SEC ICP MS characteristic of iodine compounds extracted from eggs and salt
Extraction method, based on 30 mM Tris buffer solution (pH = 7.5), was developed to gently extract water soluble forms and analyze them with size exclusion chromatography supported by ICP MS detection. It is the simplest method to gather information about iodine speciation, with minimized risk of form transformation due to the presence of oxidizing or reducing agents. As the first step before the analysis of extracts of chicken egg white and yolk, the effect of boiling on iodine forms was investigated. The standard solutions containing iodate and iodide were boiled for 5, 10 and 30 min according to preparation of eggs. The SEC analysis presented two well-separated forms, indicating that both iodine species were stable regardless of duration of boiling (not shown). Noteworthy is the fact that separation of iodine forms was possible despite their molecular size was not within separation range of size exclusion column—Fig. 2a. The effect can be explained with the influence of ionic interactions as the secondary separation mechanism [28, 29].
The chromatograms of buffer extracts of chicken egg white and yolk consists of the same main iodine peak at t
r
= 34 min, which was identified as an iodide by measuring of retention times for external standards (Fig. 2c) and internal standard addition method. For samples preconcentrated 10 times by means of lyophilization, high molecular weight (HMW) iodine compound (>70 kDa) was found (Fig. 2c). The presence of the HMW peak was recorded only for one batch of eggs with highest content of investigated element (6% of total peak areas for iodine); no correlation to the method of farming was possible to find. This is in agreement with other reports, showing that iodine can bind with HMW compounds [24].
The simulation of digestion process was carried out in two-step procedure consisting of the simulation of: (1) gastric and (2) intestinal digestions. Chromatograms obtained for enzymatic digestion of egg yolk and white were similar to those obtained for Tris extracts after lyophilization. However, additional preconcentration step to observe HMW fraction of iodine (5% of total peak area) was unnecessary. It can be suggested that there is no conversion of iodide during enzymatic hydrolysis, and it is the major compound present in the egg samples as well as in kitchen salt (data not shown).
The stability of iodine species during digestion was checked for standard solutions. As the result of the first step, reduction of iodate to iodide was found, which was observed as an appearance of the third peak and the disappearance of the second one on the SEC ICP MS chromatogram (Fig. 2). After the second step, iodine remained as an iodide. According to the literature, the pH below 7.0 and the presence of reducing compounds in the sample induces the reduction of iodate to iodide [30]. After complete simulation of gastrointestinal digestion of iodine standard solutions, the recovery was from 98 to 101% in reference to fresh standard solution analyzed by SEC ICP MS. The lack of HMW iodine compounds on the chromatograms indicates no affinity of iodide and iodate to enzymatic proteins used during gastric and intestinal digestions. However, ICP MS calibration for determination of iodine in Tris, gastric and gastrointestinal extracts of eggs and iodized kitchen salt samples should be carried out using iodide standards prepared in extracting media to compensate matrix effect; pH of the solution should be higher than 7.5 to ensure its good stability.
Determination and bioavailability of iodine
To estimate the bioavailability of iodine from chicken eggs, samples were treated with enzymatic solutions simulating human digestive juices. The mass balance of iodine was established by calculating the total amount of iodine in extract and sediment against total amount of iodine in mineralized sample (Fig. 1).
The mineralization method, based on HNO3 with addition of H2O2, was chosen due to the higher oxidation potential of H2O2 than HClO4
\( \left( {E_{{{\text{H}}_{2} {\text{O}}_{2} }}^{0} = + 1.78\,{\text{V}}\;{\text{and}}\;E_{{{\text{HClO}}_{4} }}^{0} = + 1.23\,{\text{V}}} \right) \) [31]. Additionally, the degradation products of hydrogen peroxide are not considered as a troublesome during ICP MS measurements in contrast to chloride and chlorine generated from perchloric acid. The method was validated using reference material of chicken egg powder (Table 2). The presence of iodate during ICP MS analysis is favorable as it is more stable in acidic solutions of HNO3, which is most commonly used to prepare samples and to wash the apparatus. Contents of accumulated iodine in supplemented and regular chicken eggs are presented in Table 2 and allowed to conclude that concentration of iodine in yolk is usually higher than in white. This finding is in agreement with the literature [32]. The vast differences between total amounts of iodine in eggs allow to believe that chicken eggs have great supplementation potential.
Table 2 Total iodine content in the studied egg samples (average results for 3 samples)
The efficiency of Tris buffer (pH = 7.5) extraction method was established following ICP MS calibration in 10-times-diluted extraction media and established against total amount of iodine in egg white and yolk as 22 ± 2% and 9 ± 1%, respectively. The low efficiency was probably caused by the presence of water-insoluble proteins fraction that settled out of a suspension to the bottom of the liquid. The sediment was subsequently removed during centrifugation. Additionally, after longer time of egg boiling (30 min), iodine was not detected in the extracts, probably due to more advanced protein denaturation. In case of inorganic character of kitchen salt, the concentration of iodine in kitchen salt dissolved in Tris buffer was considered as a total amount of iodine in this sample (Table 2
).
The recovery of iodine was also established for simulated gastrointestinal extraction of chicken egg white (33 ± 5%) and yolk (10 ± 3%). In case of egg boiled for 30 min, the recovery was about twice lower, indicating the problem of proteins temperature denaturation. This is in agreement with resistance of HMW iodine compound to enzymatic digestion noticed by SEC ICP MS. It should be pointed out that recovery for iodine from iodized kitchen salt was established as a 100 ± 5%, which indicates the significant influence of matrix and form of iodine on its bioaccessibility.
The comparison of iodine amount in each fraction versus total amount in egg yolk and white allowed to conclude that iodine loss was below 5%, which is in the range of the uncertainty. Additionally, Tukey’s test was applied for multiple comparisons of the results obtained for different groups of eggs. The level of significance was 0.05, and honestly significant difference (HSD) was obtained for each group of eggs (Table 2
). The level of HSD is low enough to show that in each case, the difference of iodine concentration in different type of egg sample is greater than the standard error and that various ways of feeding carried at different farms considerably influence the level of iodine in eggs.
In typical 60 g egg, water content in yolk and white was assayed as 50 and 88%, respectively, by comparison of weight of both fractions of egg (n = 5) before and after lyophilization (until stable mass). Recalculated (from dry mass to real material) element contents are presented in Table 3. Assuming bioaccessibility of it from yolk as 33%, it was found that only about 40 μg was bioaccessible from the iodine-enriched egg after 10 min of boiling and the level decreased after 30 min to <20 μg.
Table 3 Iodine content recalculated per whole egg (60 g weight) containing 22 g of yolk