rXXXX American Chemical Society A dx.doi.org/10.1021/ac202165e | Anal. Chem. XXXX, XXX, 000–000 ARTICLE pubs.acs.org/ac Fenton Digestion of Milk for Iodinalysis C. Phillip Shelor, † Catrina A. Campbell, † Martina Kroll, † Purnendu K. Dasgupta,* ,† Tammy L. Smith, ‡ Amir Abdalla, ‡ Mike Hamilton, ‡ and Terri Wiley Muhammad ‡ † Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, Texas 76019-0065, United States ‡ Kansas City District Laboratory, U.S. Food and Drug Administration, Lenexa, Kansas 66214-3338, United States b S Supporting Information I odine is an essential micronutrient. The evaluation of iodine nutrition is of great importance, especially in infants; neurode- velopment is governed by iodine-containing thyroid hormones. 1 In a newborn, the thyroid holds only a 24 h reserve of the necessary iodine; 2,3 fresh supplies must come from intake. Recently, con- cerns have arisen about U.S. infant iodine nutrition. 4,5 We have been particularly interested in infant iodine nutrition. 6À8 In adults, measurement of urinary iodine (UI), preferably 24 h iodine excretion, is the benchmark measure of iodine status; 9 creatinine adjustments of spot samples are not always reliable. 10 Our own experience indicates that collecting urine in infants is a significantly more difficult task. Aside from obvious displeasure of the subjects, the success rate tends to be low and gender-biased. Many baby care products, from diaper rash ointments to baby wipes, also contain significant concentrations of an iodinated antifungal compound, 3-iodo-2-propynyl butylcar- bamate (IPBC; see ref 11 for a partial listing of baby care products containing IPBC). Elemental iodine analysis, as in inductively coupled plasma mass spectrometry (ICPMS), often practiced for iodine determinations, 12 can be affected by contamination of the urine with IPBC present on the skin, complicated by the fact that IPBC readily penetrates through the skin. 13 The determination of iodine in breast milk or formula given to an infant provides an alternative, allowing facile, uncontaminated sample collection. Most laboratories use one of two methods for iodinalysis of biological samples. 14 The SandellÀKolthoff (SÀK) method 15 relies on the catalytic effect of the iodide ion on the decoloriza- tion of yellow Ce 4+ by As 3+ . There can be interferences; digestion to mineralize these is essential prior to analysis. 15,16 The other method is ICPMS, typically conducted in the isotope dilution (ID) mode with 129 I as the isotopic tracer. 12 For urine samples, simple dilution and tracer addition are sufficient for ICPMS. For milk samples, digestion or sample preprocessing is needed. ’ SAMPLE DIGESTION METHODS AND ALTERNATIVES An alkaline digestion procedure 17 to measure protein-bound iodine has been much used. The protein is precipitated as the zinc conjugate, 18 excess alkali added, and slow overnight eva- poration to dryness conducted at 85À95 °C. The sample is then ashed (2.5 h at 600 °C) and extracted with dilute HCl. Pre-ashing thermal losses are minimized by vessel design and using KOH as the base, 19 but glass vessels are etched during ashing and cannot be reused due to iodine loss on the surface. 19 More recently, the use of tetramethylammonium hydroxide has been advocated for alkaline ashing; such methods are still to be widely adopted. 20 Acid digestion procedures have a different problem: losses as HI or I 2 must be avoided. Fortunately, oxidation to HIO 3 occurs readily with oxidizing acids. In traditional perchloric acid digestion (H 2 SO 4 , HNO 3 , and HClO 4 21 or HNO 3 and HClO 4 22 ), radio- tracer studies show that iodine loss is <20%. 22 Traditional HClO 4 digestion 21 was later updated 23 by a procedure that involves overnight digestion with HNO 3 ,H 2 SO 4 , and H 2 SO 4 at ambient conditions in a fume hood followed by refluxing and Received: August 16, 2011 Accepted: September 20, 2011 ABSTRACT: Iodine is an essential micronutrient especially important in the neurode- velopment of infants. Spot samples of urinary iodine (UI) are used as an epidemiologic index of adult iodine nutrition. Individual infant iodine nutrition is of vital importance, but infant urine is difficult to collect, much less a 24 h sample. Monitoring the intake provides a pragmatic solution for determining infant iodine nutrition. Because of the high solids content of milk and the possible existence of iodine in an organically bound form, sample digestion is obligatory. The U.S. Food and Drug Administration, for example, uses wet ashing by HClO 4 ; special precautions and fume hoods are required. We present a method of Fenton digestion of human and bovine milk samples and infant formula. No specialized equipment or hazardous reagents are used; measurement is made by isotope dilution inductively coupled plasma mass spectrometry. In Fenton digestion, Fe(II) and H 2 O 2 oxidizes the sample. In an interlaboratory study, excellent agreement (r 2 = 0.9934) was observed with results obtained by HClO 4 digestion and SandelÀKolthoff kinetic colorimetry. Average recoveries of iodide, triiodothyronine, and thyroxine ranged between 100% and 101%. Following digestion, iodine was found to exist entirely as iodide. Control of pH is imperative if loss cannot be corrected for by isotope dilution. Loss was below 20% for all samples when the pH was between 2.25 and 2.5