Automated Extraction of a Drugs of Abuse Panel from Human Urine Using Biotage Extrahera LV-200 and Microelution SPE Prior to UPLC-MS/MS Analysis

Significance of the Topic

The reliable detection of a broad spectrum of drugs of abuse in urine is critical in clinical toxicology, forensic investigations and workplace testing. Automated microelution SPE coupled with UHPLC-MS/MS improves sample throughput, reduces solvent consumption and minimizes variability, addressing key challenges in high-volume laboratories.

Objectives and Overview

This study aimed to develop and validate an automated extraction workflow for a multi-class panel of illicit and prescription drugs in hydrolyzed human urine. It compared manual and automated processing using mixed-mode strong cation exchange microelution plates on the Biotage Extrahera LV-200, evaluating recovery, precision, linearity and limits of quantitation.

Methodology

One-milliliter urine samples were spiked with deuterated internal standards and hydrolyzed with β-glucuronidase at 60 °C for 2 h. Samples were diluted in ammonium acetate buffer (pH 5) and loaded onto 2 mg mixed-mode SCX microelution plates. The SPE protocol included conditioning (methanol), equilibration (4 % phosphoric acid), sequential washes (phosphoric acid and 50:50 water/methanol) and elution with dichloromethane/methanol/ammonium hydroxide (78:20:2). Eluates were evaporated, reconstituted in water/methanol (90:10, 0.1 % formic acid) and analyzed by UHPLC-MS/MS.

Instrumentation

• Automated SPE: Biotage Extrahera LV-200
• Manual SPE: Biotage PRESSURE+ 96 manifold
• Evaporation: Biotage SPE Dry concentrator
• UHPLC: Shimadzu Nexera with Restek Raptor Biphenyl column (100 × 2.1 mm, 2.7 µm)
• MS/MS: Shimadzu 8060 Triple Quadrupole with ESI

Results and Discussion

Both workflows produced mean recoveries above 60 % with RSDs below 5 % (n=7) across more than 30 analytes. Calibration curves spanning 1–1000 pg/mL achieved R² > 0.999. Lower limits of quantitation ranged from 1 to 250 pg/mL. Automated extraction of 96 samples required approximately 25 minutes (excluding hydrolysis and evaporation), matching or exceeding the performance of manual SPE in reproducibility and throughput.

Benefits and Practical Applications

• High sample throughput with low solvent and consumable usage
• Robust, reproducible extraction for diverse drug panels
• Reduced hands-on time and operator variability
• Scalable solution for routine clinical, forensic and QA/QC laboratories

Future Trends and Possibilities

Emerging advancements may include further SPE miniaturization, direct on-line coupling to LC-MS systems, expanded target lists for novel psychoactive substances and AI-driven workflow optimization. Green chemistry approaches and integrated robotic platforms will continue to enhance efficiency and sustainability.

Conclusion

The automated mixed-mode SCX microelution SPE method implemented on the Biotage Extrahera LV-200 delivers consistent, high-throughput extraction of a comprehensive drugs of abuse panel in urine, with excellent recoveries, precision and sensitivity, streamlining laboratory operations.

References

• Biotage AB. Automated Extraction of Drugs of Abuse from Urine Using Biotage Extrahera LV-200 and Microelution SPE Prior to UPLC-MS/MS Analysis. Application Note AN964; 2021.