Structure elucidation and quantification of the reduction products of anticancer Pt(iv) prodrugs by electrochemistry/mass spectrometry (EC-MS).

Pt(iv) prodrugs are a class of promising anticancer agents, which are activated by reduction to the active Pt(ii) species. Consequently, the reduction process is a crucial parameter. Herein, a new approach using electrochemistry (EC) coupled to liquid chromatography (LC) and electrospray ionization-mass spectrometry (ESI-MS) or inductively coupled plasma (ICP)-MS was applied. This enabled getting insights into the differences in the reduction and ligand release of platinum(iv) complexes with varying equatorial core structures.

13C-labelled yeast as internal standard for LC-MS/MS and LC high resolution MS based amino acid quantification in human plasma.

Extracts from isotopically labelled organisms can be a versatile source for isotopically labelled chemical compounds providing ideal internal standards in mass spectrometry based assays. In this work, the application of 13C enriched yeast (Pichia pastoris) for accurate absolute metabolite quantification in human samples was investigated. >99% 13C enriched Pichia pastoris was produced via fermentation and extracted employing established protocols. Quantitative assays based on LC-triple quadrupole mass spectrometry (QQQ-MS) and LC-high resolution mass spectrometry (HRMS) were validated using the Standard Reference Material, SRM 1950 - metabolites in frozen human plasma. 14 amino acids (as given in the certificate) were quantified using separations by reversed phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC). The latter chromatographic separation provided retention and selectivity for the amino acid panel, while the studied approaches employing RPLC relied on the selectivity of the MS detection. Cross-validation using the different MS platforms showed that in all cases the application of in-vivo labelled standards resulted in a significant improvement of trueness and precision. LODs and LOQs ranged, regardless of the detection system and addition of internal standards, in the same order of magnitude. The linear dynamic range of the employed detection systems was enhanced at least for one order of magnitude for several analytes when the internal standards were applied.

Triapine-mediated ABCB1 induction via PKC induces widespread therapy unresponsiveness but is not underlying acquired triapine resistance.

Although triapine is promising for treatment of advanced leukemia, it failed against solid tumors due to widely unknown reasons. To address this issue, a new triapine-resistant cell line (SW480/tria) was generated by drug selection and investigated in this study. Notably, SW480/tria cells displayed broad cross-resistance against several known ABCB1 substrates due to high ABCB1 levels (induced by promoter hypomethylation). However, ABCB1 inhibition did not re-sensitize SW480/tria cells to triapine and subsequent analysis revealed that triapine is only a weak ABCB1 substrate without significant interaction with the ABCB1 transport function. Interestingly, in chemo-naive, parental SW480 cells short-time (24 h) treatment with triapine stimulated ABCB1 expression. These effects were based on activation of protein kinase C (PKC), a known response to cellular stress. In accordance, SW480/tria cells were characterized by elevated levels of PKC. Together, this led to the conclusion that increased ABCB1 expression is not the major mechanism of triapine resistance in SW480/tria cells. In contrast, increased ABCB1 expression was found to be a consequence of triapine stress-induced PKC activation. These data are especially of importance when considering the choice of chemotherapeutics for combination with triapine.

Characterization of metal-tagged antibodies used in ICP-MS-based immunoassays.

A detailed characterization of metal-tagged antibodies is the prerequisite for the implementation of quantitative concepts in inductively coupled plasma-mass spectrometry (ICP-MS)-based bioanalysis or future medical diagnosis. In this paper, the common modification with bifunctional ligands containing maleimide residues as a reactive group was investigated in detail via size exclusion chromatography (SEC)-ICP-MS and liquid chromatography-time-of-flight (LC-TOF)-MS to determine the preservation of the antibody structure after tagging. Mouse monoclonal IgG modified with metal-coded tags (MeCATs) was used as a model system. Several antibody fragments were identified carrying different numbers of metal tags. In a second step, a functionality test was performed with isolated fragments where the antigen specificity was tested in a dot blot immunoassay.

Metabolic profiling of amino acids in cellular samples via zwitterionic sub-2 µm particle size HILIC-MS/MS and a uniformly 13C labeled internal standard.

