Fast determination of plasma catecholamines and metanephrines by solid-phase extraction and liquid chromatography–tandem mass spectrometry applicated to clinical assay

Significance of the Topic

Plasma catecholamines and their O-methylated metabolites are critical biomarkers for the diagnosis of neuroendocrine tumors such as pheochromocytomas, paragangliomas and pediatric neuroblastomas. Accurate, sensitive and rapid quantification of dopamine, epinephrine, norepinephrine and their methoxylated products supports timely clinical decision-making and enhances laboratory throughput in challenging complex biological matrices.

Objectives and Study Overview

The study aimed to develop and validate a streamlined assay combining solid-phase extraction (SPE) and ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) for simultaneous quantification of six target analytes in human plasma. Key goals included achieving low limits of quantification, robust linearity and high precision compatible with routine clinical workflows.

Methodology and Instrumentation

The analytical process comprised:

• Sample Preparation: Human plasma spiked with standards was purified using an anion-exchange SPE plate (Evolute Express 96-Well). Deuterated internal standards compensated for matrix effects.
• Chromatography: Separation was performed on a pentafluorophenyl (PFP) UHPLC column (Nexera LC40 system) using a water/methanol gradient with suitable additives. Flow rate was 0.25 mL/min, column temperature 30 °C and injection volume 5 µL.
• Mass Spectrometry: A Shimadzu LCMS-8060NX triple quadrupole system operated in MRM mode with ultra-fast polarity switching (5 ms) and up to 555 transitions/sec. Quantifier and qualifier transitions were monitored for selectivity.
• Calibration and Quantification: Linear ranges spanned 1–100 nmol/L with correlation coefficients above 0.99. Low limits of quantification were determined at 0.1–0.5 nmol/L for all analytes.

Key Results and Discussion

The method delivered complete chromatographic separation in 9 minutes per run. Calibration models achieved r² values > 0.991. Intra-day precision (n = 30) showed RSDs below 6.5 %, and control sample accuracies ranged from 92 to 111 %. No significant carryover was detected. The combination of fast extraction and rapid LC-MS/MS analysis demonstrated high throughput potential without compromising data quality.

Benefits and Practical Applications

This assay supports sensitive detection of catecholamines and metanephrines at clinically relevant concentrations, enhancing diagnostic confidence. The streamlined workflow and use of deuterated internal standards ensure reproducibility and robust compensation for matrix variability, making the method suitable for routine endocrine, neurological and pediatric diagnostics.

Future Trends and Opportunities

Further developments may include full automation of SPE-LC-MS/MS workflows, miniaturized sample preparation formats, expansion of targeted panels to encompass additional neurochemical biomarkers and integration with laboratory information management systems. Advances in high-resolution MS and data processing could further improve sensitivity, throughput and personalized medicine applications.

Conclusion

The validated SPE-UHPLC-MS/MS method achieves rapid, precise and sensitive quantification of plasma catecholamines and their methoxylated derivatives. Its strong performance metrics and compatibility with clinical laboratory operations make it a valuable tool for neuroendocrine disorder screening and monitoring.

References

• Finkielstain G. (Paula), Jha S., Merke D. Chapter 9 – Adrenal disorders, in Biochemical and Molecular Basis of Pediatric Disease, Fifth Edition, 2021; pp 267–296.