Extraction of Urinary Catecholamines and Metanephrines Using EVOLUTE® EXPRESS WCX SPE Prior to LC-MS/MS Analysis

Importance of the Topic

The accurate measurement of urinary catecholamines and metanephrines is essential for diagnosing neuroendocrine disorders and monitoring metabolic pathways. These compounds often occur at trace levels in complex biological matrices, making reliable sample preparation and high-sensitivity detection critical for clinical and research laboratories.

Objectives and Study Overview

This application note introduces a mixed-mode weak cation exchange (WCX) solid phase extraction (SPE) method to isolate epinephrine, norepinephrine, dopamine and their O-methylated metabolites from urine. The goals were to simplify workflow, eliminate conditioning steps, achieve reproducible recoveries and integrate seamlessly with LC-MS/MS analysis.

Methodology and Instrumentation

A rapid load-wash-elute protocol was developed using EVOLUTE EXPRESS WCX 10 mg 96-well plates. Key steps include:

• Pretreatment: 75 µL urine mixed with deuterated internal standards and 250 mM ammonium acetate.
• Washes: 10 mM ammonium acetate followed by isopropanol to remove matrix interferences.
• Elution: Direct elution with water/isopropanol (85:15) containing 0.1% formic acid; no evaporation required.
• Instrumentation: Shimadzu Nexera UHPLC with ACE Excel 1.7 µm C18-PFP column (100×2.1 mm); AB Sciex 5500 triple quadrupole operating in positive electrospray MRM mode; Biotage PRESSURE+ 96 manifold or automated Biotage Extrahera system.

Main Results and Discussion

Recovery at top calibration levels ranged from 69% to 108% with RSDs below 9% for all analytes. Linearity was established over clinically relevant concentration ranges with correlation coefficients above 0.99. Both manual and automated workflows produced comparable performance. The streamlined SPE procedure reduced hands-on time, solvent consumption and potential handling errors.

Benefits and Practical Applications

• High throughput: 96 samples processed in under one hour with automation.
• Elimination of conditioning and equilibration steps reduces consumables and preparation time.
• Direct elution without evaporation preserves analyte integrity and minimizes carry-over.
• Applicable to clinical diagnostics, pharmacokinetic studies and QA/QC in pharmaceutical and research settings.

Future Trends and Opportunities

Advances in automated SPE, novel sorbent chemistries and microelution formats will drive even higher throughput and lower detection limits. Integration of online SPE with UHPLC-MS/MS and data analytics is expected to enable real-time biomarker monitoring for personalized diagnostics.

Conclusion

The EVOLUTE EXPRESS WCX SPE method offers a fast, robust and scalable solution for extracting urinary catecholamines and metanephrines prior to LC-MS/MS. Its simplified load-wash-elute workflow delivers high recoveries, excellent precision and seamless integration with both manual and automated platforms.