Submitted to: Journal of Chromatography A
Publication Type: Peer reviewed journal
Publication Acceptance Date: 5/1/2003
Publication Date: 8/15/2003
Citation: Chen, Q., Li, B.W. 2003. Separation of phytic acid and other related inositol phosphates by high-performance ion chromatography and its applications.Journal of Chromatography A. 1018:41-52. Interpretive Summary: Phytic acid (InsP6) is a naturally occurring constituent and the major storage form of phosphorus in cereals, legumes, nuts and other crops. Historically, it was considered solely as an antinutrient because it can bind essential dietary minerals such as calcium, iron, and zinc, and decrease their bioavailability in humans. In the past few years, studies have shown that InsP6 has also beneficial health effects, such as protection against cancers, heart diseases, diabetes, and kidney stones. During food processing and digestion, InsP6 can be degraded to other inositol phosphates by enzymatic or nonenzymatic means. Several isomers of inositol phosphates have shown other important physiological functions, such as anti-inflammatory effects and prevention of diabetes complications. Therefore, in order to understand fully the physiological effects of individual inositol phosphates, it is crucial to establish an accurate and reliable method that can be used for the analysis of different isomeric forms of inositol phosphates including InsP6,. In this paper, we described a high-performance anion-exchange chromatographic method using an acidic gradient elution program developed in our laboratory. Applying this method, we studied the hydrolysis pathway for InsP6 under various conditions, and prepared an in-house reference standard solution. This knowledge will be useful for other analysts in generating food composition data on phytic acid and related inositol phosphates.
Technical Abstract: A high-performance anion-exchange chromatographic method was developed for the separation of phytic acid and other inositol phosphates (myo-inositol bis-, tris-, tetrakis-, and pentakisphosphates) with gradient elution and ultraviolet absorbance detection after post-column derivatization. With the acidic eluents, the combination of anion exchange and ion suppression retention mechanisms led to the separation of 35 inositol phosphates (excluding enantiomers) into 27 peaks for the first time, and the retention behaviors of all myo-inositol bis- to hexakisphosphate isomers were studied. Based on the investigations of nonenzymatic hydrolysis of phytic acid under different conditions by using this method, an in-house reference standard solution was produced, which can be used for method development. In addition, by applying this method to in vitro kinetic studies, at least one new enzymatic hydrolysis pathway of phytic acid was found, and one rule of enzymatic dephosphorylation of inositol phosphates (position effect) was proposed and another one (neighboring effect) was confirmed. The principle of the proposed identification approach for several inositol phosphate isomers based on hydrolysis products study will be applicable to other natural products analysis, for which standards are very expensive or not available.