MSACL 2019 EU Abstract

Development of a Candidate Higher Order Reference Measurement Procedure for the Analysis of Metanephrines in Plasma Using Micro-Flow LC-MS/MS

Dima AlMekdad (1,2), Leon Barron (1), David Cowan (1), Chris Hopley (2)
(1) Dept. Analytical, Environmental & Forensic Sciences, King's College London, London, UK (2) UK National Measurement Laboratory at LGC, Teddington, UK

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Dima AlMekdad (Presenter) King's College London

Presenter Bio: Dima graduated with a BSc in Pharmacy and Pharmaceutical Chemistry from the University of Kalamoon (Deratia, Syria) in 2010. After working as a pharmacist for couple of years, she pursued an MSc in Analytical Toxicology at King’s College London (London, UK) in 2012. During her degree, she did her research project placement at the National Measurement Laboratory hosted at LGC (Teddington, UK). After graduating she started working in the same laboratory focusing on the delivering traceable measurements of immunosuppressants in whole blood. In 2015, she started doing her PhD at King’s College London in standardisation of laboratory medicine focusing on two applications: plasma metanephrines analysis and tacrolimus in whole blood analysis. She is now working part time and studying part time and aiming to graduate her PhD in 2020.

Relevant Financial Disclosures (within past 24 months) Salary Part time work at National Measurement Laboratory at LGC Conflict mitigation NOT REQUIRED.

Introduction: Metanephrine and normetanephrine are the metabolites of adrenaline and noradrenaline, respectively. The determination of elevated levels of these compounds in plasma is used to diagnose the presence of pheochromocytoma and paraganglioma by a number of different methods, but mainly based on liquid chromatography coupled to mass spectrometry (LC-MS). A need for standardisation and harmonisation of plasma metanephrines (METs) analysis has been identified by the international pheochromocytoma and paraganglioma research support organization (PRESSOR). This is particularly challenging due to the low uncertainty and traceability required at the very low concentrations these compounds are typically measured at in plasma; normetanephrine 23-192 pg/mL and metanephrine 6-88 pg/mL(1).
Objectives: The aim is to develop a traceable, exact matched isotope dilution mass spectrometry (EM-IDMS) based higher order reference analytical method for the analysis of plasma METs.
Methods: We present the evaluation and optimisation of a micro-flow LC system as a solution to reaching the performance required for a higher order reference method. Agilent 1260 infinity micro-flow LC was coupled to an Agilent 6490 triple quadrupole mass spectrometer. The set up involved the micro-flow pump (maximum 100 µL/min), the micro-autosampler and a micro-flow column (1.0 x 150 mm, 1.8 µm) connected via a column coupler directly into the MS source. Plasma sample preparation clean-up was performed using a mixed mode cation exchange 96 well solid phase extraction plate.
Results: Very low matrix suppression effects were observed and high injection volumes were feasible up to 8 µL. Evidently, micro-flow LC method demonstrated up to a seven-fold increase in signal-to-noise ratio with a significantly improved detection limits compared to the same method using standard scale LC flow. Two independent human plasma materials were analysed using the candidate reference measurement procedure. The measurements of the frozen human plasma for normetanephrine was 137 pg/mL and for metanephrine was 31 pg/mL with expanded measurement uncertainties (k = 2) of 4-7% and 5-6% respectively for different sample units. The lyophilised human plasma material measurements of normetanephrine was 97 pg/mL and for metanephrine was 45 pg/mL with expanded measurement uncertainties (k = 2) of 5% and 6% respectively.
Conclusion: A candidate higher order reference measurement procedure has been developed for the analysis of plasma METs that achieves low measurement uncertainty and traceability at concentrations below 150 pg/mL with expanded uncertainties ranging 4-7% for normetanephrine and metanephrine. This is the first step towards the standardisation of this analytical test which would ultimately result in improved diagnosis accuracy and better patient health care. Reference: (1) Rao D, Peitzsch M et al. doi:10.1530/EJE-17-0077.