Validation of an LC–MS bioanalytical method for quantification of phytate levels in rat, dog and human plasma

• Direct analytical method for the quantification of phytic acid (IP6).
• Only methodology suitable for phytic acid analysis in plasma samples of 3 species.
• Validated according to the guidelines of the FDA for bioanalytical methods.
• Useful method. Applied for development of IP6 as drug for cardiovascular disease.
• This drug is ready to enter phase I clinical trials with dialysis patients.

Myo-inositol hexakisphosphate (phytate, IP6) is a naturally occuring compound whose determination in biological matrices is chanllenging. Several benefitial properties have been attributed to IP6 in parallel with the development of suitable analytical methodologies for its analytical determination in urine and some tissues. However, there is a lack of appropriate tools for its determination in plasma samples. In this paper, a direct, sensitive and selective bioanalytical method for the determination of IP6 based on LC–MS is presented. It is the first method published to quantify IP6 in plasma matrices directly through its molecular weight, being consequently a highly specific methodology. The method has been validated in rat, dog and human plasma, according to the acceptance criteria laid down in the FDA guidance Bioanalytical Method Validation. Accuracy and precision were not greater than 15% at medium and high concentrations and not greater than 20% at the LLOQ concentration. The mean absolute recovery obtained ranged from 78.74 to 102.44%, 62.10 to 87.21% and 61.61 to 86.99% for rat, dog and human plasma respectively. The LLOQ was 500 ng mL−1 due to the presence of endogenous IP6 in blank plasma samples and the limit of detection was within the range 30–80 ng mL−1.