Screening, Identification, and Quantitation of 102 Drugs in Human Whole Blood by LC/Q-TOF and LC‑QQQ

Význam tématu

Forensic toxicology increasingly demands rapid, reliable profiling of drugs of abuse and therapeutic compounds in whole blood. Whole blood analysis overcomes limitations of urine testing by directly measuring parent drugs and metabolites, enabling more accurate interpretation in postmortem, clinical and legal investigations. High-throughput workflows that combine sensitive extraction with advanced mass spectrometry are essential to meet growing case loads and ensure robust quantitative and qualitative results.

Cíle a přehled studie / článku

This study establishes and validates an end-to-end workflow for simultaneous screening, identification and quantitation of 102 target analytes (66 drugs of abuse plus metabolites and 36 medicinal drugs) in human whole blood. The approach integrates in-cartridge protein precipitation, lipid removal using Agilent Captiva EMR—Lipid cartridges, high-resolution LC/Q-TOF for suspect screening and identification, followed by triple quadrupole LC-QQQ for accurate quantitation. Key performance metrics, including screening sensitivity at 10 and 50 ng/mL and quantitative accuracy, are critically evaluated.

Použitá instrumentace

• Agilent 1290 Infinity II LC system with binary pump, high-performance autosampler and column compartment
• Agilent Positive Pressure Manifold 48 (PPM-48) and multitube vortexer for in-cartridge PPT
• Agilent Captiva EMR—Lipid 3 mL cartridges for lipid cleanup
• Agilent 6545 Q-TOF with JetStream ESI source for high-resolution screening
• Agilent 6490 triple quadrupole LC/MS with Jet Stream iFunnel ESI source for quantitation
• ZORBAX Eclipse Plus C18 analytical and guard columns (100×2.1 mm and 2.1×5 mm, 1.8 µm)

Použitá metodika

Sample preparation involved adding cold acetonitrile/methanol (95/5) to 0.5 mL whole blood directly in EMR—Lipid cartridges, followed by controlled positive pressure precipitation and two-step elution (ACN/water mixes), nitrogen drying and reconstitution. Screening data acquisition used Agilent All Ions MS/MS DIA with four collision energies (0, 10, 20, 40 V) over m/z 50–1000 in positive and m/z 40–1000 in negative mode. Quantitation was performed in dynamic MRM mode on LC-QQQ, optimizing precursor/product transitions, collision energies and retention windows for each analyte. Calibration standards and QCs were prepared in matrix, covering 0.5–200 ng/mL (grouped by analyte class) with 1/x² weighting.

Hlavní výsledky a diskuse

• In-cartridge PPT plus EMR—Lipid delivered average recoveries rising from 62% (offline PPT) to 86% with RSD <10%.
• Additional elution improved recoveries by 10–20% for challenging compounds (e.g., 6-acetylmorphine, ritalinic acid).
• Q-TOF screening identified 91% of compounds at 10 ng/mL and 98% at 50 ng/mL; buprenorphine and fentanyl were confirmed at 1 ng/mL with no false negatives in blind spikes.
• LC-QQQ quantitation yielded LOQs of 0.5–5 ng/mL, with 95% of analytes in the 70–120% recovery window, 98% with RSD <20% and 93% with matrix effects within 60–140%. Calibration curves showed R² >0.99 for most targets. Accuracy (100 ±20%) and precision (RSD <20%) criteria were met at all QC levels.

Přínosy a praktické využití metody

The validated workflow offers forensic and clinical laboratories a streamlined, robust solution for high-throughput drug screening and quantitation in whole blood. Combining minimal sample handling with powerful MS platforms reduces turn-around time, lowers risk of matrix interferences and supports reliable reporting for forensic, workplace and clinical toxicology applications.

Budoucí trendy a možnosti využití

Emerging applications include expanding suspect lists via larger high-resolution spectral libraries, automated DIA data processing with AI-driven algorithms, miniaturized and multiplexed sample prep formats, and adaptation to other biological matrices (saliva, tissue). Integration with digital reporting and laboratory information systems will further streamline forensic toxicology workflows.

Závěr

This study demonstrates a fully validated, end-to-end LC/Q-TOF and LC-QQQ workflow for simultaneous screening, identification, and accurate quantitation of 102 drugs in whole blood. The approach delivers high sensitivity, robust recoveries, minimal matrix effects and excellent precision, establishing a reference method for modern forensic toxicology laboratories.

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