OR/15/070 Appendix 2
| 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. |
Background
The purpose of the UV/Vis validation method is to describe the process used to validate the procedure and provide confidence in the robustness, accuracy and reproducibility of the method.
Scope
By using the UV/Vis method outlined in this report, concentrations of phytic acid will be measured in a range of staple African food sources. The method will predominantly focus on the quantification of foods containing high levels of phytate (>600 mg kg-1).
Test samples
The validation method will use wheat, rice and lentil samples. Pakistan wheat and Malawi rice samples were obtained during field work by BGS, whereas lentil data was obtained using a store bought product (UK). These contrasted samples showed phytate concentrations between 900–12000 mg kg-1. Resultantly, a wide range of phytate concentrations were tested within the validation method.
Calibration standards
As part of the Megazyme® phytic acid assay kit, a stock solution (50 µg/mL phosphorus), was provided. This was then diluted to create a range of calibration standards containing 0, 0.5, 2.5, 5.1 and 7.5 µg/mL of phosphorus.
Validation tests
To validate the method, both grain and rice samples were analysed, in quintuplicate, over 3 separate analytical runs. This allowed the determination of both within and between run variations. A number of runs were also carried out by a second analyst to allow the evaluation of analyst variation.
A stock solution of phytic acid was used to evaluate method accuracy at 7 and 80% of the top calibration standard during a separate run.
To validate the limit of detection (LOD) stated by Megazyme®, a blanks were run at n=10. The standard deviation (SD) was then calculated allowing the LOD and LOQ to be determined using the 3 and 10x SD respectively.
To determine spike recovery, 2 mL of a 100 000 mg kg-1 standard was added to a known sample (6442 mg kg-1 phytic acid), during extraction. This created a spiked addition of 9090 mg kg-1. Analysis was then carried out for n=5 spiked samples to determine an average spiked concentration and hence percentage recovery.
Acceptance criteria
To confirm the validation of the method, the following criteria must be attained,
- Oat flour percentage difference ≤10%
- Accuracy of 7 and 80% calibration standards ≥90%;
- Within and between run variation ≤10%;
- Spike recovery percentage ≥90%;
- Confirmation of no analyst variation at the 95% confidence level.