Dirk Schaumlöffel

Determination of element species at trace levels using capillary electrophoresis-inductively coupled plasma sector field mass spectrometry

The development and analytical characterization of a coupled capillary electrophoresis-inductively coupled plasma sector field mass spectrometer (CE-ICP-SFMS) system for the simultaneous determination of different species of As, Sb, Se and Te at trace levels are described. The species are separated by CE using a 55 cm×75 µm id fused silica capillary column. A newly developed interface with a special, low dead volume spray chamber allows the optimization of the fluid mechanical properties, thus preventing a suction effect between the nebulizer and the CE capillary. The ICP-SFMS instrument runs in the low resolution mode and is equipped with a grounded platinum electrode at the ICP torch in order to achieve the highest possible sensitivity. The CE-ICP-SFMS system developed provides stable electrophoretic conditions and allows reproducible separations to be obtained with RSDs below 3% for the migration times and below 8% for the peak areas. Excellent peak shapes down to 4 s and short analysis times of a few minutes are other important features of this combined instrument. Detection limits in the low µg L n –1 n region for compounds and in the picogram to femtogram range for the isotopes are achieved from standard solutions.

Absolute peptide quantification by lutetium labeling and nanoHPLC-ICPMS with isotope dilution analysis.

The need of analytical methods for absolute quantitative protein analysis spurred research on new developments in recent years. In this work, a novel approach was developed for accurate absolute peptide quantification based on metal labeling with lutetium diethylenetriamine pentaacetic acid (Lu-DTPA) and nanoflow high-performance liquid chromatography-inductively coupled plasma isotope dilution mass spectrometry (nanoHPLC-ICP-IDMS). In a two-step procedure peptides were derivatized at amino groups with diethylenetriamine pentaacetic anhydride (DTPAA) followed by chelation of lutetium. Electrospray ionization mass spectrometry (ESI MS) of the reaction product demonstrated highly specific peptide labeling. Under optimized nanoHPLC conditions the labeled peptides were baseline-separated, and the excess labeling reagent did not interfere. A 176Lu-labeled spike was continuously added to the column effluent for quantification by ICP-IDMS. The recovery of a Lu-DTPA-labeled standard peptide was close to 100% indicating high labeling efficiency and accurate absolute quantification. The precision of the entire method was 4.9%. The detection limit for Lu-DTPA-tagged peptides was 179 amol demonstrating that lutetium-specific peptide quantification was by 4 orders of magnitude more sensitive than detection by natural sulfur atoms present in cysteine or methionine residues. Furthermore, the application to peptides in insulin tryptic digest allowed the identification of interfering reagents decreasing the labeling efficiency. An additional advantage of this novel approach is the analysis of peptides, which do not naturally feature ICPMS-detectable elements.

Analysis for metal complexes with metallothionein in rat liver by capillary zone electrophoresis using ICP double-focussing sector-field isotope dilution MS and electrospray MS detection

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 nserved 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.

Selenopeptide mapping in a selenium-yeast protein digest by parallel nanoHPLC-ICP-MS and nanoHPLC-electrospray-MS/MS after on-line preconcentration

ICP collision cell MS was optimized for the detection and retention-time marking of selenium-containing peptides in nanoHPLC (75 μm column) after on-line 100-fold preconcentration on a capillary (300 μm id) precolumn. The mobile phase composition, gradient and flow rate were chosen to allow electrospray-MS/MS to be successfully run in parallel in identical separation and preconcentration conditions in order to produce two matching sets of chromatograms: an element-specific one and a molecule-specific one. Knowledge of the retention time of a Se-containing peptide of interest allowed efficient data mining in the corresponding ES-MS chromatogram and the identification of minor Se-species. A third chromatogram was run to obtain collision-induced dissociation data for the target peptides. The performance of the method was demonstrated for a comprehensive on-line characterization of a mixture of peptides in a tryptic digest of a Se-containing protein fraction isolated by size-exclusion chromatography from a selenium yeast extract. The method allowed the identification of whole series of Se/S substitutions in individual peptides and, in some cases, sequencing of isomers differing in the position of selenomethionine residues in the amino acid sequence.

