Iodine speciation in human serum by reversed-phase liquid chromatography-ICP-mass spectrometry

Reversed-phase liquid chromatography-inductively coupled plasma mass spectrometric hyphenation was used for iodine speciation in human serum. First investigations showed that iodine species nearly quantitatively were eluted in the void volume. The result indicated that protein-linked thryoid hormones were not interacting with the stationary phase, thus being not retained. Investigations were performed about T4-TBG (thyroxin-thyroxin-binding globulin) complex generation and its retention during chromatography. It was shown that T4-TBG was not retained on the column. Therefore, a protease treatment was introduced for serum sample preparation. The analysis of “normal” sera (after protease) gave reasonable results lying in the range published in literature: I-:11; di-iodothyrosine (DIT): 2.1; mono-iodothyrosine (MIT): 1.6; reversed tri-iodothyronine (rT3): 3.9; T3: 5.9; T4: 60; each micrograms iodine per liter. The method also proved to recognize abnormalities in a pathologic serum, having rT3 as the predominant species. In this case the method obviously was superior compared to standard immunoassay methods, as it is monitoring the iodine in the species (physiologically active iodine species), whereas immunoassay methods may sometimes detect deiodinated (inactive) compounds.     

Determination of selenium and iodine in human thyroids.

This study focuses on the determination of selenium and iodine in human thyroids. The glands were digested using nitric acid in a microwave oven. Selenium was determined by inductively coupled plasma optical emission spectrometry (ICP-OES) using a new sample introduction system consisting of a reduction system coupled to a hydride generation nebulizer (DHGN). Iodine was determined by using the Sandell-Kolthoff procedure. The detections limits were 0.2 ng/mL and 0.3 ng/mL for the determination method of selenium and iodine, respectively. The amount of iodine in the whole gland was 3.44 +/- 1.11 microg/g. The lowest iodine level was 2.34 microg/g and the highest 5.21 microg/g. The lowest selenium concentration for a single sample was 505 +/- 51 ng/g and the highest 1495 +/- 204 ng/g depending on the fraction of the gland selected.     

Reduction of iodate in iodated salt to iodide during cooking with iodine as measured by an improved HPLC/ICP–MS method

Background: Iodate is a strong oxidant, and some animal studies indicate that iodate intake may cause adverse effects. A key focus of the safety assessment of potassium iodate as a salt additive is determining whether iodate is safely reduced to iodide in food. Objective: To study the reduction of iodate in table salt to iodide and molecular iodine during cooking. Materials and Methods: Fifteen food samples cooked with and without iodated salt were prepared in duplicate. The iodine in the cooked food was extracted with deionized water. The iodine species in the extracts were determined by using an improved high-performance liquid chromatography/inductively coupled plasma–mass spectrometry (HPLC/ICP–MS). The cooking temperature and the pH of the food were determined. Results: The conversion rate of iodate in iodated salt to iodide and molecular iodine was 96.4%±14.7% during cooking, with 86.8%±14.5% of the iodate converted to iodide ions and 9.6% ±6.2% converted to molecular iodine to lose. The limit of detection, limit of quantification, relative standard deviation and recovery rate of the method HPLC/ICP–MS were 0.70 μg/L for I⁻ (0.69 μg/L for IO₃⁻), 2.10 μg/L for I⁻ (2.06 μg/L for IO₃⁻), 2.6% and 101.6%±2.6%, respectively. Conclusion: Almost all iodate added to food was converted into iodide and molecular iodine during cooking. The improved HPLC/ICP–MS was reliable in the determination of iodine species in food extracts.     

The application of preirradiation combustion and neutron activation analysis technique for the determination of iodine in food and environmental reference materials

The pre-irradiation combustion (PC) of samples to liberate iodine, followed by trapping the iodine on charcoal and quantifying the element by neutron activation analysis (NAA), has been used at the National Institute of Standards and Technology for the determination of iodine in biological materials. The applicability of this technique to numerous environmental and dietary matrices is illustrated by analysis of a range of certified reference materials (CRMs) and a powdered grass material that was prepared as an in-house reference material (RM). Because of the combustion step involved, samples with low or no fat content (e.g., cereal products, selected botanical specimens, and nonfat milk powder) and inorganic materials (e.g., coal fly ash and dried sediments) are more suited for analysis by this method. In general, the results for several types of samples obtained by this method agreed with those obtained by a second radiochemical (R) NAA, as well as by a third method using inductively coupled plasma mass spectrometry (ICP-MS). PC-NAA is a useful technique for determining iodine in biological and environmental samples, especially for verification of iodine results obtained from other methods.     

