Seasonal and regional variations of iodine in Danish dairy products determined by inductively coupled plasma mass spectrometry Erik H. Larsen,* Pia Knuthsen and Marianne Hansen Institute of Food Research and Nutrition, Danish Veterinary and Food Administration, 19 Mørkhøj Bygade, DK-2860 Søborg, Denmark. E-mail: ehl@vfd.dk Received 24th August 1998, Accepted 11th November 1998 The content of iodine in 72 samples of Danish dairy products was determined by direct flow injection (FI ) sample introduction of whole milk into the ICP-MS instrument, or by bomb ashing of cream and cheese samples prior to the ICP-MS measurement. The performance of the FI-based method was superior to the bomb ashing method in terms of the limit of detection which was 9 ng g-1 and 60 ng g-1 for the two methods, respectively, and in repeatability which was 8.4 and 45 ng g-1, respectively.Both methods of analysis were accurate as demonstrated by analyses of the CRM 063R Skim Milk Powder.The iodine content of the milk samples varied between 42–162 ng g-1. A geographical diVerence in the iodine content showed that milk from Jutland contained less iodine than milk from Sealand. This can be explained by the lower natural iodine content in the drinking water resources in Jutland. A temporal diVerence showed a general increase in iodine concentration in milk from all regions during the winter months over the summer months. This can be explained by the use of iodine-enriched fodder during the winter months.The poorer repeatability of the analyses of cream and cheese samples obscured any possible geographical and temporal variation of iodine in these samples. Iodine is an essential element to man, and a constituent of the Experimental thyroid hormones. Deficiency of iodine causes goitre.1 Like Samples and sampling many other countries Denmark is a naturally iodine-deficient region and the average daily intake of iodine2 is about The sampling was planned to illustrate the average contents 50–100 mg, which is less than the Nordic recommendation3 at of a range of nutrients including iodine in dairy products 150 mg.Because iodine enrichment of food or table salt has sold in Denmark, and also to include possible regional and been allowed only recently, the occurrence of non-toxic goitre seasonal variations in the iodine content of the samples.10 A among the Danes is relatively high.2 total of 24 samples of each of whole milk (3.5% fat), cream In Denmark, milk products (27%), water and other (38% fat) and Danbo cheese (firm cheese, 45% fat in dry beverages (26%), fish (16%) and eggs (10%) are the main matter) were taken throughout several months in 1995 from contributors to the dietary iodine intake.2 The Danish Food three dairies which are located in Northern and Central Monitoring Programme4,5 has shown that the iodine content Jutland (Hjørring, Hobro and Bjerringbro), in Southern of dairy products sampled in 1985 and 1990 varied according Jutland (Tyrstrup, Christiansfeld and Ribe) and, in the case to the season of the year and according to the geographical of milk and cream, additionally on the island of Sealand origin of the milk.The dietary iodine intake was lowest in (Slagelse). All dairies received the raw milk predominantly Jutland and highest on Sealand.2 This variation could be from regional farmers, and the samples were taken from the ascribed to geographical diVerences mainly in the iodine ordinary productions at the dairies.The samples were frozen content of potable water. Therefore, in order to be able to immediately after arrival in the laboratory and kept at assess the variation in the iodine intake with the diet in the -18 °C until the time of analysis. late 1990s, data on the geographical and seasonal variation in the iodine content of particularly milk products and water are important. Standard substances and chemicals A variety of analytical methods including spectrophotometry6 and neutron activation analysis7 have been used for An aqueous standard stock solution at 1000 mg ml-1 of iodine was prepared from potassium iodate Volumetric Standard the determination