Comparison of Epinephrine and Norepinephrine Analysis Using Core Enhanced Technology Accucore HPLC Columns with HILIC and aQ Stationary Phases

Importance of the Topic

Epinephrine and norepinephrine are critical catecholamine hormones and neurotransmitters whose accurate quantification in biological fluids is essential for diagnosing stress-related conditions, neuroendocrine tumors, and monitoring therapeutic interventions. High-performance liquid chromatography (HPLC) separations that combine speed, resolution, and compatibility with standard instrumentation improve clinical and research workflows.

Objectives and Study Overview

This study compared the performance of Thermo Scientific Accucore aQ and Accucore HILIC superficially porous HPLC columns for separating epinephrine and norepinephrine. It aimed to evaluate retention behaviour, separation efficiency, analysis time, and backpressure requirements under isocratic conditions.

Methodology

• Sample Preparation: Prepared 500 µg/mL standards of each analyte in 50:50 methanol:water, diluted to 50 µg/mL in the respective mobile phase.
• Chromatographic Conditions:
  • HILIC phase: Isocratic elution with 92:8 acetonitrile/100 mM ammonium formate (pH 3.2), flow rate 3.5 mL/min, column temperature 40 °C, injection volume 10 µL.
  • aQ phase: Isocratic elution with 5 mM ammonium formate (pH 3.2), flow rate 0.6 mL/min, column temperature 30 °C, injection volume 2 µL.
• Detection: UV absorbance at 230 nm.

Used Instrumentation

• Thermo Scientific HPLC system.
• Accucore HILIC column, 2.6 µm, 150 × 4.6 mm.
• Accucore aQ column, 2.6 µm, 100 × 2.1 mm.
• Fisher Scientific HPLC-grade solvents and reagents.
• Thermo Scientific Premium vials for sample handling.

Main Results and Discussion

• Retention and Separation: The aQ column provided minimal retention (0.37 min for epinephrine, 0.33 min for norepinephrine) with marginal peak resolution. The HILIC column achieved strong retention (4.01 min and 4.60 min) and baseline resolution in under 5 minutes.
• Backpressure: Measured at approximately 326 bar for aQ and 250 bar for HILIC, both well within the limits of conventional HPLC systems.
• Chromatographic Performance: Core enhanced particle technology delivered high-efficiency separations with lower backpressure compared to fully porous sub-2 µm materials.

Benefits and Practical Applications

• Rapid, high-resolution separation of catecholamines using standard HPLC equipment.
• Reduced instrument stress due to lower backpressure.
• Compatibility with routine clinical and QA/QC workflows.
• Simplicity of isocratic methods for reliable day-to-day operation.

Future Trends and Potential Applications

• Integration with mass spectrometry for enhanced sensitivity and selectivity.
• Expansion of superficially porous HILIC phases to other polar analytes.
• Development of gradient HILIC methods for complex biological samples.
• Adoption in high-throughput clinical and research laboratories.

Conclusion

Accucore HILIC columns offer superior retention and baseline separation of epinephrine and norepinephrine compared to Accucore aQ phases, while maintaining acceptable backpressure for standard HPLC systems. This makes HILIC columns well-suited for rapid and reliable catecholamine analysis in clinical and industrial settings.