Liquid chromatography-mass spectrometry measurements of blood diphosphoinositol pentakisphosphate levels

Abstract

Diphosphoinositol pentakisphosphate (InsP₇) is an inositol pyrophosphate generated by inositol hexakisphosphate kinases (IP6Ks) that regulate diverse biological functions in cells. To date, we have a limited understanding of the InsP₇ biology owing to limited data on InsP₇ levels in blood or other tissues. Given the significant role of InsP₇ in maintaining biological homeostasis, further advancement in InsP₇ measurement is essential. In this study, we report a highly sensitive liquid chromatography with tandem mass spectrometry method for determining InsP₇ levels in whole blood, which is an easily accessible tissue and for which knowledge on InsP₇ levels is limited. We applied a perchloric acid-based method to increase the extraction efficiency of InsP₇ from the cells. Subsequently, we combined a YMC-Triart C18 metal-free column, ion-pair reagents, EDTA, methylenediphosphonic acid, and N,N,N',N'-Ethylenediaminetetrakis(methylenephosphonic acid) to minimize InsP₇ adsorption to the detection system. We prepared blood quality control samples that were highly exposed to IP6K inhibitor, showing minimum InsP₇ levels, to decrease the lower quantification limit of InsP₇. Furthermore, we applied rat plasma, which was found to show no InsP₇ levels, as a surrogate matrix. This setting resulted in the highly sensitive detection of InsP₇ levels in blood obtained from rats, with a quantification sensitivity of 1 ng of InsP₇ per mL of blood. The current method demonstrated acceptable accuracy (100 ± 15%) and precision (coefficient of variation ≤ 15%) in rat blood. Using this method, we revealed endogenous basal levels of InsP₇ (37.4 ng/mL) in blood obtained from normal rats, and decreased levels of InsP₇ (4.1-21.7 ng/mL) in blood obtained from IP6K inhibitor-administered rats. In summary, we established a highly sensitive method for measuring InsP₇ and revealed its levels in rat blood. The current findings may help in understanding InsP₇ biology.

Keywords: Blood; Inositol hexakisphosphate kinase; Inositol polyphosphate; InsP(7); Liquid chromatography-tandem mass spectrometry.