Urinary Catecholamines, Metanephrines, and 3-Methoxytyramine in a Single LC/MS/MS Run

Significance of the Topic

Accurate measurement of urinary catecholamines, metanephrines, and 3-methoxytyramine is critical for diagnosing and monitoring neuroendocrine tumors, hypertension disorders, and metabolic syndromes. A streamlined LC/MS/MS workflow enhances laboratory throughput, reduces sample preparation complexity, and improves data reliability in clinical and research settings.

Objectives and Study Overview

This work presents the development and validation of a single-run LC/MS/MS assay for simultaneous quantitation of dopamine, epinephrine, norepinephrine, metanephrine, normetanephrine, and 3-methoxytyramine in human urine. Key goals included:

• Implementing a unified solid phase extraction (SPE) protocol to simplify sample cleanup
• Achieving low nanogram-per-milliliter sensitivity across a wide dynamic range (1.56–1,000 ng/mL for catecholamines; 4.69–3,000 ng/mL for metanephrines)
• Demonstrating excellent accuracy, precision, and linearity in a clinical research context

Methodology and Instrumentation

Sample Preparation

• Urine (0.5 mL) is spiked with deuterated internal standards and complexed with diphenyl-boronate agent at pH 7.5–9.5
• Agilent Bond Elut Plexa SPE cartridges are conditioned, washed, and samples are loaded, washed, and eluted with 5% formic acid in water
• Total assay time per sample is minimized via off-line SPE and single injection

LC/MS/MS Conditions

• Agilent 1290 Infinity LC with Pursuit PFP column (2 × 150 mm, 3 µm) at 40 °C
• Mobile phase A: 0.2% formic acid in water; B: methanol; gradient from 0%–60% B over 2.5 min, total run time 9 min
• Injection volume: 20 µL; flow rate: 0.3 mL/min; autosampler at 4 °C
• Agilent 6460 Triple Quadrupole with Jet Stream ESI+ and MassHunter software
• Multiple reaction monitoring transitions optimized for each analyte with deuterated internal standards

Main Results and Discussion

Chromatographic separation achieved baseline resolution of critical isobaric pairs (epinephrine vs. normetanephrine; metanephrine vs. 3-methoxytyramine). Absolute recoveries ranged from 53% to 112%, but internal standard correction yielded relative recoveries of 94%–104%. Calibration curves were highly linear (R² > 0.9997 for catecholamines; R² > 0.9999 for metanephrines/3-MT). Intraday and interday precision (CV) were below 6% at all levels, and accuracy stayed within ±8%. Bio-Rad Lyphocheck quality controls agreed with HPLC reference ranges, demonstrating method robustness.

Benefits and Practical Applications

This method allows clinical and research laboratories to:

• Perform comprehensive urinary catecholamine profiling in a single run
• Reduce manual sample handling and potential sources of error
• Maintain high throughput with <10 min cycle times
• Ensure reliable quantitation suitable for diagnostic investigations and longitudinal monitoring

Future Trends and Potential Applications

Emerging directions include:

• Automation of SPE workflows via online SPE coupling
• Extension to additional neurochemical markers and conjugated metabolites
• Integration with high-resolution mass spectrometry for broader untargeted screening
• Application to other biological matrices such as plasma and cerebrospinal fluid

Conclusion

The described LC/MS/MS assay delivers sensitive, precise, and high-throughput quantitation of six key urinary analytes using a single SPE and chromatographic run. It meets stringent clinical research criteria and supports routine laboratory implementation.

Used Instrumentation

• Agilent 1290 Infinity LC system with binary pump, thermostatted autosampler, and column compartment
• Agilent Pursuit PFP analytical column and MetaGuard guard column
• Agilent 6460 Triple Quadrupole Mass Spectrometer with Jet Stream ESI source
• Agilent MassHunter Software B.06.00 for data acquisition and quantitative analysis

References

1. Whiting MJ. Simultaneous measurement of urinary metanephrines and catecholamines by liquid chromatography with tandem mass spectrometric detection. Ann Clin Biochem. 2009;46:129–136.
2. Talwar D, et al. Extraction and separation of urinary catecholamines as their diphenyl boronate complexes using C18 solid-phase extraction sorbent and HPLC. J Chromatogr B. 2002;769:341–349.
3. Anon. Extraction of Catecholamines from Urine. AN1071A; Dr. Wéber Consulting KFT, Hungary.