A study on the stability of urinary free catecholamines and free methyl-derivatives at different pH, temperature and time of storage

Abstract

The goal of our study was to test the relative stability of urine, unconjugated, free catecholamines and the methyl derivatives. We measured the change in concentrations in commercially available urines after storage at various pH values, temperatures and time, from days up to 10 weeks. Samples of commercial control urines were adjusted to pH 2.0, 4.0, 6.0 and 8.0 and aliquots stored at ambient temperature (20-26 degrees C), 4 degrees C and -18(o)C. The free catecholamines (cats) and the free methyl derivatives (mets) were measured after 1, 2, 3 and 6 days and 1, 2, 3 and 10 weeks using the automated sample trace enrichment dialysis (ASTED) procedure with reversed phase ion pair high performance liquid chromatography (HPLC) and coulometric detection. Free catecholamines were relatively stable, with <15% loss of concentration, when stored at pH 6.0 or less for at least 4 days and up to 10 weeks at pH 2.0 at either 4(o)C or -18(o)C. At pH 8.0, the concentration fell to <60% after 48 h and at a pH of 6.0 or 8.0, up to 90% was lost within the first week at 4(o)C and 25(o)C. More than 40% of free normetadrenaline and metadrenaline were lost after 1-2 weeks when stored at 20-25(o)C and pH 8.0. After 10 weeks at pH 4.0, 6.0 and 8.0, up to 90% loss was observed at 25(o)C. Free cats were stable at pH 2.0 and 4.0 at -18(o)C and the free mets were stable at -18(o)C over the entire time period studied and at all pHs. In the analysis of free catecholamine and the free methyl derivatives, urine samples should be acidified to a pH range 2.0-3.0 to ensure stability and hence the correct analysis.