Analytical Biochemistry 454 (2014) 36–37
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Analytical Biochemistry
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Notes & Tips
Optimized ferrozine-based assay for dissolved iron
Thomas M. Jeitner ⇑
Neurosciences, Winthrop University Hospital, Suite 502, 222 Station Plaza North, Mineola, NY 11501, USA
a r t i c l e i n f o a b s t r a c t
Article history: The following report describes a simple and optimized assay for the detection of iron in solution based on
Received 16 September 2013 the binding of this metal by ferrozine. This assay accurately measures between 1 and 200 lM sample iron
Received in revised form 15 February 2014 concentrations within 2½ hours.
Accepted 25 February 2014
Available online 12 March 2014
Keywords:
Iron
Ferrozine
Disciplines as diverse as the food, environmental, and biological
sciences require assays for the measurement of iron concentra-
tions. The binding of ferrous iron by 3-(2-pyridyl)-5,6-bis(4-phe-
nylsulfonic acid)-1,2,4-triazine (ferrozine) causes the resulting
complex to absorb light with an extinction coefficient of
27,900 M 1 cm 1 at 562 nm, and allows for the convenient deter-
mination of iron in solution [1]. Since its introduction in 1970
[1], ferrozine has served as the basis of numerous assays for the
quantification of solvated iron [2–5]. The variety of these assays,
however, makes the task of selecting an appropriate method
difficult. This difficulty is compounded by the ambiguous composi-
tion of some of these assays. For example, the final pH of the
reaction mixture is often not reported. While the absorbance of
the iron–ferrozine complex remains unchanged in the pH range
from 4 to 9 [1], a number of the assays employ multiple acids with
no apparent consideration of the effects of these species on the
final pH of the solutions. Acidities less than pH 4 alter both the
linearity and the magnitude of color development in the ferro-
zine-based assay [1]. Given these concerns, the following study
was undertaken to optimize the ferrozine-based assay for dis-
solved iron.
Most of the published ferrozine-based assays employ ascorbic
acid to reduce the ferric iron in samples to its ferrous state
[2,4,5]. The ascorbic acid is often fortified with a strong acid to pre-
serve the reduced state of the former acid. One molar solutions of
unbuffered ascorbic acid, however, have a pH of 2.5 and are stable
on ice for several weeks. Frozen aliquots of 1 M ascorbic acid are
similarly inert for several months. Addition of 50 mM ascorbic acid
to 30 lM ferric iron was sufficient to reduce this metal to its fer-
⇑ Fax: +1 516 663 4710.
E-mail address: Tjeitner@Winthrop.org
http://dx.doi.org/10.1016/j.ab.2014.02.026
0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
Ó 2014 Elsevier Inc. All rights reserved.
rous state in 100 mM potassium acetate buffers ranging from pH
5 to 7.5, as indicated by development of equal amounts of optical
absorbance at these pH values, when combined with 10 mg/mL
ferrozine (data not shown). The concentrations of the stock solu-
tions used in these experiments were 1 M ascorbic acid, 0.2 mM
ferric chloride, and 50 mg ferrozine/mL 500 mM potassium acetate
buffer. These solutions were diluted into a 300 lL volume per well
of a 96-multiwell plate. The pH of the potassium acetate buffers
was adjusted with acetic acid, which has a pKa comparable to that
of ascorbic acid (i.e., pKa of acetic acid = 4.74 and pKa of ascorbic
acid = 4.17). Thus, the addition of ascorbic acid would be expected
to further acidify the reaction mixture. Under the final conditions
chosen for the assays—potassium acetate at a concentration of
100 mM and a pH of 5.5 – the addition of 50 mM ascorbic acid
resulted in a final pH of 4.42. Using this reaction mixture, the
absorbance at 562 nm due to the combination of iron and ferrozine
developed in a linear fashion with respect to iron concentrations,
as shown in Fig. 1. The color development depicted in Fig. 1 was
linear over the range of 225 pmol to 45 nmol iron which equates
to 1 to 200 lM sample iron concentrations. This range indicates
that this assay is as sensitive as the other reported protocols
[2,4,5]. The sensitivity of the assay was also improved by allowing
the color to develop for 24 h at 20 °C rather than recommended
periods of up to an hour (Fig. 1). Twenty-four hour incubations in-
creased the optical densities for all measured amounts of iron by
40% relative to the intensities observed after 40 min incubation
at 20 °C (P < 0.01, paired t test). Further incubations longer did
not increase the color of the reaction (data not shown). Viollier
et al. [3] also noted that the optical density of their ferrozine assay
increased slowly at 20 °C after an early and rapid generation of
color. The slow rate of color development in ferrozine assays is
explicable given that the reaction involves the simultaneous
 Notes & Tips / T.M. Jeitner0? / 454 (2014) 36–37 37

