ABSTRACT
IN the early and mid 1900s, the quantitative analysis of phytate (InsP6)was based on precipitation with ferric chloride, first described by Heubner and Stadler in 1914 [1], or purification using anion-exchange chromatography [2-4]. A disadvantage of these methods is the lack of specificity in distinguishing between InsP6 and its degradation products. Because inositol phosphates with three to five phosphate groups (InsP3-InsP5) as well as InsP6 have been shown to be nutritionally significant [5-10], it is of great importance to have a reliable method for the determination of the individual inositol phosphates. There are also difficulties in determining low InsP6 levels using the precipitation and anion-exchange methods. The amount of detectable inositol phosphates should be at least in the nanomolar range, due to the low inositol phosphate concentrations in biological samples. Thus, a sensitive analytical method for the determination of InsP6 is required. With the development of ion-pair HPLC procedures [11-12] and capillary electromigration methods [13-14], it became possible to study InsP6 and some of its hydrolysis products during food processing and digestion. These methods are relatively easy to handle with a short and simple procedure, but they do not differentiate isomeric forms of inositol phosphates. The various isomers have been shown to have different biochemical functions, so the precise stereochemistry of the inositol phosphates is of considerable significance. It is, therefore, desirable to have an analysis method that not only has the capability to separate inositol phosphates with different numbers of phosphate groups, but also to separate the different isomeric forms of the degradation products. During the last few years, a number of isomer-specific
ion exchange chromatography (HPIC) methods with gradient elution for determination of inositol phosphates in biological samples have been developed [15-20].
