|Reason, D A, Watts, M J, and Devez, A. 2015. Quantification of phytic acid in grains. (Inorganic Geochemistry, Centre for Environmental Geochemistry). British Geological Survey Internal Report, OR/15/070.|
The procedure for the quantification of phytic acid using UV/Vis spectroscopy is described in Appendix 1. This also contains instructions on the preparation of standards and specific solutions required for the analysis. Appendix 2 outlines the validation plan devised to test the rigidity and reproducibility of the method, respectively. The stability of each reagent used was described previously in the Methods section, with storage duration/conditions described for each reagent summarised in Appendix 1.
The validation process began with the analysis of four samples covering a range of typical phytic acid concentrations from rice and grain samples (1534–10 964 mg kg-1). Each sample was analysed in triplicate for n=5. Percentage relative standard deviation (%RSD), was used to determine both within and between run variations. Standards at 7 and 80% of the top calibration standard (7.5 µg/mL phosphorus equating to 29598 mg kg-1 phytic acid) at (n=5) were analysed to confirm accuracy and precision. Since standards were prepared using a phosphorus solution, calculations were required to convert these values to phytic acid concentrations prior to analysis. Phytic acid concentrations for the 7 and 80% of the highest calibration standard were 1790 and 23 679 mg kg-1, respectively. This corresponded to phosphorus concentrations of 517 and 6000 mg kg-1. A rice sample spiked with a phytic acid standard was also used to measure extraction recovery performance. A further two analytical runs were carried out by a second operator to establish whether analyst variation had a significant influence on performance criteria.
* Note: 5% of the top calibration standard resulted in a solution below the limit of quantification; hence a higher standard (7%) was used to overcome sensitivity issues.
Detection limits (DL), were quoted as 400 mg kg-1 in the assay procedure provided by Megazyme® using a smallest absorbance difference of 0.05. Using techniques described by Gonzalez and Herrador (2007), our own detection limits (DL) were obtained using standard deviations (SD), of 10 blank solutions. The limit of detection (LOD) and the limit of quantification (LOQ) were calculated using a value of three and ten times SD, resulting in values of 413 mg kg-1 and LOQ = 1408 mg kg-1, respectively (Gonzalez et al. 2007).
- Gonzalez, A G, and Herrador, M A. A practical guide to analytical method validation, including measurement of uncertainty and accuracy profiles. (2007). Trends Analytical Chemistry, 26, 227–238.