Post-column Isotope Dilution Analysis Research Articles

Quantitative multiplexed analysis of MMP-11 and CD45 in metastatic breast cancer tissues by immunohistochemistry-assisted LA-ICP-MS.

Breast cancer is the leading cause of cancer death in woman and tremendous efforts are undertaken to limit its dissemination and to provide effective treatment. Various histopathological parameters are routinely assessed in breast cancer biopsies to provide valuable diagnostic and prognostic information. MMP-11 and CD45 are tumor-associated antigens and potentially valuable biomarkers for grading aggressiveness and metastatic probability. This paper presents methods for quantitative and multiplexed imaging of MMP-11 and CD45 in breast cancer tissues and investigates their potential for improved cancer characterization and patient stratification. An immunohistochemistry-assisted laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) method was successfully developed and optimized using lanthanide-tagged monoclonal antibodies as proxies to determine spatial distributions and concentrations of the two breast cancer biomarkers. The labeling degree of antibodies was determined via size exclusion-ICP-tandem mass spectrometry (SEC-ICP-MS/MS) employing online calibration via post-column isotope dilution analysis (IDA). The calibration of spatial distributions of labeled lanthanides in tissues was performed by ablating mold-prepared gelatin standards spiked with element standards. Knowledge of labeling degrees enabled the translation of lanthanide concentrations into biomarkers concentrations. The k-means clustering was used to select tissue areas for statistical analysis and mean concentrations were compared for sets of metastatic, non-metastatic and healthy samples. MMP-11 was expressed in stroma surrounding tumor areas, while CD45 was predominantly found inside tumor areas with high cell density. There was no significant correlation between CD45 and metastasis (P=0.70); however, MMP-11 was significantly up-regulated (202%) in metastatic samples compared to non-metastatic (P=0.0077) and healthy tissues (P=0.0087).

Quantitative assessment of the metabolic products of iron oxide nanoparticles to be used as iron supplements in cell cultures

Iron nanoparticles (NPs) metabolism is directly associated to human health due to their use as anemia treatment and should be studied in detail in cells. Here we present a speciation strategy for the determination of the metabolic products of iron oxide nanoparticles coated by tartaric and adipic acids in enterocytes-like cell models (Caco-2 and HT-29). Such methodology is based on the use of SDS-modified reversed phase high performance liquid chromatography (HPLC) separation using inductively coupled plasma-mass spectrometry (ICP-MS) as Fe selective detector. Post-column isotope dilution analysis is used as quantification tool by adding 57Fe as isotopically enriched standard. To assess the separation capability of the method, two different iron nanostructures: iron sucrose nanoparticles -Venofer®- used as model suspension and iron tartrate/adipate-modified nanoparticles, both of about 4 nm (core size) were evaluated. The two nanostructures were injected into the system showing good peak profiles and quantitative elution recoveries (>80%) in both cases. In addition, both nanoparticulate fractions could be based-line separated from ionic iron species, which needed to be complexed with 1 mM citrate to elute from the column. Exposed cells up to 0.5 mM of iron tartrate/adipate-modified nanoparticles were specifically treated to extract the internalized NPs and the extracts examined using the proposed strategy. The obtained results revealed the presence of three different fractions corresponding to nanoparticle aggregates, dispersed nanoparticles and soluble iron respectively in a single chromatographic run. Quantitative experiments (column recoveries ranging from 60 to 80%) revealed the presence of the majority of the Fe in the nanoparticulated form (>75%) by summing up the dispersed and aggregate particles. Such experiments point out the high uptake and low solubilization rate of the tartrate/adipate NPs making these structures highly suitable as Fe supplements in oral anemia treatments.

