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 microM for HP and 4.2 microM 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.
Free University, Department of General and Analytical Chemistry, Amsterdam, The Netherlands.
J R
JR
Veraart
S J
SJ
Kok
J M
JM
te Koppele
C
C
Gooijer
H
H
Lingeman
N H
NH
Velthorst
U A
UA
Brinkman
D002021:Buffers; D003094:Collagen / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007834:Lasers; D013050:Spectrometry, Fluorescence / Q000379:methods*
D002021:Buffers; D003094:Collagen