Improved assay for the quantification of hydroyproline (collagen) in animal models of Idiopathic Pulmonary Fibrosis

Introduction: Animal models of bleomycin-induced lung fibrosis are widely used to evaluate the efficacy of novel treatments for Idiopathic Pulmonary Fibrosis (IPF). The quantification of fibrosis can be determined by measuring hydroxyproline which is present largely in collagen.  However, commercial assays for hydroxyproline (HP) use chemical derivatisation followed by UV–visible detection and are subject to matrix effects leading to reduced accuracy. The objective of this study was to develop a direct assay of HP quantification not affected by lung homogenate matrix effects. Methods: Hydrolysed lung homogenates were analysed using cation exchange chromatography coupled with tandem mass spectrometry detection (LC-MS/MS).  Samples were diluted and quantified against a calibration curve prepared in a surrogate matrix containing HP.  Hydroxyproline-D_­­3 was used as the internal standard.  The data are summarised from 15 independent studies. Results: HP detection was linear over a wide range of HP concentrations (10 µg/mL to 800 µg/mL, correlation coefficient ranged from 0.996 to 0.999). Average coefficient of variation varied as a function of HP concentration and was 8.5% at 10.0 µg/mL and 5.0% at 800 µg/mL.  Limited lung matrix effects were observed on lung homogenates spiked with HP standards. Conclusion: The LC-MS/MS assay described demonstrated specificity and reproducibility for the accurate quantification of HP in lung homogenates from rodent models of bleomycin-induced lung fibrosis.