Validation of an HPLC-fluorescence method for the simultaneous free and glycosylated pyridinium crosslinks determination in urine

BACKGROUND: Pyridinium crosslinks, released during bone resorption, are excreted in urine as free pyridinoline (Pyr) and deoxypyridinoline (DPyr), or bound to peptide or to sugars, as galactosyl-pyridinoline (Gal-Pyr) and glucosyl-galactosyl pyridinoline (GluGal-Pyr). Commonly, only total Pyr and D-Pyr urinary amounts (free + bound forms) are evaluated. METHOD: We developed and validated an analytical method based on HPLC-fluorescence for the evaluation of the collagen crosslinks Pyr and DPyr (free and total), GluGal-Pyr and Gal-Pyr in the urine of healthy women (n = 20; aged 27-41) and girls (n = 20; aged 5-10). Urine, spiked with an unnatural D-Pyr homologue, as IS, was solid-phase extracted prior to HPLC analysis. The use of this IS and of pure Pyr, D-Pyr, GluGal-Pyr and Gal-Pyr, synthesized to be used as primary calibrators, guarantees the specificity of the method and the correct crosslinks quantification. Total Pyr and D-Pyr amounts were also evaluated after urine hydrolysis. RESULTS: The method demonstrates good selectivity, sensitivity, linearity, precision, accuracy, recovery and stability for all measured crosslinks. Pyr and D-Pyr, both free and total, and GluGal-Pyr amounts were significantly higher in girls than in women (p < 0.0001). Gal-Pyr, evaluated in girls for the first time, was under its lower quantification limit (< 21.20 pmol/mL) in women. CONCLUSIONS: The quantification of free and glycosylated pyridinium crosslinks might provide more information on the degradation of various types of collagen, respect to the measurement of total Pyr and D-Pyr alone. Moreover, this validated method could be a useful non-invasive technique for studying pathological conditions characterized by modified glycosylation enzyme activity and for other clinical investigations on bone fragility.