Gunda Koellensperger

Determination of Pt, Pd and Rh by inductively coupled plasma sector field mass spectrometry (ICP-SFMS) in size-classified urban aerosol samples

Pt, Pd and Rh concentration levels related to particle size distribution were measured in Viennese aerosol. The urban aerosol was studied over a period of 1 month during summer 2002 in a heavy traffic area. A 6-stage cascade impactor collected classified particles with aerodynamic equivalent diameter (aed) <10 µm. Total suspended particulate matter (aed <30 µm) was assessed by filtering air (open face sampling head). The analysis of Pt, Pd and Rh in aerosol samples required a different strategy for each element to obtain accurate results and maximum sensitivity. Sample preparation was carried out by microwave-assisted acid digestion. For the determination of Pd an anion-exchange procedure was implemented. The digested samples were measured by ICP-SFMS in combination with ultrasonic nebulization and membrane desolvation, utilizing the high-resolution capabilities of this instrument for the determination of Rh. Pt and Pd were quantified by isotope dilution (IDMS), Rh by external calibration. Excellent detection limits of 0.07, 0.06 and 0.05 pg m−3 (impactor substrates) and 0.18, 0.22, 0.14 pg m−3 (open face sampling head substrates) for Pd, Pt and Rh, respectively, could be achieved. Since platinum group elements (PGE) are emitted from car catalysts as a result of mechanical processes, Pt, Pd and Rh were mainly found in the coarse fraction of urban aerosol. Summing up the concentrations assessed for the impactor stages (aed <10 µm) revealed a weekly average of 4.3 ± 1.7, 2.6 ± 0.6 and 0.4 ± 0.1 pg m−3 for Pt, Pd and Rh respectively. Concentration levels of samples collected with the open face sampling head (aed <30 µm) over the period of one week were significantly higher with average values of 38.1 ± 6.3, 14.4 ± 3.1 and 6.6 ± 2.4 pg m−3. Moreover, the presence of a collective of small particles containing the three investigated PGE was shown. Investigation of the impactor samples revealed an analogue size distribution for Pt, Rh and Pd with a maximum at 1–2.15 µm aed. These particles are toxicological relevant, as they penetrate deeply in human lungs (particles of 0.1–2.5 µm aed are deposited in the lung alveoli).

Two dimensional separation schemes for investigation of the interaction of an anticancer ruthenium(III) compound with plasma proteins

On-line 2-dimensional size exclusion/anion exchange chromatography was coupled to inductively coupled mass spectrometry with dynamic reaction cell technology (SEC-IC-ICP-MS) in order to characterize the interaction of the ruthenium-based anticancer drug KP1019 with human plasma proteins in vitro and, for the first time, in vivo. In SEC-ICP-MS studies the drug was found to bind exclusively to the protein fraction of 60–80 kDa in human plasma samples (clinical study/phase 1). The respective size fraction was collected and subsequently analyzed by reversed phase chromatography coupled to electrospray mass spectrometry (LC-ESI-MS) confirming the presence of the two well known transporter proteins, i.e. human serum albumin (HSA) and transferrin (Tf). Hence, for in vivo investigation of KP1019 interaction with HSA and Tf a fully automated SEC-IC-ICP-MS approach was applied. The stoichiometry of the KP1019 protein binding was determined through the molar Ru/S ratio. Human apo-Tf standards incubated with different stoichiometric equivalents of KP1019 were used for species-specific and species-unspecific calibration of the molar Ru/S ratio. Competitive in vitro incubation of KP1019 to both HSA and Tf for ca. 10 h showed that <20% of the overall KP1019 was found to bind to Tf in an equimolar mixture of Tf and HSA. This fraction dropped to 2% in the incubated mixture containing a 10-fold excess of HSA compared to Tf. This implied that less than 2% of KP1019 were expected to bind to Tf in human plasma samples, as they usually contain an even higher excess of HSA. Indeed, the concentration of the KP1019/Tf-adduct was below the limit of quantification in the plasma sample taken from a cancer patient treated with KP1019. Its fraction of the overall KP1019-content was estimated to be <1%. The KP1019/HSA stoichiometry in the plasma samples could be correlated with the individual infusions administered to the cancer patient within 26 days, the largest value being 1.4 (in the related sample, the KP1019/Tf stoichiometry was estimated to be in the range of 0.3–0.5). The KP1019/Tf ratio is in the expected range for a sufficient uptake via the transferrin pathway.

