Chromatographic conditions evaluation for phytic acid (IP6) determination in rice bran samples by HPLC

Abstract

Phytic acid (PA) or myo-inositol-1,2,3,4,5,6-hexakisphosphoric acid (IP6) can be hydrolyzed into inositol phosphates, such as inositol pentaphosphate (IP5), inositol tetraphosphate (IP4), inositol triphosphate (IP3) and possibly inositol di- and monophosphates (IP2 and IP1) by phytases (enzyme) during grains and seeds storage and fermentation. Chromatographic conditions of mobile phase, sample diluent, column and ionic par, for IP6 determination in defatted rice bran and purified PA from rice bran, were studied in this research. Linearity, repeatability, recovery, and limit of detection (LOD) and limit of quantification (LOQ) of the method were evaluated. The best resolution occurred with the use of both C18 columns (Shim-pack CLC-ODS and Novapak) at a 45 °C oven temperature and mobile phase A (32.50% methanol and 1.45% TBAH) and sample diluent da (32.50% methanol and 1.45% TBAH) and db (32.10% methanol and 1.20% TBAH). The R2 of calibration curve was 0.9988, confirming the linearity in the PA range of 1.5 to 10 mg mL-1 (r = 0.9993; p < 0.0000). Intra-day repeatability presented relative standard deviations (RSD) of 3.43, 2.37 and 1.88% for 8, 5, and 3 mg mL-1 of PA concentrations, respectively. Inter-day intermediate precision RSD values were 0.93, 1.38, and 3.50% for each PA concentration, 8, 5, and 3 mg mL-1 respectively. RSD values were lower than 4% for both studies, demonstrating adequate repeatability and intermediate precision for the analytical method proposed. A 91% IP6 recovery was obtained and the LOD and LOQ were 0.05 and 0.15 mg mL-1, respectively. The IP6 content in defatted rice bran and purified PA from rice bran samples were 3.90 and 420 mg 100 g-1, respectively.