Validation of a high-performance liquid chromatographic assay for lysylpyridinoline in urine: a potential biomarker of bone resorption.

ABSTRACT

We developed and validated a high-performance liquid chromatographic (HPLC) method for quantifying the bone-specific collagen crosslink, lysylpyridinoline (LP), in urine, LP was purified from cortical bone and characterized by spectrophotometry, HPLC, and 1H NMR spectroscopy. Our HPLC detected urinary LP independently of the sample volume and the range of quantification was between 63 nM and 1 microM. Average recovery of added LP standard to human urine samples was 106 +/- 21%. Mean inter- and intraassay CVs, respectively, for urines containing low, medium, and high concentrations of the crosslink LP were 7.4 and 6.3%. This analytical method is more efficient than previously published HPLC assays for LP because of the significant 24-h reduction in urinary sample preparation time. There was agreement between urinary LP concentrations measured with this method and the Metra Pyrilinks-D enzyme immunoassay (r2 = 0.714). These results emphasize the importance of using a thoroughly standardized HPLC assay as the "gold standard" for comparison of results with newly developed immunoassays.