Extraction of Illicit and Prescribed Drugs from Enzyme-Hydrolyzed Urine Using ISOLUTE® HYDRO DME+ Prior to UPLC-MS/MS Analysis

Significance of the Topic

Monitoring illicit and prescribed drugs in urine is essential for clinical toxicology, workplace testing, forensic investigations and therapeutic drug monitoring. Achieving low limits of quantitation (LOQ) while maintaining high throughput and robust sample cleanup remains a challenge. The integration of enzyme hydrolysis with solid‐phase extraction and UPLC-MS/MS analysis offers a streamlined workflow that enhances sensitivity, reduces matrix effects and meets regulatory guidelines such as SAMHSA and EWDTS.

Objectives and Study Overview

The primary goal of this study was to develop and validate a simple, high-efficiency sample preparation method for 25 target analytes—including amphetamines, opioids, benzodiazepines and other prescription drugs—from enzyme-hydrolyzed urine. Using ISOLUTE HYDRO DME+ Dual Mode Extraction plates, the protocol aimed to:

• Perform in-situ β-glucuronidase hydrolysis without post-treatment transfers
• Remove matrix components and residual enzyme effectively
• Achieve LOQs below established cut-off values
• Enable direct injection into UPLC-MS/MS with optional evaporation for enhanced sensitivity

Methodology and Instrumentation

Sample Preparation:

• Mix 500 μL urine with 25 μL internal standard (1 ng/μL), equilibrate 1 h at room temperature
• Add enzyme solution (β-glucuronidase) and ammonium acetate buffer (50 mM, pH 5.0), vortex
• Incubate 100 μL of this mixture in ISOLUTE HYDRO DME+ wells for 2 h at 60 °C for hydrolysis and cleanup
• After cooling, add 600 μL acetonitrile and perform 5 aspiration/dispense cycles for thorough mixing
• Elute analytes under ~5 psi positive pressure into a 96-well collection plate
• Optionally evaporate and reconstitute in small volumes of methanolic HCl for enhanced sensitivity

Chromatographic and Mass Spectrometric Conditions:

• UPLC: Restek Raptor Biphenyl column (100 x 2.1 mm, 2.7 μm), 0.4 mL/min gradient with 2 mM ammonium formate/formic acid in water and methanol
• Injection: 1 μL, partial loop
• MS/MS: Waters Premier XE triple quadrupole with ESI in positive MRM mode; source at 150 °C, desolvation at 450 °C; transitions optimized per analyte (r² > 0.99 for calibration curves)

Instrumentation Used

• ISOLUTE HYDRO DME+ 400 mg 96-well plates (Biotage)
• Biotage PRESSURE+ 96 Positive Pressure Manifold
• Biotage SPE Dry 96 sample evaporator (optional)
• Waters ACQUITY UPLC system
• Waters Premier XE triple quadrupole MS with ESI interface

Main Results and Discussion

Recovery and Precision:

• Recoveries ≥ 65% for most analytes; lower recoveries still met LOQ criteria
• RSD values < 10%, demonstrating reproducibility (n = 7)

Linearity and Sensitivity:

• Calibration over 10–400 ng/mL yielded r² > 0.99 for representative compounds
• LOQs met or exceeded SAMHSA/EWDTS requirements for drugs of abuse testing

Matrix Removal:

• Urea and creatinine breakthrough reduced by > 90% compared to simple acetonitrile crash
• Efficient elimination of hydrolysis enzyme and other endogenous interferents, improving signal‐to‐noise ratios

Benefits and Practical Applications

• Combined hydrolysis and cleanup in a single plate reduces handling and potential sample loss
• High analyte sensitivity without mandatory evaporation accelerates throughput
• Suitable for routine workplace and clinical drug monitoring, forensic analyses and QA/QC labs

Future Trends and Opportunities

• Automation of plate-based workflows for higher throughput and reduced hands-on time
• Miniaturized extraction formats to conserve sample and reagents
• Coupling with high-resolution mass spectrometry for broader analyte panels and unknown screening
• Integration of data analytics and cloud-based reporting for remote monitoring

Conclusion

The described method using ISOLUTE HYDRO DME+ plates prior to UPLC-MS/MS provides a robust, sensitive and streamlined workflow for the extraction of illicit and prescribed drugs from enzyme-hydrolyzed urine. It delivers high recoveries, reproducible precision and clean extracts that comply with regulatory LOQs while supporting high sample throughput.

References

• Biotage Application Note AN915, 2019