HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF POLYETHYLENE GLYCOLS AS THEIR ALPHA,OMEGA-BIS(1-NAPHTHYLURETHANE) DERIVATIVES AND SIGNAL MONITORING BY FLUORESCENCE DETECTION

Polyethylene glycols (PEGs) of average molecular mass (M-r) 600, 1000 and 3000 were converted to their alpha,omega-bis(1-naphthylurethane) d erivatives with 1-naphthylisocyanate and subjected to high-performance liquid chromatography on a Si 80 bare silica gel stationary phase. Si gnal monitoring was done by fluorescence detection at wavelengths of 2 32 nm for excitation and 358 nm for emission. A binary acetonitrile-wa ter gradient effected good separation of PEG 600 and PEG 1000 into a w ide variety of individual oligomers and almost baseline separation was achieved for PEG 600 and PEG 1000. In contrast, PEG 3000 requires a t ernary acetonitrile-water-tetrahydrofuran gradient for both efficient elution and acceptable satisfactory signal resolution. Although separa tion of PEG 3000 into individual oligomers was substantially lower tha n that observed for PEG 600 and PEG 1000, the chromatograms impressive ly reveal the oligomeric composition of the polyether sample. Using se rial dilutions of the alpha,omega-bis(1-naphthylusethane) derivatives, the detection limits for PEG 600, PEG 1000 and PEG 3000 are approxima tely 0.1 ppm, respectively. Using 50 mu l of sample dissolved in eithe r water or physiological saline, a detection limit of 1 mu g/ml was ac hieved corresponding to an absolute amount injected of 10 ng. Taken to gether 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 permeabili ty and resorption in the living organism. Furthermore, with PEG 3000 a s model compound, incorporation of two 1-naphthyl substituents to the corresponding alpha,omega-bis(1-naphthylurethane) derivative was confi rmed by matrix assisted laser desorption ionization time-of-flight mas s spectrometry (MALDI-TOF-MS). (C) 1998 Elsevier Science B.V. All righ ts reserved.