Determination of Urinary Total Hydroxyproline by HPLC with UV and EC Detectors

Abstract

Hydroxyproline is found in high concentrations in connective tissue proteins. It is remarkably useful to detect it because variation in urine levels of hydroxyproline is associated with various diseases. High performance liquid chromatography methods, in which ultraviolet and electrochemical detectors were used, were developed and validated for the determination of hydroxyproline in urine. Both methods included acid hydrolysis and derivation. The most appropriate time, temperature and pH were identified to optimize derivatization processes. Limits of detection were calculated as 1.57 mu g/mL, 0.9 mu g/mL and limits of quantification were 4.76 mu g/mL, 2.73 mu g/mL for UVD and ECD methods, respectively. Precision and accuracy of the methods were obtained <= 10.9 % (RSD), <= 11.5% (RE). The recoveries were found 108 +/- 6% and 102 +/- 7% for UVD and ECD methods, respectively. A strong, positive and linear correlation was found between hydroxyproline values obtained from UVD and ECD methods (p<0.01, r=+0.98). After optimization and validation of these methods, OHP levels in urine samples which were obtained from non-smokers and smokers were analyzed and, observed OHP/creatinine levels were compared. Results of both methods showed that total urinary OHP values of smokers were significantly higher than non-smokers (p<0.05).