Selenoprotein P as the major transporter for mercury in serum from methylmercury-poisoned rats

Selenium (Se) has been found to promote weight gain, decrease hepatic damage, but redistribute mercury (Hg) in brains and livers in methylmercury (MeHg)-poisoned rats. The aims of the present work were to examine the effects of Se on the levels of Hg in serum and the role of serum selenoproteins in binding with Hg in MeHg-poisoned rats. The concentration of Se, Hg and MeHg were studied using ICP-MS and CVAFS. The Hg- and Se-binding selenoproteins were separated and quantified using affinity chromatography with post-column isotope dilution analysis using both enriched 78Se and 199Hg. It was found that Se treatment reduced Hg levels in serum in MeHg-poisoned rats. Among the three separated selenoproteins, the amounts of SelP-bound Hg and Se increased to 73% and 93.6%, from 64.4% and 89.3% of the total Hg and Se, respectively after Se treatment, suggesting that SelP acts as a major transporter for Hg and pool for Se in serum. Over 90% of the total Hg was MeHg in serum, and the molar ratios of MeHg to Se as 1:4 and 1:9 in the formed MeHg-Se-SelP complex in the control and the Se treatment group, respectively. The elevated Se level binding with SelP facilitated the Hg extraction from tissues and organs, as well as its redistribution in brains and livers through blood circulation in the MeHg-poisoned rats. Together, our findings provide direct evidence that serum SelP is the major Hg transporter in MeHg-poisoned rats.

Quantitative selenium speciation by HPLC-ICP-MS(IDA) and simultaneous activity measurements in human vitreous humor

A post-column isotope dilution analysis (IDA) methodology was applied to carry out quantitative speciation of selenium in human vitreous humor samples by size exclusion chromatography (SEC) coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS). Two main selenium species detected by SEC-ICP-MS were foundto be associated to protein complexes. The expected molecular weights for both selenium-bound complexes were confirmed by MALDI-TOF(MS) and the results matched well with the theoretical mass of a GPx monomer (M, 22kDa) and tetramer (T, 88kDa). The quantification of the two detected selenium-bound complexes by post-column IDA showed that the total content of selenospecies in vitreous humor was approximately 3.2 ± 1.8ppb Se. Moreover, in most of the analyzed vitreous humor samples, the majority of the selenium was associated to higher molecular weight GPx biomolecules. In an attempt to assess if the enzymatic activity was associated with a given selenium-bound GPx protein, the antioxidant enzyme activity was assayed for the two separated GPx species. Only for GPx (T) was a linear relationship between activity and total Se concentration found by ICP-MS.

Elemental mass spectrometry for Se-dependent glutathione peroxidase determination in red blood cells as oxidative stress biomarker

Glutathione Peroxidase-1 (GPx1) is an enzyme playing an important role in the defense against oxidative stress which is associated with many pathological conditions. Thus, changes in the expression of this enzyme in different human tissues and fluids could be an indicator used for oxidative status assessment. Since most analytical methods for GPx1 determination are based on relative activity measurements, here we propose the absolute quantification of this protein in red blood cells through the measurement of Se present as selenocysteine in its structure. For this purpose, the sample is treated for hemoglobin precipitation and further fractionated by size-exclusion liquid chromatography (SEC) with ICP-MS for final detection. Purity of the species in the fraction obtained by SEC was assessed by orthogonal chromatographic separation based on a reversed phase mechanism (RP) of the peptides obtained by tryptic digestion of the corresponding fraction. The use of ICP-MS detection after such capillary-RP-HPLC permitted us to detect a single Se-containing peptide that was further confirmed by electrospray ionization mass spectrometric detection (ESI-MS) to belong to GPx1. Once the purity of such fraction was addressed, the quantitative analysis of GPx1 was conducted by Se post-column isotope dilution analysis after SEC separation in different samples of human red blood cells. When the concentration results obtained via SEC-ICP-MS for different protein standards are plotted versus the activity measurements (using the spectrophotometric H2O2/NADPH/GR method) a good correlation curve is obtained. Such results permit us, from ICP-MS measurements, to obtain simultaneously the GPx1 absolute concentration as well as the activity by interpolation in the previously obtained curve.

