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Validation of a gold standard method for iodine quantification in raw and processed milk, and its variation in different dairy species

  • G. Niero
    Affiliations
    Department of Agronomy, Food, Natural Resources, Animals and Environment, University of Padova, Viale dell'Università 16, 35020 Legnaro (PD), Italy
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  • M. Franzoi
    Affiliations
    Department of Agronomy, Food, Natural Resources, Animals and Environment, University of Padova, Viale dell'Università 16, 35020 Legnaro (PD), Italy
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  • V. Vigolo
    Correspondence
    Corresponding author
    Affiliations
    Department of Agronomy, Food, Natural Resources, Animals and Environment, University of Padova, Viale dell'Università 16, 35020 Legnaro (PD), Italy
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  • M. Penasa
    Affiliations
    Department of Agronomy, Food, Natural Resources, Animals and Environment, University of Padova, Viale dell'Università 16, 35020 Legnaro (PD), Italy
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  • M. Cassandro
    Affiliations
    Department of Agronomy, Food, Natural Resources, Animals and Environment, University of Padova, Viale dell'Università 16, 35020 Legnaro (PD), Italy
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  • C. Boselli
    Affiliations
    Istituto Zooprofilattico Sperimentale del Lazio e della Toscana “M. Aleandri”—National Reference Centre for Ovine and Caprine Milk and Dairy Products Quality (CReLDOC), Via Appia Nuova 1411, 00178 Rome, Italy
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  • G. Giangolini
    Affiliations
    Istituto Zooprofilattico Sperimentale del Lazio e della Toscana “M. Aleandri”—National Reference Centre for Ovine and Caprine Milk and Dairy Products Quality (CReLDOC), Via Appia Nuova 1411, 00178 Rome, Italy
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  • M. De Marchi
    Affiliations
    Department of Agronomy, Food, Natural Resources, Animals and Environment, University of Padova, Viale dell'Università 16, 35020 Legnaro (PD), Italy
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Open ArchivePublished:March 20, 2019DOI:https://doi.org/10.3168/jds.2018-15946

      ABSTRACT

      Adequate milk consumption significantly contributes to meeting the human iodine recommended daily intake, which ranges from 70 µg/d for infants to 200 µg/d for lactating women. The fulfilment of iodine recommended daily intake is fundamental to prevent serious clinical diseases such as cretinism in infants and goiter in adults. In the present study iodine content was measured in raw and processed commercial cow milk, as well as in raw buffalo, goat, sheep, and donkey milk. Iodine extraction was based on 0.6% (vol/vol) ammonia, whereas iodine detection and quantification were carried out through an inductively coupled plasma mass spectrometer analyzer. Among processed commercial cow milk, partially skimmed pasteurized milk had the greatest iodine content (359.42 µg/kg) and raw milk the lowest (166.92 µg/kg). With regard to the other dairy species, the greatest iodine content was found in raw goat milk (575.42 µg/kg), followed by raw buffalo (229.82 µg/kg), sheep (192.64 µg/kg), and donkey milk (7.06 µg/kg). Repeatability of milk iodine content, calculated as relative standard deviation of 5 measurements within a day or operator, ranged from 0.96 to 1.84% and 0.72 to 1.16%, respectively. The overall reproducibility of milk iodine content, calculated as relative standard deviation of 45 measurements across 3 d of analyses and 3 operators, was 4.01%. These results underline the precision of the proposed analytical method for the determination of iodine content in milk.

