Effect of pH on stability and solid phase extraction of urinary free metadrenaline measurement by liquid chromatography tandem mass spectrometry

BackgroundMeasurement of urine free metadrenalines offers potential diagnostic and practical advantages over urinary fractionated metadrenalines in detection of phaeochromocytoma and paraganglioma, including sample collection without acid preservative. Here, we evaluate stability with and without sample acidification as well as pH implications for analysis by solid-phase extraction (SPE) and liquid chromatography tandem mass spectrometry.MethodsSpot urine samples were adjusted to pH 3 or unacidified on day of collection and stored at room temperature, 4°C or -20°C, for up to 28 days to assess changes in free metadrenaline concentrations over time. Extraction of unacidified versus acidified urine was examined by comparing peak areas and measuring concentrations present in sample eluents according to two SPE methodologies.ResultsFree metadrenalines remained stable in urine with or without acidification for up to 28 days, with mean reduction in concentrations of <10% for all storage conditions. Measured concentrations progressively increased without acidification at room temperature at low concentrations but remained constant when spiked with pathological concentrations. Peak areas were up to 97-fold lower in acidified than unacidified samples when extracted using weak cation exchange (WCX). On average 64% of analyte eluted in the flowthrough in acidified samples relative to 1.5% without acidification. By contrast, over 99% was retained in the extract using polar extraction at either pH.ConclusionUrine free metadrenalines remain stable at room temperature for up to 28 days and are more efficiently extracted without use of acid preservative if using WCX methodology.