Ann cu« Biochem 1989; 26: 144-147

A systematic evaluation of bathophenanthroline,

ferrozine and ferene in an ICSH-based method for
the measurement of serum iron
D P DERMAN, A GREEN, T H BOTHWELL, B GRAHAM, L McNAMARA,
A P MACPHAIL and R D BAYNES
From the M RC Iron and Red Cell Metabolism Research Unit, Department of Medicine, University of
the Witwatersrand Medical School, Park town 2193, Johannesburg, South Africa

SUMMAR Y. The chromogenic substrates ferrozinc and ferenc were compared to

bathophenanthroline disulphonic acid for the measurement of iron concentrations in
aqueous and serum samples in an assay based on that of the Iron Panel of the
International Committee for Standardisation in Haematology. Ferrozine and ferene
were more sensitive than bathophenanthroline. Copper at physiological concentra-
tions in plasma caused only minimal positive interference with all three chromogenic
substrates when thioglycollic acid was used as the reducing agent, but when ascorbic
acid was used significant positive interference occurred with ferrozine and ferene.
Interference due to contaminating haem was comparable with all agents. Bilirubin and
carotene produced no interference. Profound reductions in colour development were
noted with EDTA plasma.

Iron deficiency is a prevalent problem in the
developing areas of the world, with infants and
women being at particular risk.' It is therefore
desirable to measure serum iron concentrations
as inexpensively as possible using the smallest
possible volumes of serum. In this connection,
newer chromogenic substrates such as ferrozine
and ferene? 9 are both more sensitive and cheaper
than sulphonated bathophenanthroline, which is
the agent currently recommended by the Iron
Panel of the International Committee for Stan-
dardisation in Haematology (lCSH).IO These two
agents were therefore systematically compared
with bathophenanthrolinc in the present study.
Materials and methods
Ferrozine (monosodium 3-(2-pyridyl)-5, 6-bis-(4-
phenylsulphonic acid)-I ,2,4-triazine) and batho-
phenanthroline disulphonic acid (4,7 diphenyl-l,
IO-phenanthroline disulphonic acid) were
obtained from Sigma Chemical Corporation (St
Louis, MO, USA). Ferenc (disodium 3-(2-pyri-
dyl)-5, 6-bis [2-(5-furyl sulphonic acid)]-1,2,4-
Correspondence: Professor Thomas Bothwell, Depart-
ment of Medicine, University of the Witwatersrand
Medical School, 7 York Road, Parktown 2193, Johan-
nesburg, South Africa.
144
triazine) was a gift from Diagnostic Chemical,
Charlottetown, Port Edward Island, Canada.
Cupric sulphate (CuS04.5H 20) and concentrated
hydrochloric acid were obtained from BDH
(Poole, Dorset, UK). Neocuprin (2,9 dimethyl-I,
10 phenanthroline) and anhydrous sodium ace-
tate were obtained from Merck (Darmstadt, West
Germany). All reagents were of analytical grade.
The listed iron contents of the sodium acetate
and cupric sulphate were 0.0005°1., and 0·005%
respectively. Polished certified electrolytic iron
wire of purity greater than 99·9% was obtained
from J T Baker Chemical Co (Phillipsburgh, NJ,
USA). Spectrophotometric readings were made
using a Varian DMS 100 UV-visible spectro-
photometer (Varian Techtron Pty Ltd, Mulgrave,
Victoria, Australia). The absorbance of batho-
phenanthroline-based assays was measured at
535 nM,while 562 nM and 593 nMwere found to be
optimal for the ferrozine- and ferene-based assays
respectively. It should be noted that the iron-
ferrozine and iron-ferene absorbance peaks were
narrower than that of iron-bathophenanthroline,
a feature which might make them less suitable for
use with some filter instruments. The basic
method used was modified from that of the Iron
Panel of the ICSH.IO Details of the revised
recommended method appear in the Appendix.
 Serum iron measurement withferrozine orferene
Results
Absorbance as a function of pH was investigated
for the three chromogenic substrates. The range
of pH values was obtained by adding differing
volumes of concentrated hydrochloric acid and
sodium hydroxide. Whereas the pH range for
bathophenanthroline was broad (2'0-9'5), the
ranges for both ferrozine and ferene were more
restricted, with a sharp fall as the pH rose above
6·0. Interpretation of the absorbance curves was
complicated by the fact that the reductant thio-
glycollic acid formed a coloured complex with
iron above pH 5·2. The final curves were therefore
corrected by subtracting the absorbances that
were obtained at the relevant pHs when the
chromogens were not present (Fig. I). The maxi-
mum absorbances due to thioglycollic acid were
0·026 at 535 nM, 0·022 at 562 nM and 0·015 at 593
nM.
When the chromogenic substrates were each
used in a concentration of 0·25 g/1 (i.e. ferrozine
0·53 mrnol/L, ferene 0·51 mmol/L and bathophe-
