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International Dairy Journal 34 (2014) 62e64

Contents lists available at SciVerse ScienceDirect

International Dairy Journal


journal homepage: www.elsevier.com/locate/idairyj

Short communication

Determination of iodide in high-dose dairy ingredient premixes


by high performance liquid chromatography
David C. Woollard a, Harvey E. Indyk b, *
a
b

Eurons NZlabs, 35 ORorke Road, Penrose, Auckland 1642, New Zealand


Fonterra Waitoa, SH26, Waitoa 3341, New Zealand

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 13 March 2013
Received in revised form
24 April 2013
Accepted 3 May 2013

Anhydrous premixes for inclusion in infant formulations and other nutritional products were analysed
for iodide content using high performance liquid chromatography with UV detection at 230 nm. Separation was achieved on a 3 mm Phenomenex C18 column with hexadecyl-trimethylammonium chloride
ion-pair in a 50:50 (v/v) acetonitrile:buffer (pH 5.5) mixture. Optimisation of the method has shown
quantitative recovery from a variety of matrices, with acceptable repeatability in the 3e4% range. The
contribution of sample heterogeneity and sample size to precision is discussed with the conclusion that
large subsamples (25e50 g) are required to facilitate a reliable quality-control method. The method is
applicable to premixes with iodide content of 20e2000 mg g1.
2013 Published by Elsevier Ltd.

1. Introduction
Iodine is an essential non-metallic element required in low, agedependent quantities (90e150 mg day1) for the adequate formation of thyroxin hormone. To full nutritional requirements of the
general population, and to prevent iodine-deciency disorders
such as hypothyroidism, goitre and cognitive impairment, table salt
is often fortied with iodide salts. However, iodine sufciency is
still a global problem, with many reports recommending higher
dietary intake (Prez-lpez, 2007; WHO, 2004).
In the case of infant and adult nutritional dairy-based formulations, iodine, as potassium iodide, is usually incorporated into the
bulk ingredients as part of a complex nutritional premix, where it is
titrated into a carrier such as lactose or maltodextrin. Many infant
formulae are manufactured by dissolving premixes prior to spraydrying of the nal product, thereby alleviating many homogeneity issues associated with dry-blended formulations.
There is a wide selection of methods for iodide testing that have
been reviewed (Edmonds & Morita, 1998; Paquette, Leveson, &
Thompson, 2012; Shelor & Purnendu, 2011). The analytical community can adopt one of two approaches to nutritional product
testing, targeting either iodide (Sertl & Malone, 1993; Tanner,
Barnett, & Mountford, 1993) or total iodine (Paquette et al., 2012),
and in premix products both approaches should be equivalent.

* Corresponding author. Tel.: 64 9 526 4514.


E-mail addresses: david.woollard@eurons.co.nz (D.C. Woollard), harvey.indyk@
fonterra.com (H.E. Indyk).
0958-6946/$ e see front matter 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.idairyj.2013.05.003

For premixes, where iodate and organic iodine are absent,


determination of the iodide anion is sufcient to complete the
quality-control determinations of iodine status. IDF ISO 14378:2009/
IDF 167:2009 describes a method using high performance liquid
chromatography-electrochemical detection (HPLC-ECD) to determine iodide in milk powder formulations involving the ion-pair reagent hexadecyl-trimethylammonium chloride (HDTA), and a similar
separation strategy has been developed in this study. The method
presented avoids any preliminary iodide purication step, while high
iodide levels facilitate direct UV detection.
2. Material and methods
2.1. Chromatography
Analysis was performed on an Agilent, Waldbronn, Germany
1100 HPLC equipped with a G1322A degasser, G1311A quaternary
pump, G1313A auto-sampler, G1316A column oven and G1315A
diode array detector. A 3 mm Phenomenex, Torrance CA, USA C18(2)
150 mm  3 mm column was operated at 0.5 mL min1 and injection volumes were 10 mL. The mobile phase consisted of 5 mL
HDTA, supplied as a 25% (w/w) solution from Aldrich, Steinheim,
Germany (cat 29,273-7), in a 1:1 (v/v) mixture of phosphate buffer
(50 mM KH2PO4, pH 5.5) and acetonitrile, and was allowed sufcient time for column equilibration.
Detection was performed at 230 nm and 210 nm, with the
higher wavelength used for quantitation, and the wavelength ratio
utilised for conrming peak identity. Quantitation was accomplished against potassium iodide (Merck Cat 1.05043.0500; 76.45%

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D.C. Woollard, H.E. Indyk / International Dairy Journal 34 (2014) 62e64

