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

Importance of the Topic

The accurate measurement of plasma catecholamines and their O-methylated metabolites (metanephrines) is critical for both clinical research and diagnostic applications, such as monitoring stress responses, diagnosing pheochromocytoma, and investigating neurochemical disorders.

Polar nature and low abundance of these analytes pose significant challenges in traditional reversed-phase LC-MS/MS methods, often requiring ion-pairing reagents and electrochemical detection to achieve sufficient retention and resolution.

Hydrophilic interaction chromatography (HILIC) combined with mixed-mode solid-phase extraction (SPE) offers a promising alternative that enhances selectivity, reduces matrix effects, and accelerates sample throughput.

Study Objectives and Overview

This study aimed to develop and validate a rapid, sensitive, and simultaneous assay for six key analytes—dopamine, norepinephrine, epinephrine, 3-methoxytyramine, metanephrine, and normetanephrine—in human plasma.

The workflow integrates Oasis WCX 96-well μElution plates for sample preparation with an ACQUITY UPLC BEH Amide column operated in HILIC mode, coupled to a Xevo TQ-S triple quadrupole mass spectrometer.

The method was evaluated for recovery, matrix effects, linearity, accuracy, precision, and endogenous concentration determination using standard addition.

Methodology and Instrumentation

Sample preparation:

• Human plasma (250 µL) spiked with 50 mM ammonium acetate buffer and deuterated internal standards.
• Conditioned Oasis WCX μElution plates used for mixed-mode weak cation exchange SPE.
• Washing steps: 20 mM ammonium acetate, then 50:50 acetonitrile:isopropanol.
• Elution with 85:15 acetonitrile:water containing 2% formic acid in two 25 µL steps, affording 5× on-column enrichment without evaporation.

Chromatography and detection:

• ACQUITY UPLC BEH Amide Column (1.7 µm, 2.1 × 100 mm) at 30 °C with a 4.0-min gradient elution.
• Mobile phases: A – 95:5 water:acetonitrile with 30 mM ammonium formate, pH 3.0; B – 15:85 water:acetonitrile with 30 mM ammonium formate, pH 3.0.
• Xevo TQ-S MS in positive ESI mode; compound-specific cone voltages and collision energies for MRM transitions.
• Data acquisition and processing via UNIFI Scientific Information System.

Main Results and Discussion

Chromatographic separation achieved baseline resolution between epinephrine and normetanephrine despite identical formulas, preventing cross-interference.

Calibration linearity:

• 3-methoxytyramine, metanephrine, normetanephrine, and dopamine: 10–2,000 pg/mL (R² ≥ 0.9993).
• Epinephrine and norepinephrine: 50–10,000 pg/mL (R² ≥ 0.9990).

Recovery and matrix effects:

• Mean recoveries of 76.4% across analytes (54%–90%).
• Matrix effects averaged –6.9%, with the highest suppression observed for dopamine (–22%) and norepinephrine (–23%).

Quality control:

• QC samples at four concentration levels demonstrated accuracies within ±10% and precision (%CV) < 10% for all analytes.

Endogenous concentrations measured in pooled plasma: 3-MT (7 pg/mL), metanephrine (31.7 pg/mL), normetanephrine (70.6 pg/mL), dopamine (0 pg/mL), epinephrine (29.4 pg/mL), norepinephrine (360.9 pg/mL).

Benefits and Practical Applications

This method delivers rapid (4 min cycle) and simultaneous analysis of six polar analytes with minimal sample handling.

Key advantages:

• No need for evaporation or reconstitution, reducing sample loss and contamination risk.
• Small elution volume enables on-column concentration, enhancing sensitivity to 10 pg/mL.
• Robust removal of matrix interferences via mixed-mode SPE combined with HILIC separations.
• Applicable to clinical research, drug monitoring, and diagnostic assays for neurochemical and endocrine disorders.

Future Trends and Potential Applications

Integration of HILIC-SPE workflows with high-throughput autosamplers and 384-well formats to increase sample throughput.

Expansion to include additional polar metabolites, such as serotonin and its derivatives, by tailoring SPE chemistries and MS parameters.

Implementation of microfluidic SPE cartridges and ultrafast HILIC columns to further reduce cycle times and solvent consumption.

Application to other biological matrices (urine, cerebrospinal fluid) for broader neuroendocrine and metabolomic profiling.

Conclusion

A mixed-mode SPE and HILIC-MS/MS assay was established for simultaneous quantification of plasma catecholamines and metanephrines.

The method demonstrates excellent linearity, sensitivity, accuracy, and precision while minimizing matrix effects and sample preparation time.

This workflow supports clinical research and diagnostic needs for rapid neurochemical profiling.

Reference

1. Cubbon S, Antonio C, Wilson J, Thomas-Oates J. Metabolomic applications of HILIC-LC-MS. Mass Spectrom Rev. 2010;29(5):671–84.
2. Jian W, Edom RW, Xu Y, Weng N. Recent advances in application of hydrophilic interaction chromatography for quantitative bioanalysis. J Sep Sci. 2010;33(6-7):681–97.
3. Xu RN, Rieser MJ, El-Shourbagy TA. Bioanalytical hydrophilic interaction chromatography: recent challenges, solutions and applications. Bioanalysis. 2009;1(1):239–53.
4. Jian W, Xu Y, Edom RW, Weng N. Analysis of polar metabolites by hydrophilic interaction chromatography–MS/MS. Bioanalysis. 2011;3(8):899–912.
5. Hemström P, Irgum K. Hydrophilic interaction chromatography. J Sep Sci. 2006;29(12):1784–821.
6. Danaceau JP, Chambers EE, Fountain KJ. Hydrophilic interaction chromatography (HILIC) for LC-MS/MS analysis of monoamine neurotransmitters. Bioanalysis. 2012;4(7):783–94.
7. Peaston RT, Graham KS, Chambers E, van der Molen JC, Ball S. Performance of plasma free metanephrines measured by liquid chromatography-tandem mass spectrometry in the diagnosis of pheochromocytoma. Clin Chim Acta. 2010;411(7-8):546–52.