Rapid analysis of pyridinoline and deoxypyridinoline in biological samples by liquid chromatography with mass spectrometry and a silica hydride column.

Abstract

Pyridinoline and deoxypyridinoline crosslinks are biomarkers found in urine for collagen degradation in bone turnover. For the first time, a rapid, sensitive, and ion-pairing free method is described for the analysis of pyridinoline and deoxypyridinoline using ultra-high performance liquid chromatography with Cogent Diamond Hydride column and detection by Q Exactive hybrid quadrupole-orbitrap high resolution accurate mass spectrometry. The separation was achieved using both isocratic and gradient conditions and run time <5min under isocratic conditions of 20% acetonitrile in water containing 0.1% formic acid. Pyridoxine was used as an internal standard and relative standard deviation of the retention times of both pyridinoline and deoxypyridinoline were <1%. The limit of detection was 0.082±0.023μM for pyridinoline and 0.118±0.052μM for deoxypyridinoline. The limit of quantitation was 0.245±0.070μM for pyridinoline and 0.354±0.157μM for deoxypyridinoline. The method was validated by the detection and quantitation of both pyridinoline and deoxypyridinoline in skin and urine samples.