Reduction of Cisplatin-Induced Nephrotoxicity in Vivo by Selenomethionine: The Effect on Cisplatin–DNA Adducts

Cisplatin is one of the most effective chemotherapeutic agents, although its clinical use is limited by severe renal toxicity. This toxicity seems to be related to the accumulation of the drug in kidney tissues, leading to renal failure. For this reason, several compounds have been evaluated to ameliorate the nephrotoxicity induced by cisplatin. In the present investigation, we report the effect of the oral administration of selenomethionine before intraperitoneal cisplatin treatment. The preadministration of this Se species has been shown to have an important effect in reducing renal damage induced by cisplatin by increasing the excreted urea and improving creatinine clearance. Quantification of the level of DNA--cisplatin adducts in kidney and liver tissues was carried out by postcolumn isotope dilution analysis using liquid chromatography-inductively coupled plasma (LC-ICP-MS) as speciation set up. The level of DNA--cisplatin adducts in rats given Se-methionine in the drinking water before cisplatin administration was considerably lower in kidney tissues with respect to the animals drinking only water. Such effects were not observed in liver tissue. Initial speciation studies of Pt and Se conducted in kidney tissues of exposed animals by HPLC-ICP-MS have revealed the presence of cisplatin as part of a complex with Se-methionine, which can be eventually excreted into urine. This Pt--Se complex could explain the observed reduction of the kidney damage in Se-methionine-treated animals.

Full quantification of selenium species by RP and AF-ICP-qMS with on-line isotope dilution in serum samples from mercury-exposed people supplemented with selenium-enriched yeast

Accurate determination of selenium (Se) species in biological samples is a critical issue because Se commonly occurs at low levels and in diverse species. The method for the full quantification of Se species in serum samples was proposed through combined ion-pair reverse-phase (RP) chromatography and affinity chromatography (AF) hyphenated to inductively coupled plasma-(quadrupole) mass spectrometry (ICP-qMS) with post-column isotope dilution analysis (IDA) and a collision cell technique (CCT). Different Se species like inorganic Se (Se4+ and Se6+), selenocystine (SeCys), selenomethionine (SeMet), selenoprotein P (SelP), selenoalbumin (SeAlb) and glutathione peroxidase (GPx) can be separated and quantified. The proposed methodology was used to qualitatively and quantitatively study the dynamic distribution of Se species in human serum samples from the Hg-contaminated area after supplementation with 100 μg of Se daily as Se-enriched yeast for 180 days. SelP takes up almost half and even more of the total Se and increases with the Se administration. The repeatability in terms of relative standard deviation (R.S.D. %, n = 10) is 6% for GPx and SelP and 5% for SeAlb. The detection limits are 0.1 μg Se L−1 for GPx and other non-retained Se compounds, 1.0 μg Se L−1 for SelP and 1.2 μg Se L−1 for SeAlb, 1.3 μg Se L−1 for inorganic Se; 1.2 μg Se L−1 for SeCys; 1.1 μg Se L−1 for SeMet, respectively.

Selenium speciation in rat colon tissues

A study of selenium (Se) speciation in rat colon tissues is presented. Four different procedures for the extraction of Se compounds were evaluated in terms of recovery and species preservation. Total Se in tissue and extracts was determined by ICP-MS and isotope dilution analysis. The selected and optimized protocol allowed an extraction of 43% of Se, while continuously bubbling nitrogen in the solution during the procedure was mandatory to prevent the oxidative degradation of selenoproteins. Speciation analysis was then performed on the extracts using size exclusion- and anion exchange-HPLC for species separation. A number of Se compounds were detected in rat colon extracts, and individually quantified by coupling HPLC-ICP-MS and species-unspecific on-line (post-column) isotope dilution analysis. Among the isolated selenospecies, the two major proteins glutathione peroxidase type 2 and thioredoxin reductase type 1 have been potentially identified by their molecular weight using MALDI-TOF-MS.

Gas Chromatography-Combustion-Mass Spectrometry with Postcolumn Isotope Dilution for Compound-Independent Quantification: Its Potential to Assess HS-SPME Procedures

