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S124 Food and Nutrition Bulletin, vol. 34, no. 2 (supplement) © 2013, The United Nations University.
Test kit for quantification of iron S125
determination of iron content in sauces [6, 7], but Portable device for measurement of iron
unfortunately no rapid quantitative test is available for concentration
ongoing monitoring at the production level to ensure
adequate fortification. A simple, rapid, and efficient The portable device consists of two units, the portable
method of monitoring the content of iron in fortified device (iCheck-Iron) and the disposable reagent vial
fish or soy sauces would therefore be extremely useful. (iEx IRON) in which the reaction is performed (fig. 1).
To meet this need, a small German enterprise, Bio- The disposable reagent vials contain 2 mL of reagents,
Analyt, has developed a portable device to simply and distributed in two phases, a water phase and an organic
rapidly quantify iron concentration in fortified foods solvent phase; both are needed for completion of the
(iCheck-Iron, hereafter referred to as the “portable reaction. Both the measuring unit and the reagent vials
device”). The objective of the study reported here was are commercially available (www.bioanalyt.com). The
to assess the ability and accuracy of the portable device portable device determines the concentration of iron
for measuring iron contents in iron-fortified fish sauce (intrinsic iron from the food matrix and extrinsic iron
and soy sauce using a four-step procedure: 1) determi- from fortification) in sauces by a photometric proce-
nation of working and linear range, 2) intraassay preci- dure. For quality control purposes, the device conducts
sion, 3) day-to-day precision, and 4) person-to-person an auto-control to verify that the emitter and receptor
precision. Finally, measurements were performed on are working correctly.
fish sauce and soy sauce fortified at two levels of iron During the study, the reagent vials and measuring
and compared with results obtained by atomic absorp- units were stored at room temperature (20° to 30°C)
tion spectrophotometry, commonly regarded as the prior to analysis. The recommendations and instruc-
gold standard method for mineral analysis. tions of BioAnalyt were followed except with respect
to the incubation times. For analysis, 0.4 mL of each
properly diluted prepared solution was injected into the
Materials and methods iEx IRON vial. To ensure that no air bubble was in the
syringe, the lab technician released any air bubbles by
Fortification of the fish sauce and soy sauce holding the syringe upwards and flipping the syringe
with his fingers. Once the injection was complete, the
In order to test the adequacy and accuracy of the port- lab technician vigorously shook the iEx IRON vial up
able device when used to measure iron levels in sauces, and down for 10 seconds and repeated this several
the Reproductive and Child Health Alliance (RACHA), times during the incubation period (0.3 hour, 1 hour,
which manages a national fortification program in
Cambodia, collected samples of soy sauces and fish
sauces from Cambodian manufacturers; 26 bottles of
soy sauce were collected from three manufacturers and
20 bottles of fish sauce from four manufacturers. The
intrinsic iron content of these sauces was measured
by atomic absorption spectrophotometry; the results
varied from 7.4 to 102.9 mg iron/L in soy sauces and
from 0.8 to 40.7 mg iron/L in fish sauces. The iron
compounds, ferrous sulfate (FeSO4, H2O), ferrous
fumarate (C4H2FeO4), and sodium iron ethylenedi-
aminetetraacetate (NaFeEDTA) (C10H12N2O8FeNa,
3H2O), were provided by DSM, a chemical company
certified by the Global Alliance for Improved Nutrition
(GAIN), and added to the sauces in the laboratories
of the Institut de Recherche pour le Développement
(IRD). Stock solutions at 250 mg iron/L were prepared
using each iron compound (ferrous sulfate, ferrous
fumarate, and NaFeEDTA) by dissolving appropriate
quantities of the compound either in ultrapure water
(milli-Q) or in fish or soy sauces using volumetric
flasks. This concentration (250 mg iron/L) was chosen
as representative of those usually used in programs
for the fortification of fish sauce and soy sauce. These
stock solutions were then diluted according to the FIG. 1. Prototype of the portable device that was assessed as
method described to reach concentrations in the port- described in this paper. As an indication of size, a US 25-cent
able device measurement range, i.e., 1 to 10 mg iron/L. coin (quarter) has been placed in the picture
S126 A. Laillou et al.
