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

Reliable measurement of catecholamines and metanephrines in human plasma is critical for the diagnosis and monitoring of disorders such as pheochromocytoma, neuroblastoma, and autonomic dysfunction. Automated, low-volume solid-phase extraction (SPE) workflows prior to UHPLC-MS/MS offer high sensitivity, reproducibility, and throughput while minimizing sample volume and operator variability.

Objectives and Study Overview

This application note compares manual processing with the Biotage PRESSURE+ 96 positive pressure manifold and an automated workflow using the Biotage Extrahera LV-200 system. The goal was to develop and validate a low-volume SPE method for six analytes—norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine, and 3-methoxytyramine—spiked into plasma treated with common anticoagulants.

Methodology

Sample pretreatment involved centrifugation of 200 µL plasma, addition of internal standards and sodium citrate buffer (pH 7). EVOLUTE EXPRESS WCX 10 mg plates were conditioned with methanol and ammonium acetate buffer, loaded with pretreated plasma, then subjected to three sequential washes to remove aqueous, neutral organic, and lipophilic interferences. Analytes were eluted with water/isopropanol containing formic acid, dried under nitrogen in a TurboVap 96 Dual, and reconstituted in water/methanol with formic acid.

Instrumentation

• Biotage Extrahera LV-200 automated SPE system
• Biotage PRESSURE+ 96 positive pressure manifold
• TurboVap 96 Dual evaporator
• Shimadzu Nexera UHPLC
• Avantor ACE Excel 1.7 µm C18-PFP column with RESTEK Raptor ARC-18 guard
• AB SCIEX Triple Quad 5500 MS with TurboIonSpray in positive ESI mode

Main Results and Discussion

Both manual and automated methods achieved average recoveries of 65–83% across analytes, with intra- and inter-day precision below 10% RSD. Linearity was excellent (r²>0.995) over 10–500 pg/mL. Estimated LOQs ranged from 1 to 20 pg/mL depending on analyte and anticoagulant. Matrix factor studies demonstrated minimal signal suppression or enhancement, and signal recovery was consistent between methods. Automated processing of 96 samples required approximately 60 minutes (excluding evaporation), matching manual performance but reducing hands-on time.

Benefits and Practical Applications

• High sensitivity suitable for clinical reference intervals
• Low sample volume (200–500 µL) conserves patient specimens
• Robust removal of matrix interferences extends column life
• Automatable workflow increases throughput and reproducibility
• Applicable to diverse anticoagulant matrices without reoptimization

Future Trends and Perspectives

Emerging strategies include further miniaturization of SPE formats, direct injection of elution solvents to shorten turnaround, integration of on-line SPE-UHPLC-MS, and application of this workflow to other low-abundance biomarkers. Advances in high-throughput automation and data processing will support large-scale clinical studies and personalized diagnostics.

Conclusion

The presented low-volume SPE method delivers robust, sensitive, and reproducible extraction of six catecholamines and metanephrines from human plasma. Automated implementation on the Biotage Extrahera LV-200 streamlines sample preparation, matching manual performance while reducing labor and potential errors.

References

• Biotage Application Note AN963.v2, 2022