Speciation of Al in human serum by convective-interaction media fast-monolithic chromatography with inductively coupled plasma mass spectrometric detection

A new analytical procedure using anion-exchange separation support based on convective-interaction media (CIM) was developed for the speciation of Al in human serum. The separation of proteins was performed on a weak anion-exchange CIM diethylamine (DEAE) fast-monolithic disk. To prevent co-elution of low molecular mass (LMM) Al species with high molecular mass (HMM) Al compounds on CIM disk serum proteins were first separated from LMM-Al species by the use of size exclusion chromatography (SEC). For this purpose 1 mL of serum was injected onto SEC (Superdex 75 HR 10/30) column. Isocratic elution using 0.05 M TRIS-HCl+0.03 M NaHCO(3) was applied and separation of proteins was followed by UV detection at 278 nm. It was experimentally proven that proteins were eluted in 5.5 mL peak that was collected into a polyethylene cup. A 0.1 mL of the sample aliquot was then injected onto the CIM DEAE disk. The separation of serum proteins was obtained in 10 min by applying linear gradient elution from 100% buffer A (0.05 M TRIS-HCl+0.03 M NaHCO(3)) to 100% buffer B (A+1M NH(4)Cl) and followed by UV detection at 278 nm. Separated Al species were detected on-line by inductively coupled plasma mass spectrometry (ICP-MS). Well-resolved protein peaks were obtained. It was experimentally proven that 90+/-3% of Al in spiked serum of renal patient was eluted under the transferrin peak. The proposed speciation procedure removes LMM-Al species and enables reliable determination of the concentration and composition of Al bound to proteins by CIM DEAE-ICP-MS when the concentration of Al in serum is higher than 5 ng mL(-1). In comparison to chromatographic columns CIM disks enable faster separation and simpler manipulation during cleaning procedure and coupling to ICP-MS.     

The effects of iodine level and source on iodine carry-over in eggs and body tissues of laying hens

In the presented study the effect of different iodine (I) levels and sources in hen feed on the iodine concentration of different tissues, blood serum, and eggs of laying hens was studied. For this purpose, two experiments were conducted with 30 laying hens each. In these experiments feed was enriched with KI and Ca(IO(3))(2), respectively, at 0 (Control), 0.25, 0.5, 2.5 and 5 mg I/kg feed, resulting a analysed iodine level from 0.44 to 4.20 mg/kg feed. After four weeks experimental feeding the iodine concentrations of thyroid glands, blood, meat, liver, abdominal fat and eggs were measured with inductively coupled plasma mass spectrometry. The experimental treatment did not affect hen performance. The iodine supplementation significantly increased the iodine concentration of eggs (144-1304?μg/kg), thyroid glands (3367-5975?μg/g), blood serum (16-67?μg/kg) and liver (13-43?μg/kg). Meat (about 14?μg I/kg) and abdominal fat (about 12?μg?I/kg) were not significantly affected by iodine treatment. Comparative regression analyses showed that at a similar iodine intake, the supply via KI resulted in significantly higher iodine deposition into eggs than Ca(IO(3))(2). Due to the high carry-over of iodine into eggs, eggs may considerably contribute to the iodine supply of the consumers.     

Identification of anionic selenium species in Se-rich yeast by electrospray QTOF MS/MS and hybrid linear ion trap/orbitrap MSn

An analytical approach allowing the identification of unknown selenium metabolites in selenium-rich yeast was described. Anion-exchange HPLC of the Se-metabolome fraction co-eluting with salts in size-exclusion chromatography allowed the separation of nine selenium species (excluding isomers and selenate) as monitored by inductively coupled plasma mass spectrometry (ICP MS). The individual fractions were analyzed by electrospray QTOF MS/MS and hybrid linear ion trap/Orbitrap MSn after sample introduction by reversed-phase nanoHPLC and by hydrophilic interaction LC (HILIC), respectively. Out of the nine detected species, eight were identified on the basis of accurate mass measurements and collision induced dissociation/fragmentation information. Seven Se-species (selenohomolanthionine, γ-Glu-selenocystathionine, 2,3-DHP-selenocystathionine, N-acetyl-selenocystathionine, 2,3-DHP-selenohomolanthionine, Se-methyl-selenoglutathione, and 2,3-DHP-Se-methylselenocysteine) were reported for the first time in Se-rich yeast, five of them have never been reported in any biological sample before.     