and 24 hours for ferrous sulfate and ferrous fumarate of the food matrix. The concentration of intrinsic iron
and 24 hours for NaFeEDTA). in these sauces was estimated by atomic absorption
spectrophotometry. Each sauce fortified with 250 mg
Reference method iron/L using one of the three iron compounds (ferrous
sulfate, ferrous fumarate, or NaFeEDTA) was diluted
Measurement by the reference method, atomic absorp- to reach the expected extrinsic iron concentrations of
tion spectrophotometry, was performed using a Per- 1.5 (instead of 1 mg/L due to the limit of detection),
kin-Elmer Atomic Analyst 800 (SpectrAA) with a 2.5, 5, 7.5, and 10 mg iron/L. Table 1 presents the
deuterium background corrector. Iron was extracted final expected iron concentrations taking into account
with a closed-vessel microwave digestion system intrinsic and extrinsic iron from each food matrix.
(ETHOS-1, Milestone, Italy) from about 1 mL of The portable device measurements were carried out
sample (stock solutions or sauces) in a 7:1 nitric acid/ after 0.3 hour, 1 hour, 17 hours, and/or 24 hours of
hydrogen peroxide mixture. The closed vessels were incubation.
placed in a microwave oven and digested at 1,200 W
power for 30 minutes. The elements were identified Variability of the measurements in laboratory conditions
by air–acetylene flame. Standard reference materials, To estimate intraassay, interassay, and interperson
BCR-679 White Cabbage and BCR-191 Brown Bread variation, the iron concentration of the diluted fish
(from IRMM, Institute for Reference Materials and sauce (containing the three different iron compounds)
Measurements, European Commission), were used as and soy sauce (containing only NaFeEDTA) at 5.0 mg
controls with iron SpectrAA measurements. of extrinsic iron/L (corresponding to 5.50 mg/L in
The coefficients of variation obtained with these two soy sauce and 5.56 mg/L in fish sauce) (table 1) was
reference materials were 5.74% and 5.62%, respectively, measured in triplicate by the same technician eight
with distances from the reference value of –1.24% in times on the same day (intraassay), by one technician
the case of white cabbage and –4.62% in the case of on three different days (interassay), and by three dif-
brown bread. ferent technicians on the same day (interperson), with
different incubation times (1 hour and 24 hours). The
Procedure for assessment of the portable device mean and coefficient of variation (CV) of the results
and the mean percentage of expected value were
The procedure used to assess the performance of the calculated.
portable device consisted of the three steps described
below. Comparison of the portable device with the reference
method
Linearity of the portable device To compare the results obtained with the portable
Assessment of a potential iron compound effect. Linearity device with those obtained with the atomic absorption
was determined by measuring in triplicate five standard spectrophotometer (SpectrAA), four unfortified fish
aqueous solutions with iron concentrations of 1, 2.5, 5, sauces and three unfortified soy sauces from Cambodia
7.5, and 10 mg iron/L (according to the portable device were fortified with ferrous fumarate, ferrous sulfate,
range); the five standard solutions were prepared for and NaFeEDTA at 200 and 400 mg iron/L (the range
the three different iron compounds (ferrous sulfate, of fortification levels used in Vietnam, Cambodia, and
ferrous fumarate, and NaFeEDTA) by appropriate dilu- China) by adding 32 and 64 mg, respectively, in the case
tion of the aqueous stock solutions at 250 mg iron/L. of ferrous fumarate and ferrous sulfate, and 80 and 160
The portable device measurements were carried out mg, respectively, in the case of NaFeEDTA, to 50 mL of
after 0.3 hour (20 minutes) of incubation for ferrous sauce (soy sauce only with NaFeEDTA). Then the sauce
sulfate, 0.3 and 1 hour for ferrous fumarate, and 24 samples were diluted 50 times to reach iron concentra-
hours for NaFeEDTA. tions in the linear range of the portable device between
Assessment of a potential food matrix effect. One fish 2 and 10 mg iron/L.