      Key words

      INTRODUCTION

      Iodine is a fundamental element for human health, with particular regard to thyroid activity and thyroid hormone functionality (
      • Schomburg L.
      • Köhrle J.
      On the importance of selenium and iodine metabolism for thyroid hormone biosynthesis and human health.
      ). Iodine is involved in direct and indirect regulation of a wide array of cellular metabolic pathways and plays a primary role in the regular development of fetus and in infant growth (
      • Underwood E.J.
      Trace Elements in Human and Animal Nutrition.
      ). Dietary intake is the unique source of iodine, and apart from iodized salt, iodine-rich foods are sea fish, crustaceans, milk, and dairy products. The recommended daily intake for humans depends on age and physiological status, and ranges from 70 µg/d for infants to 200 µg/d for lactating women (
      • Zimmermann M.B.
      • Andersson M.
      Prevalence of iodine deficiency in Europe in 2010.
      ;
      • EFSA
      Scientific opinion on the Dietary Reference Values for iodine.
      ). Low iodine intake leads to poor development of neurological and physical functions in children, as well as to serious clinical outcomes such as goiter in adults (
      • EFSA
      Scientific opinion on the Dietary Reference Values for iodine.
      ). Because less seafood is being consumed for cultural, economic, and health reasons (
      • Oken E.
      • Kleinman K.P.
      • Berland W.E.
      • Simon S.R.
      • Rich-Edwards J.W.
      • Gillman M.W.
      Decline in fish consumption among pregnant women after a national mercury advisory.
      ;
      • Verbeke W.
      • Vanhonacker F.
      • Sioen I.
      • Van Camp J.
      • De Henauw S.
      Perceived importance of sustainability and ethics related to fish: a consumer behavior perspective.
      ), iodized salt, milk, and dairy products have become the major sources of iodine in many European countries (
      • Girelli M.E.
      • Coin P.
      • Mian C.
      • Nacamulli D.
      • Zambonin L.
      • Piccolo M.
      • Vianello-Dri A.
      • Gottardo F.
      • Busnardo B.
      Milk represents an important source of iodine in schoolchildren of the Veneto region, Italy.
      ;
      • Watutantrige Fernando S.
      • Barollo S.
      • Nacamulli D.
      • Pozza D.
      • Giachetti M.
      • Frigato F.
      • Redaelli M.
      • Zagotto G.
      • Girelli M.E.
      • Mantero F.
      • Mian C.
      Iodine status in schoolchildren living in northeast Italy: the importance of iodized-salt use and milk consumption.
      ).
      Iodine content in cow milk is mostly influenced by sanitizers used for teat dipping procedures (
      • Flachowsky G.
      • Franke K.
      • Meyer U.
      • Leiterer M.
      • Schöne F.
      Influencing factors on iodine content of cow milk.
      ;
      • van der Reijden O.L.
      • Galetti V.
      • Hulmann M.
      • Krzystek A.
      • Haldimann M.
      • Schlegel P.
      • Manzocchi E.
      • Berard J.
      • Kreuzer M.
      • Zimmermann M.B.
      • Herter-Aeberli I.
      The main determinants of iodine in cows' milk in Switzerland are farm type, season and teat dipping.
      ) and iodine level in feed (
      • Norouzian M.
      • Azizi F.
      Factors affecting iodine content in dairy cow's milk - A review.
      ). The latter, in turn, might be highly variable according to season, farming system, dietary supplements, and level of iodine antagonists (
      • van der Reijden O.L.
      • Galetti V.
      • Hulmann M.
      • Krzystek A.
      • Haldimann M.
      • Schlegel P.
      • Manzocchi E.
      • Berard J.
      • Kreuzer M.
      • Zimmermann M.B.
      • Herter-Aeberli I.
      The main determinants of iodine in cows' milk in Switzerland are farm type, season and teat dipping.
      ). Supplementing iodine rates in feedstuffs has positive effects on animal health as it is necessary for adequate thyroxin hormone activity, allowing normal fetus development and calf growth (
      • Weiss W.P.
      • Wyatt D.J.
      • Kleinschmit D.H.
      • Socha M.T.
      Effect of including canola meal and supplemental iodine in diets of dairy cows on short-term changes in iodine concentrations in milk.
      ).
      • van der Reijden O.L.
      • Galetti V.
      • Hulmann M.
      • Krzystek A.
      • Haldimann M.
      • Schlegel P.
      • Manzocchi E.
      • Berard J.
      • Kreuzer M.
      • Zimmermann M.B.
      • Herter-Aeberli I.
      The main determinants of iodine in cows' milk in Switzerland are farm type, season and teat dipping.
      observed that the use of iodine-containing disinfectant was associated with an increase of about 40 μg/L of iodine content in bulk milk.
      • Schöne F.
      • Leiterer M.
      • Lebzien P.
      • Bemmann D.
      • Spolders M.
      • Flachowsky G.
      Iodine concentration of milk in a dose–response study with dairy cows and implications for consumer iodine intake.
      reported that doses of 0.2, 1.3, 5.1, and 10.1 mg of iodine supplement per kg of DM are reflected in increased milk iodine content, which averaged 101, 343, 1,215, and 2,762 µg/kg, respectively.
      Given the importance of milk and dairy product consumption to achieve the daily iodine requirement in humans, some methods for iodine quantification in milk have been reported (
      • Hedayati M.
      • Ordookhani A.
      • Daneshpour M.S.
      • Azizi F.
      Rapid acid digestion and simple microplate method for milk iodine determination.
      ;
      • Reid H.J.
      • Bashammakh A.A.
      • Goodall P.S.
      • Landon M.R.
      • O'Connor C.
      • Sharp B.L.
      Determination of iodine and molybdenum in milk quadrupole ICP-MS.
      ), but very few have described a simple extraction protocol and validation procedure to quantify iodine. Even less information is available with regard to the accuracy and sensitivity of available methods. Furthermore, even if iodine content in cow milk has been previously studied (
      • Sanchez L.F.
      • Szpunar J.
      Speciation analysis for iodine in milk by size-exclusion chromatography with inductively coupled plasma mass spectrometric detection (SEC-ICP MS).
      ;
      • Hedayati M.
      • Ordookhani A.
      • Daneshpour M.S.
      • Azizi F.
      Rapid acid digestion and simple microplate method for milk iodine determination.
      ;
      • Walther B.
      • Wechsler D.
      • Schlegel P.
      • Haldimann M.
      Iodine in Swiss milk depending on production (conventional versus organic) and on processing (raw versus UHT) and the contribution of milk to the human iodine supply.
      ), little information on iodine content in processed milk (
      • Stevenson M.C.
      • Drake C.
      • Givens D.I.
      Further studies on the iodine concentration of conventional, organic and UHT semi-skimmed milk at retail in the UK.
      ) and in milk of dairy species other than cows is available in the literature. Therefore, the objective of the present study was to validate a simple, fast, and robust gold standard method based on alkaline extraction and inductively coupled plasma (ICP) MS for the quantification of iodine in raw and processed commercial cow milk, and in raw buffalo, goat, sheep, and donkey milk.

