Development of a Specific Fluorescence Post-column Derivatization Method Coupled with Ion-Pair Chromatography for Phytate Analysis in Food

Phytate in plants (inositol phosphates, InsPs) affects mineral bioavailability. However, methods for their quantification may lead to variable results, and some are nonspecific (spectrophotometric techniques). In this study, ion-pair high-performance liquid chromatography (HPLC) was coupled with post-column derivatization to allow fluorescence detection (FLD, λ_excitation324/λ_emission364 nm) of InsPs. The fluorescence derivatization reaction, the main input of this study, was based on a ternary complex among phytate, iron(III), and 1,10-phenanthroline. Phytic acid (InsP₆) and three other InsPs (InsP_3-5) were analyzed in peanuts and soybean products after extraction in HCl 0.66 M, followed by purification on strong anion exchange cartridges. The novel method, named IP-HPLC-FLD, selectively separated InsP_3-6 with linear ranges between 600 and 2000 mg 100 g^-1 (<i>R</i>² > 0.99). The limits of detection and quantification were between 120-180 and 340-540 mg 100 g^-1, respectively. As the relative standard deviations were under 10%, the IP-HPLC-FLD method is suitable for phytate analysis.