Untersuchungen über den Eiweißbedarf der Mastente unter Berücksichtigung einer Lysin-, Methionin- und Vitamin-B-Ergänzung des Mastfutters: (Kurze Originalmitteilung)

In the presented study the effect of different iodine (I) levels and sources in hen feed on the iodine concentration of different tissues, blood serum, and eggs of laying hens was studied. For this purpose, two experiments were conducted with 30 laying hens each. In these experiments feed was enriched with KI and Ca(IO₃)₂, respectively, at 0 (Control), 0.25, 0.5, 2.5 and 5 mg I/kg feed, resulting a analysed iodine level from 0.44 to 4.20 mg/kg feed. After four weeks experimental feeding the iodine concentrations of thyroid glands, blood, meat, liver, abdominal fat and eggs were measured with inductively coupled plasma mass spectrometry. The experimental treatment did not affect hen performance. The iodine supplementation significantly increased the iodine concentration of eggs (144–1304 μg/kg), thyroid glands (3367–5975 μg/g), blood serum (16–67 μg/kg) and liver (13–43 μg/kg). Meat (about 14 μg I/kg) and abdominal fat (about 12 μg I/kg) were not significantly affected by iodine treatment. Comparative regression analyses showed that at a similar iodine intake, the supply via KI resulted in significantly higher iodine deposition into eggs than Ca(IO₃)₂. Due to the high carry-over of iodine into eggs, eggs may considerably contribute to the iodine supply of the consumers.     

Determination of tin in biological samples using gaseous hydride generation-inductively coupled plasma-atomic emission spectrometry

A highly sensitive method determining for sub-microgram/gram levels of tin in biological samples is described. Tin hydride reduced by sodium borohydride and trichloroacetic acid solution was introduced into inductively coupled plasma after separation of liquid and excess hydrogen by an improved gas/liquid separator, and emission intensity was measured at a wavelength of 189.989 nm. Samples were decomposed by a nitric acid-perchloric acid mixture and analyzed after dilution by a standard addition technique. The relative standard deviation was 1.2% for a 10 ng/ml tin standard solution with a detection limit of 30 pg/ml.     

Acquisition of improved reference values for cesium,iodine, strontium,thorium, and uranium in selected NIST reference materials

As part of a study on the ingestion and organ content of some trace elements of importance in radiological protection, additional work has been undertaken to acquire improved reference values for cesium, iodine, strontium, thorium, and uranium in four selected reference materials provided by the US National Institute of Standards and Technology. The materials are SRM-1548 Total Diet, SRM-1548a Typical Diet, SRM-1486 Bone Meal, and RM-8414 Bovine Muscle. A coordinated study was undertaken with the help of seven selected laboratories in five countries. Instrumental and radiochemical neutron activation analysis and inductively coupled plasma-mass spectrometry were the analytical main techniques used.     

Characterization of a rapid and reliable method for iodide biomonitoring in serum and urine based on ion chromatography–ICP-mass spectrometry