A quadrupole GC-MS instrument with an electron ionization (EI) source has been modified to enable application of postcolumn isotope dilution analysis for the standardless quantification of organic compounds injected in the gas chromatograph. Instrumental modifications included the quantitative conversion of the separated compounds into CO(2), using a postcolumn combustion furnace, and the subsequent mixing of the gas with a constant flow of (13)CO(2) diluted in helium. The online measurement of the (12)CO(2)/(13)CO(2) (44/45) ratio in the EI-MS allowed us to obtain quantitative data without resorting to compound-specific standards. Validation of the procedure involved the analysis of standard solutions containing different families of organic compounds (C(9)-C(20) linear hydrocarbons, BTEX and esters) obtaining satisfactory results in all cases in terms of absolute errors (<6%) and precision (<4% RSD). The developed procedure showed excellent linearity over the range assayed (2 orders of magnitude) and adequate detection limits for carbon containing compounds (0.8 pg C s(-1)). The generic value of this compound-independent calibration approach was assessed by studying the quantitative performance of Head Space-Solid Phase Microextraction (HS-SPME). The proposed compound-independent quantification by EI-MS permits comparison of the performance of different fibers by assessing analyte recoveries with extreme robustness, simplicity, and precision.

Quantitative Analysis and Simultaneous Activity Measurements of Cu, Zn-Superoxide Dismutase in Red Blood Cells by HPLC−ICPMS

The interest on accurate and precise determination of metalloproteins such as Cu, Zn-superoxide dismutase (Cu, Zn-SOD) involved in the redox balance of living cells is increasing. For this purpose, analytical strategies that provide absolute protein concentration measurements have to be developed. The determination of Cu, Zn-SOD through the measurement of the Cu associated to the protein, which provides its enzymatic activity, by liquid chromatography with online inductively coupled plasma mass spectrometric (ICPMS) detection is described here. Postcolumn isotope dilution analysis (IDA) of Cu has been applied for quantification after evaluation of the column recovery for the total Cu and also Cu-SOD that turned out to be quantitative. When the concentration results obtained via IDA using high-performance liquid chromatography (HPLC)-ICPMS are plotted versus the activity measurements (using the spectrophotometric pyrogallol autoxidation method) a good correlation curve is obtained. Such results permit us, from ICPMS measurements, to obtain simultaneously the Cu, Zn-SOD absolute concentration as well as its enzymatic activity by interpolation in the previously obtained curve. This possibility was explored in real samples (red blood cells of control individuals and patients with metallic total hip arthroplasty) obtaining a good match between direct enzymatic activity measurements and those obtained by interpolation in the correlation curve. The actual protein identification in the red blood cell extract was conducted by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and two matrixes were compared in order to preserve as much as possible the protein-metal interactions during the MALDI process. Interestingly, using a solution containing trihydroxyacetophenone in citrate buffer permitted us to observe some metal-protein interactions in the MS spectrum of the intact Cu, Zn-SOD from red blood cells.

Evaluation of different analytical strategies for the quantification of sulfur-containing biomolecules by HPLC-ICP-MS: Application to the characterisation of 34S-labelled yeast

Different analytical strategies, such as Compound Independent Calibration (CIC), post-column Isotope Dilution Analysis (IDA) and post-column Isotope Pattern Deconvolution (IPD) were evaluated for the determination of sulfur-containing biomolecules. CIC graphs were obtained for the sulfur-containing compounds sulfate, cysteine, glutathione and methionine separated by isocratic HPLC using four different ICP-MS instruments. Instruments evaluated included two quadrupole based instruments (XSeries II and HP-4500), with and without a collision cell respectively, one single collector double focusing (Element 2) and one multicollector double focusing (Neptune). When isocratic HPLC separations were employed, CIC using sulfate standards could be applied for the determination of small peptides and amino acids in their native form. When gradient elution HPLC separations were performed, CIC was no longer suitable and enriched stable isotopes (33S) were evaluated to compensate for gradient-derived changes in the sensitivity for sulfur in the ion source. It was observed that post-column IDA using 33S was suitable for the absolute quantification of sulfate, cysteine, glutathione and methionine by HPLC-ICP-MS when gradient elution was performed. Unfortunately, traditional equations used for post-column IDA can no longer be applied when two different sulfur enriched isotopes are employed. In the characterisation of 34S-labelled yeast we will have three different sulfur isotope patterns: natural abundance sulfur impurities, 34S used as “labelling” tracer and 33S used as post-column “quantification” tracer. So, for the characterisation of 34S-labelled yeast we have developed a post-column IPD procedure that allowed us to obtain the “pattern specific molar flow chromatograms” and, hence, to discriminate between “natural abundance” and “34S-enriched” sulfur species in the 34S-labelled yeast.

