Plasma Free Metanephrines Quantitation with Automated Online Sample Preparation and Liquid Chromatography—Tandem Mass Spectrometry

Significance of the Topic

Plasma free metanephrine and normetanephrine are essential biomarkers for diagnosing and monitoring pheochromocytoma. High specificity and sensitivity of LC-MS/MS enable accurate quantification in clinical research settings.

Objectives and Study Overview

This work aimed to implement a fully automated online sample preparation using TurboFlow technology coupled with LC-MS/MS for plasma free metanephrine analysis. The main goal was to reduce preparation time and costs while preserving analytical performance relative to an established offline SPE method.

Methodology and Instrumentation

• Sample Preparation: 0.5 mL human plasma or charcoal-stripped serum spiked with deuterated internal standards, protein precipitation with 10% trichloroacetic acid, centrifugation, and injection of 100 µL supernatant.
• Online Extraction: TurboFlow Cyclone MCX-2 column using perfluoroheptanoic acid as ion-pairing reagent.
• Chromatography: Thermo Scientific Hypercarb column (50 × 3 mm, 5 µm) at 70 °C; total runtime 12 minutes.
• Mass Spectrometry: TSQ Vantage triple quadrupole with HESI-II source operated in SRM mode, integrated via a Transcend TLX-1 system.

Main Results and Discussion

• Recovery: Absolute extraction efficiency of 56–62%; relative recoveries of MN and NMN ~91% and ~98%.
• Linearity and Accuracy: Linear ranges of 6.3–455.4 pg/mL for MN and 12.6–954.5 pg/mL for NMN; accuracy between 80.6% and 101.7%.
• Sensitivity: LLOQs of 6.3 pg/mL (MN) and 12.6 pg/mL (NMN) with acceptable precision (CV ≤ 17.2%).
• Precision: Intra- and inter-assay CVs of 2.0–10.5% at low and high QC levels.
• Matrix Effects: No significant ion suppression or enhancement observed in human plasma.
• Specificity and Carryover: Complete chromatographic resolution of epinephrine and NMN; no carryover detected.

Benefits and Practical Applications

• Online automation reduced sample preparation time by over 50% compared to offline SPE.
• Eliminates the need and expense of SPE cartridges in routine workflows.
• Maintains robust analytical performance suitable for high-throughput clinical and pharmaceutical laboratories.

Future Trends and Possibilities

Automation in sample preparation is expected to expand, with development of more selective extraction phases, microflow LC, and multiplexed workflows. Integration with high-resolution MS instruments could further improve specificity and extend metabolite coverage in clinical and bioanalytical research.

Conclusion

The TurboFlow-LC-MS/MS method delivers a fast, sensitive, and cost-effective solution for quantifying plasma free metanephrines. It matches or exceeds the performance of traditional offline SPE protocols while greatly simplifying sample handling, making it ideal for clinical research applications.

Reference

1. He X, Kozak M. Quantitative Measurement of Plasma Free Metanephrines by Ion-Pairing Solid Phase Extraction and LC-MS/MS with Porous Graphitic Carbon Column. Thermo Scientific Application Note AN539.
2. He X, Gabler J, Yuan C, Wang S, Shi Y, Kozak M. Quantitative Measurement of Plasma Free Metanephrines by Ion-pairing Solid Phase Extraction and LC-MS/MS with Porous Graphitic Carbon Column. J Chromatogr B Analyt Technol Biomed Life Sci. 2011;879(23):2355–2359.
3. Yuan C, Kosewick J, He X, Kozak M, Wang S. Sensitive Measurement of Serum 1,25-dihydroxy-vitamin D by Liquid Chromatography/Tandem Mass Spectrometry after Removing Interference with Immunoaffinity Extraction. Rapid Commun Mass Spectrom. 2011;25(9):1241–1249.
4. He X, Kozak M. Development of a Liquid Chromatography–Tandem Mass Spectrometry Method for Plasma-Free Metanephrines with Ion-pairing Turbulent Flow Online Extraction. Anal Bioanal Chem. 2012;402(9):3003–3010.