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1 The world leader in serving science
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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• Iodine • Physiological Role • Dietary Limits • In Infant Formula
• Sample Preparation
• Detection Method
• Current Study
Outline
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• 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.
Got Iodine?
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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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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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• 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
Limits for Infant Formula
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Sponsored by the International Formula Council and AOAC DRAFT AOAC SMPR 2012.XXX Version 2; June 18, 2012
Method Name: Determination of Iodine in Infant Formula and Adult/Pediatric Nutritional Formula
Approved by: Stakeholder Panel for Infant Formula and Adult Nutritionals
Adult/Pediatric Formula
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
Infant Formula
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
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Method Performance Requirements
Analytical Range 5–1000 *
Limit of Quantitation ≤ = 5 *
Repeatability (RSDr) 5–1000 * ≤ = 8%
Recovery 90 to110% of mean spiked recovery over the range of the assay
Reproducibility (RSDr) 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
Initiative to Evaluate Methods and Establish a Standard Method
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Robust Sample Preparation to Extract the Iodide from the Formula
Sensitive Analytical Method for Measuring Iodide/Iodine
Iodine Determination
&
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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-to-
feed (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
Centrifuge at 5000 rpm for 5 min
Filter 0.2 micron
Thermo Scientific™ Dionex™ On Guard™ II RP Cartridge
Injection
(control is 1 h in dark at room temperature)
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Iodide in Milk and Soy-Based Infant Formulas
Samples: Iodide A. Standard 0.040 mg/L B. Infant Formula Brand 1 0.027 C. Infant Formula Brand 2 0.042 D. Infant Formula Brand 3 0.023 E. Infant Formula Brand 4 0.035 F. Soy Infant Formula 0.046 Peaks: 1. Iodide 2. Thiocyante
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• 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.
Iodine Speciation
J. Anal. At. Spectrom. 1999, 14, 1697
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Amount Present (mg/L)
Spiked after Sample Prep
(mg/L)
Amount Detected (mg/L)
Recovery (mg/L)
Milk-Based Infant Formula Brand 2
Aliquot 1 0.047 0.050 0.099 104%
Aliquot 2 0.051 0.050 0.108 113%
Milk-Based Infant Formula Brand 4
Aliquot1 0.035 0.050 0.092 109%
Aliquot 2 0.034 0.050 0.091 115%
Soy-Based Infant Formula Aliquot 1 0.038 0.050 0.091 111%
Iodide Recoveries
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Microwave Digestion
Sample Iodide (mg/L)
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
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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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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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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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