Extraction of a Drugs of Abuse Panel from Human Urine Using Biotage® Mikro ABN SPE Microelution Plates Prior to UPLC-MS/MS Analysis

Importance of the Topic

Monitoring drugs of abuse in human urine is vital for clinical diagnostics, forensic investigations, workplace testing and compliance monitoring. Reliable detection of a broad panel of compounds at low concentrations enhances decision making in healthcare and legal settings. The use of efficient sample preparation workflows reduces analysis time and solvent consumption, supporting high-throughput laboratories.

Aims and Study Overview

This study evaluates a streamlined solid phase extraction (SPE) procedure using Biotage Mikro ABN microelution plates to isolate 49 common drugs of abuse from urine before UPLC-MS/MS analysis. Key objectives include:

• Achieving consistent recoveries (>80%) and low variability (RSD <10%) across the analyte panel.
• Establishing linear calibration ranges (1–1000 pg/mL) with r2 >0.99.
• Comparing total processing times and performance against conventional SPE formats.

Methodology and Instrumentation

Sample preparation involved:

• Hydrolysis: 1 mL urine spiked with deuterated internal standards, diluted and incubated with β-glucuronidase (60 °C, 2 h).
• SPE cleanup: Conditioning and equilibration of Biotage Mikro ABN plates, sample loading (400 µL), two wash steps (0.1% ammonium hydroxide and H2O/MeOH 90:10), elution with DCM/MeOH (90:10, 30 µL).
• Evaporation: Nitrogen/Gas concentrator at 40 °C for 5 min.
• Reconstitution: 30 µL H2O/MeOH (90:10) with 0.1% formic acid.

Used instrumentation:

• Biotage Mikro ABN Plate, 2 mg microelution format.
• Biotage PRESSURE+ 96 Positive Pressure Manifold.
• Biotage SPE Dry Sample Concentrator or TurboVap for evaporation.
• Shimadzu Nexera UHPLC with Restek Raptor Biphenyl column (100×2.1 mm, 2.7 µm).
• Shimadzu 8060 Triple Quadrupole MS with electrospray ionization.

Main Results and Discussion

The optimized protocol delivered recoveries mostly above 80% with RSDs below 10% for 49 drugs of abuse. Representative chromatograms demonstrated clear separation at 1 ng/mL. Calibration curves across 1–1000 pg/mL yielded r2 values >0.99 and low limits of quantification (as low as 1 pg/mL for select analytes). Eliminating conditioning and equilibration steps (Load-Wash-Elute approach) reduced processing time by ~5 min without affecting recovery.

Compared with a 10 mg SPE format, total extraction time decreased from ~33 min to ~22 min, and recoveries for certain benzodiazepines improved.

Benefits and Practical Applications

• High-throughput capability with 96-well microelution plates.
• Reduced solvent consumption and shorter evaporation times (30 µL elution).
• Robust performance across a large analyte panel suitable for clinical and forensic testing.
• Scalability and compatibility with automated positive pressure manifolds.

Future Trends and Potential Applications

Further advances may include integration with fully automated liquid handling platforms, expansion to additional metabolite panels, and coupling with high-resolution MS for untargeted screening. Miniaturized SPE formats and greener solvents will support sustainable workflows. Multiplexed assays and artificial intelligence–driven data analysis are emerging tools to enhance throughput and interpret complex drug profiles.

Conclusion

The Biotage Mikro ABN microelution SPE method offers a fast, reliable and solvent-efficient approach for extracting a comprehensive drugs of abuse panel from human urine. High recoveries, excellent linearity and significant time savings underline its suitability for routine clinical, forensic and workplace testing.