Determination of Tl(I) and Tl(III) by IC-ICP-MS and application to Tl speciation analysis in the Tl hyperaccumulator plant Iberis intermedia

Speciation of thallium was investigated in a Tl hyperaccumulator plant, Iberis intermedia, by ion chromatography (IC) and size-exclusion chromatography (SEC) coupled with on-line ICP-MS detection. The leaves, stems and roots of the plant were extracted with a buffer solution (pH 6.2) containing DTPA and ammonium acetate. DTPA was used to complex unstable Tl(III) to form the stable Tl(III)–DTPA anionic complex. The two species, Tl(I) and Tl(III)–DTPA, were separated by using two separation mechanisms, anion exchange chromatography and SEC, with 100 mmol L−1 ammonium acetate (pH 6.2) as eluant. The anion exchange chromatograms indicated that the chemical form of Tl present in extracts of both fresh and freeze-dried samples of the roots, stems and leaves is predominantly Tl(I), and this was confirmed by size-exclusion chromatography and electrospray mass spectromety.

Multi-element speciation of metalloproteins in fish tissue using size-exclusion chromatography coupled "on-line" with ICP-isotope dilution-time-of-flight-mass spectrometry.

An on-line isotope dilution (ID) method in combination with the coupling of size-exclusion high-performance liquid chromatography (SE-HPLC) to an axial inductively coupled plasma time-of-flight mass spectrometer (ICP-TOFMS) was applied to the quantitative speciation of Cu, Zn and Cd in carp and eel cytosols. Quantitative information on the distribution of essential and toxic metals between the different molecular weight fractions in kidney cytosols of control and Cd exposed carp was obtained with species-unspecific spiking by post-column addition of the enriched isotopes 65Cu, 67Zn and 106Cd. The isotope ratios 63Cu/65Cu, 64Zn/67Zn and 114Cd/106Cd, were monitored by ICP-TOFMS in transient signal mode. The determination of the total cytosolic element concentration was performed by flow injection conventional nebulization ICP-TOFMS. Speciation differences were encountered between control and Cd-stressed carp, indicating that quantitative speciation studies might be useful for ecotoxicological and biomonitoring purposes. Application of this methodology to quantitation of metals associated with metallothioneins (MT) in European eel, sampled at three different sites in Flanders, Belgium was also addressed. A significant increase of the amount of Cd, Cu and Zn bound to hepatic MT was observed with the increase of the total metal and MT concentrations of hepatic eel tissue.

Rapid and simple UPLC-MS/MS method for precise phytochelatin quantification in alga extracts

AbstractQuantitative phytochelatin (PC) analysis is, due to oxidation sensitivity of the PCs, matrix effects, and time consuming sample preparation, still a challenging analytical task. In this study, a rapid, simple, and sensitive method for precise determination of native PCs in crude extracts of the green alga Chlamydomonas reinhardtii was developed. Algae were exposed 48xa0h to 70xa0μM Cd. Coupling of ultra performance liquid chromatography and electrospray ionization tandem mass spectrometry with multi-reaction mode transitions for detection permitted the required short-time, high-resolution separation and detection specificity. Thus, under optimized chromatographic conditions, 10 thiol peptides were baseline-separated within 7xa0min. Relative detection limits in the nanomolar range in microliter sample volumes were achieved (corresponding to absolute detection limits at femtomol level). Next to glutathione (GSH), the most abundant cadmium-induced PCs in C. reinhardtii, namely CysGSH, PC2, PC3, CysPC2, and CysPC3, were quantified with high reproducibility at concentrations between 15 and 198xa0nmolxa0g−1 fresh weight. The biological variation of PC synthesis of nine independently grown alga cultures was determined to be on average 13.7%.n FigureA rapid UPLC-MS/MS method was developed for thiol peptide quantification in micro litre sample volumes at the nanomol level. For the first time reproducible quantification of six thiol peptides (GSH, CysGSH, PC2, PC3, CysPC2 and CysPC3) in crude extracts of Clamydomonas reinhardtii was possible

CZE-ICP-MS separation of metallothioneins in human brain cytosols: comparability of electropherograms obtained from different sample matrices