      MATERIALS AND METHODS

      Reagents and Analytical Apparatus

      Ultrapure 30% ammonia (vol/vol) was purchased from Nova Chimica (Cinisello Balsamo, Italy). Ultrapure water (18.3 MΩ/cm resistivity at 25°C) was produced with Sartorius Arium 611 UV (Monza Brianza, Italy). Iodine used for calibration curves and rhodium used as internal standard were purchased from Sigma-Aldrich (St. Louis, MO) at the highest available purity. Reference certified skim milk powder ERM BD150 was purchased from the European Joint Research Centre (Geel, Belgium).
      Milk samples were analyzed for fat, protein, casein, lactose, and MUN content using a MilkoScan FT6000 (Foss Electric A/S, Hillerød, Denmark), and for SCC using a Fossomatic (Foss Electric A/S, Hillerød, Denmark) in the laboratory of the Breeders Association of Veneto Region (ARAV, Padova, Italy). Milk iodine extraction was carried out in the laboratory of the Department of Agronomy, Food, Natural Resources, Animals and Environment of the University of Padova (Legnaro, Italy). Iodine detection and quantification were carried out through an ICP-MS analyzer (Spectro MS, Spectro Analytical Instruments, Kleve, Germany) in the laboratory of Eurolab (Bassano del Grappa, Italy).
      All solutions and solvents, including 30% ammonia and ultrapure water, were tested for iodine contamination through ICP-MS analyses. In both reagents, iodine content was lower than the limit of detection of the instrument.

      Sample Collection and Preparation

      Milk samples were collected between May and June 2018. Bulk cow raw milk (RM) samples (n = 5) were purchased in 5 different vending machines (Padova, Italy). Commercial processed cow milk samples, namely whole pasteurized milk (WM, n = 5), partially skimmed pasteurized milk (SM, n = 5), whole UHT milk (WUM, n = 5), and partially skimmed UHT milk (SUM, n = 5), were purchased in local stores (Padova, Italy). In addition, bulk buffalo (n = 5), sheep (n = 5), goat (n = 5), and donkey (n = 2) RM samples were collected in different farm tanks (Roma, Italy).
      A solution of 0.6% ammonia (vol/vol) was prepared by diluting 20 mL of 30% ammonia in ultrapure water, up to 1 L of final volume. To promote homogenization, milk samples were warmed at room temperature for 1 h, gently inverted for 20 times, and successively diluted (1:24) in 0.6% ammonia solution in disposable 50-mL plastic tubes. The mixture was incubated in a water bath at 90°C for 1 h to promote iodine extraction. After cooling at room temperature, samples were filtered using a 0.45-µm syringe filter. Finally, 5 mL of the filtered solution was diluted (1:1) in 0.6% ammonia, up to a final volume of 10 mL. The resulting solution was 50-fold diluted compared with the starting milk to keep expected sample salinity below 0.2%, as recommended for ICP-MS trials (
      • Beauchemin D.
      Encyclopedia of Spectroscopy and Spectrometry.
      ). Overall, milk sample preparation took about 1.5 h for a batch of 20 samples.

