|Development of micro-flow liquid chromatography mass spectrometry reference method for metanephrines in plasma
|Advances in Clinical Analysis
|Ms Dima AlMekdad
|Mr Chris Hopley
Dr Leon Barron
Dr Mark Parkin
Mrs Emily Whyte
|LGC and King s College London
Abstract Information :
Metanephrine, normetanephrine and 3-methoxytyramine are the metabolites of adrenaline, noradrenaline and dopamine, respectively. The determination of elevated levels of these compounds in plasma is used to diagnose the presence of pheochromocytoma and paraganglioma. A need for standardisation and harmonisation of plasma metanephrines (METs) analysis has been identified by the international pheochromocytoma and paraganglioma research support organization (PRESSOR). As the UK designated National Measurement Institute for chemical and Bio-analysis, we aim to develop an isotope dilution mass spectrometry (IDMS) based 'higher order' reference analytical method. This is particularly challenging due to the low uncertainty and traceability required at the very low concentrations (<100 pg/mL) these compounds are measured at.
To ensure traceability and low measurement uncertainty, a signal to noise of higher than 100:1 measured peak to peak is desirable at concentration of 100 pg/mL in matrix. We present the evaluation and optimisation of a micro-flow LC system as a potential solution to reaching the performance required. Agilent 1260 infinity micro-flow LC was coupled to an Agilent 6490 mass spectrometer. The set up involved only the micro-flow flow pump (maximum 100µl/min), the µ-autosampler and a micro-flow column (1.0 x 150 mm) connected via a column coupler directly into the MS source.
Sensitivity improvements were assessed against an Agilent 1290 infinity pump using a column (3.0 x 150 mm) connected to the same mass spectrometer. Thus far, very low matrix effects were observed and high injection volumes were feasible. Evidently, micro flow LC method demonstrated up to seven folds increase in signal to noise ratio with the potential for a significantly improved LOD.