A novel analytical method using hydrophilic interaction liquid chromatography combined with electrospray tandem mass spectrometry for metabolic profiling of free, underivatized amino acids is presented. The separation uses a zwitterionic modified silica-based stationary phase with 1.8-µm particle size functionalized with ammonium sulfonic acid groups. Quantification is based on external standard calibration using a Pichia pastoris cell extract grown on uniformly (13)C labeled glucose as an internal standard. The absolute limits of detection in the cellular matrix were in the subpicomolar range. Measurement accuracy was assessed by analyzing NIST Standard Reference Material 2389a, which provides certified values for 17 amino acids. The recovery of the amino acids ranged between 65 % (proline) and 120 % (lysine), with excellent repeatability precision below 2.5 % (n = 5). Only, cystine showed poor recovery (29 %) and repeatability precision (13 %). Generally, the long-term precision obtained by hydrophilic interaction liquid chromatography-tandem mass spectrometry was excellent, being on average less than 9 % over 20 h of measurement time. Moreover, the novel separation method had average repeatability and reproducibility of the chromatographic peak width over time periods of 20 h and 6 months of 8 and 15 %, respectively, demonstrating its high robustness in routine analysis of cellular samples. Large concentration differences depending on the amino acid were found in the cell extracts, typically ranging from 0.002 nmol per milligram of cell dry weight (cystine) to 56 nmol per milligram of cell dry weight (arginine and glutamic acid).

Measurement uncertainty of isotopologue fractions in fluxomics determined via mass spectrometry.

Metabolic flux analysis implies mass isotopomer distribution analysis and determination of mass isotopologue fractions (IFs) of proteinogenic amino acids of cell cultures. In this work, for the first time, this type of analysis is comprehensively investigated in terms of measurement uncertainty by calculating and comparing budgets for different mass spectrometric techniques. The calculations addressed amino acids of Pichia pastoris grown on 10% uniformly (13)C labeled glucose. Typically, such experiments revealed an enrichment of (13)C by at least one order of magnitude in all proteinogenic amino acids. Liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) analyses were performed. The samples were diluted to fit the linear dynamic range of the mass spectrometers used (10 µM amino acid concentration). The total combined uncertainties of IFs as well as the major uncertainty contributions affecting the IFs were determined for phenylalanine, which was selected as exemplary model compound. A bottom-up uncertainty propagation was performed according to Quantifying Uncertainty in Analytical Measurement and using the Monte Carlo method by considering all factors leading to an IF, i.e., the process of measurement and the addition of (13)C-glucose. Excellent relative expanded uncertainties (k = 1) of 0.32, 0.75, and 0.96% were obtained for an IF value of 0.7 by LC-MS/MS, GC-MS, and LC-TOFMS, respectively. The major source of uncertainty, with a relative contribution of 20-80% of the total uncertainty, was attributed to the signal intensity (absolute counts) uncertainty calculated according to Poisson counting statistics, regardless which of the mass spectrometry platforms was used. Uncertainty due to measurement repeatability was of importance in LC-MS/MS, showing a relative contribution up to 47% of the total uncertainty, whereas for GC-MS and LC-TOFMS the average contribution was lower (30 and 15%, respectively). Moreover, the IF actually present also depends on the isotopic purity of the carbon sources. Therefore, in the uncertainty calculation a carbon source purity factor was introduced and a minor contribution to the total uncertainty was observed. The results obtained by uncertainty calculation performed according to the Monte Carlo method were in agreement with the uncertainty value of the Kragten approach and showed a Gaussian distribution.

Quantitative profiling of in vivo generated cisplatin-DNA adducts using different isotope dilution strategies.

Platinum compounds are the major group of metal-based chemotherapeutic drug used in current practice and still a topic of intense investigation. The relative contribution of structurally defined cisplatin adducts with DNA to induce apoptosis and the cellular processing of these lesions is still poorly understood mostly due to the lack of sensitive and accurate analytical tools for in vivo studies. In this regard, two novel sensitive and selective strategies are proposed here to quantify cisplatin-DNA adducts generated in Drosophila melanogaster larvae and in head and neck squamous cell carcinoma cultures. The methods involve the isolation and enzymatic digestion of the DNA in the samples exposed to cisplatin and further quantification by high-performance liquid chromatography with inductively coupled plasma mass spectrometric detection (HPLC-ICPMS). Two different strategies, based on isotope dilution analysis (IDA), have been attempted and evaluated for quantification: species-unspecific (the postcolumn addition of a 194Pt-enriched solution) and the species-specific (by means of a synthesized isotopically enriched cisplatin (194Pt) adduct). For the second approach, the synthesis and characterization of the cisplatin adduct in a custom oligonucleotide containing the sequence (5'-TCCGGTCC-3') was necessary. The adducted oligo was then added to the DNA samples either before or after enzymatic hydrolysis. The results obtained using these two strategies (mixing before and after enzymatic treatment) permit to address, quantitatively, the column recoveries as well as the efficiency of the enzymatic hydrolysis. Species-specific spiking before enzymatic digestion provided accurate and precise analytical results to clearly differentiate between Drosophila samples and carcinoma cell cultures exposed to different cisplatin concentrations.

Monitoring, removal and risk assessment of cytostatic drugs in hospital wastewater.