      Iodine Detection and Quantification

      Instrument settings, chemical parameters, and physical conditions adopted for ICP-MS analyses (Table 1) followed the recommendations provided by
      • Spectro ICP-MS Report
      The analysis of drinking water.
      . Standard solutions were prepared freshly on each day of analysis in 0.6% ammonia, and iodine quantification was obtained with 5-point calibration curves (R2 ≥ 0.99; i.e., 0, 1, 10, 20, and 40 μg/L). The same standards were measured after each quantification round to check the effective stability of the analysis. All samples and iodine standard solutions were added with 10 µL of 1 mg/kg rhodium solution as internal standard, to cope with the underestimation due to the losses of the target element. Five technical replicates were performed for each sample as well as for each standard solution used for calibration. Therefore, iodine quantification was calculated as the mean value of 5 technical replicates, both for milk samples and iodine standard solutions. Relative standard deviation calculated on technical replicates was defined as instrumental repeatability (
      • Khan N.
      • Jeong I.S.
      • Hwang I.M.
      • Kim J.S.
      • Choi S.H.
      • Nho E.Y.
      • Choi J.Y.
      • Park K.S.
      • Kim K.S.
      Analysis of minor and trace elements in milk and yogurts by inductively coupled plasma-mass spectrometry (ICP-MS).
      ).
      Table 1Parameter settings of inductively coupled plasma emission mass spectrometer analyzer for iodine detection and quantification in milk
      ParameterSetting
      Power1,425 W
      Cool gas flow rate12 L/min
      Auxiliary gas flow rate1.2 L/min
      Nebulizer flow rate0.92 L/min
      Sample aspiration rate1 mL/min
      Sample coneNickel
      Spray chamberCyclonic (baffled)
      Plasma torchQuartz, fixed 1.5-mm injector tube
      Base interval/integration time20 ms/30 s
      Technical replicates5

      Method Validation

      Time-related repeatability was defined as the relative standard deviation (RSDr) of 5 consecutive measurements on 5 aliquots of the same milk sample, separately processed within the same day. Time-related reproducibility was calculated as the relative standard deviation (RSDR) of 15 measurements on 15 aliquots of the same milk sample, separately processed after 24, 48, and 72 h. Similarly, operator-related repeatability was assessed as RSDr of 5 consecutive measurements on 5 aliquots of the same milk sample, separately processed by the same person. Operator-related reproducibility was assessed as the RSDR of 15 measurements on 15 aliquots of the same milk sample, separately processed by 3 different operators who handled 5 aliquots each (
      • Franzoi M.
      • Niero G.
      • Penasa M.
      • Cassandro M.
      • De Marchi M.
      Technical note: Development and validation of a new method for the quantification of soluble and micellar calcium, magnesium, and potassium in milk.
      ;
      • Niero G.
      • Penasa M.
      • Berard J.
      • Kreuzer M.
      • Cassandro M.
      • De Marchi M.
      Technical note: Development and validation of an HPLC method for the quantification of tocopherols in different types of commercial cow milk.
      ). Horwitz and Horwitz ratios (HorRat) were used as comparison between measured and expected reproducibility values. Horwitz value represents the expected RSD in reproducibility conditions, whereas HorRat is the ratio between the observed RSDR and the Horwitz value. The overall relative standard deviation (RSDO) was calculated on iodine quantification carried out by 3 different operators, who handled 5 experimental replicates in 3 different days of analyses, for a total of 45 measurements on 45 aliquots. Thus, RSDO express the overall method reproducibility (
      • Landon G.
      • Bouvier-Capely C.
      • Legrand A.
      • Sontag T.
      • Finance G.
      • Saint-Hilaire M.
      • Rebière F.
      • Millot X.
      • Renaud-Salis V.
      • Agarande M.
      Validation and comparison of two calibration methods for the measurement of stable iodine in the urinary matrix by ICP-MS: Standard addition vs. external calibration.
      ).
      For recovery trials and calculation, 200 mg of certified skim milk powder was dissolved in 20 mL of 0.6% ammonia solution and gently shaken for 10 min before being processed as previously described. Five aliquots were analyzed within 1 d, and for 3 consecutive days. Relative standard deviation of repeatability and RSDR were calculated within day and across 3 d of analyses, as previously described for milk samples. Recovery was expressed as the percentage ratio between measured and certified iodine value (
      • Franzoi M.
      • Niero G.
      • Penasa M.
      • Cassandro M.
      • De Marchi M.
      Technical note: Development and validation of a new method for the quantification of soluble and micellar calcium, magnesium, and potassium in milk.
      ;
      • Niero G.
      • Penasa M.
      • Berard J.
      • Kreuzer M.
      • Cassandro M.
      • De Marchi M.
      Technical note: Development and validation of an HPLC method for the quantification of tocopherols in different types of commercial cow milk.
      ).
      Limit of detection (LOD; expressed as µg/kg), was assessed using 10 independent blank measurements and calculated as
      LOD = 2 × t95 × σ × (1 + 1/n)1/2,


      where t95 is the Student's 95% 1-sided confidence interval, σ is the standard deviation of the blank measurements at the same interval of iodine signal normalized for the method dilution, and n is the number of blank measurements (
      • Todorov T.I.
      • Gray P.J.
      Analysis of iodine in food samples by inductively coupled plasma-mass spectrometry.
      ). Limit of quantification (LOQ; expressed as µg/kg), was calculated as 3 × LOD (
      • Armbruster D.A.
      • Pry T.
      Limit of blank, limit of detection and limit of quantitation.
      ).

