Determination of Amphetamine and Derivatives in Urine

Importance of the Topic

An accurate measurement of amphetamine and its derivatives in urine is critical for forensic toxicology, sports anti-doping, and clinical diagnostics. These psychoactive stimulants pose health risks and are excreted mainly via urine, a complex matrix that demands efficient sample cleanup. Modified QuEChERS extraction coupled with capillary electrophoresis–mass spectrometry offers a rapid, sensitive, and cost-effective approach to detect trace levels of these compounds.

Objectives and Study Overview

The aim of this work was to establish a fast and reliable method for simultaneous quantification of amphetamine (AM), methamphetamine (MAM), methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA), and phentermine (PTM) in urine using a modified QuEChERS pretreatment and CE-MS/MS analysis.

Methodology and Instrumentation

Sample preparation involved a modified QuEChERS protocol: 10 mL urine extracted with 10 mL acetonitrile (pH ~12.4 by NaOH), followed by addition of MgSO4 and NaCl, centrifugation, and filtration through a 0.2 µm membrane. Separation was performed on an Agilent 7100 CE with a PVA-coated capillary (50 µm ID × 60 cm) using 0.1 M formic acid (pH 2.4) at 25 kV and 25 °C. Injection was hydrodynamic (5 s at 100 mBar), and detection used an Agilent 6430 triple quadrupole MS in positive ESI-MRM mode with optimized transitions and energy parameters.

Key Results and Discussion

The PVA coating effectively suppressed electroosmotic flow and minimized analyte–wall interactions, yielding sharp peaks and baseline separation within 7 minutes. Calibration curves were linear from 1 to 500 ng/mL (R2 > 0.995). Limits of detection ranged from 0.01 to 0.02 ng/mL, with quantification limits between 0.03 and 0.07 ng/mL. Recovery studies at 10, 20, and 50 ng/mL showed accuracies of 90–115% and RSD < 5.4%.

Benefits and Practical Applications

• Rapid analysis with run times under 7 minutes
• High sensitivity and selectivity suitable for forensic and doping control laboratories
• Reduced solvent usage and waste via modified QuEChERS extraction
• Minimal sample volume requirement and streamlined workflow for high-throughput screening

Future Trends and Applications

Emerging developments may include integration of CE-MS with high-resolution mass spectrometry, automated microfluidic sample handling, and expansion to other biological matrices. Continued refinement of green sample preparation techniques and multiplexed assays will enhance throughput and sustainability.

Conclusion

The proposed CE-MS/MS method combined with modified QuEChERS sample preparation delivers fast, accurate, and sensitive detection of amphetamines and derivatives in urine. Its simplicity, low reagent consumption, and high performance make it a valuable tool for routine forensic and clinical analysis.

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