Direct Analysis of Drug of Abuse by BioSPME

Significance of the Topic

Urine testing for drugs of abuse demands sensitive and selective methods to detect low-level analytes and confirm identities directly from biological matrices, reducing false positives and streamlining analysis.

Objectives and Study Overview

This study evaluates newly developed biocompatible SPME LC Tips with C18 coating for direct extraction of illicit drugs and metabolites from urine. Representative analytes include cocaine, benzoylecgonine, cocaethylene, norfentanyl, methadone, and EDDP. Calibration was performed with matrix-matched standards and isotopically labeled internal standards, followed by analysis of ELISA-positive human urine samples via LC–MS/MS.

Methodology

• Calibration standards prepared at 20–1000 ng/mL in urine with 200 ng/mL deuterated internal standards.
• Sample preparation: 500 µL urine adjusted with 50 µL 2 mM ammonium formate (pH 3.7) to control pH variability.
• SPME protocol: 10 min conditioning in methanol, 10 min equilibration in water, 10 min extraction in urine, 30 min desorption into 200 µL 20 mM ammonium formate (90:10 methanol:water) with agitation.
• LC–MS/MS conditions: Ascentis Express RP Amide column (10 cm × 2.1 mm, 2.7 µm), mobile phase 10 mM ammonium formate (75:25 water:acetonitrile), flow rate 0.2 mL/min, 35°C, ESI+ MRM detection, 2 µL injection.

Used Instrumentation

• SPME LC Tips with C18-bonded silica stationary phase on pipette-tip format.
• Agilent 1100 HPLC system coupled to an ABI 3200 QTrap mass spectrometer.

Main Results and Discussion

High linearity (r>0.99) was achieved for all analytes across the calibration range, demonstrating quantitative equilibrium-based extraction. Real urine samples showed concentrations from non-detectable to over 33,000 ng/mL, indicating robustness across broad concentration spans. Batch processing allows simultaneous extraction of multiple samples without extending analysis time, and minimal sample volumes (<500 µL, often <100 µL) are sufficient.

Benefits and Practical Applications

• Rapid workflow: complete extraction and analysis under 60 minutes, regardless of batch size.
• Low sample volume requirements: typically less than 100 µL per sample.
• Selective extraction of drugs and polar metabolites from complex matrices (urine, serum, plasma, saliva, whole blood).
• Quantitative performance with high linearity across relevant concentrations.

Future Trends and Applications

Integration of SPME LC Tips into automated high-throughput platforms will enhance laboratory efficiency. Development of new fiber chemistries may extend analyte coverage. Coupling with advanced mass spectrometers can improve sensitivity and selectivity. Application to diverse biological matrices and emerging biomarkers is anticipated.

Conclusion

The SPME LC Tips technique offers a fast, selective, and quantitative approach for direct analysis of drugs of abuse in urine, meeting stringent sensitivity and specificity requirements while minimizing solvent use and sample volume.