M. L. Fernández Sánchez

Speciation of inorganic selenium and selenoaminoacids by on-line reversed-phase high-performance liquid chromatography–focused microwave digestion–hydride generation-atomic detection

A high-performance liquid chromatographic–microwave digestion–hydride generation system coupled on-line with three atomic detectors (atomic absorption, inductively coupled plasma atomic emission and inductively coupled plasma mass spectrometry) has been developed and investigated for selenium species separation and determination. Total inorganic selenium, selenomethionine and selenoethionine are separated by reversed-phase chromatography prior to on-line microwave digestion of selenocompounds with a KBrO3–HBr mixture to form continuously SeIV, which is finally transformed into H2Se, also in a continuous manner, with a merging flow of sodium borohydride. Detection limits obtained for each Se species in water and urine using the three atomic detectors have been worked out and compared (i.e., for SeIV DLs were 6.8 µg l–1 by AAS, 30 µg l–1 by ICP-AES and 0.16 µg l–1 by ICP-MS).The integrated flow system, HPLC–MW digestion–HG-atomic detection, proposed here allows, in a single injection, reliable speciation in urine of the selenoaminoacids tested versus total inorganic selenium. Further speciation of the overlapped inorganic SeIV and SeVI peaks is accomplished by a second injection of the urine sample to determine only SeIV by avoiding microwave heating. Results on Se speciation in human urine have shown that more speciation information of the actual species, perhaps unknown, present in real samples could be gathered by resorting to the use of two, or more, atomic detectors coupled to the same separation scheme.

Organic and inorganic selenium speciation in urine by on-line vesicle mediated high-performance liquid chromotography–focused microwave digestion–hydride generation–inductively coupled plasma mass spectrometry

A vesicle mediated high performance liquid chromatographic (HPLC)–microwave digestion (MW)–hydride generation (HG) system coupled on-line with atomic absorption spectrometry (AAS) and inductively coupled plasma mass spectrometry (ICP-MS) has been assessed for selenium species separation and detection. Selenocystine, selenomethionine, selenoethionine, selenourea, SeIV and SeVI are separated by vesicle mediated chromatography prior to on-line selenocompounds microwave digestion with a KBrO3–HBr mixture to generate SeIV continuously, which is finally transformed into SeH2, in a continuous manner with a merging flow of sodium tetrahydroborate(III). Analytical characteristics of this coupling are compared with those obtained coupling HPLC–ICP-MS via conventional nebulisation. Detection limits (DLs) obtained for selenocystine, selenomethionine, selenoethionine, selenite and selenate in spiked human urine, when ICP-MS was used as detector, ranged between 1.0 and 5.3 µg l–1 (51–267 pg), while precision ranged between±3.4 and±8.4%. This continuous system, vesicle mediated HPLC–MW–HG–atomic detection, allows the separation and detection of selenocystine, selenourea, selenomethionine, selenoethionine, selenite and selenate in human urine. The analytical versatility of such coupling (with ICP-MS as the atomic detector) allows basal selenium speciation in urine. Three different normally occurring selenium species in human urine, simply diluted (1+1), have been found. The relative sophistication of the vesicle mediated–HPLC–MW–HG–ICP-MS interface, versus HPLC–ICP-MS via conventional nebulisation, can be justified because of its considerably higher sensitivity for most of the selenocompounds assayed, lower matrix interferences and the possibility of simultaneous interference free 77Se and 78Se monitoring.

Urinary selenium speciation by high-performance liquid chromatography–inductively coupled plasma mass spectrometry: advantages of detection with a double-focusing mass analyser with a hydride generation interface

A detailed comparison of the performance of inductively coupled plasma mass spectrometry (ICP-MS), with quadrupole and double-focusing instruments for the speciation of selenium in urine has been carried out. Selenium sensitivity about 23-59 times higher with double-focusing ICP-MS detection was observed, but limits of detection were only 1-8.7 times better because of background noise. Selenium species separation has been carried out by both reversed-phase and vesicle-mediated high-performance liquid chromatography (HPLC), coupled on-line with the detector via conventional nebulization and via on-line focused microwave digestion-hydride generation. A remarkable improvement in sensitivity (28-110 times better for (77)Se depending on the chromatographic system) and elimination of interference problems from the urinary matrix or the components of the mobile phases were achieved when an on-line microwave digestion-hydride generation interface was used, but the background noise was much higher than with conventional nebulization. Therefore, the limits of detection were not as low as expected from such improvement in the sensitivity. More selenocompounds can be separated, and a slight improvement in the sensitivity and limits of detection was obtained when the vesicle-mediated HPLC system was used as compared with reverse-phase chromatography. However, the use of several complementary chromatographic systems, such as reverse-phase HPLC, is recommended to bring some light on the selenocompounds present in basal human urine. Comparative data of rat urine speciation are also given.

Speciation of mercury by continuous flow liquid-liquid extraction and inductively coupled plasma atomic emission spectrometry detection

An accurate, precise, sensitive and automated non-chromatographic method for methylmercury speciation based on a selective continuous liquid-liquid extraction of methylmercury, into xylene, as bromide and cold mercury vapour generation directly from the organic phase and final ICP-AES mercury detection is proposed. Both separation steps, liquid-liquid and gas-liquid are accomplished in a continuous mode and on line with ICP-AES as detector. The detection limit attained for methylmercury was 4ng·ml−1 (as mercury). The precision of the determination at a concentration level around 20 times the detection limit was +-5%. The proposed methodology has been applied successfully to the speciation of methylmercury and inorganic mercury in spiked sea water and spiked urine samples.

