Determination and quantitation of catecholamines and metanephrines in urine with an efficient sample preparation and LC-MS/MS workflow for clinical research use

Importance of the Topic

Quantifying catecholamines and metanephrines in urine is essential for diagnosing and monitoring disorders such as pheochromocytoma and other neuroendocrine tumors. Reliable urinary measurements support early detection and guide therapeutic decisions in clinical research settings.

Goals and Study Overview

This study aimed to establish a streamlined, economical LC–MS/MS assay for the simultaneous quantitation of dopamine, epinephrine, norepinephrine, metanephrine, and normetanephrine in urine. Key performance metrics included sensitivity, linearity, accuracy, precision, and matrix effect assessment across a wide concentration range.

Methodology

Urine samples were processed via liquid–liquid extraction using ethyl acetate and a boronate complexing reagent at pH 9.5. After vortex mixing, centrifugation, evaporation under nitrogen, and reconstitution in aqueous mobile phase, samples were analyzed by LC–MS/MS employing scheduled SRM for each analyte and its stable-isotope internal standard.

Instrumentation Used

• Thermo Scientific UltiMate 3000 RS binary gradient HPLC system
• Acclaim Mixed-Mode WCX-1 column (2.1 × 150 mm, 3 µm) at 40 °C
• Thermo Scientific TSQ Endura triple quadrupole mass spectrometer with heated electrospray ionization

Key Results and Discussion

The assay displayed linearity from 0.4 to 900 ng/mL with R² > 0.998 and a lower limit of quantification (LLOQ) of 0.4 ng/mL. Intra- and inter-assay precision were below 15 % RSD at low concentrations and below 10 % RSD at higher levels. Accuracy bias remained within ±20 % for all analytes. Matrix effects ranged between 85 % and 130 % across mid-range calibrators. Chromatographic separation was achieved within a 10-minute gradient, and dual SRM transitions per compound ensured robust specificity.

Benefits and Practical Applications

• Avoids solid-phase extraction to reduce cost and simplify sample handling
• High throughput ideal for large-scale clinical research workflows
• Stable-isotope internal standards enhance quantification confidence

Future Trends and Opportunities

Further advances may include microflow LC or high-resolution MS integration for enhanced sensitivity. Automating the extraction protocol can improve reproducibility and throughput. Expanding the analyte panel to related metabolites could widen clinical diagnostic capabilities.

Conclusion

The developed LC–MS/MS method provides a rapid, accurate, and cost-effective solution for urinary catecholamine and metanephrine analysis in clinical research. Its robust performance and simplified workflow support high-volume testing and reliable biomarker assessment.

References

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