Liquid chromatography-mass spectrometry measurements of blood diphosphoinositol pentakisphosphate levels.

Diphosphoinositol pentakisphosphate (InsP) 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 InsP biology owing to limited data on InsP levels in blood or other tissues. Given the significant role of InsP in maintaining biological homeostasis, further advancement in InsP measurement is essential. In this study, we report a highly sensitive liquid chromatography with tandem mass spectrometry method for determining InsP levels in whole blood, which is an easily accessible tissue and for which knowledge on InsP levels is limited. We applied a perchloric acid-based method to increase the extraction efficiency of InsP 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 InsP adsorption to the detection system. We prepared blood quality control samples that were highly exposed to IP6K inhibitor, showing minimum InsP levels, to decrease the lower quantification limit of InsP. Furthermore, we applied rat plasma, which was found to show no InsP levels, as a surrogate matrix. This setting resulted in the highly sensitive detection of InsP levels in blood obtained from rats, with a quantification sensitivity of 1 ng of InsP 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 InsP (37.4 ng/mL) in blood obtained from normal rats, and decreased levels of InsP (4.1-21.7 ng/mL) in blood obtained from IP6K inhibitor-administered rats. In summary, we established a highly sensitive method for measuring InsP and revealed its levels in rat blood. The current findings may help in understanding InsP biology.