of iodine in food.Owing to its high selectivity and sensitivity, ICP-MS is a useful modern detector for (Merck, Darmstadt, Germany). Working standard solutions at 10 mg ml-1 were prepared daily from the stock solution by the determination of iodine in biological samples.Furthermore, sample preparation and manipulations can be dilution with water. Water (>18 MV cm-1) was produced in a Millipore Super-Q apparatus (Millipore, Milford, MA, reduced to a minimum8 or completely omitted if direct analysis of liquid samples using flow injection (FI ) sample introduction USA). Nitric acid, pro analysi, which was sub-boil distilled in an all-quartz apparatus (Hans Ku� rner, Rosenheim, Germany) into the ICP-MS instrument is employed.9 The aims of this paper are two-fold.Firstly, the figures of and perchloric acid, Suprapur (Merck, Darmstadt, Germany), were used for the wet ashings of the cream and cheese samples. merit obtained during routine use of a recently developed method of analysis for iodine in food8 are given. Secondly, An aqueous solution containing 0.07 mol l-1 of tetramethylammonium hydroxide (TMAH) and 0.05 mol l-1 of potassium the iodine content in Danish milk, cream and cheese are reported and discussed with emphasis on geographical and hydroxide was used to dilute the milk samples prior to the direct iodine analysis.seasonal variations. J. Anal. At. Spectrom., 1999, 14, 41–44 41Sample preparation sample introduction, a Meinhard TR-30-K3 glass concentric nebulizer (Meinhard, Santa Ana, CA, USA) was used through- The samples of milk (0.5 g) were mixed with 9.5 ml of the out in combination with a low dead-volume cyclonic glass alkaline diluent in the ICP-MS autosampler vials and were spray chamber (Glass Expansion, Victoria, Australia).This analysed with no further pretreatment. nebuliser and spray chamber assembly improved the ICP-MS The cream and cheese samples (0.3 and 0.5 g, respectively) signal-to-noise ratio for iodine by approximately 50% comwere dissolved by pressurised ashing using a mixture of 3.5 ml pared with a conventional Scott type double-pass spray nitric and 1.5 ml perchloric acid as described in detail by chamber.8 Larsen and Ludwigsen.8 The PTFE-lined steel bombs were heated at 160 °C for 4 h.The acid mixture oxidised potentially Contamination control and quality assurance volatile iodine species present in the sample to non-volatile species such as iodate. In contrast to iodide these oxidised Prior to using new PTFE liners in the pressure bombs for analytical work, the iodine content therein was reduced by species did not exhibit any memory and adhesion eVects in the ICP-MS instrument. The wet-ashed residue was diluted to treatment by the nitric acid–perchloric acid mixture at 160 °C for 4 h.The acid residue was discarded and the liners rinsed 50 ml by water prior to determination of iodine. three times with water. During routine analytical work the remaining iodine contamination of the PTFE material was ICP-MS determination of iodine kept to a minimum by extraction with base between each The diluted milk sample was pumped from the AS-90 analytical run.For this purpose an aqueous solution of 1% autosampler to fill the 500 ml sample loop of the FIAS 400 FI (m/v) TMAH was run into the liners which were heated at unit. The alkaline diluent was used as carrier solution for 90 °C in a laboratory oven for 12 h. Following this treatment introduction of the milk samples with no further pretreatment the base was discarded and the clean liners were air dried. into the ELAN 5000 ICP-MS instrument (Sciex Perkin-Elmer, On each day of analysis the 14 analytical places available Concord, Ontario, Canada).9 The determination of the iodine comprised eight unknown samples of similar composition, one content in the cream and cheese sample digests was carried of which was determined in duplicate, four blanks which were out by conventional continuous sample aspiration into the taken through the entire procedure and one determinatioof ICP-MS.The instrumental settings used are given in Table 1.the CRM 063R Skim Milk Powder11 (Commission of the