J. I. García Alonso

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.

Determination of selenium in biological materials by isotope dilution analysis with an octapole reaction system ICP-MS

An inductively coupled plasma quadrupole mass spectrometer (ICP-QMS) with an octapole reaction system (ORS) was used for the determination of selenium by isotope dilution analysis (IDA) in biological reference materials and serum samples from healthy subjects. The potentially interfering argon dimers (40Ar38Ar+ and 40Ar2+) at the selenium masses 78 and 80 were almost eliminated by the use of hydrogen as reaction gas. Thus, the detection limits were improved five times for 78Se and the measurement of 80Se (the main selenium isotope with an abundance of 49.61%) is now possible. An enriched 77Se solution was prepared and characterized by reverse IDA and isotope ratios measured were 78Se/77Se and 80Se/77Se. Instrumental parameters were optimised in order to obtain optimal precision and accuracy in the isotope ratio measurement of Se by ORS-ICP-QMS. The precision achieved for the isotope ratio measurements reached 0.2% (RSD for n = 5) for both ratios. Systematic errors, including detector dead time (47 ± 2 ns), mass bias effects (about 3%) and spectroscopic interferences due to the presence of bromine in the samples, were corrected. The accuracy of the measured selenium isotope ratios was improved by correcting the intensity signals of the selenium isotopes for SeH+ formation (about 3%). The proposed IDA method has been applied to the determination of Se in biological reference materials (serum, urine and tissues) and the results showed good agreement with the certified values.

Biosynthesis of isotopically enriched selenomethionine: application to its accurate determination in selenium-enriched yeast by isotope dilution analysis-HPLC-ICP-MS

Isotope dilution analysis is proposed for the determination of selenomethionine (SeMet) in Se-enriched yeast material by high performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS) using a 77Se-enriched selenomethionine spike obtained by yeast growth on a 77Se-rich culture medium. Different methods of yeast enrichment with selenium were evaluated to determine the most effective one. The use of two different culture media, amount of selenium added, amount of carbon source (glucose) and harvest time were evaluated in order to characterize the total selenium incorporated by the yeast cells. The yeast proteins were hydrolysed with protease XIV, and the isotopically enriched SeMet isolated by anion exchange chromatography. The isotopic composition of the SeMet in the spike solution was determined by on-line HPLC-ICP-MS and reverse isotope dilution was used for the characterization of the spike by means of a natural SeMet standard. In the analysis of the Se-enriched yeast material, the sample was spiked with 77Se-enriched SeMet, and hydrolyzed with a non-specific enzyme (protease XIV) at 37 °C for 20 hours or using an ultrasonic probe for 1 min. Both 78Se/77Se and 80Se/77Se isotope ratios were measured by ICP-MS with collision and reaction cell after the separation of selenomethionine by HPLC. The method was applied to the determination of selenomethionine in a candidate yeast reference material (SEAS project), and the results obtained were in agreement with those reported by other laboratories.

Potential of micelle-mediate procedures in the sample preparation steps for the determination of polynuclear aromatic hydrocarbons in waters

Abstract The analytical potential of using micelle-mediated procedures for the sampling and preconcentration of polynuclear aromatic hydrocarbons (PAHs) in water was investigated. The effect of micellar systems on the stability of several PAH-containing water samples, stored in different container materials, were studied. It was found that the presence of micelles in the sample was as effective as acetonitrile at a concentration of 40% (v/v) in preventing loss of PAHs due to adsorption on the surface of PTFE or borosilicate glass containers. In addition, the usefulness of such micellar systems for extraction and preconcentration was demonstrated by the observed increase in the recovery of PAHs from waters containing micelles on a typical C18 cartridge. The possible advantages of micelles over organic solvents for sampling and preconcentration of PAHs in water-borne environmental samples is also discussed.

Spectrofluorimetric determination of niobium with morin enhanced by cetyltrimethylammonium bromide micelles

Abstract The effect of surfactants on the fluorescence of the niobium—morin system is described. Cationic surfactants strongly enhanced the intensity (e.g., cetyltrimethylammonium bromide (CTAB) gives an 80-fold increase), while anionic and non-ionic surfactants are without effect. The formation of 1:1 and 1:3 (Nb:morin) complexes is demonstrated spectrophotometrically. The conditional stability constants for these complexes in CTAB micelles are β 1 = (1.14 ± 0.01) × 10 4 l mol −1 amd β 3 = (5.66 ± 0.02 × 10 10 l 3 mol −3 . The micellar-enhanced fluorimetric method has a 1 μg l −1 detection limit, and is highly selective. The r.s.d. for the determination of 50 μg l −1 Nb is 3.5%.

