OR/15/070 Methodology

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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.

This section provides a description of the experimental procedure used in the quantification of phytic acid in foodstuffs. Included is an overview of the extraction of both phytic acid and inorganic phosphorus from the grain samples, as well as the equipment required for analysis. A screenshot of the calculation software is also included below (Figure 1) that was used to determine phytic acid concentrations from absorbance measurements.

Phytic acid was extracted from grain samples using a 0.66 M solution of HCl. Following extraction, a number of enzymatic reactions were used to release inorganic phosphorus (Pi) from the extracted phytic acid as described by Megazyme®. To produce a solution quantifiable by UV/Vis spectroscopy, Pi was reacted with ammonium molybdate before redox chemistry formed a final solution containing molybdenum blue.

To quantify phytic acid in each sample, a Lambda 35 spectrometer from Perkin Elmer was used to measure absorbance of molybdenum blue at 655 nm. Proportionality between Pi and molybdenum blue allowed the software to calculate the concentration of Pi and hence phytic acid in the original sample.

Using this UV/Vis method it was possible to analyse up to 48 samples per day. With software calculating phytic acid concentrations directly from absorbance results (Figure 1). No additional staff time was required for data interpretation. This not only decreased costs but also increased time efficiency and greatly simplified the analytical process.

Figure 1    Megazyme phytic acid calculation software.
A. EQUIPMENT
      UV/Vis spectrometer Vortex mixer
      Water bath (stable at 40°C) Microcentrifuge and 1.5 mL tubes
      Glassware Micro‐cuvettes (1.5 mL)
      Analytical balance Timer
      Pipettes (20 µL to 5 mL) Megazyme calculation software
B. REAGENTS
      Megazyme® phytic acid assay kit Powdered ascorbic acid
      Concentrated Sulfuric acid Sodium hydroxide pellets
      Hydrochloric acid Powdered ammonium molybdate
      Powdered trichloroacetic acid
C. STABILITY OF REAGENTS
The Megazyme phytic acid assay kit provided solutions stable for over two years at 4°C:
      Two buffer solutions (pH 5.5 and 10.4) Phytase suspension
      Alkaline phosphatase Phosphorus standard solution
      Oat flour reference material
Other reagent solutions not supplied included the following,
      Ascorbic acid (10% w/v)/Sulfuric acid (1 M) Stable for one week at 4°C
      Ammonium molybdate (5% w/v) Stable for one month at 4°C
      Trichloroacetic acid (50% w/v) Stable for 6 months at 4°C
      Hydrochloric acid (0.66 M) Stable at room temperature
      Sodium hydroxide (0.75 M) Stable at room temperature

The supplied phosphorus standard was used to prepare a five phosphorus concentrations from 0 to 7.5 µg of phosphorus for calibration, including DI water, which were stable for one week at 4°C. The method employs a colour change in sample solutions as a result of the reaction of ascorbic acid and ammonium molybdate solutions in a 5:1 ratio. Due to its instability the complex/sample was prepared on the day of analysis (Appendix 1).