sauce and one soy sauce were selected to test the effect
TABLE 1. Expected iron concentration in iron-fortified soy and fish sauces (includ-
ing intrinsic and extrinsic iron)
Fortification level (mg iron/L)
1.5 2.5 5 7.5 10
Total iron concentration Soy sauce 1.65 2.75 5.50 8.24 10.99
(mg iron/L) Fish sauce 1.67 2.78 5.56 8.34 11.12
Test kit for quantification of iron S127
12
Statistical analysis
(mg/L)
6
necessary, data were subjected to analysis of variance
(ANOVA) and Fisher’s LSD tests were used to compare 4
means at the 5% significance level, using Statgraphics
Plus, version 5.1, software. To compare the methods, 2
besides plotting the two data sets and calculating the
Pearson coefficient and the linear regression equation 0
0 2 4 6 8 10 12
and paired Student’s t-tests, the Bland–Altman plot
Theoretical Fe concentration (mg/L)
was used [8]. This method consists of plotting the dif-
ferences between results obtained by the new method FIG. 2. Iron concentrations measured by iCheck-Iron in
(portable device) and the reference method (SpectrAA) standard aqueous solutions of different iron fortificants com-
against the mean of results obtained by both methods. pared with theoretical iron concentrations (calculated using
The limits of agreement (LOA) were calculated using the percentage of iron provided by the fortificant)
Δ – 2s = LOAlow
10 mg iron/L. If the incubation time was increased to 1
Δ + 2s = LOAhigh hour, the results were significantly improved and were
where close to the expected values in the range of 2.5 to 7.5
Δ is the mean of the difference between the mg iron/L. For NaFeEDTA, all values obtained with
two methods, and the portable device after 0.3 and 1 hour of incubation
s is the SD of this difference. were much below the expected values. After 24 hours
of incubation, however, the portable device measure-
ments were relatively good in the whole range of 1 to
Results 10 mg iron/L; the results were slightly above expected
values for the low concentrations and slightly below
Linearity of the portable device the expected values for the highest concentrations.
The best results were obtained with the solution of
Assessment of a potential iron compound effect iron at 5 mg/L. In general, with ferrous fumarate and
The linear range of the portable device was assessed NaFeEDTA, best results were obtained for the iron con-
between 1.0 and 10.0 mg iron/L as recommended by centration of 5 mg iron/L, although at 10 mg iron/L, the
BioAnalyt. Over this range, the coefficient of linear results started to move away from the expected values,
regression (R) and the regression equations for the thus showing the upper limit of the portable device.
different iron compounds were as follows: These results show that the iron compound used for
fortification has only a small impact on the measure-
Ferrous sulfate with 0.3 hour of incubation:
ments provided by the portable device iCheck-Iron. In
R = 0.9987 and y = 1.0267x + 0.05
all cases, the CV of the measurements was below 6.5%.
At the lowest iron concentration tested (1 mg iron/L),
Ferrous fumarate with 0.3 hour of incubation:
the portable device was not capable of providing iron
R = 0.9660 and y = 0.6956x + 0.465
measurements. The effective range of measurement
therefore appeared to be above 1 mg iron/L.
Ferrous fumarate with 1 hour of incubation:
R = 0.9941 and y = 0.8424x + 0.5663 Assessment of a potential food matrix effect
For both sauces, at the lowest iron concentration tested
NaFeEDTA with 24 hours of incubation:
(1.67 mg/L), the portable device was not capable of
R = 0.9974 and y = 0.8425x + 0.9018
providing iron measurements in numerous cases, thus
As shown in figure 2, for ferrous sulfate, with 0.3 hindering the calculation of SD. From these experi-
hour of incubation, all the portable device values were ments, the effective range of measurement of the port-
very close to the expected values in the range of 2.5 able device appeared to start around 2 mg iron/L. For
to 10 mg iron/L, although slightly higher. For ferrous NaFeEDTA, fish sauce and soy sauce showed similar
fumarate, with 0.3 hour of incubation, the portable results (fig. 3A and A´). It appeared clear that a mini-
device results were around 20% lower than expected mum incubation time of 24 hours was needed, as the
in the range of 2.5 to 7.5 mg iron/L and 40% lower at slope of the linear regression improved from 0.34 to
S128 A. Laillou et al.