The method of standard additions was used for European Communities, Community Bureau of Reference, quantification of the iodine. Three spikes of iodine (as iodate) Brussels, Belgium). were added at 2, 4 and 8 ng ml-1 to the diluted milk samples and at 1, 2 and 4 ng ml-1 to the diluted wet-ashed sample Results and discussion residues. Iodate was used because iodine presumably occurred as this species following the wet ashing procedure, and was Method performance and quality assurance added at 100%, 200% and 400% of the natural iodine content to achieve optimum precision of the results.In order to assure the quality of the analyses a set of acceptance criteria was applied to the raw data. The criteria included the To minimise the risk of losses and memory eVects during magnitude and variation of the blank (contamination control ), the relative standard deviation (RSD) of the recorded ICP-MS Table 1 ICP-MS and sample introduction settings signal for each sample (conversion of volatile to non-volatile iodine species during bomb-ashing), and the correlation ICP-MS instrument— coeYcient of the standard additions calibration curve Rf power 1200 W (precision).8 Sampler and skimmer cones Platinum The results obtained for iodine in the blank solutions from Argon flow rates the analytical work are plotted in Fig. 1. Values which exceeded Outer 15 l min-1 Intermediate 0.8 l min-1 the average plus three standard deviations of the preceding Nebulizer 1 l min-1 (variable) values were not accepted for analysis and reflected incompletely Mass-to-charge ratio detected m/z 127 Conventional sample aspiration Dwell time 1000 ms Sweeps per reading 3 Readings per replicate 1 Number of replicates 10 Flow injection sample introduction Dwell time 60 ms Sweeps per reading 1 Readings per replicate 250 Number of replicates 1 Scanning mode Peak hop Spray chamber and nebuliser Glass cyclonic with a Meinhard assemblies (type TR-30-K3) concentric nebuliser Data acquisition mode Quantitative Sample introduction systems— Conventional Wash time between samples 120 s Read delay 80 s Peristaltic pump speed 1.4 ml min-1 Flow injection Injection loop volume 500 ml Carrier solution 0.07 mol l-1 Tetramethyl- Fig. 1 Control chart for procedural blank values in the diluted sample ammonium hydroxide and solution. The average value is marked as X, and the average value 0.05 mol l-1 potassium plus 3 standard deviations of the blank (X+3s) is the upper hydroxide in water rejection limit. 42 J. Anal. At. Spectrom., 1999, 14, 41–44Table 2 Figures of merit for iodine determinations by ICP-MS in cream and cheese using wet ashing or in milk using flow injection (FI ) sample introduction Wet ashing FI Blank value, x±s/ng ml-1 0.32±0.21 n.d.a (n=20) Limits of detection Samples/ng g-1 60 or 100b 9c Sample solutions/ng ml-1 0.62 0.45c Sensitivity (standard additions) Mean value/counts s-1 per ng ml-1 1477 4530 (n=9) (n=7)d Within-day variation, RSDe(%) 4.0 4.1 Between-day variation, RSD(%) 8.6 n.d.a Repeatabilityf Standard deviation, sr/ng g-1 16 3.0 Repeatability, 2.8 sr/ng g-1 45 8.4 Fig. 2 Control chart with upper and lower warning and rejection Reproducibility/ng g-1 limits for the CRM 063R Skim Milk Powder reference material. Standard deviation, sR/ng g-1 35 n.d.a Reproducibility, 2.8 sR/ng g-1 98 n.d.a Accuracy (CRM 063R Skim Milk Powder) the optimisation of the ICP-MS instrument on diVerent days Found, x±s/mg g-1 0.81±0.04 0.76±0.02 of analysis.Calibration curves must therefore be constructed (n=11) (n=12)c Certified/mg g-1 0.81±0.05 0.81±0.05 daily. The coeYcient of correlation of the standard additions calibration curve were in most cases better than 0.999. aNot determined. bFor 0.5 and 0.3 g sample intakes, respectively. cData from Stu�rup and Bu� chert. dIntegrated value, counts per ng ml-1.Otherwise a new standard additions calibration curve was eRelative standard deviation (RSD) pooled and weighed average of constructed. RSD values from separate days. fBased on six double determinations Based on double determinations, the repeatability and of samples at the 100 ng g-1 concentration