[Show abstract][Hide abstract] ABSTRACT: This text presents a novel method for the separation and detection of phosphorothioate oligonucleotides with the use of ion pair ultra high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry The research showed that hexafluoroisopropanol/triethylamine based mobile phases may be successfully used when liquid chromatography is coupled with such elemental detection. However, the concentration of both HFIP and TEA influences the final result. The lower concentration of HFIP, the lower the background in ICP-MS and the greater the sensitivity. The method applied for the analysis of serum samples was based on high resolution inductively coupled plasma mass spectrometry. Utilization of this method allows determination of fifty times lower quantity of phosphorothioate oligonucleotides than in the case of quadrupole mass analyzer. Monitoring of 31P may be used to quantify these compounds at the level of 80 μg L−1, while simultaneous determination of sulfur is very useful for qualitative analysis. Moreover, the results presented in this paper demonstrate the practical applicability of coupling LC with ICP-MS in determining phosphorothioate oligonucleotides and their metabolites in serum within 7 min with a very good sensitivity. The method was linear in the concentration range between 0.2 and 3 mg L−1. The limit of detection was in the range of 0.07 and 0.13 mg L−1. Accuracy varied with concentration, but was in the range of 3%.
[Show abstract][Hide abstract] ABSTRACT: A procedure for the simultaneous quantitative carbamidomethylation of selenocysteine (SeCys) and selenomethionine (SeMet) followed by their proteolytic release from blood proteins was developed. The fraction containing both derivatized selenoaminoacids was isolated by size-exclusion chromatography and submitted to ion-pairing HPLC-ICP MS analysis. The limit of detection was ca. 0.02 μg g−1 (dry mass) for either amino acid. The quantification of SeCys and SeMet was carried out by the method of standard additions. An internal standard of 77Se-labelled SeMet was used to control the derivatization yield and chromatographic recovery. The determination of SeCys was validated by spiking with glutathione peroxidase. An additional proof of validity was achieved by monitoring the selenium mass balance (12 series of analysis over a period of 18 months; the Se amino acids accounted for 92 ± 8% of the total Se). The method was applied to the monitoring of changes in SeCys and SeMet concentrations in lamb blood during supplementation studies (tolerance and dose effect) with selenium-rich yeast.
[Show abstract][Hide abstract] ABSTRACT: A novel generic approach based on precolumn isotope dilution nanoHPLC-ICPMS analysis was developed for the accurate absolute quantification of sulfur-containing peptides. A 34S-labeled, species-unspecific sulfur spike (sulfate), noninteracting with analyte peptides under the optimized HPLC condition, was added directly to the chromatographic eluents. Thus a generic sulfur standard permanently present during analysis was used for peptide quantification. Interference-free detection of the 32S and 34S isotopes in ICPMS was achieved by eliminating O2+ ions in a collision cell using Xe gas at 130 microL min-1. The detection limit for sulfur was 45 microg L-1 which corresponded to 1-2 pmol of individual peptides. The method was validated by the analysis of a standard peptide solution showing high accuracy (recovery 103%) and good precision (RSD 2.1%). The combination of nanoHPLC-ICP IDMS with nanoHPLC-ESI MS/MS allowed the precise quantification and identification of sulfur-containing peptides in tryptic digests of human serum albumin and salt-induced yeast protein (SIP18) at the picomole level.
[Show abstract][Hide abstract] ABSTRACT: A high-throughput flow-injection — inductively coupled plasma mass spectrometry (ICP MS) analytical method was developed for the determination of mercury in gas condensates and carbon-rich solvents. The sample (undiluted or diluted 10-fold) was introduced via a modified total consumption micronebulizer working at a flow rate of 30 μl min−1 and fitted with a singlepass spray chamber. This low flow rate and the addition of oxygen (70 ml min−1) assured the plasma stability and reduced the carbon build-up on the interface and on ion lenses. A limit of detection of 0.5 ng g−1 (2.5 μl sample) was obtained owing to the reduction of dead volume and sample dispersion (peak-width was 3 s at half-height) in the liquid pass of the nebulizer. The elimination of the memory effect reduced the washout time down to 30 s which resulted in a throughput of ca. 60 h−1. The method was validated by the analysis of 3 gas condensates by cold vapour atomic absorption spectrometry.
Spectrochimica Acta Part B Atomic Spectroscopy 09/2006; 61(9):1063-1068. DOI:10.1016/j.sab.2006.10.004 · 3.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An original approach based on successive size-exclusion and hydrophilic interaction HPLC (HILIC) was developed to purify traces of Ni species from a plant aqueous extract. The degree of purity achieved was for the first time sufficient for the identification, in a natural sample, of a number of non-covalent metal complexes by electrospray Q-TOFMS/MS. Nickel complexes with malate, citrate, histidine, EDTA and nicotianamine (NA) were identified in the roots, xylem, shoots and their protoplasts of a metal hyperaccumulator plant Thlaspi caerulescens. The quantitative recovery of the most stable of these complexes (with EDTA and NA) allowed their quantitative determination by SEC-ICP-MS.
