**3. Problems with analysis of iodine in biological matrices**

Despite a wide choice of available analytical methodologies, determination of iodide in biological matrices remains a difficult problem. Biological samples belong to so-called

A Review of Spectrophotometric and Chromatographic Methods

and Sample Preparation Procedures for Determination of Iodine in Miscellaneous Matrices 379

iodine. The procedure usually requires the use of alkaline media and a high temperature (Moxon & Dixon, 1980). A catalytic spectrophotometric method based on the Sandell-Kolthoff reaction together with many modifications and improvements of this method is a low-cost assay of iodine, however, it is not free of possible interferences (especially for foodstuffs with low range of iodine levels). Most iodine in biological media is covalently bonded and there are

high lipid content in the sample (eg. milk) can cause problems in spectrophotometric readings,

When preparing a sample for analysis, it is necessary to take into consideration also the loss of analyte due to erroneous application of decomposition procedures. Some of them, e.g. "Schoeniger combustion", require a highly homogenous sample, which is sometimes difficult to obtain (Knapp et al., 1998). There are many options for biological sample preparations, among which alkaline digestion using tetramethylammonium hydroxide

Nagengast, 1994; Schramel & Hasse, 1994). Alkaline conditions during the extraction

oxidized into volatile forms (I2 or HI). A destruction of the organic matrix using a typical digestion reagent, like HNO3, is not possible because of the losses due to volatile iodine formation (therefore, no stable sample solution can be achieved). However, acid digestion procedures (by the use of mainly H2SO4, HNO3, and HClO4) have also been applied (Fischer et al., 1986). What is more, despite its obvious weak point (loss of analyte), the US Food and

Błażewicz et al. (Błażewicz et al., 2011) used the alkaline digestion with 25 % TMAH water solution for the thyroid glands` preparation before the IC method of analysis. A diluted TMAH solution has also been used for serum samples by Schramel and Hasse (Schramel & Hasse, 1994) to analyse iodine in the serum, milk, plants and tissues by using the ICP-MS

Unfortunately, despite the abovementioned advantages, the use of TMAH solution for the digestion of samples has some disadvantages as well. Since for all modern sample pretreatment methods time is a very important factor, the long procedure time still remains a huge problem. As reported in the literature, digestion of biological materials with TMAH usually requires up to 6 hours (Gamallo-Lorenzo et al., 2005). However, the assistance of microwaves significantly shortens the time of the sample preparation step (less than 20 minutes). Such microwave-assisted alkaline digestion has been developed before the IC analysis of thyroids' samples (Błażewicz et al., 2011). It is important to monitor all conditions of the digestion procedure, especially temperature, in order to avoid the decomposition of TMAH and bursting of the closed vessels (therefore a temperature lower than 100 0C is recommended). Each time the vessels must be thoroughly cleaned with a digestion mixture in order to avoid memory effects (adsorption by the walls of containers)

**4. Spectrophotometry and chromatography as tools for iodine assessment in** 

It is known that the choice of the proper analytical method depends on the intended application, the number of samples, the cost of analysis and the technical capability.

procedure have some advantages in comparison with acidic media, where I-

, NO3 -

(Fecher et al., 1998; Fecher &

, or Fe2+). A

may be

some substances that interfere with the determination reaction (eg. SCN-

so a mineralization step is absolutely necessary before analysis.

(TMAH) is the most common before the analysis of I-

method.

and consequently the loss of analyte.

**miscellaneous matrices** 

Drug Administration (FDA, 2009) still recommends such procedures.

complex samples (with complex matrices). In such samples, the analyte content is usually much scarcer when compared with the accompanying macrocomponents. Aside from the necessity of choosing the appropriately sensitive method, it is equally important to comply with the sample preservation, pretreatment, and preparation conditions.

Historically, published values of the I2/I- concentration of both tissue and body fluids from healthy subjects have varied greatly. These great differences were attributed to numerous variables, such as age, sex, dietary habits, physiological conditions, environmental factors and numerous other X-factors. Given the delicate nature and the instability of biological samples, it has been concluded that improper sample collection methods and manipulation drastically affects the iodine content of biological matrices.

Analytical methods are often versatile in nature. Thus, in order to achieve successful and satisfactory results, the process of analysis needs to be carefully tailored to its needs. Before applying the appropriate method for a particular application, many factors have to be considered and some of them are discussed below.