Ionic liquids for extraction of metals and metal containing compounds from communal and industrial waste water

In a fundamental study the potential of ionic liquids based on quaternary ammonium- and phosphonium cations and thiol-, thioether-, hydroxyl-, carboxylate- and thiocyanate-functionalized anions has been assessed for future application in advanced sewage treatment. The elimination of the metal(oid)s Ag, As, Cd, Cr, Cu, Hg, Ni, Pb, Pt, Sn, Zn and the cancerostatic platinum compounds cisplatin and carboplatin was screened using a liquid phase micro-extraction set-up. The analytical tool-set consisted of ICP-SFMS and LC-ICP-MS for quantification of metal(oid)s and cancerostatic platinum compounds, respectively. The purity of the ILs was assessed for the investigated metal(oid)s on the base of present EU environmental quality standards and was found to be sufficient for the intended use. In model solutions at environmental relevant concentrations extraction efficiencies≥95% could be obtained for Ag, Cu, Hg and Pt with both phosphonium- and ammonium-based ILs bearing sulphur functionality in the form of thiosalicylate and 2-(methylthiobenzoate) anions, as well as with tricaprylmethylammonium thiocyanate within an extraction time of 120 min. All other metals were extracted to a lower extent (7-79%). In the case of cancerostatic platinum compounds a phosphonium-based IL bearing thiosalicylate functionality showed high extraction efficiency for monoaquacisplatin. For the first time, liquid phase micro extraction with ionic liquids was applied to industrial and communal waste water samples. The concentration of all investigated metal(oid)s could be significantly reduced. The degree of elimination varied with the initial concentration of metals, pH and the amount of suspended particulate matter.

Concentrations of selected trace elements in human milk and in infant formulas determined by magnetic sector field inductively coupled plasma-mass spectrometry

Magnetic sector field inductively coupled plasma-mass spectrometry (ICP-MS) was applied to the reliable determination of the 8 essential trace elements cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), manganese (Mn), nickel (Ni), selenium (Se), and vanadium (V) as well as the 7 nonessential and toxic elements silver (Ag), aluminum (Al), arsenic (As), gold (Au), platinum (Pt), scandium (Sc), and titanum (Ti) in 27 transitory and mature human milk samples and in 4 selected infant formulas. This advanced instrumentation can separate spectral overlaps from the analyte signal hampering significantly the determination of many trace elements by conventional ICP-MS. Moreover, superior detection limits in the picogram per liter range can be obtained with such magnetic sector field instruments. Therefore, this is the first study to report the concentrations of the elements Ag, Au, Pt, Sc, Ti, and V in human milk and in infant formulas. Concentrations of Ag (median: 0.41 µg/L; range: <0.13–42 µg/L) and Au (median: 0.29 µg/L; range 0.10–2.06 µg/L) showed large variations in human milk that might be associated with dental fillings and jewelry. Pt concentrations were very low with most of the samples below the method detection limit of 0.01 µg/L. Human milk concentrations of Co (median: 0.19 µg/L), Fe (380 µg/L), Mn (6.3 µg/L), Ni (0.79 µg/L), and Se (17 µg/L) were at the low end of the corresponding reference ranges. Concentrations of Cr (24.3 µg/L) in human milk were five times higher than the high end of the reference range. For Al (67 µg/L), As (6.7 µg/L), and V (0.18 µg/L), most of the samples had concentrations well within the reference ranges. All elemental concentrations in infant formulas (except for Cr) were approximately one order of magnitude higher than in human milk.

