Screening, Identification, and Quantitation of Drugs of Abuse in Human Whole Blood by LC/Q-TOF and LC/MS/MS

Significance of Topic

Reliable screening and quantitation of drugs of abuse in biological fluids is essential for forensic investigations, clinical toxicology and quality control. Advances in liquid chromatography coupled to both high-resolution Q-TOF and tandem quadrupole MS/MS enable rapid, sensitive and comprehensive analysis of a broad panel of substances without extensive derivatization.

Objectives and Study Overview

This work demonstrates an end-to-end workflow for over 100 prescription and illicit drugs in human whole blood. The goals were to:

• Develop a streamlined sample preparation method.
• Evaluate LC/Q-TOF for broad-scope screening and compound identification.
• Assess LC/MS/MS (triple quadrupole) for accurate quantitation.

Methodology and Instrumentation

Human whole blood samples were subjected to in-cartridge protein precipitation followed by Captiva EMR-Lipid cleanup. Two analytical platforms were applied:

• High-resolution LC/Q-TOF for non-targeted screening and structural confirmation using exact mass and fragment ions.
• Triple quadrupole LC/MS/MS in dynamic MRM mode for quantitation at low ng/mL levels.

Used Instrumentation

• Agilent 1290 Infinity II UHPLC with ZORBAX Eclipse Plus C18 (2.1×100 mm, 1.8 µm) column.
• Agilent 6490 triple quadrupole LC/MS for targeted quantitation (TQ conditions: 220°C gas temperature, 18 L/min gas flow, dMRM).
• Agilent 6546A Q-TOF LC/MS for high-resolution screening (250°C drying gas, 30 psi nebulizer, Hi-Res 10 GHz acquisition).

Main Results and Discussion

Sensitivity and reproducibility were demonstrated at both 10 ng/mL and 50 ng/mL fortification levels. Key findings included:

• Screening/identification success rates of 91% at 10 ng/mL and 98% at 50 ng/mL using LC/Q-TOF.
• Quantitative recoveries within 70–120% for 97% of analytes; relative standard deviations below 20%.
• Matrix effects in whole blood were within ±40% for 93% of compounds, indicating robust performance across diverse chemistries.

Benefits and Practical Applications

This integrated approach offers:

• Rapid sample turnaround with minimal manual steps.
• Comprehensive coverage of opioids, stimulants, sedatives, antidepressants and other abused substances.
• Reliable confirmation of identity by accurate mass and characteristic fragments.
• Precise quantitation suitable for forensic and clinical decision-making.

Future Trends and Potential Applications

Emerging directions include:

• Expanding HRMS libraries to cover novel psychoactive substances.
• Automated data processing with machine learning for faster interpretation.
• Integration with dried blood spot and micro-sampling techniques.
• Real-time screening applications in roadside and workplace testing.

Conclusion

The study validates a robust workflow combining in-cartridge cleanup with dual LC/MS platforms for comprehensive screening and quantitation of drugs in whole blood. The methods meet forensic standards for sensitivity, accuracy and throughput.

Reference

Agilent application notes 5994-2830EN; 5991-9251EN; 5991-9222EN.