Novel HPLC-ICP-MS strategy for the determination of β2-Transferrin, the biomarker of cerebrospinal fluid (CSF) leakage

Asialo-Transferrin (S(0)) or beta(2)-Transferrin is an accepted marker of cerebrospinal fluid (CSF) leakage from the subarachnoid space into the nasal or aural cavity as the result of a head trauma. At this time, the S(0) detection method of choice is isoelectric focusing on polyacrylamide gel with direct immunofixation of transferrin (Tf) and silverstaining which are time-consuming techniques (> 5 h). The aim of this study is to present an alternative methodology for determination of Tf sialoforms present in CSF samples, specifically S(0), based on the detection of the Fe associated with those forms. A double spiking methodology is developed by saturating the protein with isotopically enriched iron ((57)Fe) and conducting post-column Isotope Dilution Analysis (IDA) with (54)Fe after separation of the different sialoforms by anion exchange chromatography. The results obtained have been validated using a certified serum (ERM-DA470) and applied with satisfactory results to a pooled CSF sample.

Metal–metallothioneins like proteins investigation by heteroatom-tagged proteomics in two different snails as possible sentinel organisms of metal contamination in freshwater ecosystems

Metal speciation analysis in MLPs was carried out in two snails, Marisa cornuarietis and Pomacea bridgesi, in order to investigate them as possible sentinel organisms of heavy metal contamination. To carry out this study snails born in a non-contaminated environment were divided into two groups: a control group and a contaminated one with cadmium administered for 40 days. Subsequently, we investigated the speciation of the induced MLPs in exposed animals in relation to controls. In order to obtain the MLP fraction, cytosols from both snail species where subjected to size-exclusion fractionation, monitoring on-line the metal signal (Cd, Cu and Zn) by ICP-MS while protein elution was followed by on-line UV detection. MLP fraction was then separated by anion-exchange (AE)-FPLC using optimal chromatographic conditions for the separation of the different MLP isoforms in both snail species. Specific detection of separated metalloforms was carried out again by the hyphenation of the AE chromatographic system with ICP-MS. The determination of the amount of metal bound to MLPs was carried out by post-column isotope dilution analysis ICP-MS, finding that the snail M. cornuarietis accumulated higher concentrations of cadmium than P. bridgesi. Thus this first snail could therefore be a better candidate sentinel organism of pollution in natural waters. Identification and characterization of the isoforms separated in M. cornuarietis was carried out for the entire or intact isoforms by MALDI-TOF and then conventional triptic digestion was also carried out to identify the nature of the formed peptides. The presence identification of a MLP isoform of relatively low molecular weight in M. cornuarietis is reported.

Quantification of selenium species in petroleum refinery wastewaters using ion chromatography coupled to post-column isotope dilution analysis ICP-MS

A post-column Isotope Dilution Analysis (IDA) methodology has been used to carry out the quantitative speciation of selenium (Se) in industrial wastewaters by ion chromatography coupled to ICP-MS with an octapole reaction system (ORS). The interfering argon dimers on the 78Se and 80Se isotopes were suppressed by pressurizing the octapole chamber with 4 mL min-1 of hydrogen. The inorganic Se species selenite [Se (IV)], selenate [Se (VI)] and selenocyanate [SeCN-] were separated by ion chromatography (IC) using isocratic elution with 20 mmol L-1 Na2CO3 / 2 mmol L-1 NaHCO3 as eluent. After the separation, the eluent carbonates were converted to CO2 on-line by a membrane self-regenerating suppressor, in order to avoid clogging problems in the nebulizer or in the cones of the ICP-MS interface. Detection limits (as 3σ of the baseline noise) of 0.18, 0.13 and 1.0 ng g-1 were achieved for Se (IV), Se (VI) and SeCN- respectively. Quantification of selenium in each separated species was performed using post-column isotope dilution analysis by continuous mixing of an enriched 77Se spike solution with the effluent from the suppressor column. Finally, the 78Se/77Se or 80Se/77Se isotope ratios were monitored and the concentration of the corresponding selenium species was evaluated. The proposed methodology has been successfully applied to study the quantitative speciation of selenium in petroleum refinery wastewaters.

