A Novel Method for the Determination of Urinary Citrate Levels: Ion-Pair and High Performance Liquid Chromatography with Liquid Membrane Extraction Technique

Abstract

In the clinical evaluation, and treatment of the patients with urinary calcium oxalate stones, determination of urinary citric acid levels is as important as oxalic acid levels. Presence of specific matrix proteins, and other anions in urine complicates its measurement. Therefore we aimed to develop a routine high performance liquid chromatography (HPLC) method which could determine citric acid levels in urine, and other body fluids. One hundred patients referred from various clinical sections were included in the study, and their urinary citrate levels in spot or 24-hour urine samples were measured as required. Measurement of oxalic acid was performed with a liquid membrane technique using a 314 nmUV detector at a stationary phase of C18 column, under 25oC ambient temperature, and at a flow rate of 1.7 mL/min Internal standard method was used for the determination of citrate concentration. For citrate standards at two distinct concentrations (1.25 mmol/L, and 5 mmol/L) respective intraday, and interday coefficient of variation (CV) values were found to be 4.29 vs 5.37%, and 2.78 vs 3.13% with a linearity interval of 0.03-20 mmol/L. In three different concentrations mean recovery rates of 101.2, 100, and 99.7% were obtained, respectively, and functional sensitivity of the assay was determined as 0.009 mmol/L. In conclusion, thanks to this method we developed, problems encountered during analysis of biological samples with complex matrix appears to be solved at greater extent. Besides compared with other available methods, this HPLC assay yields results in a shorter time frame with higher cost-effectiveness, simplicity, reproducibility, and reliability Therefore, we think that it can be used routinely in the determination of citrate levels in urine, and other body fluids. © © 2014 OMU.