Extraction of Catecholamines and Metanephrines from Human Plasma using EVOLUTE® EXPRESS WCX Prior to UHPLC-MS/MS Analysis

Significance of the topic

An accurate and sensitive assay for catecholamines and metanephrines in plasma is essential for diagnosing and monitoring neuroendocrine disorders such as pheochromocytoma and Parkinson’s disease. These biomarkers occur at low concentrations in a complex biological matrix, demanding a robust sample preparation workflow to ensure quantitative recovery, minimal matrix interference, and clinically relevant limits of quantitation.

Objectives and study overview

This application note evaluates a solid-phase extraction (SPE) method using EVOLUTE EXPRESS WCX to isolate six analytes—norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine, and 3-methoxytyramine—from human plasma treated with various anticoagulants. The goal is to achieve high recovery, precise quantitation, and compatibility with UHPLC-MS/MS analysis.

Methodology and instrumentation

• Sample pre-treatment: Centrifuge plasma at 6,000×g for 10 min, spike with deuterated internal standards, and dilute 1:1 with 10 mM sodium citrate pH 7.
• SPE protocol (96-well EVOLUTE EXPRESS WCX, 30 mg sorbent): Condition with methanol and 10 mM ammonium acetate pH 6; load 600 µL sample; wash aqueous interferences with ammonium acetate, neutral organics with 80:20 MeOH/H₂O, and lipophilics with dichloromethane; elute with 85:15 H₂O/2-propanol containing 0.1% formic acid; dry and reconstitute in 95:5 H₂O/MeOH with 0.1% formic acid.
• UHPLC: Shimadzu Nexera with Avantor ACE Excel 1.7 µm C18-PFP column (100×3.0 mm) and Raptor ARC-18 guard; gradient from 2 mM ammonium formate (0.05% formic acid) in water to 0.5 mM ammonium fluoride in methanol at 0.5 mL/min, 30 °C.
• MS/MS: AB SCIEX Triple Quad 5500, ESI+ mode, scheduled MRM.

Main results and discussions

• Extraction recoveries exceeded 90% for most analytes (61–103% across matrices), with intra- and inter-day precision below 5% RSD.
• Calibration linearity was excellent over 10–500 pg/mL (r² > 0.995) and LOQs ranged from <0.01 to 0.12 nmol/L depending on the analyte.
• Matrix factors and signal factors across lithium/heparin, sodium/heparin, EDTA, and citrate plasma were within 0.8–1.1, demonstrating minimal matrix effects.
• Phospholipid profiling confirmed efficient removal of lipid interferences compared to protein precipitation.

Benefits and practical applications

• High throughput 96-well format with consistent performance across common anticoagulants.
• Clinically relevant sensitivity without derivatization, enabling direct quantitation in routine diagnostics.
• Reduced system maintenance due to effective removal of matrix and phospholipids.

Future trends and opportunities

• Automation and on-line SPE coupling to further increase throughput and reproducibility.
• Adaptation to other polar biomarker panels and miniaturized sample volumes.
• Incorporation of novel sorbent chemistries for simultaneous broad-spectrum metabolite extraction.

Conclusion

The EVOLUTE EXPRESS WCX SPE method delivers reliable, high‐recovery extraction of catecholamines and metanephrines from plasma, supports sensitive UHPLC-MS/MS quantitation, and demonstrates robustness across anticoagulant matrices. This workflow simplifies clinical bioanalysis and offers a platform for expanding targeted assays in metabolomics.

Reference

• Biotage, 2016. Simultaneous Extraction of Catecholamines and Metanephrines from Plasma Prior to Analysis using LC-MS/MS. Poster P152, MSACL-EU 2016.