A. Color development due to 4.5 to 45 nmol Fe

0.20
3.0

Absorbance at 562 nm
2.5 0.15

2.0
0.10
1.5

1.0 0.05
37ºC
20ºC
0.5
Absorbance at 562 nm

40 min 0.00
1440 min 0 45 90 135 1440
0.0 Time (min)
0 10 20 30 40
Fig.2. Effect of warming on the absorption of the ferrozine–iron complex at
B. Color development due to 0.225 to 4.5 nmol Fe 562 nm. The effects of warming at 37 °C on the formation of the ferrozine–iron
complex due to 9 nmol iron are shown. With the exception of the amount of iron
and the changes in temperature, the conditions of these reactions were as described
0.25
in the legend for Fig. 1. Each symbol represents the mean and SEM of four
independent experiments which in turn were performed in quadruplicate.
Performing the reactions at 37 °C significantly increased optical density by
0.20 15 min and thereafter (P < 0.01, t test).

0.15
studies, we recommend a 135 min reaction at 37 °C beginning with
addition of 15 lL 1 M ascorbic acid to 225 lL of sample followed by
0.10 60 lL of 50 mg ferrozine/mL 500 mM potassium acetate buffer, pH
5.5. Under these conditions, the final reactant concentrations are
50 mM ascorbic acid, 10 mg /mL ferrozine (20.3 mM), and
0.05
100 mM potassium acetate, pH 5.5 in a total volume of 300 mL.
40 min
1440 min In summary, the above describes a simple assay and sensitive fer-
0.00 rozine-based assay for iron in solution.
0 1 2 3 4
Iron (nmol) Acknowledgments

Fig.1. Absorption of the ferrozine–iron complex at 562 nm. Shown is the absorption
of ferrozine–iron complex due to the binding of (A) 0 to 45 and (B) 0 and 4.5 nmol
iron to ferrozine at 20 °C. The depicted values represent the mean and SEM data of a
representative experiment. Regression analysis established that each of the
depicted cures was linear with R2 values of 1.0. The increases in optical density
due to incubation for 1440 min were significantly greater than the absorbance
values recorded after 40 min incubation, for every measured amount of iron
(P < 0.01, paired t test). Final concentrations of the reaction mixture were 50 mM
ascorbic acid, 10 mg/mL ferrozine (20.3 mM), and 100 mM potassium acetate, pH
5.5, and the sample and final volumes were 225 and 300 lL, respectively.
binding of three molecules of the dye to each atom of iron [6]. The
effect of increasing the reaction temperature from 20 to 37 °C was
examined in an attempt to decrease time taken for maximal color
development (Fig. 2). Increasing the reaction temperature to 37 °C
caused a significant increase in the amount of the iron–ferrozine
complex relative the amount obtained at 20 °C within 15 min of
the reaction. Incubation at 37 °C for 135 min increased color devel-
opment to 95% of the maximum observed at 24 h. Based these
These studies were funded by RO3-NS074286 and the Theresa
Pantnode Santmann Foundation Award.
References
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