nanthroline 0·51 mrnol/L) the assay was linear
over the range of iron concentrations encoun-
0·15
0·\0
1IJ
u effect of interference by copper. The potential
c
o
Ll
(;
en
Ll
« acid (2'5 gil, i.e. 14·2 mmol/L) was substituted
0·05
o "--''r-r-r-r--r,-,-----,--,---,------r'"'':'''
o I 2 3 4 5 6 7 8 9 10 II
pH
FIG. 1. The absorbances of iron complexes with
bathophenanthroline at 535 nM(0), ferrozine at 562 nM
(.) and ferene at 593 nMee) over a pH range from ',5 to
10·5. Since the reductant, thioglycollic acid, also formed
a coloured complex with iron at higher pHs, the
individual cur~es have been corrected appropriately.
ThIS was achieved by subtracting the absorbances
obtained at the various pHs on samples not containing
any of the three chromogenic agents. The concentration
of iron in the test solution was 17·9 Jlmol/L.
145
tered in serum (4,5-143,1 Jlmol/L) (r= 1·000;
P < 0·000 I for all three). The absorbance readings
expressed relative to that developed with batho-
phenanthroline disulphonic acid (100,:,q were
125·1'1., (±3·6%) for ferrozine and 150·7%
(± 2'3%) for ferene. The measured extinction
coefficients were 22 369, 27 963 and 33 850
Lrrnol/cm for bathophenanthroline, ferrozine
and ferene respectively. Increasing the concentra-
tion of the chromogenic agent from 0·25 gil to
O· 5 gil (i.e. ferrozine \·06 mmol/L, ferene 1·02
mrnol/L and bathophenanthroline 1·02 rnmol/L)
did not materially alter the assay results nor did
changing the concentrations of sodium acetate
from I mol/L to 2 mol/L,
The time of measuring colour development (up
to 3 h) did not influence the concentration of iron
obtained. While there was an increase in the
abso~bance of both the test samples and blanks,
the difference between the two readings remained
constant.
The effect on the three chromogenic substrates
of contaminating copper was also tested, since it
had previously been reported to lead to over-
estimation of the serum iron concentration. 11
There was a slight increase in measured iron
concentrations with all three chromogenic sub-
strates when increasing concentrations of copper
were added and thioglycollic acid was used as the
reductant. The rise in the iron concentration was
very modest, with concentrations of copper of up
to 31· 5 Jlmol/L causing a rise in iron concentra-
tion ofless than 0·54 Jlmol/L in both aqueous and
serum samples. The addition of 0·25 gil (1'2
mrnol/L) neocuprin virtually erradicated the
interference by copper was significantly affected
by the nature of the reductant. When ascorbic
for the thioglycollic acid, the interfering copper
had a marked dose-dependent effect on iron
values when ferrozine and ferene were used as the
chromogenic substrates. For example, the addi-
tion of 31·5 Jlmol/L copper caused an increase in
iron concentrations of 2'13 and 3·22 Jlmol/L with
ferrozine and ferene respectively. In contrast,
copper had no apparent effect when bathophe-
nanthroline was used with ascorbic acid.
Values for serum iron concentrations using the
various chromogenic substrates under study were
compared on 20 serum samples. Correlations
were as follows: bathophenanthroline: ferrozine
r = 0·9997, P < 0·000 I; bathophenanthroline: fer-
ene r=0'9998, P<O·OOOI. The numerical
relationships between the methods were: batho-
phenanthroline = (ferrozine x 1·0 I) + 0·00036
146 Derman et al.
J.lIIloI/L; bathophenanthroline = (ferene x 1,03)
+0·55 J.Lmol/L.
It was found possible to scale down the serum
sample size of 2·0 mL recommended by the Iron
Panel of the ICSHIOto 0·5 mL without prejudic-
ing the accuracy or reproducibility of the method.
Sequential dilutions of test sera and the addition
of known quantities of iron yielded values very
close to those predicted. Ten repeated measure-
ments on the same serum sample yielded a
coefficient of variation (CY) of 1·7% for batho-
phenanthroline, 1·4%, for ferrozine and 2·1 o/., for
ferene. Longitudinal intersample variability was
evaluated by using the same stock solutions to
measure a serum sample thirteen times over a
five-week period. The inter-sample variability
was 2·1 % (bathophenanthroline), 3·7% (ferene)
and 2·5% (ferrozine). The addition of ethanol
(0'34 mmol/L), bilirubin (43 J.LmoI/L) dissolved in
Na2COJ (0'01 mol/L) or carotene (0'93 Ilmol/L)
did not alter the absorbances obtained in blank,
standard and serum samples, while Iyophilised
haemoglobin in a concentration of 480 mg/L did
not cause any changes in absorbances with the
three chromogenic substrates. Subsequent freez-
ing or prolonged incubation at 37°C in the
presence of the Iyophilised haemoglobin caused
measurable overestimation (freezing: 5,8%,
5,0%, and 5·5% for bathophenanthroline, ferro-
zine and ferene respectively; 37"C: 3·9%, 7·1%
and 7-4% for bathophenanthroline, ferrozine and