Isotopically-labelled compounds for validating organometallics speciation analysis

Abstract The possibilities of using isotopically-labelled species with GC-ICP-MS to validate speciation analysis of organometallic compounds in environmental samples are summarised. The analytical advantages of this unique way of quantification, based on isotope-ratio measurements in each separate species, are highlighted and compared with more conventional procedures. Particular emphasis is paid to validating published speciation procedures, which usually involve a large number of discrete steps, each a possible source of error. The preparation of isotopically-labelled butyltin species and its application to seawater and sediment organotin speciation quantitative analysis are reviewed. In addition, the impressive capability of the “multi-isotope-labelled multispecies” spikes to control every step in speciation, even the initial solid-liquid extraction of the species from a solid sample, is also addressed. Such an innovative strategy allows detection and correction for species degradation during this difficult step to follow the extraction process. Moreover, the use of such multi-isotope-labelled multispecies approaches opens the door to studies on reported extraction procedures in speciation work. This approach is shown to be extremely useful for both ensuring quantitative recoveries of the species being sought from the solid matrix and evaluating the extent of possible extraction-derived rearrangement reactions. The article reviews applications of this innovative tool to the study and validation of the reported solid-liquid extraction of butyltin species from a certified reference sediment (using different techniques, including mechanical shaking, microwave, ultrasonic and pressurized liquid extraction).

Comparison of different numerical approaches for multiple spiking species-specific isotope dilution analysis exemplified by the determination of butyltin species in sediments

An examination and comparison of all the mathematical approaches for multiple spiking species-specific isotope dilution analysis published so far in the literature is presented in this work with the determination of TBT and DBT in sediments. The basis of four different numerical approaches—Calculation of Stable Isotope Concentrations, Speciated Isotope Dilution Analysis, Species-Specific Isotope Dilution Analysis and Isotope Pattern Deconvolution—are explained and compared in terms of complexity, analytical figures of merit and specific advantages. The four methodologies have been found to provide exactly the same degradation-corrected concentrations for DBT and TBT in all samples even when different degradation extents are taking place. However, slight differences in the degradation factors have been obtained between two pairs of methods, being in all cases lower than the corresponding instrumental uncertainty of the values. The capability of extending the methodologies to a higher number of analytes by the use of additional enriched species as well as the specific advantages of the different methods is discussed. In this sense, it has been demonstrated that Calculation of Stable Isotope Concentrations and Isotope Pattern Deconvolution provide additional advantages such as the qualitative information on any non-spiked species present in the samples. In addition, using Isotope Pattern Deconvolution the performance of a new internal procedure for mass bias correction without the additional measurement of reference isotope ratios is presented.

The surfactant-sensitized analytical reaction of niobium with 8-hydroxyquinoline-5-sulphonic acid

Cationic surfactants, such as cetylpyridinium bromide (CPB), sensitize the colour reaction of Nb(V) with 8-hydroxyquinoline-5-sulphonic acid (H(2)L). The formation of a ternary complex of stoichiometry 1:3:3 (Nb-L-CPB) is responsible for the observed enhancement in absorptivity and the quenching in the fluorescence of the Nb-L chelate, when a surfactant is present. The ternary complex exhibits maximum absorption at 383 nm ( = 1.46 +/- 0.01 x 10(4) l.mole(-1).cm(-1)) at pH 5.7, and Beers law is obeyed up to 6-mug ml Nb concentration. Conditional formation constants of the niobium chelate in the presence and absence of CPB have been determined. On the basis of a detailed spectrophotometric and fluorimetric study the nature of the chromophoric reagent-surfactant interaction and the peculiar features of the sensitization by CPB are discussed.

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.

Determination of some selected polycyclic aromatic hydrocarbons in environmental samples by high-performance liquid chromatography with fluorescence detection

SummaryAnalytical methods for the determination in environmental samples, of some selected Polycyclic Aromatic Hydrocarbons (PAHs), which are included on the EPA Priority Pollutant list, have been developed and evaluated. The methodology involves the extraction of PAHs from water samples by solvent extraction with dichloromethane. Solid samples were ultrasonically extracted with acetone/hexane and the extract was cleaned up on a silica gel/alumina column. The concentrated and cleaned up extracts were analysed by HPLC on a polymeric C18 column using a gradient of acetonitrile/water as the mobile phase and fluorescence detection. Typical detection limits lie in the range of 1–30 ng ml−1 of the analytes, but after sample pretreatment detection limits of 10–300 ng l−1 were obtained. The extraction, clean-up and HPLC methodology was applied to the determination of selected PAHs in coal washings samples and the method was validated by the quantification of PAHs in a natural contaminated and a spiked sediment.