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

Author(s): McDonald L, Livie C, Smith K, Johnston S

Publication: Ann Clin Biochem, 2025, Vol. , Page 45632251342098

PubMed ID: 40301723 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the stability of free metadrenalines in acidified and unacidified spot urine specimens during storage at room temperature, 4°C and -20°C for 28 days using liquid chromatography tandem mass spectrometry. The authors also investigated the effect of extraction method on metadrenaline loss to plate flowthrough. Urine was collected from 23 healthy volunteers (details not specified) and aliquoted. On the days of collection, half of each specimen was acidified with hydrochloric acid to a pH of 3 (2.5-3.5) while the other half was not adjusted (pH 5-4-7.7). Acidified and unadjusted urine specimens from 8 volunteers were aliquoted and stored at room temperature, 4°C and -20°C for 0, 3, 7, 14, and 28 days before analysis. Samples were extracted using Oasis WCX SPE μElution plates. To investigate the effects of a different extraction method on metadrenaline levels, polarity-based extraction was performed on specimens from seven volunteers using BondElut NEXUS 96 Square-well plates. Metadrenalines were quantified by an Acquity UPLC system coupled to a Waters XEVO TQ-XS mass spectrometer in electrospray positive mode.

   Summary of Findings:

   Levels of free normetadrenaline (NMT), metadrenaline (MET) and 3-methoxytyramine (3-MT) increased with storage of nonacidified urine for 28 days, with the largest mean increases observed when nonacidified urine was stored at room temperature (77.1%, 20.7% and 57.4%, respectively). When the urine specimens were spiked with metadrenalines at pathological levels, no significant change in levels was observed after 7 days at room temperature. Further analysis found that the LC-MS/MS peak areas of metadrenalines were significantly higher (by up to 97-fold) in nonacidified urine than acidified urine (P = 0.003 for NMT, P=0.001 for MET and P=0.002 for 3-MT) when not corrected using internal standards, but the change was <26% after correction with the 6PLUS1 Multilevel Urine Calibrator Set.  Analysis of the flowthrough from extraction plates revealed that while only tiny amounts of metadrenalines (<1.5% of total) were lost from non-acidified specimens during extraction with Oasis WCX SPE plates, an average of 64% of metadrenalines were lost in the flowthrough during extraction from acidified urine using the same method. When extraction was polarity-based, the loss of metadrenalines in the flow through was minimal (<1%), regardless of specimen acidification. The authors conclude that free metadrenaline remains stable in urine for up to 28 days at room temperature and that extraction with WCX plates should not be used for acidified urine.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Small molecule	LC-MS or LC-MS/MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Biospecimen components	Spiked to pathological levels Unspiked urine
   Biospecimen Aliquots and Components	pH	Unadjusted 3
   Storage	Time at room temperature	Room temperature 4°C -20°C
   Analyte Extraction and Purification	Analyte isolation method	Oasis WCX SPE μElution plates BondElut NEXUS 96 Square-well plate
   Storage	Storage duration	0 days 3 days 7 days 14 days 28 days

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