High-performance liquid chromatography of polyethylene glycols as their α,ω-bis(1-naphthylurethane) derivatives and signal monitoring by fluorescence detection

Abstract

Polyethylene glycols (PEGs) of average molecular mass ( M r) 600, 1000 and 3000 were converted to their α,ω-bis(1-naphthylurethane) derivatives with 1-naphthylisocyanate and subjected to high-performance liquid chromatography on a Si 80 bare silica gel stationary phase. Signal monitoring was done by fluorescence detection at wavelengths of 232 nm for excitation and 358 nm for emission. A binary acetonitrile–water gradient effected good separation of PEG 600 and PEG 1000 into a wide variety of individual oligomers and almost baseline separation was achieved for PEG 600 and PEG 1000. In contrast, PEG 3000 requires a ternary acetonitrile–water–tetrahydrofuran gradient for both efficient elution and acceptable satisfactory signal resolution. Although separation of PEG 3000 into individual oligomers was substantially lower than that observed for PEG 600 and PEG 1000, the chromatograms impressively reveal the oligomeric composition of the polyether sample. Using serial dilutions of the α,ω-bis(1-naphthylurethane) derivatives, the detection limits for PEG 600, PEG 1000 and PEG 3000 are approximately 0.1 ppm, respectively. Using 50 μl of sample dissolved in either water or physiological saline, a detection limit of 1 μg/ml was achieved corresponding to an absolute amount injected of 10 ng. Taken together both, separation efficiency and detection sensitivity, this new technique, termed as a “pseudo-reversed-phase separation process”, may therefore be applicable to investigations of intestinal permeability and resorption in the living organism. Furthermore, with PEG 3000 as model compound, incorporation of two 1-naphthyl substituents to the corresponding α,ω-bis(1-naphthylurethane) derivative was confirmed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS).