Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS

Significance of the Topic

Amphetamines and related stimulants remain among the most commonly misused substances in clinical and forensic settings. Rapid, accurate screening and quantitation in biological fluids are critical for workplace drug testing, toxicology, and regulatory compliance. Traditional workflows rely on immunoassay screening followed by GC/MS analysis, which requires time-consuming derivatization and lengthy sample preparation. The introduction of direct-injection LC/MS/MS methods promises to deliver higher throughput, reduced sample handling, and improved laboratory efficiency.

Objectives and Study Overview

The primary goal was to develop and validate a fast, direct-injection LC/MS/MS method for simultaneous screening and quantitation of five amphetamines (amphetamine, methamphetamine, MDA, MDMA, MDEA) and four potential interferences (ephedrine, pseudoephedrine, norpseudo-ephedrine, phentermine) in urine. An isotopically distinct internal reference (PMPA) was included. The method follows the 2010 SAMHSA guidelines, with emphasis on eliminating derivatization and extensive clean-up, while maintaining sensitivity and robustness.

Methodology and Instrumentation

• Sample preparation: Human urine diluted 1:10 with Milli-Q water; no further pre-treatment.
• Chromatography: Shim-pack XR-ODS III UHPLC column (1.6 µm, 50 × 2 mm); mobile phases water (A) and methanol (B) with 0.1% formic acid; fast gradient (0–1.6 min B 2→14%; 1.8–2.3 min B 70%; re-equilibration to 4 min); flow rate 0.6 mL/min.
• Mass spectrometry: Shimadzu LCMS-8040 triple quadrupole with Nexera UHPLC and SIL-30A autosampler; positive ESI; drying gas 15 L/min; nebulizing gas 3 L/min; block temperature 400 °C; DL temperature 250 °C; injection volumes 0.1–1 µL.
• MRM detection: Automated optimization yielding two transitions per compound (quantifier and qualifier).

Main Results and Discussion

• Separation and detection: All ten compounds produced sharp peaks between 0.75 and 2.2 min.
• Calibration and linearity: R²>0.999 over 1–500 ppb (0.1 µL injection); comparable performance at 1 µL.
• Sensitivity: LOQs for amphetamines in source urine (10× diluted) ranged 2.1–17.1 ng/mL (MDA 53 ng/mL); interferences LOQs 17–91 ng/mL; internal reference LOQ 2.4 ng/mL.
• Precision and matrix effects: RSDs 1.6–7.9% (0.1 µL) and 1.6–7.8% (1 µL); matrix effects 79–115% at 62.5 ppb.
• Operational stability: After 120 consecutive injections (~10 h) of spiked urine, peak areas declined by ~17%, confirming robust performance without source cleaning or derivatization.

Benefits and Practical Applications

The direct-injection LC/MS/MS approach eliminates derivatization and extensive sample cleanup, reducing turnaround time and consumable costs. Ultra-small injection volumes limit source contamination, extending run periods. Sensitivity surpasses SAMHSA confirmation cutoffs (250 ng/mL), making this method suitable for forensic toxicology, workplace testing, and clinical monitoring.

Future Trends and Applications

Future developments may focus on further miniaturization, integration with full automation, and expansion to other drug classes. Continuous improvements in UFMS technology and column chemistry will enable even faster separations and higher throughput. Coupling with robotic sample handling and informatics platforms will streamline laboratory workflows.

Conclusion

A direct-injection LC/MS/MS method was successfully developed for rapid screening and quantitation of amphetamines in urine. The validated procedure demonstrates excellent linearity, sensitivity, precision, and operational stability using ultra-small injection volumes. This workflow offers a robust, high-throughput alternative to traditional GC/MS protocols in forensic and clinical laboratories.

Used Instrumentation

• Shimadzu LCMS-8040 triple quadrupole mass spectrometer
• Shimadzu Nexera UHPLC system with SIL-30A autosampler
• Shim-pack XR-ODS III UHPLC column (1.6 µm, 50 × 2 mm)

References

1. Kudo K., Ishida T., Hara K., Kashimura S., Tsuji A., Ikeda N. Direct determination of amphetamines in urine by LC/MS/MS. Journal of Chromatography B, 2007, 855, 115–120.
2. Mandatory Guidelines for Federal Workplace Drug Testing Programs, 73 FR 71858–71907, November 25, 2008.
3. Lin H.-R., Choi K.-I., Lin T.-C., Hu A. High-throughput LC/MS/MS screening of amphetamines in urine. Journal of Chromatography B, 2013, 929, 133–141.