level on separate days. reproducibility (both at the 95% confidence level ) were estimated (Table 2) according to guidelines given by the International Organization for Standardization.12 Expressed cleaned PTFE bomb inserts.If the four blanks exceeded this in relative terms, the values given for cream and cheese upper limit, the results obtained for the samples were discarded. correspond to a relative repeatability of 45% and a relative After several bomb ashings had been carried out in the same reproducibility of 98% at the 100 ng g-1 iodine concentration PTFE liner, the iodine contamination tended to increase in level. The analysis of iodine in milk by the FI-ICP-MS method that liner.This was especially pronounced when samples high provided a relative repeatability of 8.4% also at the 100 ng g-1 in iodine had been wet-ashed in the liners. In this case the concentration level. The reproducibility however, was not extraction treatment by the 1% solution of TMAH was not determined for the FI-based method. suYcient to keep the blanks at a low concentration level. Finally, the accuracy of the bomb-ashing method as well as Therefore the treatment by the acid mixture used for new that of the FI-based method of analysis was assured by PTFE inserts as mentioned in the Experimental section was analysing theCRM 063R Skim Milk Powder reference material applied for satisfactory reduction of the blank value. in parallel with the samples.The results have been shown in The limit of detection (LOD) given in Table 2 for the wet Fig. 2 and the warning and rejection limits defined as ±2 and ashing and the FI-based method was determined as three ±3 standard deviations, respectively, are also indicated.The standard deviations of all accepted blank values. The corre- standard deviation of the distribution of means which was sponding LOD values for the samples depended on the sample derived from the results of the laboratories participating in intake and the dilution factor. When using wet ashing the high the certification campaign of the CRM11 was used to establish fat content of the cream and cheese samples limited the these limits.Using this control chart it became possible to maximum possible sample intake whereby the LOD value accept or reject a single determination13 of iodine in the CRM. became relatively high. In comparison, the FI-based analytical If the value for the CRM was accepted, the analyses of the method used for milk was superior in LOD value (Table 2) unknown samples which were carried out in parallel with the due to less variation of the blank and less sample dilution.CRM were also accepted. The results given for the CRM in If conversion of volatile iodine species contained in the the control chart in Fig. 2 show that the analyses were accurate samples to non-volatile species had not occurred completely although the standard deviation of the values determined by during the bomb ashing, the RSD value of the recorded the bomb-ashing method was twice of that for the FI-based ICP-MS signal intensity would increase markedly owing to method as indicated in Table 2.memory eVects. Earlier investigations showed that a RSD value below 1.5% was acceptable as accurate results using The content and variation of iodine of dairy products CRMs were obtained.8 Consequently, if this value was exceeded the results were rejected and the analysis repeated. Whole milk. The results for iodine in milk shown in Fig. 3 varied between 42 and 162 ng g-1 with an average value of The method of standard additions was used for calibration of all samples and provided information on the within-day 101 ng g-1.The results reflect regional as well as seasonal diVerences. Milk from Sealand contained more iodine than and between-day variation of the sensitivity. The within-day RSD value of approximately 4% (Table 2) is relatively modest milk from Jnd which can be explained by the higher iodine content of potable water on Sealand. Typical contents of and allows for a more cost-eYcient addition calibration procedure in future analytical work.Using this procedure, stan- iodine in