Quantitative Determination of Drugs of Abuse in Human Plasma and Serum by LC/MS/MS Using Agilent Captiva EMR—Lipid Cleanup

Significance of the Topic

In forensic toxicology, rapid and reliable detection of drugs of abuse in blood matrices is critical for legal and clinical decision making. Removing proteins and lipids prior to LC/MS/MS analysis minimizes matrix effects and system contamination, improving accuracy and throughput.

Objectives and Study Overview

This application note describes a high-throughput LC/MS/MS method for quantifying 24 representative drugs of abuse in human plasma and serum, employing a 96-well plate format with in-well protein precipitation followed by Agilent Captiva EMR–Lipid cleanup.

• Integrate precipitation and lipid removal in a single plate
• Evaluate method performance (accuracy, precision, LOQ, linearity)
• Cross-verify across four common blood matrices

Methodology and Instrumentation

Samples and calibration/QC standards were prepared in serum and plasma (Li-heparin, Na-citrate, K2EDTA). In-well protein precipitation was performed by adding crashing solvent directly to the sample well, followed by phospholipid removal on Captiva EMR–Lipid sorbent. Elution was achieved using a positive pressure manifold. Calibration curves ranged from 0.1–0.5 to 20 ng/mL with 1/x2 weighting.

Instrumentation

• Agilent 1290 Infinity LC system (binary pump, autosampler, column compartment)
• Agilent 6490 triple quadrupole LC/MS with Jet Stream iFunnel source
• InfinityLab Poroshell 120 EC-C8 column (100 × 2.1 mm, 2.7 µm) with C18 guard
• Agilent Captiva EMR–Lipid 96-well plates, PPM-96 positive pressure manifold, centrifuge

Results and Discussion

• Phospholipid removal exceeded 99 %, significantly reducing ion suppression and carryover.
• LOQs of 0.1–0.5 ng/mL achieved for most analytes; heroin and amphetamine at 0.5 ng/mL.
• Calibration linearity R2 > 0.99; accuracy 100 ± 20 %; precision RSD < 20 % across all QCs.
• Cross-verification confirmed consistent performance in serum and all plasma anticoagulants.
• Sample-first precipitation enhanced mixing homogeneity and minimized consumable use.

Practical Applications and Benefits

• Enables processing of 96 samples in under 2 hours for high-throughput labs.
• Streamlined workflow reduces manual steps and solvent consumption.
• Robust quantitation supports DUID and routine forensic testing.
• Cleaner extracts extend LC/MS/MS uptime and reduce maintenance.

Future Trends and Opportunities

The integration of advanced sorbent chemistry with automation platforms will further enhance throughput and reproducibility. Emerging cleanup materials and microflow LC/MS approaches may extend applications to clinical toxicology, environmental monitoring, and metabolomics.

Conclusion

The validated 96-well plate LC/MS/MS method with Agilent Captiva EMR–Lipid cleanup offers a fast, reliable, and high-throughput solution for quantifying drugs of abuse in plasma and serum. Superior matrix cleanup, robust performance across matrices, and simplified sample processing make it ideal for forensic and clinical laboratories.

Reference

1. Saito, K.; et al. Analysis of Drugs of Abuse in Biological Specimens. J. Health Sci. 2011, 57(6), 472–487.
2. Moeller, M.R.; Steinmeyer, S.; Kraemer, T. Determination of Drugs of Abuse in Blood. J. Chromatogr. B. 1998, 713, 91–109.
3. Moeller, M.R.; Kraemer, T. Drugs of Abuse Monitoring in Blood for Control of Driving Under the Influence of Drugs. Ther. Drug Monit. 2002, 24, 210–221.
4. Zhao, L.; Lucas, D. Efficiency of Biological Fluid Matrix Removal Using Agilent Captiva EMR–Lipid Cleanup. Agilent Technologies Application Note. 2017.
5. Zhao, L.; Juck, M. Quantitative Determination of Drugs of Abuse in Human Whole Blood by LC/MS/MS Using Agilent Captiva EMR–Lipid Cleanup. Agilent Technologies Application Note. 2018.
6. Zhao, L.; Juck, M. Protein Precipitation for Biological Fluid Samples Using Agilent Captiva EMR–Lipid 96-well Plates. Agilent Technologies Application Note. 2018.