Rapid and Sensitive Quantitation of Metanephrines and 3-Methoxytyramine in Human Plasma Using an Agilent Ultivo Triple Quadrupole LC/MS

Importance of the Topic

Measurement of metanephrines and 3-methoxytyramine in plasma is critical for the accurate diagnosis and monitoring of catecholamine-related disorders such as pheochromocytoma and paraganglioma. A rapid, sensitive, and specific analytical method supports timely clinical decision-making and improves patient outcomes.

Objectives and Study Overview

This work describes the development and evaluation of a fast LC/MS/MS workflow using the Agilent Ultivo triple quadrupole system for simultaneous quantitation of metanephrine (MN), normetanephrine (NMN), and 3-methoxytyramine (3-MT) in human plasma. The study aimed to achieve baseline separation within four minutes, low detection limits, and robust method performance.

Methodology and Instrumentation Used

• Sample Preparation: Solid-phase extraction (SPE) with strong cation exchange (WCX) cartridges on a positive pressure manifold (PPM-48) to remove interferences and concentrate analytes.
• Chromatography: Agilent 1260 Infinity II LC system with Pursuit 3 PFP column (3.0 × 100 mm, 3 µm) and a 10-minute gradient (5%–95% methanol with 0.1% formic acid).
• Mass Spectrometry: Agilent Ultivo LC/TQ with Jet Stream ESI source in positive mode. Multiple reaction monitoring (MRM) transitions optimized via MassHunter software, with deuterated internal standards for each analyte.

Main Results and Discussion

• Sensitivity: Limits of detection were 4 ng/L for MN and 3-MT, and 20 ng/L for NMN. Limits of quantitation corresponded to 10 ng/L for MN and 3-MT, and 40 ng/L for NMN.
• Linearity and Accuracy: Calibration curves (six levels) exhibited R² > 0.999. Accuracy across the linear range was 90%–110%, with CV < 4%.
• Precision and Recovery: Intra- and interday recoveries for three QC levels ranged from 90% to 110% (RSD ≤ 5%), and interday reproducibility was ≤ 4% RSD. Carryover was negligible (< 0.02% vs highest calibrator).
• Chromatographic Performance: All three analytes were baseline-separated in under four minutes, and simultaneous analysis of native catecholamines (norepinephrine, epinephrine, dopamine) was demonstrated.

Benefits and Practical Applications of the Method

This rapid and sensitive LC/MS/MS approach offers a robust alternative to traditional HPLC-ECD or HPLC-FLD for plasma metanephrine analysis. It supports high-throughput clinical and research laboratories, enables accurate monitoring of catecholamine metabolism, and may improve turnaround times in diagnostic workflows.

Future Trends and Potential Applications

Further developments may include full multiplexed panels of catecholamine metabolites, integration with automated SPE platforms for higher throughput, and application to additional biological matrices. Advances in MS hardware and software could further lower detection limits and enhance quantitation in low-abundance scenarios.

Conclusion

The described Agilent Ultivo LC/TQ workflow combines efficient SPE cleanup with fast PFP-based chromatography and sensitive MRM detection to deliver reliable quantitation of metanephrines and 3-methoxytyramine in plasma. Its robust performance metrics make it well-suited for routine clinical research and diagnostic applications.

References

• Lee SY, Cazenave-Gassiot A, Pakkiri LS, Drum CL, Wenk MR, Bendt AK. Rapid and Sensitive Quantitation of Metanephrines and 3-Methoxytyramine in Human Plasma Using an Agilent Ultivo Triple Quadrupole LC/MS. Agilent Technologies Application Note. 2022;5994-4729EN.