Validation of a methodology for determination of polyamines in urine by High Performance Liquid Chromatography (HPLC)

Abstract

The levels of polyamines (PAs) in urine are an important biomarker of physiological and pathological phenomena for humans.Due to its importance, the validation of a PAs determination methodology was validated for (Putrescine (Pu), Cadaverine(Cdv), Spermidine (Spd), Spermine (Spm), Epinephrine (Epf) and Histamine (Hst) in urine by Reversed-phase HighPerformance Liquid Chromatography (RP-HPLC) with pre- column derivatization using dansyl chloride and UV detection at254 nm. To this aim, selectivity, working range and linearity, detection /quantification limit were determined. The precision (asrepeatability and precision under intermediate conditions), accuracy (measured as recovery) and robustness were established.The methodology was adapted from the Chilean Standard for the detection of histamine and other biogenic amines in fish andshellfish ME-711.O4-070, 2014, so it was necessary to implement adjustments for its application in urine. The results showedthat amino acids (AAs) did not cause interference in the analysis of PAs, and allow determination of concentrations in thelinear range between 4 and 28 μg/mL. However, good recovery (accuracy) was not obtained in the experiment, possiblybecause the standards used were not acetylated (42.6% Pu and 69% Cdv). The present methodology allows determining thelevels of Pu, Cdv, Spd, Spm Hst and Efd in urine samples from humans and other mammalian species.