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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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• Iodine • Physiological Role • Dietary Limits • In Infant Formula

• Sample Preparation

• Detection Method

• Current Study

Outline

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Iodine

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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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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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• 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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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: 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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Iodide Concentrations

Iodide in infant formula—acid digest at retention time vs 70 °C.

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Heating vs No Heating—Time Course

Digestion Time (h)

Iodi

de (m

g/L)

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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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Iodide Recoveries

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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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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!