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

Importance of the Topic

Accurate measurement of catecholamines and their metabolites in human plasma is essential for diagnosing and monitoring various endocrine and neurological disorders. Automated and robust sample preparation enhances throughput and consistency in clinical and research laboratories.

Goals and Study Overview

The study aims to develop and compare automated and manual workflows for extracting six catecholamines and metanephrines from human plasma using weak cation exchange microelution SPE plates prior to UHPLC MS MS analysis. Both methods are evaluated for recovery, precision, linearity, and matrix effects.

Methodology

A mixed mode weak cation exchange microelution SPE procedure was optimized. Plasma samples are pretreated with internal standards and buffered, loaded onto WCX plates, washed sequentially to remove aqueous, neutral organic, and lipophilic interferences, and eluted with water propan 2 ol with formic acid. Extracts are dried and reconstituted for UHPLC MS MS analysis. Manual processing uses a positive pressure manifold; automation employs the Biotage Extrahera LV 200.

Instrumentation

• Biotage Extrahera LV 200 and PRESSURE 96 manifold
• UHPLC system: Shimadzu Nexera with ACE Excel 1.7 C18 PFP column and Raptor ARC 18 guard cartridge
• MS MS: AB SCIEX Triple Quad 5500 with Turbo V source in positive ESI mode

Main Results and Discussion

Extraction recoveries ranged from 72 to 93% across analytes with precision below 12% RSD. Calibration curves showed excellent linearity (r>0.997) over 10 to 500 pg/mL, with LOQs between 1 and 24 pg/mL. Matrix and signal factors demonstrated minimal interference, comparable between manual and automated processes. Automated processing of 96 samples took approximately 60 minutes, delivering lower variability.

Benefits and Practical Applications

• High sensitivity and low limits of quantitation suitable for clinical reference intervals
• Reduced manual handling and improved reproducibility with automation
• Effective removal of matrix interferences for reliable quantitation
• Adaptable to different plasma anticoagulants

Future Trends and Opportunities

Integration of online SPE with UHPLC MS MS, miniaturization of workflows, and adoption of novel sorbent chemistries may further enhance throughput and sensitivity. Integration with laboratory information systems and method transfer to other platforms represents potential growth areas.

Conclusion

The described microelution SPE method offers a robust solution for automated and manual extraction of catecholamines and metanephrines, achieving high recovery, precision, and minimal matrix effects. Its implementation streamlines sample preparation and supports clinical and research applications requiring sensitive neurotransmitter analysis.