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 High Performance Liquid Chromatography (RP-HPLC) with pre- column derivatization using dansyl chloride and UV detection at 254 nm. To this aim, selectivity, working range and linearity, detection /quantification limit were determined. The precision (as repeatability 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 and shellfish ME-711.O4-070, 2014, so it was necessary to implement adjustments for its application in urine. The results showed that amino acids (AAs) did not cause interference in the analysis of PAs, and allow determination of concentrations in the linear range between 4 and 28 µg/mL. However, good recovery (accuracy) was not obtained in the experiment, possibly because the standards used were not acetylated (42.6% Pu and 69% Cdv). The present methodology allows determining the levels of Pu, Cdv, Spd, Spm Hst and Efd in urine samples from humans and other mammalian species.