Capillary electrophoresis of the collagen crosslinks HP and LP utilizing absorbance, wavelength-resolved laser-induced fluorescence and conventional fluorescence detection

A capillary electrophoretic (CE) method is presented for the determination of the collagen crosslinks hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP), Various detection techniques are compared, i.e. UV-Vis diode-array absorbance detection (DAD) and fluorescence detection both in the laser-induced fluorescence (LIF) and the conventional fluorescence mode. LIF detection was performed using a frequency-doubled Rhodamine dye laser pumped by an excimer laser, for excitation at 290 and 325 nm, The emission was measured with an intensified diode-array detector mounted on a spectrograph to obtain wavelength-resolved spectra, Relevant concentration detection limits were achieved only by using LIF detection, i.e. 200 nM of HP and LP in a 30 mM phosphate buffer (pH 2.0), Linear calibration curves were obtained from the detection limits up to the maximum concentration available, 23 mu M for HP and 4.2 mu M for LP, respectively for both fluorescence modes, The identity of the migrating compounds was confirmed by on-line recording of both the absorption and the fluorescence spectra. (C) 1998 John Wiley & Sons, Ltd.