An appropriate and controlled supply of thyroid hormones is vital for proper body function. In turn, an appropriate synthesis of T3 and T4 in the thyroid gland is dependent on a sufficient and balanced iodide concentration in blood serum. Due to widespread iodine deficiency or some cases of iodine over exposure, iodide biomonitoring in serum is important and it is that biomonitoring approach being closest to the bioavailable I⁻ supply for the thyroid gland. Therefore, this paper describes a biomonitoring method for iodide determination in serum based on ion chromatography–inductively coupled plasma mass spectrometry (IC–ICP-MS). Since in literature only very few data are available on iodide in serum but many in urine the method is also extended to I⁻ monitoring in urine. The method was additionally designed to have short analysis time (8 min) for increased sample throughput, good precision in serial measurement (serum: 4.86%; urine: 1.4%), and day-to-day determination (serum: 5.7%; urine: 2.28%), high accuracy (serum: 105%; urine: 101%) and good recovery (serum: 102%; urine: 99%) even in matrix-rich samples at low I⁻ concentration. Also, investigations were performed to elucidate whether internal standardization during chromatography, sample preparation for protein-matrix removal or matrix-matched calibration are advantageous for analytical performance. Finally, limits of detection (3σ) of 0.12 μg/L or 0.05 μg/L (serum or urine) and limit of quantification (10σ) of 0.39 μg/L or 0.17 μg/L (serum or urine) were achieved.     

Iodine levels in different regions of the human brain

BackgroundIodine is a key component of the thyroid hormones thyroxine (T4) and triiodothyronine (T3), which are crucial for proper growth and development of the human body. In particular, a great body of literature has been published on the link between thyroid hormones and brain development and functioning. However, there is a lack of knowledge on the iodine levels in the human brain. The aim of this work was to determine the brain iodine levels and to contribute to the establishment of “reference” levels for iodine in the different anatomical and functional regions of normal (i.e., subjects without neurological or psychiatric diseases) human brain.MethodsThe iodine levels were determined in 14 brain regions of 52 dead subjects without evidence of neurological or psychiatric disease (n = 728 samples). Iodine was extracted from brain samples using a standard procedure and determined by inductively coupled plasma – mass spectrometry (ICP-MS).ResultsFour subjects presented abnormally high brain iodine levels (26.0 ± 14.2 μg/g) and were excluded from the overall data analysis. The average brain iodine levels for the remaining 48 subjects was 0.14 ± 0.13 μg/g dry weight. Iodine showed very heterogeneous distribution across the different brain regions, with the frontal cortex, caudate nucleus and putamen showing the highest levels. Interestingly, these brain regions are closely related to cognitive function. Iodine levels also showed a tendency to increase with age. The high levels observed in four subjects seemed to be related to previous exposure to iodine-based contrast agents widely used in radiology and computed tomography exams.ConclusionsThis paper provides important data on iodine levels at different brain regions in “normal” people, which can be used to interpret eventual imbalances in subjects with mental disorders and neurodegenerative diseases.     

Separation and quantitation of phospholipids and their ether analogues by high-performance liquid chromatography.

The common mobile phase hexane/isopropanol/water used for separation of phospholipids on high-performance liquid chromatography silica columns poses several problems, such as incomplete separation and rapid column deterioration. By inclusion of 5 mM ammonium sulfate in the aqueous phase, we were able to substantially improve the chromatographic resolution and obtain complete separation of phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, cardiolipin, phosphatidylglycerol, and sphingomyelin. In addition, ammonium sulfate prevented column degeneration and greatly improved reproducibility. A new quantitation method for alkenylacyl, alkylacyl, and diacyl forms of phospholipids was also developed based on derivatization with [(3)H]acetic anhydride. Separation and quantitation of the radioactive acetyl diradylglycerols were performed by straight-phase HPLC coupled to a radioactive flow detector and enabled detection of the various ether analogues at the picomole level with high reproducibility. The described methods are mild and nondestructive and can therefore be easily combined with analysis of either molecular species or fatty acid and aldehyde composition of the individual phospholipids.     

Pollen morphology and localization of Ni in some Ni-hyperaccumulator taxa of Alyssum L. (Brassicaceae)

Pollen morphology of seven Alyssum L. taxa growing on serpentine soils in different places in the European Mediterranean macrobioclimate territory were studied, described and compared. Cluster analysis was performed to show similarity between species and their populations. The shape of the pollen grains varies among the species and among the grains within the same anther. The pollen grains are 3-colpate, prolate, with long and narrow colpi reaching the poles. The ornamentation of the exine varies from micro-reticulate to reticulate between the species. Pollen sterility/fertility was also calculated. The highest percentage of sterile pollen (73.76%) was calculated for Alyssum murale subsp. murale and the lowest (9.54%) for A. bertolonii subsp. bertolonii. All species are representatives of sect. Odontarrhena (C.A. Meyer) Koch well known as Ni-hyperaccumulators. Nickel and other elements present in pollen and stamen were studied by inductively coupled plasma-mass spectrometry. The stamen parts of all species were micromorphologically analyzed by scanning electron microscopy coupled to an energy-dispersive X-ray probe. Accumulation of Ni was detected in the stamens of all studied species and rarely in the pollen grains. The distribution patterns of Ni were similar among the species examined.     

