Title
Sensitive fluorimetric quantitation of pyridinium and pentosidine crosslinks in biological samples in a single high-performance liquid chromatographic run
Author
Bank, R.A.
Beekman, B.
Verzijl, N.
de Roos, J.A.D.M.
Nico Sakkee, A.
Tekoppele, J.M.
TNO Preventie en Gezondheid
Publication year
1997
Abstract
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 μm 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. Chemicals/CAS: Arginine, 74-79-3; Cross-Linking Reagents; Lysine, 56-87-1; pentosidine, 124505-87-9; Pyridinium Compounds
Subject
Biology
Biomedical Research
Collagen
Pentosidine
Pyridinium
collagen
pyridinium derivative
animal tissue
articular cartilage
bone
cattle
collagen synthesis
connective tissue
cross linking
high performance liquid chromatography
human tissue
measurement
nonhuman
priority journal
reproducibility
reversed phase high performance liquid chromatography
standard
urine
Adolescent
Arginine
Bone and Bones
Cartilage, Articular
Chromatography, High Pressure Liquid
Circadian Rhythm
Connective Tissue
Cross-Linking Reagents
Humans
Hydrolysis
Ligaments
Linear Models
Lysine
Middle Aged
Pyridinium Compounds
Reproducibility of Results
Sensitivity and Specificity
Spectrometry, Fluorescence
Tendons
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DOI
https://doi.org/10.1016/s0378-4347(97)00391-5
TNO identifier
234268
ISSN
1572-6495
Source
Journal of Chromatography B: Biomedical Applications, 703 (1-2), 37-44
Document type
article