High‐resolution mass spectrometric analysis of myo‐inositol hexakisphosphate using electrospray ionisation Orbitrap

RATIONALE: The phosphorus storage compound in grains, phytic acid, or myo‐inositol hexakisphosphate (IP6), is important for nutrition and human health, and is reportedly the most abundant organic phosphorus compound in soils. Methods for its determination have traditionally relied on complexation with iron and precipitation, acid digestion and measurement of phosphate concentration, or (31)P NMR spectroscopy. Direct determination of phytic acid (and its homologues) using mass spectrometry has, as yet, found limited application to environmental or other complex matrices. The behaviour of phytic acid in electrospray ionisation high‐resolution mass spectrometry (ESI‐HRMS) and its fragmentation, both in‐source and via collision‐induced dissociation, have not been studied so far. METHODS: The negative ion mass spectrometry and tandem mass spectrometry (MS/MS) of IP6, and the lower inositol pentakisphosphate (IP5), using an ESI‐Orbitrap mass spectrometer is described. The purity of the compounds was investigated using anion‐exchange chromatography. RESULTS: IP6 is highly anionic, forming multiply charged ions and sodium adduct ions, which readily undergo dissociation in the ESI source. MS/MS analysis of the phytic acid [M−2H](2−) ion and fragment ions and comparison with the full MS of the IP5 reference standard, and the MS/MS spectrum of the pentakisphosphate [M−2H](2−) ion, confirm the fragmentation pattern of inositol phosphates in ESI. Further evidence for dissociation in the ion source is shown by the effect of increasing the source voltage on the mass spectrum of phytic acid. CONCLUSIONS: The ESI‐HRMS of inositol phosphates is unusual and highly characteristic. The study of the full mass spectrum of IP6 in ESI‐HRMS mode indicates the detection of the compound in environmental matrices using this technique is preferable to the use of multiple reaction monitoring (MRM).