tap water were 2–8 ng ml-1 in Jutland and 10–30 ng ml-1 on Sealand.14 Within Jutland, higher iodine dards may be spiked to only one out of every 10 unknowns of similar composition. However, the somewhat higher contents were found in milk from the dairy in Southern rather than in Central Jutland. During the winter period the iodine between-days RSD in sensitivity was caused by diVerences in J.Anal. At. Spectrom., 1999, 14, 41–44 43Cream and cheese. The iodine content in cream varied from <100–217 ng g-1 with an average content at 110 ng g-1. The results for iodine in cream were close to the LOD value of 100 ng g-1. Consequently, the repeatability at 45 ng g-1 (Table 2) obscured possible regional and seasonal variations in the iodine content. The iodine content of cheese varied from <60 to 164 ng g-1 with an average value at 121 ng g-1.All samples were taken from three dairies in Jutland because the production of cheese is centralised in this part of the country. Any possible regional diVerence in the iodine content of the cheese samples was obscured by the relatively high repeatability. Consequently, the average iodine content of cheese from the three dairies has been plotted against the month of production. The results indicate that no significant seasonal diVerences can be demonstrated.Fig. 3 Regional and seasonal variations in iodine contents of milk. The repeatability has been indicated by error bars as ± one standard References deviation. y axis: Iodine content/ng g-1; x axis: Month of production. —#—, Average of the three dairies; - -×- -, Slagelse 1 B. S. Hetzel and J. T. Dunn, Ann. Rev. Nutr., 1989, 9, 21. (Sealand); - -%- -, Tyrstrup (Southern Jutland); - -6- -, Hobro 2 L. B. Rasmussen, G. Andersson, J. Haraldsdottir, E. Kristiansen, (Central Jutland).K. Molsted, P. Laurberg, K. Overvad, H. Perrild and L. Ovesen, Int. J. Food Sci. Nutr., 1996, 47, 377. 3 Nordic Nutrition Recommendations 1996, Scand. J. Nutr./ Nœringsforskning, 1996, 4/96, 40, 161. 4 National Food Agency of Denmark, Food Monitoring 1988–1992, Publication no. 232 (English version), December 1995, DK-2860 Søborg, Denmark. 5 J. S. Jacobsen and T. Leth, Food Monitoring System for Nutrients, Dairy Products, 2nd Cycle (In Danish with English summary). Report CLA 92004, 1992, National Food Agency of Denmark, Søborg. 6 P. W. Fischer, M. R. L’Abbe� and A. Giroux, J. Assoc. OV. Anal. Chem., 1986, 69, 687. 7 M. Dermelj, Z. Slejkovec, A. R. Byrne, P. Stegnar, V. Stibilj and M. Rossbach, Fresenius’ J. Anal. Chem., 1990, 338, 559. 8 E. H. Larsen and M. B. Ludwigsen, J. Anal. At. Spectrom., 1997, 12, 435. 9 S. Stu�rup and A. Bu� chert, Fresenius’ J. Anal. Chem., 1996, 354, 323. 10 J. S. Jacobsen and P. Knuthsen, Food Monitoring System for Nutrients, Dairy Products, 3rd. Cycle (in Danish with English summary). Report IFE 1998.2, 1998, Danish Veterinary and Food Administration, Søborg. Fig. 4 Average iodine contents of cheese against time of the year. The 11 P. Quevauviller, D. Van Renterghem, B. Griepink, S. T. Sparkes repeatability has been indicated by error bars as ± one standard and G. N. Kramer, The certification of the contents (mass deviation. Month 12 of production designates December of the year fractions) of Ca, Cu, Cl, I, Fe, K, Mg, P, Pb, N, Na and Zn in Skim prior to the year of sampling. y axis: Iodine content/ng g-1; x axis: Milk Powder (CRM 063R), Report EUR 15021 EN, DG XIII, Month of production. —#—, Average of the three dairies. L-2920 Luxembourg, 1993. 12 International Standard ISO 5725, 2nd edn., 1986, International content of milk from all regions was significantly higher than Organization for Standardization, Geneva, Switzerland. 13 E. H. Larsen, G. A. Pedersen and J. W. McLaren, J. Anal. At. during summer as indicated in Fig. 3. The marked decrease in Spectrom., 1997, 12, 963. iodine content from April to May coincides with a change to 14 L. B. Rasmussen and E. H. Larsen, 1998, unpublished results. outdoors grazing during the summer, while the cows’ fodder during the winter months is enriched in iodine. Paper 8/06642F 44 J. Anal. At. Spectrom., 1999, 14, 41&nd