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Iodine in Infant Formula
Lipika Basumallick and Jeffrey S. Rohrer
245th ACS National Meeting & Exposition
April 7, 2013
OT70617_E 04/13S
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Outline
• Iodine
• Physiological Role
• Dietary Limits
• In Infant Formula
• Sample Preparation
• Detection Method
• Current Study
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Iodine
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Got Iodine?
• Physiological Role
• Synthesis of thyroid hormones by the thyroid gland
• Brain and central nervous system (CNS) development
(15th week to three years)
• Regulation of metabolic processes
• Recommendation for minimum daily iodine
intake
• Excess iodine can lead to thyroid disorders
(rare).
• Supplementation of iodine is a common
practice.
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Metabolic Circuit of Iodine
The iodine content of fruits, vegetables, and grains depends on the
iodine content of the soil in which it is grown.
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Iodine Deficiency
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Recommended Iodine Intake
J. Clin. Endocrinol. Metab. 2004, 89 (7), 3421–3424.
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Iodine Intake—Upper Limit
Upper Limit: ~10-fold of recommended intake
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Infant Formula
• Highly regulated consumer food product on the market
• The Infant Formula Act (1980) specifies minimum and
maximum amounts of several nutrients, and gives U.S. Food
and Drug Administration (FDA) the authority to create and
enforce standards for commercial infant formulas.
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Limits for Infant Formula
• Minimum Iodine: 5 mg/100 kcal
• FDA Infant Formula Act, Codex Alimentarius, European Union
guidelines
• Current suggested upper limit of iodine: 75 mg/100 kcal
• FDA 1985
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Initiative to Evaluate Methods and
Establish a Standard Method
Sponsored by the International Formula Council and AOAC
DRAFT AOAC
SMPR 2012.XXX
Method Name:
Approved by:
Adult/Pediatric
Formula
Infant Formula
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Version 2; June 18, 2012
Determination of Iodine in Infant Formula and
Adult/Pediatric Nutritional Formula
Stakeholder Panel for Infant Formula and Adult Nutritionals
Nutritionally complete, specially formulated food, consumed
in liquid form, which may constitute the sole source of
nourishment, made from any combination of milk, soy, rice,
whey, hydrolyzed protein, starch, and amino acids with and
without intact protein
Breast-milk substitute specially manufactured to satisfy, by
itself, the nutritional requirements of infants during the first
months of life up to the introduction of appropriate
complementary feeding, made from any combination of milk,
soy, rice, whey, hydrolyzed protein, starch, and amino acids
with and without intact protein.
Initiative to Evaluate Methods and
Establish a Standard Method
Method Performance Requirements
Analytical Range
5–1000 *
≤=5*
Limit of Quantitation
Repeatability (RSDr)
Recovery
Reproducibility (RSDr)
5–1000 *
≤ = 8%
90 to110% of mean spiked recovery
over the range of the assay
5–1000 *
≤ = 15%
Concentration applies to: a) “ready-to-feed” liquids “as is”;
b) reconstituted powders (25 g in 200 g of water); and
c) liquid concentrates diluted 1:1 by weight.
* mg/100 g reconstituted final product
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Iodine Determination
Robust Sample Preparation
to Extract the Iodide from the Formula
&
Sensitive Analytical Method for Measuring Iodide/Iodine
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Sample Preparation Options
• Acid digestion (chloric acid, acetic acid)
• Methanol/acetonitrile
• Alkaline ashing
• Ultracentrifugation
• Oxygen combustion
• Microwave digestion (perchloric acid, nitric acid, ammonia,
tetra methyl ammonium hydroxide) with closed/open vessel
• And others
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Commonly Used Techniques for Measuring Iodine/Iodide
• Colorimetry based on catalytic reactions
• Gas chromatography
• Ion-selective electrodes
• X-ray fluorescence spectrometry
• ICP-MS, SEC-ICP-MS , ICP/isotope dilution MS, ICS-OES
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Commonly Used Techniques for Measuring Iodine/Iodide
• Radiochemical neutron activation analysis
• Ion chromatography—UV detection, electrochemical
detection, ion chromatography (IC)-MS/MS, IC-ICP-MS
• X-ray fluorescence spectrometry
• Cathode stripping voltammetry
• Flame atomic absorption spectrometry
• And more
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A Few Have Been Adopted as Official Methods
1. AOAC Method 992.24, iodide determination in ready-tofeed (RTF) milk-based infant formula by ion selective
electrode
2. LMBG 49.00-6, a German-based official method for total
iodine by ICP-MS (also adopted by the European
Committee for Standardization as EN15111:2007)
3. ISO 14378:2000, an HPLC method for the determination
of free ionic iodide in pasteurized whole milk and dried
skim milk (similar to AOAC method)
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AOAC Method 992.24
• Method for Iodide in Ready-to-Feed Milk-Based
Infant Formula Ion-Selective Method
• Preparation of Test Solution
• Transfer 100 mL RTF test portion to 250 mL volumetric flask.
