Separation of Enantiomers of Amphetamine-Related Drugs and Their Structural Isomers

Importance of the topic

The separation of enantiomers and positional isomers of amphetamine-related psychoactive compounds is crucial in forensic and toxicological analysis. These stereoisomers can exhibit distinct pharmacological and toxicological profiles, affecting both legal classification and health risk assessments. High-resolution methods capable of clear chiral separation facilitate reliable identification and quantification, supporting criminal investigations and quality control.

Aim and overview of the study

This study demonstrates the application of supercritical fluid chromatography (SFC) on the Agilent 1260 Infinity II SFC system coupled to single and triple quadrupole mass spectrometry (MS) detectors. It focuses on separating six stereoisomers of 4-, 5-, and 6-EAPB, as well as resolving the corresponding structural isomers without chiral discrimination. The objective is to establish robust, sensitive, and rapid workflows for forensic laboratories.

Methodology and used instrumentation

Analytical methods were developed on chiral CHIRALPAK AD-H (250×4.6 mm, 5 µm) and CHIRALPAK AD-3 (150×4.6 mm, 3 µm) columns using supercritical CO2 with a methanol modifier containing 0.1 % aqueous ammonia. Key parameters were:

• Column temperature: 20 °C (stereoisomers) and 30 °C (structural isomers)
• Flow rates: 3 mL/min (chiral separation) and 2.5 mL/min (achiral separation)
• Modifier concentration: 10 % MeOH for chiral, 11 % MeOH for achiral
• Run times: 7 min for six-isomer separation; 3.5 min for three-isomer separation

Coupling to MS was performed using:

• Agilent 1260 Infinity II SFC Control Module, Pump, Multisampler, DAD, Thermostat, and SFC/MS Splitter Kit
• Agilent 6495 Triple Quadrupole MS with Jet Stream and iFunnel
• Agilent 6150 Single Quadrupole MSD

Main results and discussion

The optimized chiral SFC method achieved baseline separation of all six EAPB stereoisomers between 3.2 and 5.5 min. Calibration curves from 100 ppt to 100 ppb exhibited excellent linearity (R2 > 0.999) with LOQ at 100 ppt and LOD at 30 ppt. For structural isomers, the achiral method on CHIRALPAK AD-3 resolved 4-, 5-, and 6-EAPB in under 3.5 min. Positive electrospray ionization and MRM (triple quadrupole) or SIM (single quadrupole) modes provided selective detection.

Benefits and practical applications

This SFC-MS approach offers:

• High throughput with short analysis times and reduced solvent consumption
• Superior resolution for challenging stereoisomeric separations
• Trace-level sensitivity suitable for forensic toxicology and QA/QC
• Compatibility with existing Agilent SFC and MS platforms for seamless integration

Future trends and possibilities

Advancements may include:

• Development of novel chiral stationary phases for broader chemical classes
• Integration with high-resolution MS for enhanced structural information
• Automation and AI-driven method optimization
• Miniaturized SFC systems for field-deployable forensic analysis

Conclusion

The presented Agilent 1260 Infinity II SFC-MS workflows enable rapid, sensitive, and reproducible separation of amphetamine-related isomers. These methods support forensic investigations by delivering reliable qualitative and quantitative data at trace levels.

References

• Taschwer M., Hofer M.G., Schmid M.G. Enantioseparation of benzofurans by CE with sulfobutylether β-cyclodextrin. Electrophoresis 2014, 35, 2793–2799.
• Agilent Technologies Technical Note: Use of the SFC-MS Splitter Kit G4309-68715, 2015.
• Muelek M., Godel H., Naegele E. Chiral Separation of D/L-Amphetamine. Agilent Technologies Application Note 5991-8262EN, 2017.