Analysis of a Urinary Biomarker Hydroxyproline for Clinical Assessment in Osteoporosis Patients

The various degradation products derived from bone collagen are pyridinoline, deoxypyridinoline, N-telopeptides, and hydroxyproline are excreted in the urine. Among that hydroxyproline is an important biomarker used to correlate the osteoporosis condition. A simple method of reversed phased ultra-fast liquid chromatography (RP UFLC) with a PDA detector. Hydroxyproline, a UV inactive molecule, is derivatized with fluorenyl methyl chloroformate (FMOC) to form a UV active adduct. FMOC-hydroxyproline adduct and chlorthalidone as an internal standard were spiked to healthy adult urine and processed through strata phenomenex C18 cartridges. Extracts from cartridges were collected and analysed on Phenomenex C18 column (250 mm × 4.6 mm i.d., 5 μm particle size) as a stationary phase. The mobile phase was composed of acetonitrile and diethylamine buffer (DEA) pH 9.0, 50:50 v/v ratio with a flow rate of 0.8 ml min-1. Elutes were analysed using a PDA detector at a wavelength of 265 nm. The proposed method was validated in accordance with the US-FDA guidance. In this study, the chromatographic peaks of hydroxyproline and chlorthalidone improved with a retention time of 2.5 and 6.4 min, respectively. Hydroxyproline exhibits excellent linearity with a correlation coefficient of 0.9994 (R2). A new improved, validated, and easy bioanalytical method has been used for the clinical evaluation of hydroxyproline in biological fluids. It is used to quantify the biomarker concentration (hydroxyproline) in a urine sample and correlate to disease condition (osteoporosis).