Simultaneous determination of inositol and inositol phosphates in complex biological matrices: Quantitative ion-exchange chromatography/tandem mass spectrometry

myo-Inositol (Ins) and myo-inositol phosphates (InsPs) are widely distributed in plants and animals. The evaluation of the distribution of Ins and InsPs in cells and plant sources can impact the understanding of their role in nutrition, cellular processes and diseases, and how they may be modulated by diet. We developed an anion-exchange chromatography/tandem mass spectrometry (HPLC/ESI-MS/MS) method for the separation and simultaneous quantitation of Ins and different naturally occurring phosphorylated inositol compounds. Chromatographic separation was achieved in 30 min on a commercial anion-exchange column (0.5 x 150 mm) using a gradient of 200mM ammonium carbonate buffer (pH 9.0) and 5% methanol in H₂O. Analytes were identified by selective reaction monitoring using a triple quadrupole mass spectrometer in negative ion electrospray ionization mode. Adenosine 5′-monophosphate was used as a general internal standard for quantitation. Detection is linear in the range of 0.25-400 pmol for Ins, InsP₁, InsP₄, and InsP₅, 40-400 pmol for InsP₂ and InsP₃, and 60-400 pmol for InsP₆, with a minimum r² > 0.994. The limit of detection is 0.25 pmol with a signal-to-noise ratio of 10:1 for all analytes. The intra-day and inter-day variations were within 17% at three concentration levels. Recovery values for the seven analytes spiked into extraction solution or different matrices were between 63 and 121%. Using this approach, Ins and InsPs were measured in three different plant samples and in cultured cells, illustrating significant differences in the distribution of inositol compounds in food samples compared to cells and between cell types.