It is well known that sources of errors that affect the final error of an analytical result are connected, among other things, with incorrect obtaining of the samples, their improper transport, storage and transformation, wrong methodology, wrong measurement (instruments, parameters) or human errors (Konieczka & Namieśnik, 2007). When it comes to quantitative evaluations of iodine concentrations (in all chemical forms), the proper storage of biological samples is of paramount importance. The tissues must be preserved in such a way that a potential loss of the analyte (i.e. iodine) is minimized. The choice of the tissue-fixing agents is quite wide. Formalin is a routinely used tissue-fixing agent after surgical procedures. Other recommended agents for tissue preservation include, e.g. a mixture of 50% glutaraldehyde, 16 % paraformaldehyde, and 0.2M sodium phosphate buffer solutions (i.e. original composition of Karnovsky fixative) or its modification. The other possibility to preserve the tissues is sample freezing.

Hansson at al. (Hansson et al., 2008) used X-ray fluorescence analysis (XRF) and secondary ion mass spectrometry (SIMS) for evaluation of a freezing technique for preserving samples (XRF analysis) and for evaluation of the efficacy of using aldehyde fixatives to prepare samples (SIMS analysis). There were no significant changes in the iodine content due to freezing. Freezing for 4 weeks produced no more than a 10% change in the iodine content. For all the samples fixed in an aldehyde, there was a loss of iodine. The decrease in iodine content from baseline was significant for samples fixed in aldehyde (p < 0.05). Karnovsky was the best fixative in this regard, yielding a mean 14% loss compared to 20% and 30% for glutaraldehyde and formaldehyde, respectively. For SIMS method, Fragu et al. (Fragu et al., 1992) recommended chemical fixation with a mixture of Karnovsky fixative, followed by embedding in methacrylate. This method, which was evaluated for iodine loss by Rognoni et al. (Rognoni et al., 1974), has proven suitable for preservation of substances bound to macromolecules (like iodine bound to thyroglobulin [Tg]).

The effect of sample preservation on determination of I in healthy and pathological human thyroids has also been studied (Błażewicz et al., 2011). It was pointed out that the way of tissue preservation (either in formalin or by freezing) had no significant effect on the iodine determination result (α = 0.1) by ion chromatography combined with the pulsed amperometic detection method (IC-PAD). Sample decomposition is a critical step in iodides' analysis as well. All reported methods have a digestion or ashing step prior to the final determination of

complex samples (with complex matrices). In such samples, the analyte content is usually much scarcer when compared with the accompanying macrocomponents. Aside from the necessity of choosing the appropriately sensitive method, it is equally important to comply

Historically, published values of the I2/I- concentration of both tissue and body fluids from healthy subjects have varied greatly. These great differences were attributed to numerous variables, such as age, sex, dietary habits, physiological conditions, environmental factors and numerous other X-factors. Given the delicate nature and the instability of biological samples, it has been concluded that improper sample collection methods and manipulation

Analytical methods are often versatile in nature. Thus, in order to achieve successful and satisfactory results, the process of analysis needs to be carefully tailored to its needs. Before applying the appropriate method for a particular application, many factors have to be

It is well known that sources of errors that affect the final error of an analytical result are connected, among other things, with incorrect obtaining of the samples, their improper transport, storage and transformation, wrong methodology, wrong measurement (instruments, parameters) or human errors (Konieczka & Namieśnik, 2007). When it comes to quantitative evaluations of iodine concentrations (in all chemical forms), the proper storage of biological samples is of paramount importance. The tissues must be preserved in such a way that a potential loss of the analyte (i.e. iodine) is minimized. The choice of the tissue-fixing agents is quite wide. Formalin is a routinely used tissue-fixing agent after surgical procedures. Other recommended agents for tissue preservation include, e.g. a mixture of 50% glutaraldehyde, 16 % paraformaldehyde, and 0.2M sodium phosphate buffer solutions (i.e. original composition of Karnovsky fixative) or its modification. The other