      RESULTS AND DISCUSSION

      Variation of Iodine Content Across Technological Treatments

      Descriptive statistics for iodine content, milk chemical composition, MUN, and SCC in raw and processed cow milk are reported in Table 2. Partially skimmed pasteurized milk had the greatest iodine content (359.42 µg/kg), followed by WUM (305.24 µg/kg), SUM (266.60 µg/kg), WM (253.53 µg/kg), and RM (166.92 µg/kg). To the best of our knowledge there are no scientific studies dealing with milk iodine content in relation to milk chemical composition. Because iodine compounds are soluble in water (
      • Dahl L.
      • Opsahl J.A.
      • Meltzer H.M.
      • Julshamn K.
      Iodine concentration in Norwegian milk and dairy products.
      ;
      • Moschini M.
      • Battaglia M.
      • Beone G.M.
      • Piva G.
      • Masoero F.
      Iodine and selenium carry over in milk and cheese in dairy cows: Effect of diet supplementation and milk yield.
      ), it is likely that milk iodine increases in partially skim milk, having greater soluble fraction on a volume to volume ratio as a result of the skimming process (
      • Niero G.
      • Penasa M.
      • Currò S.
      • Masi A.
      • Trentin A.R.
      • Cassandro M.
      • De Marchi M.
      Development and validation of a near infrared spectrophotometric method to determine total antioxidant activity of milk.
      ). Further than soluble or inorganic fraction, results of the present study suggest that milk iodine might be partially associated with casein micelles, as observed by
      • Franzoi M.
      • Niero G.
      • Penasa M.
      • Cassandro M.
      • De Marchi M.
      Technical note: Development and validation of a new method for the quantification of soluble and micellar calcium, magnesium, and potassium in milk.
      and
      • Visentin G.
      • Penasa M.
      • Niero G.
      • Cassandro M.
      • De Marchi M.
      Phenotypic characterisation of major mineral composition predicted by mid-infrared spectroscopy in cow milk.
      who reported favorable correlations between milk proteins and major milk minerals. Overall, the average content of iodine in WUM observed in the present study was close to values reported by
      • Stevenson M.C.
      • Drake C.
      • Givens D.I.
      Further studies on the iodine concentration of conventional, organic and UHT semi-skimmed milk at retail in the UK.
      , slightly lower than values reported by
      • Payling L.M.
      • Juniper D.T.
      • Drake C.
      • Rymer C.
      • Givens D.I.
      Effect of milk type and processing on iodine concentration of organic and conventional winter milk at retail: implications for nutrition.
      , and slightly higher than findings of
      • Dahl L.
      • Opsahl J.A.
      • Meltzer H.M.
      • Julshamn K.
      Iodine concentration in Norwegian milk and dairy products.
      . Conversely, the overall iodine content in WM was considerably lower than that reported by
      • Payling L.M.
      • Juniper D.T.
      • Drake C.
      • Rymer C.
      • Givens D.I.
      Effect of milk type and processing on iodine concentration of organic and conventional winter milk at retail: implications for nutrition.
      and
      • Stevenson M.C.
      • Drake C.
      • Givens D.I.
      Further studies on the iodine concentration of conventional, organic and UHT semi-skimmed milk at retail in the UK.
      , whereas the average iodine contents in RM and WUM were from 1.28- to 2.83-fold and 2.26- to 4.77-fold those reported by
      • Walther B.
      • Wechsler D.
      • Schlegel P.
      • Haldimann M.
      Iodine in Swiss milk depending on production (conventional versus organic) and on processing (raw versus UHT) and the contribution of milk to the human iodine supply.
      .
      Table 2Descriptive statistics of iodine content, milk chemical composition, MUN, and SCC in different commercial processed cow milks
      Milk
      RM = raw milk; WM = whole pasteurized milk; SM = partially skimmed pasteurized milk; WUM = whole UHT milk; SUM = partially skimmed UHT milk.
      Samples, nIodine, μg/kgFat, %Protein, %Casein, %Lactose, %MUN, mg/dLSCC, cells/mL
      MeanCV, %MeanCV, %MeanCV, %MeanCV, %MeanCV, %MeanCV, %MeanCV, %
      RM5166.9270.933.6913.843.332.402.492.814.782.2127.3842.0025731.60
      WM5253.5316.573.512.363.310.792.600.634.890.5528.5412.1134110.88
      SM5359.4219.361.543.023.330.942.661.053.330.9426.4412.0935118.96
      WUM5305.2426.853.472.073.381.352.672.174.862.2136.067.763974.91
      SUM5266.6021.561.554.463.451.382.741.304.941.7237.028.111444.63
      1 RM = raw milk; WM = whole pasteurized milk; SM = partially skimmed pasteurized milk; WUM = whole UHT milk; SUM = partially skimmed UHT milk.
      The greatest coefficient of variation (CV) for iodine content was observed for cow RM (70.93%), whereas processed milk showed considerably lower CV, from 16.57 to 26.85% for WM and WUM, respectively. As expected, the greatest CV for gross milk composition, including fat, protein, casein, lactose, and MUN content was calculated for RM, whereas lower CV was observed for processed and standardized milk samples. Thus, it is likely that the variation of milk iodine content is associated with the variation of milk chemical composition (and vice versa), rather than to the pasteurization procedures or to the UHT treatments; this agrees with previous findings of
      • Walther B.
      • Wechsler D.
      • Schlegel P.
      • Haldimann M.
      Iodine in Swiss milk depending on production (conventional versus organic) and on processing (raw versus UHT) and the contribution of milk to the human iodine supply.
      . Overall, it cannot be excluded that factors such as animal feeding, farming conditions, herd management, and sanitizers used for pre- and postdipping procedures might have affected milk iodine concentration. Nevertheless, because bulk milk from different herds is mixed to obtain standardized commercial milk samples, we might assume that the aforementioned sources of variation were smoothed to a negligible extent.

