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

Abstract

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 (250mm ×4.6mm i.d., 5µm particle size) as a stationary phase. The mobile phase was composed of acetonitrile and diethylamine (DEA) buffer pH 9.0 in the ratio 50:50v/v at a flow rate of 0.8 ml/min. Elutes were analyzed using a PDA detector at a detection wavelength of 265nm. The proposed method was validated as per US-FDA Guidance. In this study, the chromatographic peaks of hydroxyproline and chlorthalidone show better resolution with a retention time of 2.5 and 6.4 min respectively. Hydroxyproline shows excellent linearity with 0.9994 of the correlation coefficient (R2). The newly developed and validated bioanalytical method established is conveniently used for the clinical assessment of hydroxyproline in biological fluids & is used to quantify the biomarker concentration (hydroxyproline) in a urine sample and correlate to disease condition (osteoporosis).