Drug Analysis in Human Urine Using Agilent Chem Elut S Supported Liquid Extraction by LC/MS/MS

Significance of the Topic

The quantitative determination of drugs in urine supports clinical research, toxicology screening, and therapeutic monitoring by providing rapid, reliable measurement of multiple analytes. Efficient sample preparation is essential to remove matrix interferences, minimize handling time, and ensure reproducible results in high-throughput workflows.

Objectives and Study Overview

This application note demonstrates the use of Agilent Chem Elut S supported liquid extraction (SLE) 96-well plates for the simultaneous measurement of 24 drugs in human urine by LC/MS/MS. The study benchmarks analytical performance against traditional liquid-liquid extraction (LLE) and diatomaceous earth (DE) SLE methods, focusing on recovery, precision, and operational efficiency.

Instrumentation Used

• Agilent 1290 Infinity quaternary pump, autosampler, column compartment, and thermostat
• Agilent 6490 triple quadrupole LC/MS with iFunnel, dynamic MRM, positive ion mode
• Agilent Chem Elut S 400 µL 96-well plates and positive pressure manifold
• Poroshell 120 EC-C8 analytical column with C18 guard (50 °C, 0.5 mL/min)
• HPLC-grade methanol, acetonitrile, methyl tert-butyl ether, ammonium formate, formic acid, β-glucuronidase enzyme

Methodology

Urine (200 µL) was incubated with ammonium acetate buffer and β-glucuronidase at 40 °C for 60 min to release conjugated drugs. Samples were loaded onto Chem Elut S plates, equilibrated for 5 min, then eluted twice with 900 µL methyl tert-butyl ether under gravity and low positive pressure. Extracts were evaporated to dryness, reconstituted in 85:15 aqueous mobile phase:ACN, and analyzed by LC/MS/MS using a gradient from 5% to 95% organic over 7 min. Target compounds were measured by dynamic MRM with compound-specific transitions.

Main Results and Discussion

Calibration curves were linear from 0.1 to 20 ng/mL (R2 0.991–0.999) with limits of quantitation at 0.1 ng/mL for most analytes (0.5 ng/mL for amphetamine and heroin). Recoveries ranged from 79% to 117% (RSD <16%) for all but proadifen (55% recovery, RSD <12%). Compared to DE-based SLE and LLE, Chem Elut S delivered higher and more consistent recoveries, with fewer compounds exceeding 20% RSD. The synthetic sorbent eliminated variability inherent to natural DE media and simplified automation relative to LLE.

Benefits and Practical Applications

• Fast, high-throughput extraction with minimal emulsions or manual phase separation
• Excellent recovery and precision for a broad panel of drugs in urine
• Uniform synthetic sorbent ensures batch-to-batch consistency and robust water-holding capacity
• Compatible with automation platforms and 96-well workflows, reducing labor and turnaround time

Future Trends and Applications

Supported liquid extraction with synthetic sorbents is poised for broader adoption in bioanalytical assays, including steroid profiling, therapeutic drug monitoring in plasma or serum, and environmental contaminant analysis. Integration with automated liquid handlers and on-line extraction techniques will further streamline sample preparation and increase laboratory throughput.

Conclusion

Agilent Chem Elut S 96-well SLE plates provide a fast, simple, and reliable platform for multi-drug quantitation in urine by LC/MS/MS. The synthetic sorbent ensures consistent recovery and precision while outperforming traditional LLE and DE-based SLE in ease of use and data quality.

References

• Zhao L. Quantitative Determination of a Panel of Endogenous Steroids in Human Serum by LC/MS/MS using Agilent Supported Liquid Extraction (SLE) Chem Elut S Plate. Agilent Technologies Application Note, 5994-0949EN.