      Variation of Iodine Content Across Dairy Species

      Descriptive statistics for iodine content, milk chemical composition, MUN, and SCC in bulk buffalo, sheep, goat, and donkey RM are reported in Table 3. Buffalo, sheep, and goat milk showed similar composition to that reported by
      • Niero G.
      • Currò S.
      • Costa A.
      • Penasa M.
      • Cassandro M.
      • Boselli C.
      • Giangolini G.
      • De Marchi M.
      Short communication: Phenotypic characterization of total antioxidant activity of buffalo, goat, and sheep milk.
      in individual milk samples. In agreement with previous findings of
      • Salimei E.
      • Fantuz F.
      • Coppola R.
      • Chiofalo B.
      • Polidori P.
      • Varisco G.
      Composition and characteristics of ass's milk.
      , donkey RM had relatively low fat (0.65%) and protein content (1.78%), and comparatively high lactose content (6.27%). Iodine content in buffalo, sheep, goat, and donkey milk averaged 229.82, 192.64, 575.42, and 7.06 µg/kg, respectively. Thus, ruminant dairy species, including cow, buffalo, sheep, and goat showed iodine content in the same order of magnitude. On the other hand, notably lower iodine content was observed for donkey milk, being the only monogastric species considered in the present study. Within ruminants, the greatest iodine content observed in goat RM was associated with the lowest fat percentage. Thus, the hypothesis of greater milk iodine content in low fat milk might be confirmed also in ruminant dairy species other than cow. Differences in iodine contents across species are likely due to animal diet and physiology, nutrient absorption and transportation, herd management, farming systems, and milking procedures, as reported also for major milk minerals (
      • Flynn A.
      Minerals and trace elements in milk.
      ).
      Table 3Descriptive statistics of iodine content, milk chemical composition,
      Casein and MUN were not available for sheep and goat milk.
      MUN, and SCC in raw milk of different dairy species
      MilkSamples, nIodine, μg/kgFat, %Protein, %Casein, %Lactose, %MUN, mg/dLSCC, cells/mL
      MeanCV, %MeanCV, %MeanCV, %MeanCV, %MeanCV, %MeanCV, %MeanCV, %
      Buffalo5229.82103.657.648.454.525.113.405.224.874.9928.5239.7410847.46
      Sheep5192.6438.406.788.485.578.134.415.2657683.50
      Goat5575.4250.413.4132.833.151.804.135.3341158.18
      Donkey27.0613.140.653.291.785.560.854.886.275.1924.7092.754556.57
      1 Casein and MUN were not available for sheep and goat milk.
      Among dairy species other than cow, buffalo RM showed the greatest CV (103.65%), followed by goat RM (50.41%) and sheep RM (38.40%). Such a variability within dairy species is similar to that observed for cow RM, but considerably wider compared with processed cow milk samples (Table 2).