• Add 10 Ml 3% acetic acid solution. Dilute to volume with water and
mix well.
• Blend in blender on low speed two min.
• Filter to obtain clear filtrate, discarding the first 10 mL filtrate if cloudy.
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Sample Preparation for Current Study
10 mL formula + 2 mL 3% acetic acid + 8 mL DI water
Mix, 70 °C for 1 h
(control is 1 h in dark
at room temperature)
Centrifuge at 5000 rpm for 5 min
Filter 0.2 micron
Thermo Scientific™ Dionex™ On Guard™ II RP Cartridge
Injection
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Results
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Iodide on a Themo Scientific™ Dionex™ IonPac™ AS11
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Linear Range
Iodide Linearity 0.005–10 mg/L
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Low-Level Iodide Standard
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Iodide in Milk and Soy-Based Infant Formulas
Samples:
A. Standard
B. Infant Formula Brand 1
C. Infant Formula Brand 2
D. Infant Formula Brand 3
E. Infant Formula Brand 4
F. Soy Infant Formula
Peaks:
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1. Iodide
2. Thiocyante
Iodide
0.040 mg/L
0.027
0.042
0.023
0.035
0.046
Iodide Concentrations
Iodide in infant formula—acid digest at retention time vs 70 °C.
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Iodide (mg/L)
Heating vs No Heating—Time Course
Digestion Time (h)
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Iodine Speciation
• Iodine can be in free and bound form in infant formula.
• Room temperature acid digest extracts free iodine.
• 70 °C acid digest extracts both.
• Soy-based formula contains only free iodide.
J. Anal. At. Spectrom. 1999, 14, 1697
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Iodide Recoveries
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Iodide Recoveries
Milk-Based Infant
Formula Brand 2
Milk-Based Infant
Formula Brand 4
Soy-Based Infant
Formula
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Amount
Present
(mg/L)
Spiked after
Sample Prep
(mg/L)
Amount
Detected
(mg/L)
Recovery
(mg/L)
Aliquot 1
0.047
0.050
0.099
104%
Aliquot 2
0.051
0.050
0.108
113%
Aliquot1
0.035
0.050
0.092
109%
Aliquot 2
0.034
0.050
0.091
115%
Aliquot 1
0.038
0.050
0.091
111%
Microwave Digestion
Iodide (mg/L)
Sample
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On Label
Measured after
Microwave Digestion
Milk-Based Infant Formula Brand 1
0.1014
0.0913
Milk-Based Infant Formula Brand 2
0.0812
0.1450
Soy-Based Infant Formula
0.1010
0.0914
Method Ruggedness
• Over 800 injections of samples and standards
• Peak Area RSD <6%
• Retention Time RSD 0.6%
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Iodate as a Source of Iodine
• Iodate added to some infant formula as a source of iodine
• Need to measure Iodate to quantify total Iodine
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Iodate on a Dionex IonPac AS11?
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Convert Iodate to Iodide
• When iodate ions are added to an acidic solution containing
iodide ions, an oxidation-reduction reaction occurs.
IO3− + 5 I− + 6 H+ → 3 I2 + 3 H2O
• It is the iodine formed by this reaction that oxidizes the
ascorbic acid to dehydroascorbic acid as the iodine is
reduced to iodide ions
Ascorbic Acid + I2 → 2 I− + Dehydroascorbic Acid
• Due to this reaction the iodine formed is immediately reduced
to iodide as long as there is any ascorbic acid present.
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Iodate Spike
Chromatogram of iodide in A) infant formula and B) infant formula
spiked with iodate.
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Iodate Recovery (Room Temp)
Measuring iodate as iodide.
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Heat vs No Heat on Iodate Recovery
Recovery of iodate as iodide in milk- and soy-based infant formula.
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Conclusions
• Robust IC HPAE-PAD-based method
• Sample preparation conditions
optimized
• Extract free and bound forms of iodide
• Reduce iodate to iodide
• Broad linear range (0.005–10 mg/L)
and high precision (< 0.6% retention
time RSD and <6% peak area RSD
• Sensitive and accurate for milk- and
soy-based infant formula
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Thank you for your attention!
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