Speciation of trace elements in human serum by micro anion exchange chromatography coupled with inductively coupled plasma mass spectrometry

Speciation analysis of essential trace elements in human serum provides important information on nutritional status and homeostatic mechanisms regulating transport processes, acute phase reactions, and protection against oxidative damage. Anion exchange high-performance liquid chromatography (HPLC) combined with inductively coupled plasma mass spectrometry (ICP-MS) has proved to be a useful tool in speciation. Here we describe a fast method that can be applied to carry out the speciation of Fe, Cu, Zn, and Se in as little as 1 microl [corrected] of serum. The method employs monolithic anion exchange micro columns installed on a tandem HPLC system coupled on-line with an ICP-MS detector. The chromatographic separation is similar to those reported previously but with considerable gain in terms of time and sample requirement. Reproducibility is acceptable for most species. Using our method, we were able to find species-specific differences between different commercially available trace element reference materials. Because the method chosen to collect blood might interfere with speciation, the proposed methodology was used to compare heparinized plasma, ethylenediaminetetraacetic acid (EDTA) plasma, and serum from adult healthy volunteers. As expected, EDTA strongly affects speciation analysis (especially for Fe and Zn), whereas changes due to the use of lithium-heparin (Li-He) as anticoagulant appear to be minimized.     

On-line reversed-phase liquid chromatography hydride generation emission spectrometry: speciation of arsenic in urine of patients intravenously treated with As2O3

Hydride generation inductively coupled plasma–atomic emission spectrometry (HG ICP–AES) was used as a continuous detection system for the determination of arsenic in the eluate from a high-performance liquid chromatographic (HPLC) system. Four arsenic species [arsenite As(III), arsenate As(V), monomethylarsonate (MMA), and dimethylarsinate (DMA)] present in the urine samples of patients treated intravenously with arsenite, were analyzed separately by HPLC–HG-ICP–AES using a non-polar C₁₈ column. This analytical method allowed the sensitive determination of the arsenic species in the submicrogram per liter range. Urine samples collected on different days after arsenite administration were found to contain arsenite predominantly – monomethylarsonate and dimethylarsinate were also detected.     

Site specific radioiodination of recombinant hirudin

Recombinant hirudin variant rHV2-Lys47 was radioiodinated using the chloramine-T method. Depending on the reaction pH, the two tyrosine residues, Tyr3 and Tyr63, responded differently to iodination but without change in total iodination yield. Of the incorporated -125 iodine 80% was located on Tyr3 at pH 7.4, but 65% was found on Tyr63 at pH 4. These distinct iodination patterns suggest the existence of a pH-dependent multimerization and/or important conformational changes in the tertiary structure with pH. Each radiotracer was purified to high specific activity by simple low-pressure chromatography including gel filtration and reverse-phase separation, both on short cartridges. The method was validated by reverse-phase and anion-exchange HPLC with on-line radioactivity detection. The iodination sites were characterized following carboxypeptidase Y cleavage coupled with radio-HPLC.     

Validation of an optimized method for the determination of iodine in human breast milk by inductively coupled plasma mass spectrometry (ICPMS) after tetramethylammonium hydroxide extraction