μLC coupled to ICP–SFMS with post-column isotope dilution analysis of sulfur for absolute protein quantification

Absolute protein quantification has become an important challenge in modern bioanalytical chemistry. Among several approaches based on mass spectrometric techniques, inductively coupled plasma (ICP) as ionisation source provides element-selective and sensitive detection of heteroatoms, and thus, a potentially emerging tool in protein analysis. In this work we applied coupling of capillary liquid chromatography (μLC) and inductively coupled plasma-sector field mass spectrometry (ICP-SFMS) to the separation and determination of standard proteins. For quantification purposes, post-column isotope dilution of sulfur was applied and optimised for this type of hyphenated technique. Provided that the protein sequence is known (number of sulfur-containing amino acids, i.e. cysteines and methionines) the protein amount can then be directly calculated from the determined sulfur content in a certain protein fraction. In order to prove the reliability of the presented method, two different certified reference materials were analysed: CRM 393 (human apolipoprotein A-I) and CRM 486 (α-fetoprotein). For CRM 393 excellent agreement (37.0 ± 1.4μmol L(-1)) was obtained with the certificate (37.7 ± 1.8μmol L(-1)). However, the recovery rate for α-fetoprotein in CRM 486 was found to be about 60% indicating incomplete elution of the protein during the chromatographic separation.

Analysis of mercury-containing protein fractions in brain cytosol of the maternal and infant rats after exposure to a low-dose of methylmercury by SEC coupled to isotope dilution ICP-MS

The mercury-containing protein fractions in the brain cytosol of the maternal and infant rats after exposure to low-dose of methylmercury chloride (0.3 mg Hg kg−1 day−1) were analyzed by a SEC-ICP-MS method with the postcolumn isotope dilution analysis. The enriched spiking including 34S, 65Cu, 67Zn and 198Hg was continuously added into the eluate from the HPLC column and then the isotope-diluted fractions were on-line measured by ICP-MS. Therefore, the absolute amounts of sulfur, mercury, zinc and copper in the eluted protein fractions could be attained after calculation of the corresponding peak areas in the mass flow chromatogram. Five mercury-containing protein fractions were monitored in the maternal sample, whereas only one was in the infant sample. The mercury content in the infant sample was about 10 times lower than the one in the maternal sample. In the meantime, two copper- and four zinc-containing fractions were found in both maternal and infant samples. The detection limit for S, Cu, Zn and Hg are 11, 0.1, 1.5 and 0.2 ng, respectively. The results demonstrate that different mercury-containing protein fractions may exist in brain cytosol between maternal and infant rats and the quantitative calculation may be helpful for the toxicological study.

Stable isotope labelling and FPLC–ICP-SFMS for the accurate determination of clinical iron status parameters in human serum

Iron is involved in the function of all living cells and, in fact, many diseases arise from imbalances in iron homeostasis. Therefore, the development of analytical methodologies to improve and automate the measurement of clinical indices of iron status has increased tremendously over the years. This work describes the development of two complementary methodologies to evaluate transferrin (Tf) saturation, total iron-binding capacity (TIBC), unsaturated iron-binding capacity (UIBC) and serum iron based on the use of iron-selective monitoring by inductively coupled plasma mass spectrometry (ICP-MS). The first methodology is based on the saturation of transferrin (Tf) with natural Fe3+ followed by separation of the different sialoforms in an anion exchange column (Mono-Q) to quantify the iron in each Tf sialoform and total Tf by ICP-MS using post-column isotope dilution analysis. In the second part, the saturation is done with an iron tracer (57Fe) and the application of pattern deconvolution analysis permits the direct quantification of the Tf saturation, the serum iron and the unsaturated iron-binding capacity. These strategies are validated by using a reference serum certified for total Tf and tested also in serum samples from individuals suffering from hemochromatosis and Fe-supplemented patients. The results obtained for all the parameters related to Fe status were in good agreement with those obtained by clinical tests. The use of stable isotope labelling in connection with the on-line coupling of fast protein liquid chromatography (FPLC) to ICP-MS allows the accurate determination of several parameters of great clinical relevance in iron homeostasis by means of two independent chromatographic runs. The main advantage of the proposed methodology is the number of parameters that can be simultaneously obtained.