[Show abstract][Hide abstract] ABSTRACT: The potential of tandem mass spectrometry following matrix-assisted laser desorption ionization (MALDI) was studied for speciation of selenium. Non-peptide selenium-containing compounds were isolated from a selenized yeast aqueous extract by size-exclusion chromatography. Post-source decay (PSD) was compared with orthogonal quadrupole collision cell dissociation for the purpose of obtaining fragmentation and structural information. In the PSD mode, the use of ion gate covering the whole isotopic cluster of the parent compound allowed the immediate recognition of fragments containing Se and those in which this element was absent. The tandem mass spectra obtained by orthogonal MALDI Q-TOF were equally informative in terms of the number of fragments but suffered from a poorer sensitivity. The mass accuracy was ca. 20 times better in the oMALDI configuration than in the PSD mode. An unknown selenium compound with an m/z 388 was detected with a mass accuracy of 3 ppm according to the proposed empiric formula.
The Analyst 10/2004; 129(9):846-9. DOI:10.1039/b406650b · 4.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Two simple sample preparation methods for the speciation analysis of triphenyltin and butyltin compounds in marine biotissues, using tetramethylammonium hydroxide (TMAH) solubilization and enzymic hydrolysis, have been developed and compared with conventional acid digestion. Derivatization was carried out in situ using sodium tetraethylborate (NaBEt4) without prior separation of the analytes from the tissue matrix. Separation and detection was performed using capillary gas chromatography (GC) coupled to microwave-induced plasma atomic emission spectrometry (MIP AE) allowing detection limits of 2 ng g−1 (as tin) to be reached. The accuracy of the presented methods was demonstrated by the analysis of a fish reference material (NIES No. 11). the necessity for sample clean-up is discussed and examples of the analysis of mussel tissue are shown.
[Show abstract][Hide abstract] ABSTRACT: The major selenium compound in an aqueous extract of the most popular mushroom in Eastern Asian countries, shiitake ( Lentinula edodes), fortified with selenium (Se) was identified by means of hyphenated techniques, i.e. HPLC-inductively coupled argon plasma mass spectrometry and HPLC-electrospray ionization mass spectrometry (HPLC-ICP MS and HPLC-ESI MS). Sixty-eight per cent of the total Se in the selenized shiitake was extracted with water, and 49.8% of the Se in the water extract was eluted in the high molecular mass fraction (>40,000 kDa) before incubation at 37 degrees C. After incubation, 40.6% of the Se in the water extract was eluted in a lower molecular mass fraction and the Se eluted in the high molecular mass fraction had decreased to 14.0%, suggesting that the major selenium compound in the water extract was initially in a form bound to macromolecule(s) and was then enzymatically liberated from the macromolecule(s). The retention time of the liberated selenium compound in HPLC-ICP MS matched that of selenomethionine (SeMet), and the masses of molecular and fragment ions detected by HPLC-ESI MS also suggested that the selenium compound was SeMet. The selenized shiitake accumulated Se as SeMet, and SeMet might be bound to the water extractable high molecular mass protein(s).
[Show abstract][Hide abstract] ABSTRACT: A sheathless interface based on a total consumption micronebulizer operating at flow rates in the range 0.5-7.5 microL min(-1) was developed between capillary HPLC and ICPMS. It allowed the efficient nebulization and transport into the plasma of mobile phases containing up to 100% organic solvent without either cooling the spray chamber or oxygen addition. The coupled system was applied to selenopeptide mapping in a protein fraction isolated from a selenized yeast extract. The detection limits were 150 (80Se) and 200 fg (82Se) for a quadrupole instrument with and without a collision cell, respectively, which is a factor 100-150 less than that reported elsewhere for HPLC-ICPMS. The minimal peak broadening ( approximately 5 s at the half-height) allowed baseline resolution of a mixture containing more than 30 selenopeptides, many of which could not be separated using the conventional HPLC-ICPMS coupling.
[Show abstract][Hide abstract] ABSTRACT: Speciation of nickel was investigated in a hyperaccumulator tree, Sebertia acuminata, by liquid chromatography (LC) with parallel ICP MS and electrospray MS-MS detection. The latex of the tree was leached with water. Two separation mechanisms, size-exclusion (SEC) and capillary zone electrophoresis (CZE), were investigated for the separation of Ni-complexes. A preparative scale SEC allowed the isolation of six stable nickel species. A nickel complex with Mr 360 was identified by ES MS. The collision induced fragmentation (CID) of the m/z 360 ion suggested the presence of three carboxylic and one amino groups, and allowed the identification of a Ni-complex with nicotianamine. A modification of the SEC column with Ni2+ ions allowed the isolation of a fraction containing nickel citrate. Quantification of the species showed that in the water extract of the latex 99.4% of the nickel is complexed by citrate and 0.3% by nicotianamine.