Comparison of two CE-ICP-MS interfaces based on microflow nebulizers: application to cadmium speciation in metallothioneins using quadrupole and double focusing mass analyzers

Two different interfaces, based on microflow nebulizers, were investigated in detail and compared for metal speciation studies by CZE-ICP-(Q)MS and CZE-ICP-(DF)MS. The model species selected were rabbit liver metallothioneins (MTs). Small differences among MT sub-isoforms determine the need for an adequate interface, which is critical if the resolution achieved (e.g., by UV) is to be maintained with ICP-MS detection. HEN and MicroMist nebulizers were compared to interface the flows emerging from the CE capillary with the ICP-MS instrument. The observed analytical characteristics of the hybrid systems were compared in terms of calibration graphs, resolution, repeatability and detection limits (DLs). The separation profiles matched perfectly those obtained with UV detection for both interfaces under scrutiny. Similar performances were observed by CE-ICP-(Q)MS in terms of resolution and DLs achieved. The MicroMist interface proved to offer better performance than the HEN-based one using the ICP-(DF)MS instrument, because the introduction of an additional Ar “make-up” gas into the spray chamber is not required (very important if the HEN-based interface is used). In general terms, the MicroMist interface was easier to operate, providing the best DLs for Cd (about 2 pg in each isoform by CE-ICP-(DF)MS, a value 6 times lower than that obtained in a quadrupole system).

Sample preparation for identification of selenocompounds in urine by electrospray-MS/MS

The speciation of selenium in urine stands as a challenge to analytical chemists for two main reasons: the low physiological concentration of selenium in urine and the lack of commercially available Se-species standards for comparisons. Therefore, techniques giving direct access to structural information of the selenocompounds formed must be used to identify the chemical nature of urine Se species and sample preparation is critical for that purpose. Two sample preparation procedures to analyse selenocompounds in urine by electrospray-MS/MS have been developed here. The proposed sample treatments allow the purification of selenocompounds and consist of removal of the saline interfering matrix by different approaches followed by preparative size exclusion chromatography and final reversed-phase HPLC separation. Preconcentration of selenocompounds was achieved by several freeze-drying steps. One of the developed procedures was successfully tested in human urine spiked with selenocompounds but it was found to be unsuitable for basal samples. An alternative treatment is described that allows the detection of the main Se metabolite reported in urine samples, Se-methyl-N-acetylselenohexosamine, in rats under supplementation with selenomethionine.

Enriched stable isotopes and isotope pattern deconvolution for quantitative speciation of endogenous and exogenous selenium in rat urine by HPLC-ICP-MS

Interest in selenium metabolism and its role in health and disease continue to stimulate a lot of bioanalytical research. The use of enriched stable isotopes of Se and isotope pattern deconvolution (IPD) with an ICP-MS in lactating rats is approached here for total selenium distribution studies. A mathematical tool has been developed to calculate supplemented and endogenous total Se contents in urine and faeces by ICP-MS. Also, quantification of endogenous (natural) and exogenous seleno-species by HPLC-ICP-MS has been worked out. The proposed IPD methodology, for total determinations and for quantitative speciation, was applied to reference materials to validate total selenium quantification in faeces and quantitative selenium speciation in urine samples. Selenium apparent absorption and retention and natural and exogenous selenium distribution in urine samples were calculated. Interesting quantitative information about selenium bio-transformations and final catabolism can be obtained from careful examination of the results observed, using such analytical tools, of the urinary endogenous and exogenous selenium metabolites in urine of rats and their kinetics throughout the two weeks of selenium supplementation.

Determination of essential and toxic total elements in premature human milk by inductively coupled plasma mass spectrometry (ICP-ORC-MS), using an octopole reaction cell

The analytical potential of a quadrupole ICP-MS instrument equipped with an octopole reaction cell for the multielemental determination of essential (Cr, Mn, Fe, Co, Cu, Zn, Se and I) and toxic (Al, Cd and Pb) elements in whole premature breast human milk and premature infant formulae was demonstrated. Milk samples were digested using a microwave assisted digestion method in order to destroy the organic matrix. Different parameters affecting the octopole reaction cell were optimised in order to minimize/remove polyatomic interferences caused by argide species from the ICP. The accuracy of the proposed method was tested by analysing a milk powder reference material (BCR 063R) with satisfactory results. The detection limits in a synthetic matrix obtained using a collision cell ranged from 0.5 ng g−1 to 11 ng g−1 for the elements under study. After validation the method was applied to the multielemental analysis of premature human milk samples (after 28–32 gestational weeks) at three different lactating stages during the first month after birthday, and to formula milks commercially available for premature newborns. Results of total concentrations of the above mentioned elements in the two sample types compared here (human milk and formula milk) are discussed. Important differences between human and formula milk in their respective contents of essential elements are shown and the nutritional significance of such differences is highlighted.

Rapid determination of eight elements in cement and its raw mixes by inductively coupled plasma atomic emission spectrometry

An investigation was carried out on the influence of the operating conditions and of the analytical performance characteristics of inductively coupled plasma atomic emission spectrometry for the determination of eight elements (Si, Al, Fe, Ti, Ca, Mg, Na and K) in the raw components of cement. The aim of this investigation was to develop a rapid method of quality control for such raw materials. The results showed that this technique is sensitive enough for each element of interest and no serious interferences were detected after a detailed inter-element interference study. Rapid determination by automatic sequential analysis of the eight elements in a single solution of the sample proved to be highly satisfactory. A suitable fusion method for non-clinkerised material, normally difficult to dissolve, is also given.