Extraction of Catecholamines and Metanephrines from Human Plasma Biotage® Extrahera™ LV-200 and Low Volume SPE Prior to UHPLC-MS/MS Analysis

Significance of the Topic

The accurate measurement of catecholamines and metanephrines in human plasma is essential for the diagnosis and management of adrenal and neurological disorders. Automated sample preparation improves throughput, repeatability and reduces manual errors, supporting high-volume clinical and research laboratories.

Objectives and Study Overview

This work describes the development and validation of a mixed-mode solid phase extraction (SPE) method for six analytes (epinephrine, norepinephrine, dopamine, normetanephrine, metanephrine and 3-methoxytyramine) in plasma. Two approaches were compared: manual processing with the Biotage PRESSURE+ 96 manifold and automated processing using the Biotage Extrahera LV-200. Key performance metrics—recovery, precision, linearity, limits of quantitation and matrix effects—were evaluated for both workflows.

Methodology

Sample pretreatment involved centrifugation and spiking with stable isotope internal standards. After pH adjustment with sodium citrate buffer, analytes were extracted on EVOLUTE EXPRESS WCX 10 mg plates. The SPE sequence comprised conditioning (methanol, ammonium acetate), sample loading, aqueous and organic washes to remove proteins, phospholipids and lipids, followed by elution with water/isopropanol containing formic acid. Extracts were dried, reconstituted and analyzed by UHPLC-MS/MS under scheduled MRM conditions. Calibration used stripped plasma with an 80–500 pg/mL range and internal standard at 200 pg/mL.

Used Instrumentation

• Biotage Extrahera LV-200 automated SPE system and Biotage PRESSURE+ 96 positive pressure manifold
• EVOLUTE EXPRESS WCX 10 mg fixed-well SPE plates
• Shimadzu Nexera UHPLC with Avantor ACE Excel 1.7 μm C18-PFP column and RESTEK Raptor ARC-18 guard cartridge
• AB SCIEX Triple Quad 5500 mass spectrometer with TurboIonSpray source

Main Results and Discussion

Both manual and automated methods achieved mean recoveries of 65–83% across all analytes with precision (RSD) below 10%. Calibration curves were linear (r² > 0.995) over 10–500 pg/mL, and LOQs ranged from 1 to 20 pg/mL depending on the analyte. Matrix factors and signal suppression/enhancement were minimal, demonstrating efficient removal of endogenous interferences. Automated processing of 96 samples required ~60 minutes (excluding evaporation) and matched manual performance, confirming robustness and reproducibility.

Benefits and Practical Applications

• High throughput sample preparation for clinical diagnostic laboratories
• Reduced solvent consumption and operator variability through automation
• Low limits of quantitation meeting clinical reference intervals
• Efficient cleanup minimizing matrix effects and instrument maintenance

Future Trends and Opportunities

Advances in micro-SPE and on-line SPE-LC-MS integration may further reduce sample volumes and turnaround times. The adoption of greener solvents and novel mixed-mode sorbents can enhance selectivity and sustainability. Integration with laboratory information management systems (LIMS) and robotics will support fully automated clinical workflows. High-resolution mass spectrometry could expand analyte panels, enabling broader metabolomic profiling.

Conclusion

The validated automated SPE-UHPLC-MS/MS workflow provides a robust, high-throughput solution for quantifying catecholamines and metanephrines in human plasma. Comparable performance to manual methods, combined with significant time savings, supports its implementation in routine clinical and research environments.

Reference

• Biotage. Automated Extraction of Catecholamines and Metanephrines using Biotage Extrahera LV-200 and Low Volume SPE. Application Note AN963.v1, 2021.