Rapid and Simultaneous Analysis of Urinary Catecholamines and Metanephrines Using Mixed-Mode SPE and Hydrophilic Interaction Chromatography (HILIC) for Clinical Research

Importance of the Topic

Accurate measurement of urinary catecholamines and metanephrines is crucial for diagnosing and monitoring neuroendocrine disorders such as pheochromocytoma and paraganglioma. These compounds are highly polar and present at low concentrations in complex urine matrices, posing challenges for conventional reversed-phase LC-MS/MS and electrochemical detection techniques. Efficient, high-throughput methods that minimize matrix effects and eliminate ion-pairing reagents are in high demand in clinical research and diagnostic laboratories.

Objectives and Study Overview

This study aimed to develop a rapid, simultaneous assay for six urinary analytes (dopamine, norepinephrine, epinephrine, 3-methoxytyramine, metanephrine, normetanephrine) by combining mixed-mode weak cation exchange solid-phase extraction (SPE) with hydrophilic interaction chromatography (HILIC) coupled to tandem mass spectrometry. The goal was to achieve baseline resolution, linear quantification (0.5–500 ng/mL), and robust performance without ion-pairing additives.

Methodology and Instrumentation

Sample pretreatment involved acidifying urine with 1 N HCl, spiking with stable isotope internal standards, and buffering with ammonium acetate. Extraction was performed on Oasis WCX 96-well plates, with conditioning (MeOH, water), sample loading, washing (ammonium acetate, MeOH), vacuum drying, and elution with acidified acetonitrile/water. Chromatographic separation used a Waters ACQUITY UPLC BEH Amide column (1.7 µm, 2.1×100 mm) with a gradient from high organic to aqueous ammonium formate buffers at pH 3.0. MS detection employed electrospray positive ionization, multiple reaction monitoring, and compound-specific cone voltages and collision energies. Data acquisition and quantification were carried out in MassLynx with TargetLynx.

Instrumentation

• Waters ACQUITY UPLC I-Class System
• Waters ACQUITY UPLC BEH Amide Column, 1.7 µm, 2.1×100 mm
• Waters Oasis WCX 30 mg 96-well Plates
• Waters Xevo TQD Mass Spectrometer
• MassLynx Software with TargetLynx

Main Results and Discussion

The HILIC method achieved clear baseline separation of all six analytes in under 2 minutes, notably improving resolution between epinephrine and normetanephrine (0.22 min vs. 0.05 min in reversed-phase). Recoveries ranged from 36% (norepinephrine) to 98.5% (3-methoxytyramine) with CVs below 5%. Matrix effects were minimized (≤ –10% for most analytes; contrasted with –60% under reversed-phase). Calibration curves (0.5–500 ng/mL) displayed excellent linearity (R² ≥ 0.992). Quality control samples at four spike levels showed accuracies within 10% and CVs generally under 10%.

Benefits and Practical Applications of the Method

• Simultaneous quantification without ion-pairing reagents
• High throughput suitable for clinical research and diagnostics
• Reduced matrix suppression and improved reproducibility
• Rapid analysis (< 4 min per sample) with baseline resolution

Future Trends and Potential Applications

The approach can be extended to other polar biomarkers and integrated into automated workflows. Advances in high-resolution mass spectrometry and microfluidic SPE formats may further increase sensitivity and throughput. This method holds promise for large-scale metabolomic profiling and point-of-care platforms.

Conclusion

The combined mixed-mode SPE and HILIC-MS/MS workflow provides a fast, robust, and accurate platform for urinary catecholamine and metanephrine analysis. It overcomes limitations of traditional ion-pairing methods, delivers excellent resolution and quantitation, and is well suited for clinical and research laboratories.

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