A Complete Toxicology Screening Procedure for Drugs and Toxic Compounds in Urine and Plasma Using LC-MS/MS

Significance of the Topic

Liquid chromatography–tandem mass spectrometry (LC-MS/MS) has emerged as a powerful tool for comprehensive toxicology screening. Its high sensitivity and specificity overcome the limitations of traditional immunoassays, GC-MS, and HPLC-DAD by enabling broad compound coverage regardless of volatility or thermal stability. Automated reporting software further streamlines workflows, reducing manual data review and improving laboratory throughput.

Objectives and Study Overview

This application note details the development of a turnkey LC-MS/MS screening procedure for over 275 drugs and toxic compounds in human urine and plasma. The goals were to establish a robust sample preparation protocol, optimize chromatographic separation, build an extensive MS/MS spectral library, and implement automated data processing and reporting via ToxID software.

Methodology and Instrumentation

Samples (1 mL urine or 0.5 mL plasma) were spiked with deuterated internal standards and extracted using mixed-mode solid-phase cartridges. Chromatographic separation utilized a 50 × 2.1 mm Perfluorophenyl (PFP) column with a 13-minute gradient (water and acetonitrile with 0.1% formic acid and ammonium formate). MS analysis was performed on an LXQ ion trap with electrospray ionization in polarity-switching, scan-dependent MS/MS mode. Retention time windows and stepped collision energies facilitated acquisition of full-scan and MS2 spectra for co-eluting analytes.

Key Results and Discussion

Method prequalification in spiked urine demonstrated detection limits below 10 ng/mL for 70% of compounds, with the remainder reliably identified at 100–1000 ng/mL. Plasma performance was comparable. Clinical validation against LC-UV and immunoassay data from patient samples revealed LC-MS/MS identified a broader range of analytes, including several not detected by traditional methods.

Benefits and Practical Applications

• Broad analyte coverage: over 275 drugs and toxins in a single run.
• High sensitivity: sub-10 ng/mL detection for most compounds.
• Efficient workflow: simple SPE preparation and a 13-minute LC run.
• Automated reporting: ToxID generates summary and detailed reports, reducing manual review time.
• Flexible library expansion: new analytes can be added in under one hour via direct infusion and retention time mapping.

Future Trends and Applications

Advances in high-resolution MS and data-independent acquisition will further expand screening capabilities. Integration of machine learning for spectral interpretation and cloud-based library sharing may enhance compound coverage and throughput. Miniaturized sample prep and microflow LC could reduce solvent use and analysis time.

Conclusion

The described LC-MS/MS screening procedure, combined with automated ToxID software, offers a comprehensive, sensitive, and efficient solution for toxicology laboratories. It surpasses conventional screening methods in scope and performance, delivering rapid, reliable identification of a wide array of drugs and toxic compounds in urine and plasma.

Instrumentation

• LC: Accela pump with Hypersil GOLD PFP column (50 × 2.1 mm, 5 µm).
• MS: Thermo Scientific LXQ ion trap with Ion Max ESI source.
• Key MS parameters: capillary temperature 275 °C, spray voltage 5.0 kV, polarity switching, stepped collision energy 35% ±10%, WideBand Activation™ enabled.