A. Fish Sauce fortified with NaFeEDTA A´. Soy Sauce fortified with NaFeEDTA
12 12
Expected Expected
24h
10 24h 10
17h 17h
Fe iCheck (mg/L)
Fe iCheck (mg/L)
8 8
6 6
4 1h 4
1h
0.3h
0.3h
2 2
0 0
0 2 4 6 8 10 12 0 2 4 6 8 10 12
Expected Fe (mg/L) Expected Fe (mg/L)
B. Fish Sauce fortified with Fe fumarate B´. Soy Sauce fortified with Fe fumarate
12 12
Expected Expected
10 10
24h 24h
17h
Fe iCheck (mg/L)
Fe iCheck (mg/L)
17h
8 1h 8 1h
0.3h 0.3h
6 6
4 4
2 2
0 0
0 2 4 6 8 10 12 0 2 4 6 8 10 12
Expected Fe (mg/L) Expected Fe (mg/L)
C. Fish Sauce fortified with Fe sulfate C´. Soy Sauce fortified with Fe sulfate
12 Expected 12
17h Expected
1h 17h
0.3h
10 10
1h
Fe iCheck (mg/L)
Fe iCheck (mg/L)
8 8 0.3h
6 6
4 4
2 2
0 0
0 2 4 6 8 10 12 0 2 4 6 8 10 12
Expected Fe (mg/L) Expected Fe (mg/L)
FIG. 3. Iron measurements with iCheck-Iron in fish and soy sauces fortified with NaFeEDTA (A and A´), ferrous fumarate
(B and B´), or ferrous sulfate (C and C´) with different incubation times
Test kit for quantification of iron S129
0.82 in fish sauce and from 0.20 to 0.89 in soy sauce respectively, after 1 hour of incubation and 6.0%, 2.7%,
when the incubation time was increased from 1 hour and 7.4%, respectively, after 24 hours of incubation.
to 24 hours. Therefore, an incubation time of 24 hours Performing the measurements on different days led to
was used for any further measurement of sauce fortified a small increase in the CVs. According to the ANOVA
with NaFeEDTA. For ferrous fumarate (fig. 3B and B´), tests, in both types of sauce, whatever the iron com-
except for the lowest iron concentration, similar results pound, there was a slight difference between days.
were obtained with both sauces. The gap between This shows that results obtained from iCheck-Iron may
measured and expected values appeared to increase differ slightly from one day to another.
with iron concentration, which can be attributed to the
poor solubility of ferrous fumarate. For ferrous sulfate, Interperson precision
the results were different in soy and fish sauces, show- There was no significant effect when different opera-
ing a food matrix effect. In fish sauce, iron concentra- tors performed the measurement with the portable
tions measured with the portable device were close to device, as long as they followed the measurement pro-
the expected values independently of concentration tocol rigorously, including strong agitation 10 minutes
and incubation time. Conversely, in soy sauce, the before reading the result (p > .05).
results showed an effect of incubation time for iron
concentrations higher than 4 mg/L; the values were Comparison of the portable device with the reference
lower than expected for 0.3 and 1 hour of incubation. methods
After 17 hours, in both types of sauce, the portable
device values were very close to expected values, what- The results obtained with SpectrAA on fortified sauces
ever the concentration (fig. 3C and C´). at 250 mg iron/L were all very close to expected values
and showed low variability, with CVs from 0.3%
Variability of the measurements in laboratory to 2.2%. The mean results obtained with the port-
conditions able device on the different soy sauces (fortified with
NaFeEDTA) and fish sauces (fortified with one of the
Intraassay precision three iron compounds) were plotted against those
The means and CVs of the eight measurements con- obtained with SpectrAA (fig. 4). In figure 4A, only the
ducted within 1 day by one technician on diluted sauces data obtained after 1 hour of incubation of fish sauces
with total iron concentrations of 5.5 mg of iron/L for fortified with ferrous sulfate and ferrous fumarate were
soy sauce and 5.56 mg of iron/L for fish sauce are pre- used. In figure 4B, all data obtained after 24 hours
sented in table 2. The CV varied from 1.5% to 7.6%, of incubation were used. Paired Student’s t-tests per-
depending mainly on the iron compounds, and the formed on the differences between the results obtained
mean of the measurements was relatively close to the with the two methods showed no significant difference
expected concentration. between iCheck-Iron and SpectrAA (p > .05) after
either a 1-hour or a 24-hour incubation. The equation
Interassay precision of the correlation for 1 hour of incubation is y = 0.989x
The CV for the results of one person measuring for- + 5.112 (with ferrous sulfate and ferrous fumarate),
tified soy sauce with NaFeEDTA after 24 hours of with y being the result obtained with the iCheck-Iron.