In this study a novel method to determine iodine concentrations in human breast milk was developed and validated. The iodine was analyzed by inductively coupled plasma mass spectrometry (ICPMS) following tetramethylammonium hydroxide (TMAH) extraction at 90 °C in disposable polypropylene tubes. While similar approaches have been used previously, this method adopted a shorter extraction time (1 h vs. 3 h) and used antimony (Sb) as the internal standard, which exhibited greater stability in breast milk and milk powder matrices compared to tellurium (Te). Method validation included: defining iodine linearity up to 200 μg L⁻¹; confirming recovery of iodine from NIST 1549 milk powder. A recovery of 94–98% was also achieved for the NIST 1549 milk powder and human breast milk samples spiked with sodium iodide and thyroxine (T4) solutions. The method quantitation limit (MQL) for human breast milk was 1.6 μg L⁻¹. The intra-assay and inter-assay coefficient of variation for the breast milk samples and NIST powder were <1% and <3.5%, respectively. NIST 1549 milk powder, human breast milk samples and calibration standards spiked with the internal standard were all stable for at least 2.5 months after extraction. The results of the validation process confirmed that this newly developed method provides greater accuracy and precision in the assessment of iodine concentrations in human breast milk than previous methods and therefore offers a more reliable approach for assessing iodine concentrations in human breast milk.     

Separation of Organoarsenicals by Means of Zwitterionic Hydrophilic Interaction Chromatography (ZIC®‐HILIC) and Parallel ICP‐MS/ESI‐MS Detection

An analytical scheme was developed for the separation and detection of organoarsenicals using a zwitterionic stationary phase of hydrophilic interaction chromatography (ZIC^®‐HILIC) coupled in parallel to electrospray ionization mass spectrometry (ESI‐MS) and to inductively coupled plasma mass spectroscopy (ICP‐MS). The optimization of separation and detection for organoarsenicals was mainly focused on the influence of the percentage of acetonitrile (MeCN) used as a major component of the mobile phase. Isocratic and gradient elution was applied by varying the MeCN percentage from 78 % to 70 % MeCN and 22 % to 30 % of an aqueous solution of ammonium acetate (125 mM NH₄Ac; pH 8.3) on a ZIC^®‐HILIC column (150 × 2.1 mm id, 3.5 μm), to allow for the separation and successful detection of nine organoarsenicals (i.e., 3‐nitro‐4‐hydroxyphenylarsonic acid (roxarsone, Rox), phenylarsonic acid (PAA), p‐arsanilic acid (p‐ASA), phenylarsine oxide (PAO), dimethylarsinate (DMA), methylarsonate (MMA), arsenobetaine (AsB), arsenocholine (AsC) and trimethylarsine oxide (TMAO)) within 45 min. All analytes were prepared in the mobile phase. The flow rate of the mobile phase, the splitting ratio between ICP‐MS and ESI‐MS detection, and the oxygen addition were adapted to ensure that there appeared a stably burning inductively coupled plasma. Furthermore, the analytical method was evaluated by the identification and quantification of AsB in the reference material DORM‐2 (dogfish muscle) resulting in a 95‐% recovery with respect to the AsB concentration in the extract.     

Increase in liver nuclear tri-iodothyronine receptors associated with increased deoxyribonucleic acid by long-term administration of tri-iodothyronine in thyroidectomized rats

The effect of long-term administration of small amounts of tri-iodothyronine was examined on the nuclear tri-iodothyronine receptors in rat liver. The maximal binding capacity (C(max.)) and association constant (K(a)) of the receptors were determined in thyroidectomized rats given vehicle alone (group A), 2mug of tri-iodothyronine/100g body wt. (group B) or 7mug of tri-iodothyronine/100g body wt. (group C) for 2 weeks. Scatchard analyses with correction for the amount of endogenous tri-iodothyronine revealed that C(max.) values per g of liver were increased to 1.5 and 2.7 times the control value in groups B and C respectively. Since concentrations of DNA per g of liver were significantly increased in the two groups of hormone-treated rats, C(max.) values per mg of DNA were nearly the same in group B, but still increased significantly in group C compared with group A. K(a) values remained unchanged in all three groups of animals. Mitochondrial alpha-glycerophosphate dehydrogenase activity was 9.6 and 28.7 times as high in groups B and C, respectively, as in group A. Concentrations of endogenous tri-iodothyronine bound to non-histone protein were substantially increased in groups B and C, although concentrations of serum tri-iodothyronine remained rather low. The results obtained indicate that the long-term administration of tri-iodothyronine in small doses induces an increase in the nuclear receptors associated with increased DNA with and without accompanying a relative increase in the receptor concentration in thyroidectomized rats. Also the hormonal effect is closely related to the total number of the nuclear receptors and the concentrations of nuclear tri-iodothyronine bound to the receptors rather than the serum tri-iodothyronine concentrations.