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

Importance of Topic

Rapid and reliable quantitation of drugs of abuse in human urine is a cornerstone of forensic toxicology and clinical screening. Traditional liquid–liquid extraction methods can be labor-intensive, difficult to automate, and prone to variability. Supported liquid extraction (SLE) using synthetic media offers improved phase contact, simplified workflows, and consistent performance, addressing key challenges in high-throughput forensic testing.

Objectives and Overview

This application note investigates the performance of Agilent Chem Elut S 96-well plates (400 µL) for the quantitative analysis of 24 drugs of abuse in human urine by LC/MS/MS. The study compares Chem Elut S against conventional LLE and a diatomaceous-earth (DE)-based SLE approach, assessing recovery, reproducibility, limit of quantitation, and ease of use.

Used Instrumentation

• Agilent Chem Elut S 400 µL supported liquid extraction 96-well plates
• Agilent Positive Pressure Manifold PPM-96
• Agilent 1290 Infinity LC system (quaternary pump, autosampler, thermostatted column compartment)
• Agilent 6490 Triple Quadrupole LC/MS with iFunnel (dynamic MRM, positive mode)
• Agilent Poroshell 120 EC-C8 analytical column (2.1×100 mm, 2.7 µm) with EC-C18 guard
• Multitube vortexer, 96-probe liquid handler, SPE 96 evaporator, standard pipettes

Methodology

Human urine samples were enzymatically hydrolyzed with β-glucuronidase at 40 °C for 60 minutes to release conjugated analytes. Aliquots (200 µL) were loaded onto Chem Elut S plates under gentle positive pressure and equilibrated for five minutes. Analytes were eluted with two 900 µL aliquots of methyl tert-butyl ether, dried under nitrogen, and reconstituted in 85:15 ammonium formate buffer/acetonitrile. Chromatographic separation employed a gradient from 5 % to 95 % organic over seven minutes at 50 °C, 0.5 mL/min. The 6490 MS used dynamic MRM transitions optimized for each drug and isotopic internal standards.

Main Results and Discussion

Calibration curves over 0.1–20 ng/mL (0.5–20 ng/mL for amphetamine and heroin) delivered coefficients of determination (R2) from 0.991 to 0.999. For 23 of 24 analytes, mean recoveries ranged from 79 % to 117 % with %RSD below 16 %. Proadifen showed lower recovery (~55 %) despite good precision. Limits of quantitation as low as 0.1 ng/mL were achieved for most compounds. Compared to DE-based SLE and LLE, Chem Elut S provided higher and more consistent recoveries, reduced well-to-well variability, and simplified handling by eliminating emulsions and manual phase transfers.

Benefits and Practical Applications

Implementation of Chem Elut S plates for urine drug screening offers:

• High-throughput workflow compatible with 96-well automation
• Improved recovery and precision across multiple drug classes
• Reduced hands-on time and labor compared to LLE
• Consistent batch-to-batch performance versus natural sorbents
• Clean extracts with minimal matrix effects for LC/MS/MS

Future Trends and Potential Uses

Ongoing developments focus on extending synthetic SLE to other biological matrices such as plasma and serum, integrating microflow LC/MS workflows, and coupling automated extraction platforms with advanced data-processing software. Emerging trends include miniaturized plate formats, environmental applications, and AI-driven method optimization for broader forensic and clinical assays.

Conclusion

Agilent Chem Elut S supported liquid extraction plates deliver a fast, robust, and reproducible sample-preparation solution for quantitative urine analysis of drugs of abuse by LC/MS/MS. The synthetic sorbent’s high water-holding capacity and uniform performance outperform traditional LLE and DE-based SLE, enabling reliable high-throughput forensic testing.

Reference

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3. Rositano J, et al. Supported Liquid Extraction (SLE) for the Analysis of Methylamphetamine, Methylenedioxymethylamphetamine and Δ9-Tetrahydrocannabinol in Oral Fluid and Blood of Drivers. Forensic Sci Int. 2016;265:125–130.
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