incubation on three different days was 4.7%. For fish The corresponding Pearson coefficient for the relation-
sauce fortified with ferrous sulfate, ferrous fumarate, ship is R2 = 0.874, and the correlation coefficient is R =
and NaFeEDTA, the CVs were 4.3%, 2.9%, and 18.2%, 0.935. The Bland–Altman plots were drawn to further
TABLE 2. Intraassay precision (mean, SD, and CV) of iron concentration measured with the iCheck-Iron
device in fortified fish sauce and soy saucea
Expected Recovery rate (%)
Sauce and Incubation conc. Mean ± SD
fortificant time (h) (mg/L) conc. (mg/L) Mean Min–max CV (%)
Soy sauce 5.50
NaFeEDTA 24 5.65 ± 0.23 103 99–112 4.1
Fish sauce 5.56
Ferrous fumarate 1 4.90 ± 0.37 88 82–102 7.6
Ferrous fumarate 24 4.78 ± 0.34 86 80–98 7.1
Ferrous sulfate 1 5.84 ± 0.09 105 103–107 1.5
Ferrous sulfate 24 5.82 ± 0.15 105 102–109 2.6
NaFeEDTA 24 5.26 ± 0.25 95 89–101 4.8
a. Eight tests were performed within one day by one technician.
S130 A. Laillou et al.
assess the agreement between the two methods (fig. 5). Bland–Altman plot (fig. 5B) indicates that there are
The mean of the differences between the two methods data points (two soy sauces) that are outside the 2SD
(the mean lines on fig. 5) corresponds to the bias. The line. The limits of agreement are as follows: LOAlow is
Bland–Altman plot for 1 hour of incubation (fig. 5A) –73.7 mg/L and LOAhigh is + 68.9 mg/L, the mean dif-
indicates that there are no data points outside the 2SD ference between the methods being –2.3 mg/L.
line. The limits of agreement are as follows: LOAlow is When all iron fortificants are considered together,
–63.2 mg/L and LOAhigh is + 66.9 mg/L, the mean dif- the mean values of the differences (+1.8 on fig. 5A
ference between the methods being + 1.8 mg/L. and –2.4 on fig. 5B) are low compared with the order
For 24 hours of incubation, the equation of the cor- of magnitude of the studied parameter (the range of
relation (with ferrous sulfate, ferrous fumarate, and measurement was between 200 and 500 mg iron/L).
NaFeEDTA) is y = 0.8909x + 17.974, with y being the This means that there is no significant bias with
result obtained with iCheck-Iron. The corresponding iCheck-Iron measurements compared with SpectrAA
Pearson coefficient for the relationship is R2 = 0.8559, measurements.
and the correlation coefficient is R = 0.926. The
A. B.
500 500
Fe sulfate Fe sulfate
Fe fumarate Fe fumarate
NaFeEDTA
400 400
300 300
200 200
100 100
100 200 300 400 500 100 200 300 400 500
SpectrAA iron concentration (mg/L) SpectrAA iron concentration (mg/L)
FIG. 4. Linear regression between results obtained by iCheck-Iron and SpectrAA conducted in the laboratory with 1 hour
(A) or 24 hours (B) of incubation. The dashed line is the line of equality
A. B.
100 100
Difference iCheck 24h— SpectrAA
Difference iCheck 1h— SpectrAA
80 SD 80 SD
60 60
40 40
20 20
Mean Mean
0 0
–20 –20
–40 –40
–60 SD –60
SD
–80 –80
–100 –100
–120 –120
100 200 300 400 500 100 200 300 400 500
Mean iCheck 1h / SpectrAA Mean iCheck 24h / SpectrAA
Fe sulfate Fe fumarate Fe sulfate Fe fumarate NaFeEDTA
FIG. 5. Bland–Altman plots: difference versus average of values obtained by iCheck-Iron and SpectrAA conducted in the
laboratory with 1 hour (A) or 24 hours (B) of incubation. The ± 1.96 SD lines represent the 95% limits of agreement
Test kit for quantification of iron S131
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