RP-HPLC-RID analysis of InsP6 to InsP3 in Indian cereals, legumes, and their products: A comparative evaluation of PRP-1 Vs C18 column

Abstract

Phytic acid or inositol hexakisphosphate (InsP₆) and its dephosphorylated forms (InsP₅, InsP₄ & InsP₃) are integral to cellular functions and confer several health benefits. The present study was aimed to develop a cost effective and high sample throughput RP-HPLC-RID method for routine quantification of lower inositol phosphates in both raw and processed cereals and pulses. For this a suitable mobile phase composition was formulated and two columns (Macroporus Hamilton PRP-1 Vs Waters Symmetry C18) were compared in terms of system specificity, linearity, accuracy and precision. Separation of InsP₃, InsP₄, InsP₅ and InsP₆ were recorded at 2.39, 2.93, 3.83 and 5.37 min using PRP-1column while the RT were 4.67, 5.64, 6.99 and 9.14 min with C18 column. Linearity of standards (R² > 0.99), with an accuracy and precision ranging from 1 to 5 % was achieved. The LOD and LOQ of all InsPs were 5 and 15 μg/ml, respectively. In quality control sample InsP₆ was found in highest concentration (446 ± 14.71 mg/100 g) followed by InsP₅ (162 ± 8.00 mg/100 g) and InsP₄ with the least concentration of 11.63 ± 1.06 mg/100 g whereas InsP₃ was below detectable limit (BDL). The optimised method was used for profiling of InsPs in the raw and processed cereals and pulses consumed as staple foods in India. Processed foods contained lesser InsP₆ and more of lower InsP compared to raw foods. The optimised method using unique mobile phase composition was found to yield accurate results and can used for large scale analysis of cereals and pulses and estimation of mineral nutrition potential and allied health benefits.