63

iodine) external standard calibration. The stock solution (approximately 1000 mg mL1 iodide) was prepared by dissolving 130 mg KI
in 100 mL deionised water. Volumes of 1, 5 and 10 mL were diluted
in 100 mL of water:acetonitrile (50:50, v/v) to produce working
standards in the range 1e100 mg mL1.
2.2. Sample preparation
Premixes (50 g) were weighed to 3 decimal places into 1 L volumetric asks. Approximately 500 mL deionised water was added and
mixed to thoroughly disperse, 5 mL HDTA added and the solution
made to volume. The solution was brought to 40  C and sonicated
(Bandelin Sonorex 10P, Berlin, Germany) for 30 min. After cooling,
5 mL of the extract was pipetted into a 10 mL volumetric ask and
made to volume with acetonitrile. The solution was ltered (0.45 mm)
into a 2 mL HPLC vial ready for analysis. The extract was stable for at
least 1 week.
2.3. Validation
The described method was subjected to estimation of precision
and recovery.
3. Results and discussion
3.1. Chromatography
An aqueous iodide test solution of 10 mg mL1 was subjected to
retention studies using a range of available C18 columns. Although
several yielded successful chromatography, the recommended
column was selected for further studies. The UV spectrum of iodide
facilitated detection in the low UV region at the relatively high
concentrations found in premixes. Near 230 nm, the absorption was
maximised against the eluent background and was used for
quantitation, while the 230:210 nm area ratio was a useful diagnostic of iodide identity. The dose:response relationship for working standards was linear at 230 nm. Fig. 1 depicts a typical
chromatogram of a poly-vitaminised premix containing 350 mg g1
of iodide.
3.2. Sample preparation
Attention was focussed on achieving reliable iodide extraction
from premixes containing an extensive range of poorly soluble
excipients. Simple dissolution of the samples in warm water was
potentially suitable, but recoveries were found to be erratic. To
alleviate this, 5 mL HDTA was added to each sample prior to sonication, which facilitated iodide recovery, despite the remaining
undissolved premix material. The effect of ultrasonication under
various conditions on iodide recovery was investigated and 30 min
at 40  C was found to be optimum.
To facilitate ltration and minimise on-column precipitation,
extracts were diluted with an equal volume of acetonitrile.
Even with rigorous mixing during commercial manufacture of
premixes, crystalline potassium iodide is not readily distributed
evenly. Thus, 50 g test samples were preferred to minimise
analytical variation, although 25 g prepared to 500 mL was a viable
alternative.
3.3. Method validation
A range of commercial premixes were used at the 50 g level for
single laboratory validation trials and yielded the following method
characteristics:

Fig. 1. Sample chromatograms of (A) premix extract and (B) iodide standard solution,
at 230 nm and 210 nm.

3.3.1. Repeatability
The repeatability for three premixes is shown in Table 1,
together with target concentrations provided by the manufacturer.
Each premix was a blend of vitamins, minerals, and iodine as potassium iodide. Precision was satisfactory for iodide above
100 mg g1 but the HorRat was >1 at concentrations of approximately 10 mg g1. This was due to both poor dispersion of crystalline
iodide and detection sensitivity.
Table 2 illustrates the effect of sample size on repeatability. A
mid-range premix (355 mg g1 iodide) was analysed at 50 g, 25 g
and 10 g test quantities and each was dissolved to the same nal
iodide concentration. These data illustrate that routine analysis
should be performed with not less than 25 g of premix. However, if
smaller sample weights are unavoidable, then reduced precision
can be expected.

Table 1
Repeatability of analysis of three premixes at various iodide concentrations
(mg g1).a
Parameter

Premix 1

Premix 2

Premix 3

Test 1
Test 2
Test 3
Test 4
Test 5
Test 6
Mean (mg g1)
SD (mg g1)
RSDr (%)
HorRatb
Expected (mg g1)

1988
2011
2111
1951
1950
2022
2006
59.6
2.97
0.58
1615e2055

766
804
811
788
839
795
801
24.4
3.05
0.51
740e942

18.0
12.4
16.1
15.2
19.7
14.8
16.0
2.56
16.0
1.5
12.4e15.6

Sample size 50 g.
HorRat observed RSD/predicted RSD from Horwitz equation; predicted
RSD 2^(10.5logC) where C is the concentration in g g1.
b

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64

D.C. Woollard, H.E. Indyk / International Dairy Journal 34 (2014) 62e64


Table 4
Recoveries of iodide spiked in to various premixes.a

Table 2
The effect of sample size on repeatability.

Premix

Parameter

50 g

25 g

10 g

Test 1
Test 2
Test 3
Test 4
Test 5
Test 6
Mean (mg g1)
SD (mg g1)
RSDr (%)
HorRat
Expected (mg g1)

351
360
337
342
337
367
349
12.5
3.59
0.54
314e385

369
343
337
355
328
327
343
16.4
4.76
0.72
315e385

378
317
328
344
374
387
351
29
8.26
1.25
315e385

1
1005
1
557
2
611
2
50.2
3
1560
3
333
4
1560
4
101
5
2120
5
509
Mean recovery (%)
a

Table 3
Comparison of results obtained for tested premixes with the current HPLC method
or alternative techniques (mg g1).a
Premix

Current HPLC

ISEb

ICPMSc

1
2
3
4
5
6
7
8
9
10

362
891
1634
987
56.7
620
259
1019
309
133

372
785
1529
969
e
e
e

e
e
e
e

a
b
c

57.0
641
249
1001
308
141

HPLC and ISE results are the average of triplicates on different days.
ISE was based on AOAC 992.24.
ICPMS was performed at another laboratory in duplicate on the same day.

