Veraart J R, Kok S J, te Koppele J M, Gooijer C, Lingeman H, Velthorst N H, Brinkman U A
Free University, Department of General and Analytical Chemistry, Amsterdam, The Netherlands.
Biomed Chromatogr. 1998 Jul-Aug;12(4):226-31. doi: 10.1002/(SICI)1099-0801(199807/08)12:4<226::AID-BMC739>3.0.CO;2-1.
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.
本文介绍了一种用于测定胶原蛋白交联物羟赖氨酰吡啶啉（HP）和赖氨酰吡啶啉（LP）的毛细管电泳（CE）方法。比较了各种检测技术，即紫外可见二极管阵列吸光度检测（DAD）以及激光诱导荧光（LIF）和传统荧光模式下的荧光检测。LIF检测使用由准分子激光泵浦的倍频罗丹明染料激光器进行，激发波长为290和325nm。发射光用安装在光谱仪上的增强型二极管阵列探测器测量，以获得波长分辨光谱。仅通过LIF检测才达到了相关的浓度检测限，即在30mM磷酸盐缓冲液（pH 2.0）中HP和LP的检测限为200nM。对于两种荧光模式，分别从检测限到可用的最大浓度（HP为23μM，LP为4.2μM）获得了线性校准曲线。通过在线记录吸收光谱和荧光光谱来确认迁移化合物的身份。
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