Quantitative Measurement of Plasma Free Metanephrines by a Simple and Cost-Effective Microextraction Packed Sorbent with Porous Graphitic Carbon and Liquid Chromatography-Tandem Mass Spectrometry.

Xin Xiong,Rongsheng Zhao,Yuanyuan Zhang,Núria Fontanals

doi:10.1155/2021/8821276

Abstract

Plasma free metanephrines are widely used for the diagnosis of pheochromocytoma and paraganglioma (PPGL), yet quantifying metanephrines using a simple and cost-effective approach may be challenging due to preanalytical and analytical constraints. In this study, we established and validated a new method for quantitative measurement of plasma free metanephrines based on microextraction by packed sorbent (MEPS) with porous graphitic carbon (PGC) and liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). The elution step was fully compatible with HILIC mode without evaporation and reconstitution. The analytes were well resolved, and potential interferences (54 substances) were investigated. This method was linear from 24.7–2717 pg/mL for metanephrine (MN) and 24.5–4010 pg/mL for normetanephrine (NMN) with a coefficient of determination (R2) higher than 0.994. The limit of MN and NMN detection were 12.4 pg/mL and 12.3 pg/mL, respectively. The intra- and interassay impressions were ≤12.8% for spiked quality controls and ≤13.6% for commercial quality controls; the method recoveries ranged within 88.0–109.0%, respectively. The area under the receiver operating characteristic (ROC) curve was 0.848 ± 0.047 for MN and 0.979 ± 0.021 for NMN. Validation that was performed by comparing clinical specimens with various biochemical results showed that plasma free metanephrines in a seated position had comparable sensitivity and lower specificity to urinary free metanephrines, which could be compensated by combining other biochemical tests. The newly developed MEPS method resulted as a time-saving, reliable, and cost-effective microextraction technique that can be applied for a successful screening of PPGL.

Highlights

Pheochromocytoma and paraganglioma (PPGL) are rare neuroendocrine tumors characterized by the overproduction of catecholamines [1], which can lead to serious cardiovascular complications, such as hypertensive crisis, myocardial infarction, and stroke [2]. e biochemical testing is crucial for diagnosing PPGL
Cross-contamination may occur due to epinephrine/NMN isobaric analyte pairing with identical multiple reaction monitoring (MRM) transitions, 4-hydroxy-3-methoxymethamphetamine (HMMA), a metabolite of 3, 4-methylenedioxymethamphetamine (MDMA), and NMN share a common pharmacophore resulting in the same product ion after fragmentation [27]
An endogenous analyte found in all plasma samples, 3-O-methyldopa, could not be eliminated by multistage fragmentation (MRM3) [28], which may enable more specific target quantification compared with traditional MRM [29]. erefore, chromatographic separation remains essential for interferences involving unresolvable mass fragmentation

Summary

Introduction

Pheochromocytoma and paraganglioma (PPGL) are rare neuroendocrine tumors characterized by the overproduction of catecholamines [1], which can lead to serious cardiovascular complications, such as hypertensive crisis, myocardial infarction, and stroke [2]. e biochemical testing is crucial for diagnosing PPGL. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) has been widely used to measure plasma free metanephrines due to its inherent selectivity and sensitivity [8,9,10,11,12,13,14,15,16] These compounds are poorly ionizable and with high polarity, which presents at very low concentrations in a complex matrix. The ion-pairing reagent can be used to form a complex with metanephrines, which could be retained on the C18 SPE cartridges [9] In these studies, extract evaporation for concentration or use nonvolatile salts as buffers or a large volume of sample (500 μL [9] or 900 μL [12]) and injection volume (35 μL [11] or 40 μL [9]) were required to achieve the necessary sensitivity and specificity. This study evaluated its usefulness by comparing it with other metabolites, including urinary free catecholamines, metanephrines, and vanillylmandelic acid (VMA) in a routine clinical setting

Materials and Methods

Results and Discussion

Method Validation