[Show abstract][Hide abstract] ABSTRACT: An understanding of the mechanisms controlling the essentiality and toxicity of trace elements in biological systems at the molecular level depends critically on the possibility of the identification, characterization, and quantification of chemical forms of these elements involved in life processes. Hyphenated techniques based on the combination of (electro)chromatography with ICP MS have become a routine tool for the analysis for metallospecies present in biological tissues. Finer analytical information on the true (down to individual species) speciation of trace elements in living organisms can be obtained by adding additional dimensions to the separation and detection steps, consisting of a sequential use of different HPLC separation mechanisms and capillary electrophoresis at the separation level, and of the use of electrospray MS, including collision induced dissociation MS, on the detection level. The value of the instrumental analytical data is decisively enhanced by the complementary use of molecular biology approaches involving gene identification, cloning and in vitro reproduction of the metal-controlled processes. A brief summary of the recent progress in biochemical speciation analysis is presented in the context of the latest research carried out in the authors' laboratory.
[Show abstract][Hide abstract] ABSTRACT: The complementarity of ICP sector-field double-focussing (SF-DF) MS and electrospray MS detection in capillary zone electrophoresis (CZE) was studied for the identification and determination of metal complexes with metallothionein (MT) induced in the liver of a rat exposed to intravenously administered Cd2+. The MT fraction investigated was isolated by size-exclusion chromatography, preconcentrated by lyophilization and desalted. CZE-ICP MS allowed the separation of four mixed-metal complexes with one or more MT isoforms. Isotope dilution analysis was developed for the accurate quantification of the MT content in each of the species and the determination of the stoichiometry of the metal complex. A multi-isotope spike (33S,106Cd, 65Cu, 70Zn) was supplied on-line in the makup flow (6 l min−1) of a self-aspirating total consumption micronebulizer that
served as the interface. The characterization of the MT fraction was completed by electrospray MS, allowing the determination of the molecular masses of the complexes formed and the identification of the ligands as MT-1 and MT-2 isoforms.
[Show abstract][Hide abstract] ABSTRACT: HPLC-ICP-MS using different separation mechanisms: reversed-phase, anion-exchange, cation-exchange, and size-exclusion was revisited and comprehensively evaluated using a set of 14 standards of arsenic species. An analytical protocol based on three-dimensional (size-exclusion–anion-exchange–reversed-phase) chromatography, capable of isolating individual arsenocompounds from a biological sample, and electrospray tandem mass spectrometry (ES MS–MS), for their identification, was developed. Two minor arsinoylribosides could be detected in addition to As(V) and one major riboside in the algae (Hizikia fusiforme) sample investigated. The other minor species isolated showed different chromatographic behavior than the standards available while the ES MS–MS fragmentation patterns indicated the presence of an arsenoylriboside moiety.
[Show abstract][Hide abstract] ABSTRACT: An interface between capillary zone electrophoresis and inductively coupled plasma mass spectrometry (CZE-ICP-MS) based on a self-aspirating total consumption micronebulizer was evaluated for a study of metal complexation by metallothionein. The elimination of the nebulizer suction (and thus of the laminar flow) allowed a separation efficiency comparable with that reachable with on-capillary UV detection whereas the low (6 µl min−1) nebulizer aspiration rate allowed the minimization of the post-capillary dilution effect, and thus maximization of the CZE-ICP-MS sensitivity. Operating conditions were optimized with the natural rabbit liver metallothionein preparations allowing detection limits of ca. 10 ng mL−1
(as MT-bound Cd) to be obtained within 10 min at quasi-baseline resolution. Recombinant (mouse liver MT-1-Zn7) and natural (rabbit
liver MT-1-Cd7 and MT-2-Cd7) proteins were then titrated with Cd(II) and Cu(I), and Cu(I), respectively. A number of mixed metal complexes with different migration times were observed as a function of the Cu∶Cd ratio in the system.
