A sensitive HPLC assay for quantitative determination of polyamidoamine dendrimers after pre-column derivatization using FITC

Abstract

A pre-column derivatization method was developed to analyze polyamidoamine (PAMAM) dendrimers. The fluorescein isothiocyanate (FITC) derivation method improved the retention of PAMAM dendrimers on the reversed-phase column of high performance liquid chromatography (RP-HPLC). The derivation benefited the UV-Vis detection of PAMAM, although the fluorescence of FITC was quenched by PAMAM dendrimers. The primary amine-terminated PAMAM dendrimers, generation 3 (PAMAM-NH₂ (G3)), were quantitatively determined by UV-Vis spectroscopy over the concentration range of 10–80 μg mL⁻¹, with the calibration curve expressed as A = 131.6C (μg mL⁻¹) − 295.6 (R² = 0.9901) and the detection limit of 2.7 μg mL⁻¹. The binding ratio of FITC to PAMAM-NH₂ (G3) was determined as 8 : 1 by liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-IT-MS).