An attempt to differentiate HPLC-ICP-MS selenium speciation in natural and selenised Agaricus mushrooms using different species extraction procedures

Total determination and speciation analysis of Se in commercial and selenised Agaricus mushrooms have been performed to investigate the Se species naturally occurring in non-enriched mushrooms as well as those present in specimens grown in a Se-enriched medium. Mushroom aqueous and enzymatic extracts have been analysed by three complementary chromatographic separation mechanisms (size-exclusion, anion-exchange and reversed-phase) coupled to an inductively coupled plasma mass spectrometer with an octopole reaction system. Post-column isotope dilution analysis has been used on-line with the separations for quantification of the Se species eluted. The 78Se-to-77Se isotope ratio was monitored after adequate corrections for both total determinations and Se species quantitative speciation. The results showed marked differences not only in total Se contents but also in Se species found in the two types of Agaricus mushrooms investigated. Selenomethionine was detected in both of them (free in commercial mushrooms and incorporated into proteins in selenised ones) together with a number of unknown selenocompounds.

Qualitative and quantitative speciation analysis of water soluble selenium in three edible wild mushrooms species by liquid chromatography using post-column isotope dilution ICP–MS

Speciation analysis of Se in hot water extracts of three edible wild mushroom species ( Macrolepiota procera, Lepista luscina and Boletus luridus) has been performed to investigate Se species naturally occurring in wild mushrooms. Mushroom extracts have been analysed using size exclusion, reversed-phase and anion exchange chromatographies. Those chromatographic separation systems were coupled on-line to an ICP–MS, which was equipped with an octopole reaction system using H 2 as reaction gas to eliminate interfering argon dimers in Se detection. Post-column isotope dilution analysis has been used on-line with the separations to quantify Se in the eluting peaks by converting conventional intensity chromatograms into mass flow chromatograms (previous adequate mathematical corrections). 78Se/ 77Se ratio was used, after correction of intensity signals for SeH + formation, to quantify both total Se and individual selenocompounds. Extraction efficiencies in hot water between 47 and 91% were obtained for Se, depending on the mushroom considered. Selenomethionine was found to be the major compound of Se in the aqueous extracts of the three types of mushroom investigated. A number of unknown selenocompounds, all of them corresponding to low molecular weight species, were also detected and their Se content was quantified.

Quantitative speciation of selenium in human serum by affinity chromatography coupled to post-column isotope dilution analysis ICP-MS

A post-column isotope dilution analysis (IDA) methodology has been applied to carry out the quantitative speciation of selenium in human serum by affinity chromatography coupled to inductively coupled plasma mass spectrometry (ICP-MS) with an octapole reaction system (ORS). The interfering argon dimers on the 78Se and 80Se isotopes were suppressed by pressurizing the octapole chamber with hydrogen. The separation of the selenium-containing proteins was evaluated both by anion exchange chromatography (Mono Q HR 5/5) and affinity chromatography (Hi-Trap Heparin- and Hi-Trap Blue-Sepharose columns). Quantification of selenium was performed by post-column isotope dilution analysis by continuous mixing of an enriched 77Se spike solution with the eluent from the column. Finally, the 78Se/77Se or 80Se/77Se isotope ratios were monitored and the amount of selenium bound to proteins was evaluated. The chromatographic separation of selenium-containing proteins in human serum by anion exchange chromatography was not satisfactory. On the other hand, the use of affinity chromatography allowed for a rapid, precise and convenient fractionation of those proteins. Three main selenium fractions could be separated and quantified: selenoprotein P, albumin and glutathione peroxidase. Mass balance performed under different experimental conditions showed quantitative selenium recovery. The proposed methodology was used to study qualitative and quantitative speciation of selenium in human serum samples from healthy volunteers and patients on haemodialysis. The distribution of selenium between plasma glutathione peroxidase (∼20%), selenoprotein P (∼55%) and albumin (∼20%) was similar in both populations.