A Comprehensive Workflow for a Large Panel of Drugs of Abuse in Human Whole Blood by LC/MS/MS

Significance of the topic

Forensic toxicology demands rapid and accurate quantification of drugs of abuse in biological matrices. Whole blood is particularly challenging due to its high protein and lipid content, which can cause matrix ion suppression, instrument contamination, and reduced analytical sensitivity. Implementing efficient cleanup strategies is essential to ensure reliable results, high sample throughput, and minimal downtime in forensic laboratories.

Objectives and study overview

This study aimed to develop and validate a robust LC/MS/MS workflow for the quantitative determination of 67 drugs of abuse and metabolites in human whole blood. Key goals included accommodating a large sample volume (1 mL), achieving high recoveries and reproducibility, minimizing matrix effects through efficient lipid removal, and establishing method sensitivity suitable for forensic applications.

Methodology

Sample preparation combined in-cartridge protein precipitation (PPT) with cold 95/5 acetonitrile/methanol, followed by lipid cleanup using Agilent Captiva EMR–Lipid 6 mL cartridges. After initial elution, an additional wash with 80/20 acetonitrile/water ensured complete analyte recovery. Dried extracts were reconstituted in ammonium acetate buffer/acetonitrile, filtered through 0.2 μm regenerated cellulose vials, and directly injected.

Instrumentation used

• PPM-48 positive pressure manifold with Captiva EMR–Lipid 6 mL cartridges
• Agilent 1290 Infinity LC system with binary pump, autosampler, thermostatted column compartment
• Agilent ZORBAX Eclipse Plus C18 column (100 × 2.1 mm, 1.8 μm) with guard cartridge
• Agilent 6490 triple quadrupole mass spectrometer with JetStream ESI source
• Agilent MassHunter software for data acquisition and analysis

Main results and discussion

Lipid removal exceeded 99%, markedly reducing matrix ion suppression and instrument contamination. Most analytes achieved recoveries between 60 and 120% with RSDs below 20%. Calibration curves were linear (R² > 0.99) over 0.5–50 ng/mL for the majority of compounds, with method LOQs of 0.5–1 ng/mL and as low as 0.05 ng/mL for fentanyl. Accuracy (100 ± 20%) and precision (RSD < 20%) criteria were met across low, mid, and high QC levels. The workflow demonstrated high selectivity, negligible carryover, and robustness suitable for routine forensic analysis.

Benefits and practical applications

• Significant reduction of matrix effects and instrument fouling
• Large sample volume compatibility enhances detection of low-abundance analytes
• Simplified workflow with minimal manual steps improves throughput
• Reliable quantification across a broad panel of drugs facilitates comprehensive forensic screening

Future trends and potential applications

Advancements may include extension of the protocol to emerging psychoactive substances, integration with high-resolution MS for screening unknown compounds, automation of sample handling to further increase throughput, and adaptation to alternative matrices (e.g., oral fluid, tissues). The approach could also support clinical toxicology and environmental monitoring where lipid-rich matrices are encountered.

Conclusion

The combination of in-cartridge protein precipitation and Captiva EMR–Lipid cleanup, coupled with LC/MS/MS, provides a reliable, high-throughput workflow for quantitative analysis of a large panel of drugs of abuse in human whole blood. The method meets stringent forensic criteria for sensitivity, accuracy, and precision while minimizing matrix interferences and instrument downtime.

References

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• Limian Z. Quantitative Determination of Drugs of Abuse in Human Whole Blood by LC/MS/MS Using Agilent Captiva EMR–Lipid cleanup. Agilent Technologies Application Note 5991-9251EN, 2018.
• Limian Z. Quantitative Determination of Drugs of Abuse in Human Plasma and Serum by LC/MS/MS Using Agilent Captiva EMR–Lipid cleanup. Agilent Technologies Application Note 5991-9312EN, 2018.
• Limian Z.; Megan J. Protein Precipitation for Biological Fluid Samples Using Agilent Captiva EMR–Lipid 96-well Plates. Agilent Technologies Application Note 5991-9222EN, 2018.