Hansson at al. (Hansson et al., 2008) used X-ray fluorescence analysis (XRF) and secondary ion mass spectrometry (SIMS) for evaluation of a freezing technique for preserving samples (XRF analysis) and for evaluation of the efficacy of using aldehyde fixatives to prepare samples (SIMS analysis). There were no significant changes in the iodine content due to freezing. Freezing for 4 weeks produced no more than a 10% change in the iodine content. For all the samples fixed in an aldehyde, there was a loss of iodine. The decrease in iodine content from baseline was significant for samples fixed in aldehyde (p < 0.05). Karnovsky was the best fixative in this regard, yielding a mean 14% loss compared to 20% and 30% for glutaraldehyde and formaldehyde, respectively. For SIMS method, Fragu et al. (Fragu et al., 1992) recommended chemical fixation with a mixture of Karnovsky fixative, followed by embedding in methacrylate. This method, which was evaluated for iodine loss by Rognoni et al. (Rognoni et al., 1974), has proven suitable for preservation of substances bound to

thyroids has also been studied (Błażewicz et al., 2011). It was pointed out that the way of tissue preservation (either in formalin or by freezing) had no significant effect on the iodine determination result (α = 0.1) by ion chromatography combined with the pulsed amperometic detection method (IC-PAD). Sample decomposition is a critical step in iodides' analysis as well. All reported methods have a digestion or ashing step prior to the final determination of

in healthy and pathological human

with the sample preservation, pretreatment, and preparation conditions.

drastically affects the iodine content of biological matrices.

considered and some of them are discussed below.

possibility to preserve the tissues is sample freezing.

macromolecules (like iodine bound to thyroglobulin [Tg]). The effect of sample preservation on determination of I-

iodine. The procedure usually requires the use of alkaline media and a high temperature (Moxon & Dixon, 1980). A catalytic spectrophotometric method based on the Sandell-Kolthoff reaction together with many modifications and improvements of this method is a low-cost assay of iodine, however, it is not free of possible interferences (especially for foodstuffs with low range of iodine levels). Most iodine in biological media is covalently bonded and there are some substances that interfere with the determination reaction (eg. SCN- , NO3 - , or Fe2+). A high lipid content in the sample (eg. milk) can cause problems in spectrophotometric readings, so a mineralization step is absolutely necessary before analysis.

When preparing a sample for analysis, it is necessary to take into consideration also the loss of analyte due to erroneous application of decomposition procedures. Some of them, e.g. "Schoeniger combustion", require a highly homogenous sample, which is sometimes difficult to obtain (Knapp et al., 1998). There are many options for biological sample preparations, among which alkaline digestion using tetramethylammonium hydroxide (TMAH) is the most common before the analysis of I- (Fecher et al., 1998; Fecher & Nagengast, 1994; Schramel & Hasse, 1994). Alkaline conditions during the extraction procedure have some advantages in comparison with acidic media, where I may be oxidized into volatile forms (I2 or HI). A destruction of the organic matrix using a typical digestion reagent, like HNO3, is not possible because of the losses due to volatile iodine formation (therefore, no stable sample solution can be achieved). However, acid digestion procedures (by the use of mainly H2SO4, HNO3, and HClO4) have also been applied (Fischer et al., 1986). What is more, despite its obvious weak point (loss of analyte), the US Food and Drug Administration (FDA, 2009) still recommends such procedures.

Błażewicz et al. (Błażewicz et al., 2011) used the alkaline digestion with 25 % TMAH water solution for the thyroid glands` preparation before the IC method of analysis. A diluted TMAH solution has also been used for serum samples by Schramel and Hasse (Schramel & Hasse, 1994) to analyse iodine in the serum, milk, plants and tissues by using the ICP-MS method.

Unfortunately, despite the abovementioned advantages, the use of TMAH solution for the digestion of samples has some disadvantages as well. Since for all modern sample pretreatment methods time is a very important factor, the long procedure time still remains a huge problem. As reported in the literature, digestion of biological materials with TMAH usually requires up to 6 hours (Gamallo-Lorenzo et al., 2005). However, the assistance of microwaves significantly shortens the time of the sample preparation step (less than 20 minutes). Such microwave-assisted alkaline digestion has been developed before the IC analysis of thyroids' samples (Błażewicz et al., 2011). It is important to monitor all conditions of the digestion procedure, especially temperature, in order to avoid the decomposition of TMAH and bursting of the closed vessels (therefore a temperature lower than 100 0C is recommended). Each time the vessels must be thoroughly cleaned with a digestion mixture in order to avoid memory effects (adsorption by the walls of containers) and consequently the loss of analyte.
