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Background: Pyridinium cross-links Pyridinoline (PYD) and Deoxypyridinoline (DPD) are established markers of collagen degradation. Measurement of PYD and DPD can be used to evaluate changes in bone turnover in patients with metabolic bone disease and to monitor response to anti-resorptive treatment. Objective: To develop a method to extract and measure urine free PYD (fPYD) and free DPD (fDPD) by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). The method was used to quantify urine samples from 172 healthy individuals and 63 patients diagnosed with metabolic bone disease. Method: Acidified urine samples were extracted using solid phase extraction with cellulose slurry. PYD and DPD were separated by reversed-phase, ion-paired chromatography prior to MS/MS detection. Results: The fully validated method showed good agreement with other laboratories in the UK National External Proficiency Scheme (UK NEQAS). The method was compared against two commercial immunoassays for fDPD and pyridinium cross-links, r2 were 0.906 and 0.816 respectively. Urine concentrations of fDPD/Cr and fPYD/Cr were significantly higher in the patients than healthy individuals (p<0.001). An average (±SD) fDPD:fPYD ratio of 0.29 (±0.08) was consistently observed across all subgroups. A markedly increased fDPD:fPYD ratio of 8.9 was observed in a patient with type VI Ehlers-Danlos Syndrome (EDS). Conclusion: Simultaneous measurement of two free pyridinium cross-links provides a valuable, cost effective assessment tool for use in the diagnostic work-up of patients with metabolic bone disease. Improvements in sample extraction efficiency have increased assay specificity and analysis throughput. The use of the fDPD:fPYD ratio can assist in the diagnosis of type VI EDS

LC–MS/MS application for urine free pyridinoline and free deoxypyridinoline: Urine markers of collagen and bone degradation