3.3.2. Intermediate precision


Intermediate precision was evaluated over fteen days
involving three analysts, two HPLC instruments and 25 g portions
of a commercial premix containing an expected level of 340 mg g1
iodide. Duplicates were run daily and yielded a between-day intermediate precision (RSDiR) estimate of 4.27% (HorRat 0.64) and
an observed mean of 331.6 mg g1 (n 15). A repeatability RSDr of
4.14% (HorRat 0.62) was determined from within-day duplicates,
in agreement with independent repeatability experiments
(Tables 1 and 2). The minimal difference between these two precision estimates suggests that sampling error is the major component of total analytical error.
3.3.3. Accuracy
Since iodide absorption in the low-UV region does not allow for
spectral selectivity, chromatographic resolution is mandatory. A
wide range of iodide-free commercial vitamin-mineral premixes
were tested, with no underlying interferences observed. Certied
reference materials were not available, but a comparison with other
techniques is shown in Table 3. Ion-selective electrode (ISE) estimations were made at this laboratory and ICP-MS analyses at an
external laboratory based on Paquette et al. (2012).
When the protocol was followed as described, premixes spiked
with known levels of iodide showed an average recovery of 96.9% as
shown in Table 4. Iodide is known to be labile to oxidation under
certain circumstances, but the presence of ion-pair at commencement of the extraction stabilised the ion in the presence of other
excipients.
3.3.4. Limit of detection
HPLC with UV detection of iodide has reduced sensitivity
compared with electrochemical detection. However, the method

Iodide
added
(mg g1)

Iodide
measured
(mg g1)

Recovery
(%)

Other excipientsb

985
540
599
47.1
1509
323
1522
96
2089
496

98.0
96.9
98.0
93.8
96.7
97.0
97.6
95.0
98.5
97.4
96.9

FSV WSV
FSV WSV
FSV WSV
FSV WSV
FSV WSV
FSV WSV
FSV
FSV
WSV
WSV

MIN
MIN
MIN
MIN

Single tests for each premix, (n 1).


FSV, fat-soluble vitamins; WSV, water-soluble vitamins; MIN, minerals.

detection limit MDL [sd  t(n1, 0.01)] as estimated from replicate


analysis of a premix sample containing iodide near the detection
limit (see Table 1), was measured as 8.6 mg g1 (n 6). This
parameter sets the detection limit at the 99% condence level,
minimising false-positive errors. The described HPLCeUV method
is therefore suitable for the measurement of iodide in premixes at
levels >20 mg g1 with 3 mm C18 columns and 10 mL injection
volumes.
Analytically, such low iodide concentrations are readily
measured, but the associated random errors might not suit premix
manufacturers. The major problem with verifying the composition
of premixes with low iodide content is the difculty in blending a
nite number of potassium iodide crystals into a large bulk of
carrier. The only way of improving quality control decisions below
20 mg g1 is to take an impractically large sample for testing.

4. Conclusion
A reliable method has been developed and validated for use
with a wide range of vitaminemineral blends containing potassium
iodide. Iodide is extracted by dissolution and ltration and subjected to ion-pair HPLC with UV detection. The ion-pair system has
excellent capability for retaining iodide and separating it from
potential interfering factors. The method has sufcient sensitivity
for analysis of common commercial premixes used as additives in
infant formulations and other nutritional products.

References
Edmonds, J. S., & Morita, M. (1998). The determination of iodine species in environmental and biological samples. IUPAC publication. Pure and Applied Chemistry, 70, 1567e1584.
Paquette, L. H., Leveson, A. M., & Thompson, J. J. (2012). Determination of total
iodine in infant formula and nutritionals by inductively coupled plasma/mass
spectrometry. Single laboratory validation. Journal of AOAC International, 95,
169e179.
Prez-lpez, F. R. (2007). Iodine and thyroid hormones during pregnancy and
postpartum. Gynecological Endocrinology, 23, 414e428.
Sertl, D., & Malone, W. (1993). Liquid chromatographic method for determination of
iodine in milk: collaborative study. Journal of AOAC International, 76, 711e719.
Shelor, C. P., & Purnendu, K. D. (2011). Review of analytical methods for the quantication of iodine in complex matrices. Analytica Chimica Acta, 702, 16e36.
Tanner, J. L., Barnett, S., & Mountford, M. K. (1993). Analysis of milk-based formula
phase IV, iodide, linoleic acid and vitamin D and K: US Food and Drug
Administration e infant Formula Council: collaborative study. Journal of AOAC
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B. Takkouche, & H. Allen (Eds.), WHO global database on iodine deciency (pp. 1e
48). Geneva, Switzerland: World Health Organisation.