Quantification of bone turnover markers in control human urine by HPLC-fluorescence using a new proposed internal standard

The aim of this study is to validate an HPLC-fluorescence method for the quantification of free pyridinoline (Pyd), free deoxypyridinoline (D-Pyd), indices of bone degradation, together with galactosyl (Gal-Pyd) and glucosyl-galactosyl (Glu-Gal-Pyd) pyridinolines in non-hydrolyzed healthy women urine (n=7, age=34±7). For the first time, a synthesized D-Pyd superior homologue is proposed as internal standard. For all these compounds, the method is linear in the range 0-170 pmol injected and no matrix effect is found. The coefficients of variation for intra- and inter-day repeatability are between 3.1 and 10.4%. The limit of detection is around 2.3 pmol injected. Free D-Pyd, free Pyd and Glu-Gal-Pyd evaluated in urine account for 40.5±4.7, 215.1±25.0 and 96.4±20.1 pmol/mL (mean±SD), respectively. Gal-Pyd is absent in control urines or under our detection limit. The addition of the new proposed internal standard to urine before any pre-analytical step, allows the unequivocal quantification of the selected bone resorption indices. Moreover, the correct HPLC identification of all selected analytes is guaranteed by the availability of pure corresponding synthesized standards.