Model based engineering of Pichia pastoris central metabolism enhances recombinant protein production

The production of recombinant proteins is frequently enhanced at the levels of transcription, codon usage, protein folding and secretion. Overproduction of heterologous proteins, however, also directly affects the primary metabolism of the producing cells. By incorporation of the production of a heterologous protein into a genome scale metabolic model of the yeast Pichia pastoris, the effects of overproduction were simulated and gene targets for deletion or overexpression for enhanced productivity were predicted. Overexpression targets were localized in the pentose phosphate pathway and the TCA cycle, while knockout targets were found in several branch points of glycolysis. Five out of 9 tested targets led to an enhanced production of cytosolic human superoxide dismutase (hSOD). Expression of bacterial β-glucuronidase could be enhanced as well by most of the same genetic modifications. Beneficial mutations were mainly related to reduction of the NADP/H pool and the deletion of fermentative pathways. Overexpression of the hSOD gene itself had a strong impact on intracellular fluxes, most of which changed in the same direction as predicted by the model. In vivo fluxes changed in the same direction as predicted to improve hSOD production. Genome scale metabolic modeling is shown to predict overexpression and deletion mutants which enhance recombinant protein production with high accuracy.

SEC-ICP-DRCMS and SEC-ICP-SFMS for determination of metal–sulfur ratios in metalloproteins

Simultaneous determination of Fe/S and Mn/S ratios on transient signals was performed by size exclusion chromatography, hyphenated to inductively coupled plasma mass spectrometry with dynamic reaction cell technology (SEC-ICP-DRCMS), in order to characterize metalloprotein samples by their metal/sulfur ratio. Oxygen was used as the cell gas. The method eliminates the effect of polyatomic isobaric interferences at m/z = 32 by detecting sulfur as the product oxide ion 32S16O which is less interfered. Using the same reaction gas conditions, Fe and Mn were measured at m/z 54, 56 and 55, respectively. SEC-ICP-DRCMS measurements (injection volume 20 µL, SEC flow 300 µL min−1) resulted in excellent limits of detection (LOD). 4.3, 0.4, 2 and 0.6 ng g−1 were assessed for 32S16O, 55Mn, 54Fe and 56Fe, respectively. Reference measurements were carried out by size exclusion chromatography inductively coupled plasma sector field mass spectrometry (SEC-ICP-SFMS), setting the mass resolution at 4500. LODs of 14, 0.5 and 0.4 ng g−1 were obtained for 32S, 55Mn and 56Fe, respectively. The metal/sulfur ratios of 5 commercially available metalloproteins were determined (myoglobin, haemoglobin, cytochrom c, arginase and Mn superoxide dismutase from E. coli). Two proteins were characterized after in-house heterologous expression in a host organism (Mn superoxide dismutase from Anabaena PCC 7120, catalase-peroxidase from Synechocystis PCC 6803). Different calibrants (i.e., Fe3+, Mn2+, SO42−, methionine, myoglobin) for the assessment of inter-elemental ratios have been employed. It was found that calibration using metalloprotein myoglobin is preferable to inorganic standards in terms of uncertainty of measurement. However, all metal/sulfur ratios of the investigated proteins obtained by the different methods agreed within their total combined uncertainty.

Application of HPLC-ICP-MS to speciation of cisplatin and its degradation products in water containing different chloride concentrations and in human urine

Cisplatin, mono- and diaquacisplatin were measured in aquatic samples and in diluted urine of a cancer patient by HPLC-ICP-MS. On-line IDMS was applied for accurate, species unspecific quantification. Limits of detection of 0.74, 0.69 and 0.65 µg L−1 (3 s criterion) were calculated for cisplatin, monoaqua- and diaquacisplatin, respectively. Degradation kinetics of 6 × 10−6 M cisplatin were determined over a period of 48 h in solutions containing 100, 50 and 0 mg L−1 chloride, showing the suitability of the HPLC-ICP-MS method for kinetic model studies. The first order rate constants k1 of cisplatin aquation for the three chloride concentrations were 1.79 × 10−5, 1.68 × 10−5 and 2.06 × 10−5 s−1. For cisplatin anatation (second order reverse reaction), rate constants of k−1 = 6.5 × 10−3, 5.8 × 10−3 and 4.1 × 10−3 M−1 s−1 could be assessed. At low chloride levels, no equilibrium was established between cisplatin and its degradation products. It was found that the intermediately formed mono- and diaquacisplatin-products started to decay after several hours. Diluted urine of a cancer patient contained the parent drug cisplatin and a considerable fraction of highly active monoaquacisplatin, as well as several unknown platinum species.