ferene respectively). When fresh haemoglobin
was employed similar results were obtained. The
presence of EDTA in the sample markedly inhi-
bited colour development, especially in the pres-
ence of either ferrozine or ferene. The inhibition
could not be overcome by adding excess zinc prior
to testing.
Discussion
The present findings demonstrate that the newer
chromogenic substrates, ferene and ferrozine, are
very similar to bathophenanthroline disulphonic
acid in terms of accuracy, stability, reproducibi-
lity and assay conditions. Since the operating pH
in the assay is in the region of4'5, the fall off in the
absorbance with ferene and ferrozine with
increasing pH was not of any practical signifi-
cance. In addition, the normal operating condi-
tions for the assay (pH 4·5,1,5 mol/L Na-Acetate
and 0·25 giL chromogenic substrate) appeared
optimal for both ferene and ferrozine. The two
newer substrates were also comparable to batho-
phenanthroline in terms of interference from
other pigments and reagents (alcohol, bilirubin,
carotene and haemoglobin). EDTA had a
marked inhibitory effect on the measurement,
which means that EDTA plasma should not be
used with any of these chromogenic agents.
In the presence of thioglycollic acid, copper
that was added to an iron solution and to a serum
sample caused a modest rise in the measured iron
concentration. However, even at high concentra-
tions of copper (63 J.LmoI/L) the measured rise was
still less than 2 J.Lmol/L. When ascorbic acid was
used as reductant there was significantly more
copper interference (4,47 J.Lmol/L at a copper
concentration of 63·0 J.LmoI/L) but this was noted
only with ferrozine and ferene and not with
bathophenanthroline.
Since it is desirable to measure the serum iron
concentrations on as small a volume of serum as
possible, the newer chromogenic agents ferrozine
and ferene offer potential advantages. In addi-
tion, ferrozine and ferene are less expensive than
the previously recommended chromogenic sub-
strate, bathophenanthroline disulphonic acid.
Given their comparable assay performance they
should replace bathophenanthroline disulphonic
acid in the recommended methods for measure-
ment of serum iron concentration. On their
performance in the current investigation, the use
of either reagent would seem equally appropriate.
Appendix
BASIC METHOD
Render all water iron-free by passing water
distilled in a glass apparatus through a suitable
deioniser (e.g. Elgastat Spectrum System C deio-
nizer, Elga, Bucks, UK).
SOLUTIONS
Protein precipitant
Prepare a solution of 100 g/L trichloracetic acid
and 30 g/L thioglycollic acid by dissolving 50 g
trichloracetic acid in 200-300 mL water contain-
ing 43 mL concentrated hydrochloric acid (0,5
rnol/L) and 15 mL concentrated thioglycollic
(mercaptoacetic) acid. Make up to a final volume
of 500 mL with the addition of water. If stored in
a dark brown bottle, the solution will remain
stable for at least 2 months.
Chromogen solution: bathophenanthroline
sulphonate. ferrozine or ferene
Prepare a 1·5 molar Na-acetate solution contain-
ing 0·25 g/L, bathophenanthroline, ferrozine or
ferene by dissolving 61·5 g Na-acetate and 125 mg
of the chromogen in 200-300 mL water and make
 Serum iron measurement withferrozine or ferene
up to a final volume of 500 mL in a volumetric
flask with the addition offurther water. Store this
solution in a dark brown bottle wrapped in
aluminium foil; it will maintain its potency over at
least an 8-week period.
Stock iron standard solution (17'9 mmol]
L; 1 mgjmL)
Place I g dry, polished certified electrolytic iron
wire in a 1 litre volumetric flask containing 20 mL
concentrated hydrochloric acid and heat over a
boiling water bath. When it is dissolved make up
to volume with water. Prepare a working iron
standard solution 35·78 1lmoljL by placing 2 mL
of the stock iron in a 1 litre volumetric flask and
make up to volume with 0·005 mol/L redistilled
HC!.
TEST METHOD
Add O'5 mL serum, working standard solution or
reagent blank to 0·5 mL of the protein precipitant
solution. Agitate the mixture thoroughly for 45 s
either by hand or with a vortex mixer. Allow to
stand for at least 5 min and then centrifuge at
1500 g for 20 min. Add 0·5 mL to an equivalent
volume of the chromogenic substrate and mix.
After allowing to stand for at least 10 min,
measure the absorbance spectrophotometrically
at the appropriate wavelength (535 nM for batho-
phenanthroline, 562 nM for ferrozine and 593 nM
for ferene). Calculate the results as follows:
Serum iron concentration
Abs serum unknown-Abs blank
Abs iron standard - Abs blank x 35·78
= concentration of iron in 1lmoljL
147
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Accepted/or publication 2 November 1988