      Repeatability

      Instrumental repeatability ranged from 0.01 to 2.01%. Table 4 reports mean iodine content and RSDr, calculated within each day of analysis and each operator on 5 aliquots of the same cow RM sample. Five aliquots were handled in the first day of analysis (d 1) by the first operator (operator 1); the mean milk iodine content was 65.82 µg/kg and the RSDr was 0.96%. Relative standard deviation of repeatability within day of analysis ranged from 0.96% (d 1) to 1.84% (d 2). Relative standard deviation of repeatability within operator was even lower than that within day of analysis, ranging from 0.72 to 1.16% for operator 3 and operator 2, respectively. Table 5 reports mean iodine content and RSDr, calculated within 1 d of analysis on 5 aliquots of 1 cow WM, SM, WUM, and SUM sample, and 1 buffalo, sheep, goat, and donkey RM sample. Among commercial cow milk samples, the greatest repeatability was achieved for WUM (0.37%). Slightly lower repeatabilities were obtained for SUM (1.07%), SM (1.11%), and WM (2.49%). Relative standard deviation of repeatability calculated on RM of other dairy species ranged from 1.07 to 8.81% for goat and donkey milk, respectively.
      Table 4Mean (μg/kg), relative standard deviation of repeatability (RSDr, %; n = 5) within day and operator, relative standard deviation of reproducibility (RSDR, %; n = 15), expected Horwitz RSDR (%), and Horwitz ratio (HorRat) across days and operators for iodine content in cow raw milk
      ValidationSamples, nAliquots, no.MeanRSDrRSDRHorwitz RSDRHorRat
      Day2.6724.190.11
       11565.820.96
       21562.261.84
       31564.291.20
      Operator1.0224.090.04
       11565.820.96
       21565.521.16
       31566.280.72
      Table 5Mean (μg/kg) and relative standard deviation of repeatability (RSDr, %; n = 5) within day for iodine content in processed commercial cow milk, and raw buffalo, sheep, goat, and donkey milk
      Milk
      RM = raw milk; WM = whole pasteurized milk; SM = partially skimmed pasteurized milk; WUM = whole UHT milk; SUM = partially skimmed UHT milk.
      Samples, nAliquots, no.MeanRSDr
      Cow milk
       RM1565.820.96
       WM15270.432.49
       SM15366.591.11
       WUM15254.380.37
       SUM15313.861.07
      Buffalo RM15178.292.81
      Sheep RM15243.165.48
      Goat RM15591.061.07
      Donkey RM157.268.81
      1 RM = raw milk; WM = whole pasteurized milk; SM = partially skimmed pasteurized milk; WUM = whole UHT milk; SUM = partially skimmed UHT milk.
      Overall, RSDr values reflected fulfilling repeatability for iodine quantification, being lower than 5.48% for all milk samples, with the only exception of donkey RM. Relative standard deviations of repeatability observed in the present study were close to those obtained by other authors for the quantification of whey proteins and milk tocopherols through HPLC (
      • Sturaro A.
      • De Marchi M.
      • Masi A.
      • Cassandro M.
      Quantification of whey proteins by reversed phase-HPLC and effectiveness of mid-infrared spectroscopy for their rapid prediction in sweet whey.
      ;
      • Niero G.
      • Penasa M.
      • Berard J.
      • Kreuzer M.
      • Cassandro M.
      • De Marchi M.
      Technical note: Development and validation of an HPLC method for the quantification of tocopherols in different types of commercial cow milk.
      ), for the determination of soluble and colloidal milk minerals through inductively coupled plasma optical emission spectrometry (
      • Franzoi M.
      • Niero G.
      • Penasa M.
      • Cassandro M.
      • De Marchi M.
      Technical note: Development and validation of a new method for the quantification of soluble and micellar calcium, magnesium, and potassium in milk.
      ), and for the assessment of milk total antioxidant activity through the spectrophotometric method (
      • Niero G.
      • Penasa M.
      • Currò S.
      • Masi A.
      • Trentin A.R.
      • Cassandro M.
      • De Marchi M.
      Development and validation of a near infrared spectrophotometric method to determine total antioxidant activity of milk.
      ). The undue RSDr observed for donkey RM is likely due to the relatively low iodine content, being greater than method LOD but lower than method LOQ (
      • Armbruster D.A.
      • Pry T.
      Limit of blank, limit of detection and limit of quantitation.
      ).

