Bank R A, Beekman B, Verzijl N, de Roos J A, Sakkee A N, TeKoppele J M
TNO Prevention and Health, Division of Vascular- and Connective Tissue Research, Leiden, The Netherlands.
J Chromatogr B Biomed Sci Appl. 1997 Dec 5;703(1-2):37-44. doi: 10.1016/s0378-4347(97)00391-5.
A high-performance liquid chromatographic assay was developed for pyridinium crosslinks and pentosidine in mature collagen of a wide variety of connective tissue hydrolysates by a simple two-step isocratic assay using a reversed-phase column. The crosslinks (including the internal standard pyridoxine) were optimally detected by their native fluorescence by switching wavelengths of the detector during the assay. The method resulted in highly sensitive and accurate measurements, without need for precleaning of the samples: crosslink levels in 200 microm thin slices of the various zones of articular cartilage were easily quantified. The detection limit was as low as 0.4 pmol for the pyridinolines and 0.05 pmol for pentosidine. The intra-assay and inter-assay coefficients of variation were as low as 2% (pyridinolines) and 5% (pentosidine); calibration curves for all compounds were linear over a concentration range larger than two orders of magnitude. With our chromatographic system, the diglycosylated form of hydroxylysylpyridinoline in unhydrolyzed urine was separated as well.
采用反相柱，通过简单的两步等度测定法，开发了一种用于测定多种结缔组织水解产物成熟胶原蛋白中吡啶交联物和戊糖苷的高效液相色谱分析法。在测定过程中，通过切换检测器波长，利用交联物（包括内标吡哆醇）的天然荧光对其进行最佳检测。该方法无需对样品进行预净化，即可实现高灵敏度和准确的测量：关节软骨各个区域200微米薄片中的交联水平很容易定量。吡啶啉的检测限低至0.4皮摩尔，戊糖苷的检测限低至0.05皮摩尔。批内和批间变异系数分别低至2%（吡啶啉）和5%（戊糖苷）；所有化合物的校准曲线在大于两个数量级的数据范围内呈线性。在我们的色谱系统中，未水解尿液中双糖基化形式的羟赖氨酸吡啶啉也能被分离出来。
