emulsions and reduced prep time versus traditional LLE.
• Compatibility with manual and automated workflows ensures reproducibility.
• Covers a wide panel including amphetamines, opioids, benzodiazepines, cocaine, LSD and others.

Future Trends and Opportunities

• Inclusion of novel psychoactive substances and additional biomarkers in nail testing.
• Further automation and integration with online SPE for end-to-end processing.
• Miniaturization and coupling to high-resolution MS for metabolite profiling.
• Standardization of nail reference materials to enhance inter-laboratory comparability.

Conclusion

The optimized protocol delivers robust, sensitive extraction of a comprehensive drugs-of-abuse panel from human nails, supporting both manual and automated workflows with consistent recoveries, low variability and sub-pg/mg quantitation limits, facilitating reliable UPLC-MS/MS analysis in routine toxicology.

References

• Biotage Application Note AN916: Streamlined Sample Preparation of a Drugs of Abuse Panel in Human Nails, 2019.