      Reproducibility

      Relative standard deviation of reproducibility, Horwitz RSDR, and HorRat calculated on 15 aliquots of the same cow RM sample across 3 d of analyses and across 3 operators are reported in Table 4. The RSDR calculated across 3 d of analyses was greater than RSDR across 3 different operators, being 2.67 and 1.02%, respectively. Therefore, a slight decline in accuracy was found across days when compared with the accuracy observed within day, in accordance with the results of
      • Niero G.
      • Penasa M.
      • Currò S.
      • Masi A.
      • Trentin A.R.
      • Cassandro M.
      • De Marchi M.
      Development and validation of a near infrared spectrophotometric method to determine total antioxidant activity of milk.
      . Nevertheless, RSDR values lower than 22.00% are considered acceptable for chemicals between 10 and 100 µg/kg (
      • Thompson M.
      • Fearn T.
      What exactly is fitness for purpose in analytical measurement?.
      ). On the other hand, reproducibility performances calculated across operators did not evidence notable discrepancies when compared with the within operator repeatability. Such findings suggest that the greatest source of variability for milk iodine analyses is the day of analysis, whereas almost negligible effects are associated with the operator in charge of milk iodine extraction and quantification. Horwitz ratios calculated across days and operators were 0.11 and 0.04, respectively. Since HorRat values lower than 1 are considered acceptable (
      • Horwitz W.
      • Albert R.
      The Horwitz ratio (HorRat): A useful index of method performance with respect to precision.
      ), results of the present study indicate that the method proposed to quantify milk iodine content is reproducible.
      Finally, RSDO calculated on quantification obtained by 3 different operators, who handled 5 aliquots in 3 different days of analyses (for a total of 45 experimental replicates), was 4.01%, with an overall HorRat value of 0.17. The RSDO was greater than RSDr and RSDR, and this was somewhat expected since RSDO summarizes all sources of variability, including all days of analysis and all operators together. Still, RSD below 5% is considered acceptable (
      • Niero G.
      • Penasa M.
      • Currò S.
      • Masi A.
      • Trentin A.R.
      • Cassandro M.
      • De Marchi M.
      Development and validation of a near infrared spectrophotometric method to determine total antioxidant activity of milk.
      ), meaning that extraction and quantification of milk iodine can be carried out by different operators in different days, affecting to a minor extent the reliability of analytical process (
      • Thompson M.
      • Fearn T.
      What exactly is fitness for purpose in analytical measurement?.
      ).

      Recovery, LOD, and LOQ

      Recoveries calculated on certified skim milk within single day of analyses varied between 87.41 and 98.90%, whereas the overall recovery calculated as the mean value across 3 d of analyses was 92.55%. Overall, the iodine content determined in the present study for certified skim milk was in accordance with the 95% confidence interval reported in reference material certificate of analysis. Relative standard deviation of repeatability for iodine quantification on certified skim milk ranged from 3.06 to 5.50%, whereas RSDR across 3 d of analyses was 6.47%. Recovery results, as well as RSDr and RSDR calculated on recovery trials, were comparable to findings of
      • Dahl L.
      • Opsahl J.A.
      • Meltzer H.M.
      • Julshamn K.
      Iodine concentration in Norwegian milk and dairy products.
      who studied iodine content in Norwegian milk and dairy products.
      Limit of detection of iodine content calculated on known standard solution and on 50-fold diluted milk sample was 0.1 µg/kg. Therefore, iodine LOD in the entire milk sample was equal to 5 µg/kg. Limit of quantification of iodine calculated as 3 times the LOD was 0.3 µg/kg on standard solution and on 50-fold diluted sample, and 15 µg/kg for the entire milk sample. Other than iodine content in donkey milk, the minimum content of iodine observed in the present study was 7.23-fold that of LOD, and 2.41-fold that of LOQ; thus, adequate confidence level and sensitivity may be assumed.

      CONCLUSIONS

      The present study provided a fast and reliable protocol for iodine quantification in processed commercial and raw cow milk, and in raw buffalo, sheep, goat, and donkey milk. Milk sample preparation is based on ammonia, takes about 1.5 h for a batch of 20 samples, and does not require specific laboratory equipment such as a microwave digester. The method was repeatable and reproducible among different days of analyses and operators, and can be used for quantification of samples containing more than 15 µg of iodine per kg of milk. The variation of iodine content in commercial cow milk was likely related to milk chemical composition, rather than to technological treatments. Across the 5 dairy species considered in our study, raw goat and donkey milk showed the greatest and the lowest iodine content, respectively. Further research is needed to gain knowledge on how and to which extent milk iodine content is driven by herd management including feeding strategies and milking procedures, environmental effects, breed, and animal physiological status such as lactation stage and parity order.

      ACKNOWLEDGMENTS

      The authors thank the laboratory of the Breeders Association of Veneto Region (ARAV, Padova, Italy), Eurolab (Bassano del Grappa, Vicenza, Italy), and Luciano Magro and Massimo Cagnin (University of Padova) for technical support.

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