Reversed-phase UPLC-MS/MS Analysis of Plasma Catecholamines and Metanephrines For Clinical Research

Significance of the topic

• Accurate quantification of plasma catecholamines and their O-methylated metabolites (metanephrines) plays a vital role in diagnosing and researching neuroendocrine and cardiovascular disorders.
• Their high polarity presents a challenge for conventional reversed-phase LC-MS/MS, leading many labs to adopt more complex HILIC methods.

Objectives and study overview

This work aimed to develop and validate a streamlined reversed-phase UPLC-MS/MS workflow for quantitative analysis of six key catecholamines and metanephrines in human plasma, delivering fast turnaround, robust performance, and minimal matrix effects.

Methodology and instrumentation

• Sample preparation: 250 µL plasma mixed with ammonium acetate buffer and internal standard, loaded onto Oasis WCX µElution 96-well plates for mixed-mode weak cation exchange SPE.
• Plate processing: Sequential washes with ammonium acetate and methanol, elution with acidified methanol, evaporation, and reconstitution in water:acetonitrile:formic acid (97:2:1).
• Chromatography: ACQUITY UPLC I-Class PLUS System with Flow-Through Needle and ACQUITY UPLC HSS PFP Column (2.1 × 100 mm, 1.8 µm) using a water/acetonitrile gradient (0.1% formic acid), flow rate 0.5 mL/min, injection volume 5 µL.
• Mass spectrometry: Xevo TQ-S micro operated in positive-mode MRM; comparison of standard electrospray vs. UniSpray™ Ion Source for sensitivity gains.

Key results and discussion

• Linearity: Calibration curves linear from LLOQs (1–10 pg/mL) up to 20,000 pg/mL with R² > 0.999 for all analytes.
• Sensitivity and precision: LLOQs met acceptance criteria (±20% accuracy, ≤20% CV); between- and within-batch precision generally <10% at low and mid concentrations, <6% at higher levels.
• Recovery and matrix effects: Consistent extraction recoveries across five plasma lots (<20% CV) despite moderate absolute recoveries for some analytes; negligible matrix suppression/enhancement.
• Chromatographic resolution: All six analytes eluted within 1.5 minutes; baseline separation achieved for isobaric epinephrine and normetanephrine by selecting alternative precursor ions.
• Interference removal: Common co-medications (metformin, midodrine, L-DOPA) fully resolved from target peaks.
• Enhanced sensitivity: UniSpray ionization improved metanephrine signal intensity approximately five-fold compared to standard electrospray.

Benefits and practical applications

• Rapid throughput: Total cycle time <4 minutes simplifies high-volume clinical research studies.
• Robust performance: Mixed-mode SPE and unique PFP stationary phase deliver reproducible results with minimal sample transfer steps.
• Wide dynamic range: Enables accurate quantitation from trace to elevated concentrations relevant for physiological and pathological studies.

Future trends and prospects

• Integration of novel ionization sources (e.g., UniSpray) to further enhance sensitivity for low-abundance biomarkers.
• Expansion of multiplex panels to include additional neurotransmitter metabolites or conjugates, leveraging the high selectivity of PFP phases.
• Automation and miniaturization of sample preparation workflows using µElution formats to increase throughput and reduce solvent consumption.

Conclusion

A robust reversed-phase UPLC-MS/MS protocol has been established for clinical research quantification of plasma catecholamines and metanephrines, combining mixed-mode SPE, a tailored PFP column, and sensitive MRM detection. The method offers fast analysis, excellent linearity, precision, and minimal matrix interference, supporting high-throughput investigations into neuroendocrine function.

Reference

Danaceau J.P., Qianqian L., Balloch S., Calton L.J. Reversed-phase UPLC-MS/MS Analysis of Plasma Catecholamines and Metanephrines for Clinical Research. Tech. Brief. Waters Corporation, 2020.