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

Analysis of catecholamines and metanephrines in plasma is essential for clinical diagnosis of neuroendocrine disorders such as pheochromocytoma and for monitoring stress-related biomarkers. Reliable quantification at low concentration levels supports accurate patient assessment and research into catecholaminergic pathways.

Objectives and Overview of the Study

This study aims to develop and validate an automated microelution SPE workflow using Biotage Extrahera LV-200 and to compare it with manual processing on the Biotage PRESSURE+ 96 manifold. Objectives include optimizing sample preparation, assessing recovery, precision, linearity, limits of quantitation, and evaluating matrix effects for six target analytes.

Methodology and Instrumentation

Sample preparation uses weak cation exchange mixed-mode SPE on Biotage Mikro WCX microelution plates. Plasma is pretreated with sodium citrate buffer, spiked with stable isotope-labeled internal standards, and subjected to sequential conditioning, equilibration, loading, washing (aqueous, organic, lipophilic), elution, evaporation, and reconstitution. Chromatographic separation employs UHPLC on a C18-PFP column with a gradient of ammonium formate/formic acid aqueous and ammonium fluoride methanol mobile phases. Detection is by triple quadrupole MS in positive ESI mode using scheduled MRM transitions.

Main Results and Discussion

Recoveries ranged from 72% to 93% across the six analytes, with precision (RSD) below 12% for both manual and automated methods. Calibration curves exhibited coefficients of determination above 0.997, and estimated LOQs between 1 and 24 pg/mL. Matrix factors and signal factors indicated minimal interference, apart from a slightly lower signal factor for norepinephrine. Automated processing reduced RSD values and maintained comparable performance while enabling processing of 96 samples in approximately 60 minutes.

Benefits and Practical Applications

• High reproducibility and low limits of quantitation suitable for clinical diagnostics.
• Streamlined, high-throughput workflow reduces hands-on time and variability.
• Effective removal of matrix interferences ensures reliable quantification.
• Adaptable to different anticoagulants (heparin, EDTA, citrate) for flexible sample handling.

Future Trends and Opportunities

Emerging sorbents and microfluidic SPE formats may further reduce sample and solvent volumes. Direct injection approaches could accelerate turnaround if matrix effects can be managed. Integration with high-resolution MS and AI-driven data analysis promises deeper insights into metabolic pathways and real-time clinical monitoring.

Conclusion

The automated Biotage SPE method delivers robust, reproducible extraction of catecholamines and metanephrines from human plasma, matching manual performance while enhancing precision and throughput. It supports low-level quantitation, minimal matrix effects, and efficient processing for clinical and research laboratories.

Used Instrumentation

• Biotage Extrahera LV-200 automated SPE system
• Biotage PRESSURE+ 96 positive pressure manifold
• Biotage Mikro WCX 2 mg microelution plates
• TurboVap 96 Dual evaporator
• Shimadzu Nexera UHPLC system
• Avantor ACE Excel 1.7 µm C18-PFP column with RESTEK Raptor ARC-18 guard cartridge
• AB SCIEX Triple Quad 5500 mass spectrometer with Turbo-V source and TurboIonSpray probe

Reference

No external literature references were provided in the source document.