A Simplified, Mixed-Mode Sample Preparation Strategy for Urinary Forensic Toxicology Screening by UPLC-MS/MS

Significance of the Topic

In forensic toxicology, efficient and reliable sample preparation is essential to detect a wide range of drug classes in urine. Simplifying extraction workflows while ensuring sensitivity and reducing matrix effects can improve laboratory throughput and lower operational costs.

Objectives and Study Overview

This work aims to present a streamlined mixed-mode SPE strategy combined with UPLC-MS/MS to screen 38 compounds—opioids, stimulants, benzodiazepines, metabolites—in under 30 minutes. The goal is to maintain high recovery, fast analysis, and robust performance for comprehensive forensic toxicology panels.

Methodology

• Sample Pretreatment: 50 µL urine mixed in-well with 0.5 M ammonium acetate, β-glucuronidase enzyme, and 4% H₃PO₄ in Oasis MCX µElution plates for enzymatic hydrolysis and acidification.
• Extraction: Load by vacuum, single wash (200 µL 20% MeOH in 0.02 N HCl), elution with 2×50 µL ACN/MeOH + 5% NH₄OH, evaporation at 40 °C under N₂, reconstitution in 50 µL 2% ACN/1% formic acid.
• Chromatography: ACQUITY UPLC I-Class with BEH Phenyl column (1.7 µm, 2.1×100 mm), gradient from 5% to 62.5% organic over 5 min, 0.6 mL/min, 40 °C.
• Mass Spectrometry: Xevo TQD with positive ESI, MRM transitions optimized per analyte, individualized cone and collision voltages.

Used Instrumentation

• Waters ACQUITY UPLC I-Class System
• Waters ACQUITY UPLC BEH Phenyl Column, 1.7 µm, 2.1×100 mm
• Waters Xevo TQD Mass Spectrometer
• Waters Oasis MCX µElution Plates
• MassLynx Software with TargetLynx Application Manager

Main Results and Discussion

• Chromatographic separation of 38 analytes—including polar glucuronides—in under 4 min with baseline resolution of isobaric compounds.
• Recoveries >80% for 37 analytes (average ~100%); %CV <10% for 37 compounds.
• Limits of detection between 1 and 10 ng/mL, suitable for semi-quantitative forensic screening.
• SPE workflow reduced from six to three steps, enabling a 96-well plate to be processed in ~30 min.

Benefits and Practical Applications

• Accelerated sample preparation and analysis time.
• Reduced solvent consumption and disposal costs.
• Enhanced sensitivity, specificity, and reproducibility.
• Lower matrix effects, extended column lifetimes, and decreased instrument downtime.

Future Trends and Applications

• Automation of in-well hydrolysis and SPE for higher throughput.
• Direct analysis of glucuronide conjugates without enzyme treatment.
• Adoption for novel psychoactive substances and integration with high-resolution MS.

Conclusion

This simplified mixed-mode SPE and UPLC-MS/MS method delivers rapid, robust, and sensitive screening of a comprehensive urinary toxicology panel. By consolidating extraction steps and leveraging water-wettable sorbent properties, the workflow minimizes complexity and supports reliable forensic analyses.

References

• Danaceau JP, Chambers E, Fountain KJ. Advantages of CORTECS C18 2.7 µm and XBridge Phenyl XP 2.5 µm Columns for the Analysis of a Comprehensive Panel of Pain Management Drugs for Forensic Toxicology. Waters Application Note. 2014; Literature Code 720005185en.
• Wang P, Stone JA, Chen KH, Gross SF, Haller CA, Wu AH. Incomplete recovery of prescription opioids in urine using enzymatic hydrolysis of glucuronide metabolites. J Anal Toxicol. 2006 Oct;30(8):570–5.