Determination of the polyamines in human toenails as 1-(5-fluoro-2,4-dinitrophenyl)-4-methylpiperazine derivatives using high-performance liquid chromatography

A high-performance liquid chromatography (HPLC) assay with UV detection was developed for the determination of five polyamines, i.e., 1,3-diaminopropane (DAP), putrescine (PUT), cadaverine (CAD), spermidine (SPD) and spermine (SPM). Primary (NH2) and secondary (NH) amines in the polyamine structures were first labeled with 1-(5-fluoro-2,4-dinitrophenyl)-4-methylpiperazine (PPZ) at 60 °C for 50 min in a mixture of 0.1 M borax (pH 9.3) and acetonitrile. The resulting polyamine derivatives were separated within 16 min using an ODS column and gradient elution with a mixture of water-(acetonitrile:methanol = 6:4, v/v). The separated polyamine derivatives were sensitively detected with a UV detector. The detection limits were 5 fmol-100 fmol. Good linearity was achieved with the calibration curves, which were obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS), 1,6-diaminohexane (DAH), against the injected amounts of each polyamine (0.5-500 pmol for DAP, PUT and CAD, 5-500 pmol for SPD and 50-500 pmol for SPM, r2 > 0.999). The proposed method was applied to analyze toenails of healthy volunteers. The structures of polyamines in toenails were identified from the protonated-molecular ions obtained by HPLC-ESI-MS. The mean concentrations of PUT, CAD, SPD and SPM in 1 mg of human toenails (n = 6) were 29.27, 4.42, 3.15 and 6.77 pmol, respectively. The proposed method provides a good accuracy and trace detection of the polyamines in toenails. For these reasons, this analytical technique is applicable for determining various biological compounds in toenails.