Interactions between ABC‐transport proteins and the secondary Fusarium metabolites enniatin and beauvericin

Enniatins (ENN) and beauvericin (BEA) exert cytotoxic properties. Here, we observed that their impact on Ca(2+)-homeostasis can be reversed by exogenous ATP. Thus, we investigated whether membrane-located ATP-binding cassette (ABC) transporters influence ENNs- and BEA-induced cytotoxicity. In short-term exposure assays breast cancer resistance protein (ABCG2)-overexpression weakly but significantly reduced the cytotoxic activity of BEA but not ENNs. In contrast, multidrug resistance-associated protein-1 (ABCC1)- and P-glycoprotein (ABCB1)-overexpression was not protective under identical conditions. ABCG2-mediated resistance against BEA was reversible by ABCG2 modulators. In long-term exposure assays, ABCG2 and ABCB1 significantly protected against ENNs- and to a lesser extent BEA-induced cytotoxicity. Moreover, both fusariotoxins potently inhibited the ABCG2- and ABCB1-mediated efflux of specific fluorescent substrates, with BEA being more effective. Additionally, ATPase and photoaffinity-labelling assays proofed interaction of both substances with ABCG2 and ABCB1. Remarkably, 2 years selection of KB-3-1 cells against both fusariotoxins resulted only in two-fold ENNs but negligible BEA resistance. Interestingly, the selected sublines displayed upregulation of multidrug resistance proteins and crossresistance to other chemotherapeutics. Summarizing, ABCG2 and ABCB1 slightly but significantly protect human cells against ENNs- and BEA-induced cytotoxicity. However, both mycotoxins potently interact with ABCB1 and ABCG2 transport functions suggesting influences on bioavailability of xenobiotics and pharmaceuticals.

Quantification of cisplatin, carboplatin and oxaliplatin in spiked human plasma samples by ICP-SFMS and hydrophilic interaction liquid chromatography (HILIC) combined with ICP-MS detection

Therapeutic drug monitoring addresses the determination of the free and intact drug in patient blood. The free fraction of cancerostatic platinum compounds (CPC) is determined by measurement of total platinum levels in plasma ultrafiltrate. In this work, as a starting point, the distribution of cisplatin, carboplatin and oxaliplatin among different blood compartments, i.e. whole blood, plasma, residue (pellet fraction), plasma-ultrafiltrate and protein-residue fraction, was assessed in an ex vivo experiment via ICP-SFMS measurement of total platinum after microwave digestion. The results clearly showed that the distribution is independent of the initial concentration but dependent on the used drug. The platinum in the plasma-ultrafiltrate amounted to 16.8 ± 1.3%, 56.8 ± 0.1% and 10.4 ± 1.0% for cisplatin, carboplatin and oxaliplatin, respectively. Acetonitrile precipitation of plasma samples delivered corresponding results simplifying and accelerating (5 min vs. 150 min for plasma ultrafiltration and total digestion) sample preparation. This method could be ideally combined with a novel speciation analytical approach using hydrophilic interaction liquid chromatography (HILIC) with ICP-QMS detection. A comparison of the total platinum levels to species selective results revealed that 88.0 ± 12.0% of cisplatin and 106.4 ± 7.7% of carboplatin were present as parent drug, whereas only 34.3 ± 0.4% of oxaliplatin were present in the intact state.

U13C cell extract of Pichia pastoris – a powerful tool for evaluation of sample preparation in metabolomics

Quantitative metabolic profiling is preceded by dedicated sample preparation protocols. These multistep procedures require detailed optimization and thorough validation. In this work, a uniformly (13)C-labeled (U(13)C) cell extract was used as a tool to evaluate the recoveries and repeatability precisions of the cell extraction and the extract treatment. A homogenous set of biological replicates (n = 15 samples of Pichia pastoris) was prepared for these fundamental experiments. A range of less than 30 intracellular metabolites, comprising amino acids, nucleotides, and organic acids were measured both in monoisotopic (12)C and U(13)C form by LC-MS/MS employing triple quadrupole MS, reversed phase chromatography, and HILIC. Recoveries of the sample preparation procedure ranging from 60 to 100% and repeatability precisions below 10% were obtained for most of the investigated metabolites using internal standardization approaches. Uncertainty budget calculations revealed that for this complex quantification task, in the optimum case, total combined uncertainty of 12% could be achieved. The optimum case would be represented by metabolites, easy to extract from yeast with high and precise recovery. In other cases the total combined uncertainty was significantly higher.