A novel metal–dye system for urinary phytate detection at micro-molar levels in rats

Abstract

At present, beneficial properties of phytate have been observed. However, due to the special characteristics of this molecule and its low concentrations in physiological samples, the analytical determination of phytate in these samples becomes a complex and tedious problem. Therefore, the aim of this paper was to develop a simple and reliable methodology for phytate analysis in rat urine. In this study, two diets were administered to Wistar rats, namely: UAR-A03 (standard diet containing 1% phytate) and AIN-76A (purified diet with undetectable phytate). 24 h urine samples were collected and analysed for phytate by employing a procedure based on the purification and preconcentration of phytate by solid-phase extraction prior to detection with Al(III)–xylenol orange. All variables for both the solid phase extraction and colorimetric assay were optimized. The working linear range for colorimetric detection was 0–12 μM phytate. The limit of detection was 0.044 μM and the limit of quantification was 0.188 μM. The relative standard deviation (RSD) for 1 μM phytate was 1.46%. When the standard rat chow was replaced by the phytate-poor rat chow, the urinary phytate concentrations fell to very low values after 15 days. These concentrations were restored to normal values when the standard diet was newly supplied. A recovery study was conducted with satisfactory results (88%). The results obtained in the present study, using the new metal–dye system, are comparable to the results obtained with an alternative method of detection. All these results demonstrate the suitability of the new method for phytate analysis in rat urine.