Mayank Tandon, Thirumeignanam, D. and Dr. S.N. Rai.

Ferric Reducing
Antioxidant Power (FRAP) Assay. Dairy Cattle Nutrition Division, N.D.R.I.,
Karnal, India.
ESTIMATION OF TOTAL ANTIOXIDANT ACTIVITY
Total antioxidant activity is measured by ferric reducing antioxidant power (FRAP)
assay of Benzie and Strain (1999). FRAP assay uses antioxidants as reductants in a
redox-linked colorimetric method, employing an easily reduced oxidant system present
in stoichiometric excess.
Principle
At low pH, reduction of ferric tripyridyl triazine (Fe III TPTZ) complex to ferrous form
(which has an intense blue colour) can be monitored by measuring the change in
absorption at 593nm. The reaction is non specific, in that any half reaction that has
lower redox potential, under reaction conditions, than that of ferric ferrous half reaction,
will drive the ferrous (Fe III to Fe II) ion formation. The change in absorbance is
therefore, directly related to the combined or “total” reducing power of the electron
donating antioxidants present in the reaction mixture.
Reagents
FRAP Reagent
a) Acetate buffer 300 mM pH 3.6: Weigh 3.1g sodium acetate trihydrate and add
16 ml of glacial acetic acid and make the volume to 1 L with distilled water.
b) TPTZ (2, 4, 6-tripyridyl-s- triazine) (M.W. 312.34) 10 mM in 40mM HCl (M.W.
36.46)
c) FeCl3. 6H2O (M.W. 270.30) 20 mM
The working FRAP reagent was prepared by mixing a b & c in the ratio of 10:1:1 at the
time of use.
Standard: Ascorbic Acid (M.W. 176.13) 1000 µ M
Procedure
Sample (100 µl) (Plasma/ Milk/ Urine/ Feed Extract etc.) is mixed with 3 ml of working
FRAP reagent and absorbance (593 nm) is measured at 0 minute after vortexing.
Thereafter, samples are placed at 370C in water bath and absorption is again measured

FRAP Assay 1
after 4 minutes. Ascorbic acid standards (100µM-1000µM) were processed in the same
way.
Solutions Blank Standard Test
Sample (Plasma/ -- -- -- -- 100 μl
Milk/ Urine/ Feed
Extract etc.)
Standard -- -- 100 μl -- --
(Ascorbic acid)
Working FRAP 3000 μl 3000 μl 3000 μl
Solution

- Mix well
- Blank the analyzer/ spectrophotometer with Blank
- Measure the OD of Standard and Test at zero minute and again after four
minutes at 593 nm.
Calculation: Results were calculated as follows.

FRAP value of Sample (µM)

= (Change in absorbance of sample from 0 to 4 minute / Change in absorbance of standard
from 0 to 4 minute) X FRAP value of standard (1000 µM)

Note: FRAP value of Ascobic acid is 2.

Reference:
Benzie, F.F. and Strain, J.J. 1999. Ferric Reducing/ Antioxidant Power Assay: Direct
Measure of Total antioxidant Activity of Biological Fluids and Modified Version
for Simultaneous Measurement of Total Antioxidant Power and Ascorbic Acid
Concentration. Methods in enzymology. vol. 299:15-23.

FRAP Assay 2