Rapid assay for hard tissue collagen cross-links using isocratic ion-pair reversed-phase liquid chromatography.
Following a detailed study, a rapid and sensitive assay for the naturally fluorescent collagen cross-links pyridinoline and deoxypyridinoline has been developed using ion-pair reversed-phase high-performance liquid chromatography in the presence of 1-octanesulphonic acid (OSA). Pyridinoline and deoxypyridinoline were separated on an Exsil 100 ODS, 5-microns column (100 mm X 4.6 mm I.D.) using 25 mM sodium formate, 5 mM OSA and 1 mM ethylenediaminetetraacetic acid adjusted to pH 3.25, containing 20% (v/v) methanol. The mobile phase flow-rate was 1.5 ml/min. Compounds were detected by their natural fluorescence (xenon lamp; excitation wavelength 290 nm, emission wavelength 400 nm). Peak areas were linear to 25 pmol injected for pyridinoline and 20 pmol injected for deoxypyridinoline (r = 0.99). Intra-assay coefficients of variation for urinary extracts were 7.65 and 9.07% (n = 10), respectively. Limit of detection (signal-to-noise ratio = 5) was 200 fmol injected. Quantification of the cross-links in acid hydrolysates and human urine samples was possible in under 15 min.
James
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Bone and Joint Research Unit, London Hospital Medical College, Whitechapel, London, U.K.
Perrett
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Thompson
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D000476:Alkanesulfonates / Q000494:pharmacology; D017738:Alkanesulfonic Acids; D000596:Amino Acids / Q000032:analysis / Q000378:metabolism; D000779:Anesthetics, Local / Q000494:pharmacology; D002021:Buffers; D002853:Chromatography, Liquid / Q000379:methods*; D003094:Collagen / Q000032:analysis / Q000652:urine*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D005561:Formates / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D000432:Methanol / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
D000476:Alkanesulfonates; D017738:Alkanesulfonic Acids; D000596:Amino Acids; D000779:Anesthetics, Local; D002021:Buffers; D003432:Cross-Linking Reagents; D005561:Formates; C030544:formic acid; C015484:pyridinoline; D003094:Collagen; D004492:Edetic Acid; C042005:1-octanesulfonic acid; D000432:Methanol