Determination of Catecholamines by Agilent Ultivo LC/TQ with an InfinityLab Poroshell 120 Aq‑C18 Column

Significance of the Topic

The quantification of catecholamines—dopamine, epinephrine, norepinephrine and their metabolites—is critical for clinical research, neuroscience and cardiovascular diagnostics. These highly polar neurotransmitters regulate key physiological processes, and accurate measurement in complex biological matrices supports disease monitoring, pharmacokinetic studies and research into stress responses.

Objectives and Study Overview

This application note presents a validated method for simultaneous determination of six catecholamine compounds (3-MT, dopamine, epinephrine, norepinephrine, methoxy­epinephrine and methoxy­norepinephrine) in bovine serum albumin (BSA) matrices. The goals were to achieve baseline separation, high sensitivity and broad linear dynamic range using Agilent’s Ultivo LC/TQ system coupled with an InfinityLab Poroshell 120 Aq-C18 column.

Methodology and Instrumentation

Sample Preparation and Standards:

• Stock solutions (1,000 ng/mL) of each catecholamine prepared in 0.1% formic acid aqueous solution and serially diluted to 50–5,000 pg/mL.
• Matrix standards generated by spiking 100 µL of each dilution into 900 µL of 1% BSA in water (0.1% formic acid, 25 mg/mL citric acid), yielding 5–500 pg/mL working solutions.
• Internal standard solution (10 ng/mL) of isotopically labeled analogs prepared in 0.1% formic acid water.
• Protein precipitation performed by adding 80 µL of 10% trichloroacetic acid to 200 µL of matrix standard plus 5 µL internal standard, followed by centrifugation and supernatant collection.

Used Instrumentation:

• Agilent 1260 Infinity II LC with binary pump, high-throughput autosampler and column compartment.
• Agilent Ultivo LC/TQ mass spectrometer with Jet Stream Technology ion source (positive mode).
• InfinityLab Poroshell 120 Aq-C18 column (4.6 × 100 mm, 2.7 µm).
• MassHunter acquisition, qualification and quantification software; low-temperature centrifuge; vortex mixer.

LC Conditions:

• Mobile phase A: 10 mM ammonium acetate + 0.25 mM ammonium fluoride in water; B: acetonitrile.
• Gradient from 2% B to 95% B over 7 min; flow rate 0.5 mL/min; column temperature 40 °C; injection volume 25 µL.

MS Conditions:

• MRM transitions optimized for each analyte and corresponding isotope standard.
• Capillary voltage 2,000 V; nebulizer gas 45 psi; drying gas 180 °C at 5 L/min; sheath gas 370 °C at 11 L/min.

Results and Discussion

Baseline separation of all six analytes was achieved within a 7 min run time. Calibration curves were linear over 5–500 pg/mL (10–500 pg/mL for epinephrine) with correlation coefficients (R2) above 0.996. Recovery of spiked quality controls ranged from 80% to 120%, demonstrating accuracy and reproducibility in BSA matrices.

Benefits and Practical Applications

This method offers:

• High sensitivity for low-picogram quantification.
• Fast analysis with excellent peak shape and resolution.
• Robust performance in protein-rich matrices.
• Applicability to clinical research, pharmaceutical development and QA/QC laboratories.

Future Trends and Potential Applications

Advances may include automated sample preparation, further miniaturization of flow rates for enhanced sensitivity, and extension to real clinical matrices such as plasma or cerebrospinal fluid. Integration with high-throughput workflows and application to broader panels of polar metabolites will expand utility.

Conclusion

The Agilent Ultivo LC/TQ system combined with the InfinityLab Poroshell 120 Aq-C18 column provides a rapid, sensitive and reliable approach for catecholamine quantification in biomatrix simulations. Its wide dynamic range and robust performance support diverse applications in research and diagnostics.

Reference

1. Fu R.-J.; Wei T.-C. Analysis of Polar Compounds Using an Agilent InfinityLab Poroshell 120 Aq-C18 Column with Improved and Reliable Performance. Agilent Technologies Application Note, publication 5994-5555EN, 2022.