A LC-MS method for the measurement of 245 compounds of interest in toxicology with a fully-automated sample preparation

Importance of the Topic

Efficient and reliable detection of a wide range of xenobiotics in biological fluids is crucial for clinical toxicology and forensic analysis. Automating sample preparation streamlines workflows, reduces human error, and increases throughput, addressing rising demands in toxicology laboratories.

Objectives and Study Overview

This study aimed to develop and validate a fully automated LC-MS/MS method capable of identifying and quantifying 245 illicit and therapeutic compounds in blood, plasma, and urine. Key goals included assessing method robustness, calibration stability over time, and comparison with existing targeted assays.

Methodology

The workflow integrates automated extraction directly coupled to liquid chromatography-tandem mass spectrometry:

• Sample matrices: whole blood, plasma, urine
• Automated solid-phase extraction: CLAM-2000 system interfaced with LC
• Separation and detection: Shimadzu LCMS-8060 triple quadrupole MS operated in MRM Spectrum mode (up to 15 MRM transitions per analyte)

Used Instrumentation

• Shimadzu CLAM-2000 programmable liquid handler
• Shimadzu LCMS-8060 triple quadrupole mass spectrometer
• MRM Spectrum mode library search and quantitation software

Main Results and Discussion

Calibration curves remained valid for up to one month, yielding uncertainties below 20% for over 80% of compounds in a reference mix. In analysis of 188 patient samples across 312 concentration measurements, the automated method demonstrated strong correlation with existing assays (R² = 0.85, slope = 0.81). High data density did not compromise quantitative linearity (R² > 0.999 for individual analytes) or identification confidence.

Benefits and Practical Applications

• Substantial reduction in manual handling and sample preparation time
• High-throughput quantitative screening spanning diverse drug classes
• Consistent performance meeting ISO 15189 validation criteria
• Simplified workflow enabling laboratory resource optimization

Future Trends and Potential Applications

Expanding compound libraries and tighter integration with laboratory information management systems will enhance coverage and traceability. Advances in real-time data processing, machine learning–driven spectral matching, and miniaturized extraction platforms promise further gains in throughput and applicability for clinical and forensic toxicology.

Conclusion

The fully automated LC-MS/MS workflow provides robust, high-throughput quantification of 245 toxicologically relevant compounds in various matrices. Its demonstrated calibration stability and strong agreement with traditional targeted assays highlight its potential as a standardized platform for clinical and forensic toxicology laboratories.