Liquid chromatography coupled to tandem mass spectrometry for the analysis of inositol hexaphosphate after solid-phase extraction
Résumé
Inositol hexaphosphate, a naturally occurring component in cereals and plants, is the main reserve and principal carrier of phosphate. Inositol hexaphosphate is also found in biological fluids such as urine, plasma, or whole blood. Moreover, inositol hexaphosphate is studied for its pharmaceutical applications. Liquid chromatography coupled to mass spectrometry is now the reference method for small analyte determination. However, the specific quantitation of polyanionic molecules in the biological matrix is still challenging.
In this article, a bioanalytical method for the extraction and determination of inositol hexaphosphate in whole blood is described by using solid-phase extraction followed by liquid chromatography-mass spectrometry/mass spectrometry using selected reaction monitoring mode for its specificity.
Using pentylamine in excess, an ion pair is created, enhancing sensitivity by avoiding the presence of many Na adducts on the phosphate functions of inositol hexaphosphate. Moreover, hexafluoroisopropanol was added to stabilize the ion pairs that were created. Then, a specific extraction of inositol hexaphosphate by anion-exchange solid-phase extraction was developed, resulting in an extraction recovery of 89%. The linearity of the method was verified between 0.78 and 100 mu g/mL, and both accuracy and precision were greater than 85%. Finally, the endogenous rate of inositol hexaphosphate was measured in the whole blood of mice and was estimated at 2 mu g/mL.