Development and implementation of method for analysis of free metanephrines in plasma by the liquid chromatography tandem mass spectrometry for routine use in clinical laboratory.

Summary

Objective:
The development and implementation of method for the determination of free metanephrines in plasma by the liquid chromatography tandem mass spectrometry (LC-MS/MS) suitable for routine practice in diagnostic laboratory which overcomes inconveniences related with classical liquid (HPLC) or gas chromatography (GC) and immunoassay approaches, like extensive sample preparation, the step of derivatization or interfering substances.

Design:
Analytical method development. Assessment of suitability for routine practice in diagnostic laboratory.

Settings:
Department of Clinical Biochemistry – Instrumental methods section, Spadia lab, a.s., Dr. Slabihoudka 6232/11, 708 52 Ostrava

Material and Methods:
Method development and validation were performed on Agilent 6490 Triple Quadrupole LC-MS/MS system. Chromatographic separation was achieved on SeQuant^®ZIC^®HILIC HPLC 3,5 µm, 100Å, PEEK 100 x 2,1 mm metal-free HPLC Column (Merck KGaA, Germany) by gradient elution. For determination of analytical parameters lyophilised plasma calibrators (purchased from RECIPE Chemicals and Instruments GmbH, Germany) and controls (purchased from Recipe Chemicals and Instruments GmbH and Chromsystems Instruments α Chemicals GmbH, Germany) were used. The study included 40 patients with diagnosis arterial hypertension.

Results:
The analytical performance of the new developed method is satisfactory. The intra-assay and inter-assay coefficients of variation (CV) were below 10 %. Recoveries determined by standard addition were from 89 to 98 %. The limit of quantification for MN was 0.035 nmol/l, for NMN was 0.049 nmol/l. This method fulfilled requirements for validation parameters for both analytes. Comparison of three methods for quantification of free metanephrines in plasma was performed – 2-MET Plasma ELISA^{Fast Track} (Labor Diagnostika Nord GmbH, Germany), FEO Kit (VUOS a.s., Czech Republic) dedicated for HPLC-ECD systems and developed LC-MS/MS method. Statistical analysis was performed using the MedCalc 16.1 (Medcalc Software bvba, Belgium) using the Passing Bablok regression.

Conclusions:
Neuroendocrine tumors represent a clinically and etiologically diverse group of disorders. Neuroendocrine tumors, such as pheochromocytomas or paragangliomas, exhibit a persistent or pyroxysmal excessive catecholamines production. 3-O-methylated catecholamine metabolites, metanephrine (MN) and normetanephrine (NMN), are prioritized for diagnosis of pheochromocytoma due to their highest diagnostic sensitivity and specificity. Presented LC-MS/MS method overcomes classical HPLC and immunoassay approaches. Method provides simple step of sample preparation, short runtime and required analytical parameters.

Keywords:
free plasma metanephrines, pheochromocytoma, LC-MS/MS.