Measurement of Urinary Metadrenaline and Normetadrenaline by Liquid Chromatography Tandem Mass Spectrometry for the diagnosis of Phaeochromocytoma

Rachel Marrington and Craig Webster

Department of Biochemistry and Immunology, Birmingham Heart of England NHS Foundation Trust

We have developed an LC-MS/MS method for the quantification of total fractionated urinary metadrenaline and normetadrenaline.  The method described here uses an acid hydrolysis step to convert conjugated metabolites into free metadrenalines and does not require the use of solid phase extraction prior to sample analysis.  There is complete baseline separation of the catecholamines from normetadrenaline and metadrenaline which is critical because adrenaline has the same molecular weight as normetadrenaline.

Acidifed urine was used for this analysis.  DL-Normetadrenaline•HCl (alpha-D2, |beta|-D1) and DL-Metadrenaline•HCl (alpha-D2, |beta|-D1) were used as internal standards.  2M HCl was used to hydrolyse the urine samples.  The chromatographic separation was achieved using a Phenomenex Luna 3µ PFP column (150 ? 2.00 mm) with PFP 4 ? 2.0 mm guard cartridges.  20 µL of acid hydrolysed urine samples were injected directly onto the column without further pre-treatment.  An isocratic system was used with the mobile phase consisting of 97.5% 10 mM Ammonium Formate pH 3.5 and 2.5% Methanol.  The flow rate was 0.2 mL/min.   Analysis was performed using an API 3200 QTrap tandem mass spectrometer (Applied Biosystems) using an ESI ion source, with transitions of m/z 166.1?134.0 (normetadrenaline), m/z 169.2?137.1 (D3- normetadrenaline), m/z 180.2?148.0 (metadrenaline) and m/z 201.2?183.2 (D3- metadrenaline).

Linearity was exhibited across the calibration range for both normetadrenaline and metadrenaline with the limit of quantification 0.1 µmol/L and 0.05 µmol/L respectively.  Within assay precision for both normetadrenaline and metadrenaline was less than 5.5% with %CVs of less than 4% in the abnormal concentration range.  Between assay precision was less than 13%.  Neither noradrenaline nor adrenaline interfere with the assay as determined by the spiking of samples with high concentrations of the catecholamines.

In conclusion, an analytically specific and sensitive method has been developed and evaluated for the analysis of urine metadrenalines which is now in routine use.