In this work, the LC-MS-ESI-TOF method for simultaneous determination of phytates (inositol mono-, bis-, tris-, tetrakis-, pentakis-, and hexakisphosphates, abbreviated to IP1, IP2, IP3, IP4, IP5, and IP6, respectively) in food samples was developed and validated. The suitability of U-13C-labelled maize as a source for labelled internal standards for quantification of phytates was elucidated. The effectiveness of liberating IP1, IP2, IP3, IP4, and IP5 from phytic acid extracted form U-13C-labelled maize was evaluated for a variety of hydrolysis conditions, including enzymatic and acid hydrolysis. Enzymatic degradation of phytic acid using phytase (PHYZYME XP 5000 L) was very effective; phytic acid was degraded to lower phytates, but their distribution was unequal. Chemical hydrolysis was conducted under acidic conditions using hydrochloric acid and elevated temperatures up to 140 °C. The highest yields of IP4, IP5, and IP6 and of IP1, IP2, and IP3 were achieved by chemical hydrolysis at 105 °C for 7 h and 24 h, respectively. Thus, a combination of these two chemical treatments was selected for internal standard production. The developed LC-MS-ESI-TOF method was tested and successfully validated using plant-based food samples with different distribution of phytates. With this method, different forms of phytates in foods were separated and quantified simultaneously within 20 min. The high accuracy and precision of the developed method were guaranteed using respective labelled internal standards derived from U-13C-labelled maize.
Keywords: Liquid chromatography; MS-TOF; Phytates; Stable isotope-labelled internal standards; U-13C-labelled maize.
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