Extraction of Amphetamine and Designer Amphetamine Analogues from Whole Blood Using ISOLUTE® SLE+ Prior to GC/MS Analysis

Significance of the Topic

Reliable measurement of amphetamines and designer analogues in whole blood is essential in forensic toxicology, clinical monitoring, and workplace drug testing. Achieving high recovery, low detection limits and robust reproducibility supports accurate interpretation of drug exposure and compliance with regulatory standards.

Objectives and Study Overview

This application note evaluates a streamlined supported liquid extraction (SLE) workflow using ISOLUTE SLE+ 1 mL columns for simultaneous isolation of amphetamine-type stimulants from whole blood prior to GC/MS analysis. The study also compares methyl tert-butyl ether (MTBE) and dichloromethane (DCM) as elution solvents and explores simultaneous extraction of other drug classes.

Methodology and Instrumentation

Sample pretreatment involves spiking 1 mL of whole blood with deuterated internal standard, adjusting pH with 1 % ammonium hydroxide, and loading 750 µL onto the SLE column. After equilibration, analytes are eluted in two 2.5 mL aliquots of MTBE or DCM into tubes containing 0.2 M HCl in methanol to stabilize free bases. Extracts are evaporated under nitrogen or air, reconstituted in ethyl acetate, derivatized with pentafluoropropionic anhydride at 70 °C for 20 minutes, and transferred to GC vials.

Used Instrumentation

• GC: Agilent 7890A with splitless inlet and backflush capability
• MS: Agilent 5975C in selected ion monitoring (SIM) mode
• Column: Agilent J&W DB-5, 30 m × 0.25 mm ID × 0.25 µm
• Evaporation: TurboVap LV or SPE Dry concentrator at ambient temperature, 20–40 L/min

Main Results and Discussion

Recoveries for 18 amphetamine analogues spiked at 100 ng/mL exceeded 80 % with relative standard deviations below 10 % for both MTBE and DCM protocols. Calibration curves from 10 to 500 ng/mL displayed excellent linearity (r2 between 0.9939 and 0.9999). Lower limits of quantitation ranged from 10 to 100 ng/mL depending on the analyte and choice of solvent. MTBE delivered optimum performance for amphetamines, while DCM enabled concurrent extraction of barbiturates, benzodiazepines and opiates without compromising amphetamine recoveries.

Benefits and Practical Applications

• Simplified workflow without protein precipitation or emulsions
• High analyte recovery and reproducibility across multiple psychoactive drug classes
• Low LLOQs suitable for forensic and clinical toxicology
• Compatibility with high-throughput platforms and automated manifolds

Future Trends and Potential Applications

• Integration of SLE with online automation for increased sample throughput
• Expansion to novel psychoactive substances and metabolites
• Adaptation to liquid chromatography–mass spectrometry (LC-MS) and tandem MS workflows
• Miniaturization of SLE formats for microsampling and remote testing applications

Conclusion

The ISOLUTE SLE+ workflow offers a robust, high-throughput extraction method for amphetamine-type stimulants in whole blood. It achieves clean extracts, strong recoveries, low detection limits and excellent linearity, making it a valuable tool for forensic, clinical and workplace drug testing laboratories.

References

Biotage Application Note AN855, 2015. Extraction of Amphetamine and Designer Amphetamine Analogues from Whole Blood by ISOLUTE SLE+ prior to GC/MS Analysis.