[Show abstract][Hide abstract] ABSTRACT: The coupling of capillary zone electrophoresis with electrospray mass spectrometry was optimized for the direct determination of phytochelatins (PCs) in extracts obtained from cells and plants that had been exposed to metal stress. Gluthathione and phytochelatins belonging to the different families (gamma Glu-Cys)nGly (n-PC), (gamma Glu-Cys)nSer, (gamma Glu-Cys)n beta Ala and (gamma Glu-Cys)n were separated in an uncoated capillary at pH 4 using a 5 mM ammonium acetate buffer, and detected by electrospray (ES) MS in the full scan mode (300-1100 u). The use of on-line tandem MS detection in the product ion scan mode of putative protonated molecules of PCs allowed the unambiguous confirmation of the identity of the compounds detected by ES MS. The operational conditions were optimized and the figures of merit were evaluated using n-PC2, n-PC3 and n-PC4 standards purified from a mixture obtained after the reaction of glutathione in the presence of Cd2+ and the enzyme PC-synthase. The method was applied to the characterization of bioinduced ligands in cell cultures of soybeans (Glycine max) and in rice (Oryza sativa) roots without the need for a preliminary sample cleanup by size-exclusion and/or reversed phase chromatography.
The Analyst 06/2001; 126(5):624-32. DOI:10.1039/B100864L · 4.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An analytical strategy has been developed for the characterization of Cd complexes in rat liver tissue. The sample preparation included the isolation of the Cd–macromolecular fraction by semi-preparative size-exclusion chromatography, followed by a preconcentration step by lyophilization and desalting. The analysis was carried out by microbore reversed-phase HPLC with parallel ICP-MS and ESMS detection. The HPLC-ICP-MS interface was based on a Micromist nebulizer and a low dead volume cyclonic spray chamber. The HPLC-ESMS interface allowed the measurement of the molecular mass of the ligand protein owing to the dissociation of the complex by the on-line post-column acidification of the eluate. The approach allowed the detection of two major metallothionein (MT)isoforms and their identification on the basis of the molecular mass as rat liver MT-1 and MT-2. The actual number of peaks in the chromatograms was larger because of the formation of mixed Cd–Cu complexes of the same MT isoform that showed different hydrophobicities.
[Show abstract][Hide abstract] ABSTRACT: Speciation of selenium in garlic harvested in naturally seleniferous soil was investigated. The sample was leached with water and the aqueous extract was fractionated by preparative size-exclusion chromatography. Selenium was found in only one (low-molecular mass) fraction. The chromatographic purity of the fraction was verified by reversed-phase chromatography with inductively coupled mass spectrometric detection. Again, one major signal accounting for more than 95% of the total selenium was observed. The heartcut fraction containing this compound produced an intense peak in an electrospray mass spectrum with the selenium pattern centered at m/z 313 (80Se). Protonated molecular ions corresponding to the four Se isotopes gave rich fragmentation patterns by collision induced dissociation that allowed the identification of the selenium species to be γ-glutamyl-Se-methylselenocysteine without the need for an authentic standard.
[Show abstract][Hide abstract] ABSTRACT: Recent advances in the coupling of gas chromatography (GC) and high performance liquid chromatography (HPLC) with inductively coupled plasma mass spectrometry (ICP MS) and their role in trace element speciation analysis of environmental materials are presented. The discussion is illustrated with three research examples concerning the following topics: (i) development and coupling of multicapillary microcolumn GC with ICP MS for speciation of organotin in sediment and biological tissue samples; (ii) speciation of arsenic in marine algae by size-exclusion-anion-exchange HPLC-ICP MS; and (iii) speciation of cadmium in plant cell cultures by size-exclusion HPLC-ICP MS. Particular attention is paid to the problem of signal identification in ICP MS chromatograms; the potential of electrospray MS/MS for this purpose is highlighted.
Spectrochimica Acta Part B Atomic Spectroscopy 07/2000; 55(7-55):779-793. DOI:10.1016/S0584-8547(00)00210-X · 3.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The recent advances and trends in the development and applications of electrospray mass spectrometry (ESI-MS) to speciation analysis are highlighted. The use of the different operational modes of ESI-MS: elemental mode (source collision-induced dissociation (SCID)), molecular mode (MS of protonated molecule ions) and tandem MS mode (product ion scan of the collision-induced dissociation (CID) products of the protonated molecule ion), is discussed. Advantages and limitations of ESI-MS for signal identification in high performance liquid chromatography (HPLC)–ICP-MS and as a free-standing tool for species-selective analysis are highlighted. The discussion is illustrated with a number of examples related to the trace element speciation in biochemistry, viz. selenium in yeast, arsenic in seaweeds, cobalamins in pharmaceutical preparations, and Cd complexes with phytochelatins and metallothioneins in plant and animal tissues
[Show abstract][Hide abstract] ABSTRACT: Multicapillary column gas chromatography (MC GC) is presented as a novel technique for the time-resolved sample introduction of gaseous analytes in atomic spectrometry. The principles and the theory of MC GC, the coupling of MC GC with microwave-induced plasma atomic emission spectrometry and with inductively coupled plasma mass spectrometry, and features of the coupled techniques developed are discussed. Applications of MC GC with element-selective detection to species-selective analysis are reviewed. The possibility of incorporating MC columns into dedicated sample preparation accessories for elemental speciation by atomic spectrometry is evaluated.