Extraction of Cocaine and Metabolites from Whole Blood Using ISOLUTE® SLE+ Prior to GC/MS Analysis

Importance of the Topic

Reliable extraction and analysis of cocaine and its metabolites from whole blood is essential in forensic toxicology, clinical diagnostics, and anti‐doping efforts. Efficient sample preparation enhances sensitivity, reduces matrix effects, and accelerates turnaround times in demanding laboratory environments.

Study Objectives and Overview

This application note presents a streamlined protocol for isolating cocaine, benzoylecgonine, cocaethylene, and related analytes from whole blood using Supported Liquid Extraction (SLE+) prior to GC/MS analysis. The method aims to deliver clean extracts, consistent recoveries, and low limits of quantitation, while accommodating simultaneous extraction of other drug classes.

Methodology and Instrumentation

The sample preparation workflow employs 1 mL SLE+ columns for dilution‐based extraction without protein precipitation. Key steps include:

• Spiking 1 mL whole blood with internal standard (100 ng/mL), adding 1% aqueous ammonium hydroxide, and vortex mixing.
• Loading 750 µL treated blood onto the SLE+ cartridge and allowing absorption.
• Elution with two 2.5 mL portions of dichloromethane under gravity, followed by gentle vacuum.
• Evaporation to dryness under air or nitrogen and reconstitution in ethyl acetate, followed by derivatization with BSTFA + 1% t-BDMCS at 70 °C for 30 minutes.

Instrumentation for chromatographic analysis:

• Gas Chromatograph: Agilent 7890A with DB-5 column (30 m × 0.25 mm, 0.25 µm).
• Mass Spectrometer: Agilent 5975C in Selected Ion Monitoring mode.

Main Results and Discussion

Recovery experiments at 100 ng/mL demonstrated reproducible extractions with RSD values below 10%. Calibration curves spanning 10–500 ng/mL exhibited correlation coefficients (r2) between 0.9953 and 0.9995. Lower limits of quantitation ranged from 20 ng/mL for AEME to 50 ng/mL for cocaine and its major metabolites. The protocol avoided emulsion formation and delivered clean chromatograms, as evidenced by blank and spiked blood extracts.

Benefits and Practical Applications

The SLE+ approach offers significant advantages over traditional liquid–liquid extraction: reduced solvent consumption, elimination of protein precipitation steps, minimal matrix interference, and compatibility with automated platforms. Laboratories can employ this method for high-throughput toxicology screens, postmortem analyses, and therapeutic drug monitoring.

Future Trends and Potential Applications

Emerging developments in microelution SLE formats and integration with online SPE‐GC/MS systems will further streamline workflows. Adaptation to ultra-high sensitivity detectors and multiplexed analyses can expand applications to trace‐level forensic examinations and real‐time clinical monitoring.

Conclusion

The described SLE+ extraction method provides a robust, reproducible, and efficient protocol for quantifying cocaine and metabolites in whole blood. High recoveries, low LLOQs, and simplified sample handling make it a valuable tool for forensic and clinical laboratories.

References

1. Biotage Application Note AN856, 2015
2. Agilent Technologies GC/MS Operation Manuals