Quantitative Measurement of Plasma Free Metanephrines by Ion-Pairing Solid Phase Extraction and LC-MS/MS with Porous Graphitic Carbon Column

Significance of the Topic

Accurate quantification of plasma free metanephrines (MN and NMN) is crucial for diagnosing and monitoring pheochromocytoma and related disorders. High sensitivity and specificity are needed to detect low endogenous levels in clinical research and diagnostic settings.

Study Objective and Overview

The primary goal was to establish a robust LC-MS/MS method combining ion-pairing solid phase extraction (IP-SPE) and porous graphitic carbon (PGC) chromatography to measure plasma free metanephrines with high analytical performance.

Methodology

Sample Preparation

• Ion-pairing SPE using Thermo Scientific HyperSep C-18 cartridges conditioned with acetonitrile and 0.1% perfluoroheptanoic acid (PFHA).
• Plasma samples loaded, washed with 0.1% PFHA and eluted in 60% acetonitrile. Eluates were dried and reconstituted for analysis.

Used Instrumentation

• Thermo Scientific TSQ Vantage triple stage quadrupole mass spectrometer with heated electrospray ionization (HESI-II).
• Thermo Scientific Accela UHPLC system.
• Thermo Scientific Hypercarb PGC column (50 × 2.1 mm, 5 μm) maintained at 70 °C.

LC-MS/MS Conditions

Mobile phases were 1% formic acid in water with ammonium formate (aqueous) and 0.1% formic acid in acetonitrile (organic). A 7-minute gradient separated MN and NMN. MN-d3 and NMN-d3 served as internal standards.

Results and Discussion

Interference

• Epinephrine (EPI) and NMN share SRM transitions; PGC chromatography achieved baseline resolution of EPI-d3 and NMN-d3.

SPE Recovery

• Absolute recovery for analytes and internal standards ranged from 86.4% to 97.5%.
• Relative recovery for MN and NMN were 97.7% and 113.5%, respectively.

Ion Suppression

• Post-column infusion showed no significant suppression for MN-d3 and NMN-d3 in processed plasma.

Linearity and Sensitivity

• Linear range: 7.2–486.8 pg/mL for MN, 18.0–989.1 pg/mL for NMN (R2 > 0.99).
• Lower limits of quantitation: 7.2 pg/mL (MN) and 18.0 pg/mL (NMN).
• Accuracy: 92.2%–118.0% for MN, 92.1%–115.0% for NMN.

Precision

• Intra- and inter-batch CVs ranged from 2.1% to 10.9% across low and high QC levels in charcoal-stripped serum and pooled plasma.

Carryover

• No detectable carryover up to 500 ng/mL (MN) and 1000 ng/mL (NMN).

Benefits and Practical Applications

This method offers high sensitivity and specificity, rapid analysis (7-minute run), and reliable quantitation of low-level metanephrines in plasma. It supports clinical research workflows for endocrine disorder assessment and can be integrated into routine laboratory QA/QC processes.

Future Trends and Opportunities

The approach can be expanded to multiplex assays of other polar biomarkers. Advances may include microflow LC for reduced solvent use, automation of SPE for higher throughput, and integration with high-resolution mass spectrometry for broader metabolomic profiling.

Conclusion

A sensitive, precise, and rapid LC-MS/MS assay combining IP-SPE and PGC chromatography was validated for plasma free metanephrines, meeting clinical research requirements with robust performance metrics.

Reference

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 Liquid Chromatography-Tandem Mass Spectrometry with Porous Graphitic Carbon Column. J Chromatogr B Analyt Technol Biomed Life Sci. 2011;879(23):2355–2359.