An In-Well Hydrolysis Solid Phase Extraction Method Using EVOLUTE® HYDRO CX for 96 Licit and Illicit Drugs of Abuse from Urine Prior to UPLC-MS/MS

Importance of the Topic

Efficient extraction and analysis of licit and illicit drugs in urine is critical for clinical toxicology, forensic investigations, workplace testing, and anti-doping programs. In-well hydrolysis combined with solid phase extraction reduces sample handling, minimizes risk of contamination and carryover, and accelerates workflow, enabling high-throughput screening of a broad panel of analytes.

Objectives and Study Overview

This application study aimed to develop and validate a 96-well plate method for simultaneous hydrolysis and extraction of 96 drugs of abuse from urine, followed by UPLC-MS/MS quantitation. Key goals were to achieve high recovery, low matrix effects, streamlined processing, and compatibility with automation platforms.

Methodology and Instrumentation

Sample Pre-treatment and Hydrolysis

• Urine (100 µL) was pipetted into EVOLUTE HYDRO CX 96-well plates.
• Hydrolysis was performed in-well using various enzymes (eg, BGTurbo, IMCSzyme, Kura BG100) with appropriate buffer systems at 55 °C for 30 minutes.
• Samples were acidified with phosphoric acid prior to extraction.

Solid Phase Extraction

• Sequential washes with aqueous phosphoric acid and 50:50 methanol/water removed interferences.
• Elution used dichloromethane/methanol/ammonium hydroxide (78:20:2) in two 500–750 µL aliquots.
• Positive pressure manifolds (Biotage PRESSURE+ 96 or Extrahera) controlled flow and drying steps.

Post-processing

• Eluates were evaporated under nitrogen (Biotage SPE Dry 96) at 40 °C.
• Residues were reconstituted in 100 µL of 90:10 water/methanol with 0.1% formic acid.

Chromatography and Detection

• UPLC: Shimadzu Nexera x2, Restek Raptor Biphenyl 50×3 mm, 2.7 µm, 40 °C, flow 0.45 mL/min, gradient from 5% to 95% methanol with 0.1% formic acid in 9.25 minutes.
• MS/MS: Sciex 5500 QqQ, ESI positive/negative, ion source at 600 °C, 1500 V spray voltage.

Main Results and Discussion

Recovery for most analytes ranged from 60 to 100%, with stronger reverse-phase binders showing occasional losses during high-methanol washes. Matrix effects were generally below 15%, indicating effective cleanup. Hydrolysis efficiency varied by enzyme type, highlighting the importance of enzyme selection for glucuronide cleavage. Overall, the in-well hydrolysis approach achieved robust performance across drug classes.

Benefits and Practical Applications

• Integrated hydrolysis and extraction in a single plate reduced handling steps and sample transfers.
• Reduced conditioning and equilibration time improved throughput.
• Automation compatibility with PRESSURE+ 96 and Extrahera platforms enabled unattended operation.
• High recovery and low matrix effects support reliable quantitation in routine and high-volume laboratories.

Future Trends and Potential Applications

Further developments may include adaptation to high-resolution mass spectrometry, expansion to other biological matrices (eg, plasma, oral fluid), integration with robotic liquid handlers, and exploration of novel sorbent chemistries for enhanced selectivity. Emerging enzyme formulations could improve hydrolysis efficiency for challenging metabolites.

Conclusion

The EVOLUTE HYDRO CX in-well hydrolysis SPE method provides a streamlined, high-throughput solution for urinary drug screening. By combining enzymatic cleavage and SPE in a single plate format, this approach offers strong recoveries, minimal matrix interference, and seamless integration with UPLC-MS/MS workflows, meeting the demands of modern analytical laboratories.

Reference

Biotage Application Note AN886, 2017