Capillary zone electrophoresis, with its high resolution capability in the separation of different compounds, is well suited for the investigation of metal-containing proteins, especially when elemental detection is conducted using hyphenated inductively coupled plasma-mass spectrometry. A major problem in the separation of proteins in body fluids is caused by the effects of different sample matrix composition. The migration time of proteins varies significantly, depending on the nature of the matrix. Electropherograms are consequently difficult to compare and the peak identification is uncertain. Pre-analytical steps for the reduction of matrix compounds enhance the quality of the data, but the results are still unsatisfactory. This paper describes a technique for obtaining electropherograms that can be used for comparison purposes by correction of the data with the aid of time markers. A mixture of five substances (caesium chloride, arsenocholine, arsenobetaine, dimethylarsinic acid and monomethylarsonic acid) was added in a separate injection step. Ionic caesium eluted at the start of the separation and the other four markers appeared throughout and at the end of the electropherogram. All electropherograms were normalized to a reference run by recalculation of the time axis using the time markers. The method was applied to the analysis of human brain cytosols. Samples were separated after different pre-treatment steps and were compared, with special emphasis on the detection of the isoform metallothionein-3.

Optimization of capillary electrophoresis–inductively coupled plasma mass spectrometry for species analysis of metallothionein-like proteins extracted from liver tissues of Elbe-bream and Roe deer

Abstract Species analysis of metallothionein-like proteins (MLP) in liver tissues from Elbe-Bream ( Abramis brama L.) and Roe Deer ( Capreolus capreolus L.) using capillary electrophoresis (CE) combined with inductively coupled plasma mass spectrometry detection is described. In order to allow systematic development of the method, commercially available metallothionein (MT) preparations of rabbit liver were used. Optimum separation efficiency was obtained by investigating the influence of parameters such as voltage, capillary temperature, buffer concentration, buffer pH and the use of different buffer systems. Instrumental parameters such as CE capillary position, interface adjustment and contamination problems are also discussed. Separation was performed using uncoated fused silica capillaries with 75 μm i.d. and 70 cm length. The optimum conditions were found to be: Separation voltage 30 kV, positive polarity, capillary temperature 288.15 K and a buffer concentration of 100 mmol l −1 Tricine–NH 3 adjusted to pH 7.2. Sample preparation was performed so as to minimize oxidation and heavy metal contamination of the samples. The high molecular mass protein matrix was reduced by acetonitrile precipitation. For commercial MT preparations the relative standard deviations (R.S.D) in the retention times were 0.9% for MT-1 and 1.9% for MT-2; the R.S.D.s in the peak areas were less than 6% for MT-1 and 16% for MT-2, respectively. Under optimized conditions the MLPs in the real samples could be separated efficiently in less than 10 min. By comparison with the migration times of commercially available MT preparations, two of the observed peaks could be assigned to MT-1 and MT-2.

Nano-High-Performance Liquid Chromatography with Online Precleaning Coupled to Inductively Coupled Plasma Mass Spectrometry for the Analysis of Lanthanide-Labeled Peptides in Tryptic Protein Digests

Low background signals are an indispensable prerequisite for accurate quantification in bioanalytics. This poses a special challenge when using derivatized samples, where excess reagent concentrations are increasing the background signal. Precleaning steps often are time-consuming and usually lead to analyte losses. In this study, a set of labeled model peptides and a protein digest was analyzed using inductively coupled plasma mass spectrometry (ICPMS), coupled to nano ion pairing reversed-phase high-performance liquid chromatography (nano-IP-RP-HPLC). In addition, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was used for peptide identification. Peptides were labeled with lanthanide metals using bifunctional DOTA-based (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) reagents. The resulting metal excess was removed online during nano-HPLC, by trapping the labeled peptides on a C18-precolumn and washing them prior to their elution to the analytical column. Different ion pairing reagents like TFA (trifluoroacetic acid) and HFBA (heptafluorobutyric acid) were used in the study to enhance interactions of the different peptide species with the C18 material of the precolumn. HFBA even allowed the detection of a highly hydrophilic peptide that was not retained using TFA. It was shown that for the mixture of labeled model peptides, even a short 3 min washing step already enhanced the removal of the excess reagents significantly, whereas peptide losses were observable starting with a 10 min washing time. A 6 min washing time was determined to be the best parameter for lowering the lanthanide metal background while maintaining maximum peptide recovery. Alternative precleaning setups using EDTA to enhance the removal of free metal or an offline approach using solid phase extraction did not show promising results. The application of the optimized method to labeled peptides in a lysozyme digest showed results comparable to those obtained with model peptides.