Cytostatic agents are applied in cancer therapy and subsequently excreted into hospital wastewater. As these substances are known to be carcinogenic, mutagenic and toxic for reproduction, they should be removed from wastewater at their source of origin. In this study the fate and effects of the cancerostatic platinum compounds (CPC) cisplatin, carboplatin, oxaliplatin, 5-fluorouracil (5-FU) and the anthracyclines doxorubicin, daunorubicin and epirubicin were investigated in hospital wastewater. Wastewater from the in-patient treatment ward of a hospital in Vienna was collected and monitored for the occurrence of the selected drugs. A calculation model was established to spot the correlation between administered dosage and measured concentrations. To investigate the fate of the selected substances during wastewater treatment, the oncologic wastewater was treated in a pilot membrane bioreactor system (MBR) and in downstream advanced wastewater treatment processes (adsorption to activated carbon and UV-treatment). Genotoxic effects of the oncologic wastewater were assessed before and after wastewater treatment followed by a risk assessment. Monitoring concentrations of the selected cytostatics in the oncologic wastewater were in line with calculated concentrations. Due to different mechanisms (adsorption, biodegradation) in the MBR-system 5 - FU and the anthracyclines were removed < LOD, whereas CPC were removed by 60%. In parallel, genotoxic effects could be reduced significantly by the MBR-system. The risk for humans, the aquatic and terrestrial environment by hospital wastewater containing cytostatic drugs was classified as small in a preliminary risk assessment.

Platinum determination by inductively coupled plasma-sector field mass spectrometry (ICP-SFMS) in different matrices relevant to human biomonitoring.

The analytical challenges of Pt determination by ICP-SFMS posed by different human tissues and fluids have been critically assessed. Investigated samples were (1) urine, (2) serum of cancer patients sampled during chemotherapy with carboplatin, (3) microdialysates (20 micro L sample volume) collected from tumor and non-tumor tissue, and, finally-for the first time-(4) human lung tissue to study background concentrations of inhaled platinum. Sample preparation involved microwave digestion and open vessel treatment or simple dilution (microdialysates). Depending on the sample preparation and introduction systems used (microconcentric nebulization, ultrasonic nebulization with and without membrane desolvation) excellent procedural detection limits (3s criterion) of 0.35 pg g(-1) for urine, 420 pg g(-1) for serum, 400 pg g(-1) for lung tissue and 13 pg g(-1) for microdialysates could be obtained. Ultratrace concentrations of 1-40 pg g(-1), and 1000-3000 pg g(-1) were measured in urine and human lung tissue, respectively, as typical for samples in environmental studies. Quantification was carried out by IDMS and standard addition in the case of urine samples. Internal standardization could not correct for non-spectral interferences in external calibration. In the serum and microdialysates of patients during chemotherapy with carboplatin, elevated Pt levels ranging between 0.01 and 10 micro g g(-1) were determined by external calibration ((195)Pt isotope). For all investigated samples spectral interferences could be excluded by following different strategies. High-resolution control measurements ((194)Pt, (195)Pt) were performed in the case of elevated Pt levels, i.e. for microdialysates and serum samples. An Hf/Pt ratio of 0.4 was determined in human lung samples. An HfO formation ratio of 0.2% was assessed for standard solutions at the present experimental conditions, revealing that the contribution of (179)Hf(16)O, (178)Hf(17)O, (177)Hf(18)O to the (195)Pt isotope signal used for quantification was not significant.

Analysis of Ir in Köfelsit rocks by inductively coupled plasma-sector-field mass spectrometry (ICP-SFMS).

Three different analytical strategies have been evaluated for the quantification of Ir in geological samples. Glassy rock samples from Köfels and reference material WGB-1 were analyzed directly by inductively coupled plasma sector field mass spectrometry (ICP-SFMS) at mass resolution 400 using membrane desolvation and at mass resolution 9500 without membrane desolvation. Matrix separation by anion-exchange pre-concentration was also investigated. The ultrasonic nebulizer USN6000AT+ (Cetac Technologies, Omaha, NE, USA) incorporating a membrane desolvation unit was used as the sample-introduction system. Sample preparation involved complete microwave-assisted acid digestion of the silicate matrix with HNO3-HCl-HF. The results obtained by the three methods of quantification were in good agreement, showing that oxide-type interferences were effectively eliminated solely by membrane desolvation. The limits of detection were 6 pg g for low resolution measurement with use of the membrane, 15 pg g(-1) at a mass resolution of 9500, and 59 pg g(-1) for the ion-exchange procedure. The ultimate precision obtained for the Köfelsit Ir data was, however, compromised by the small sample intake (0.3 g), because of the inhomogeneous distribution of Ir in geological samples.

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 microg/L; range: < 0.13-42 microg/L) and Au (median: 0.29 microg/L; range 0.10-2.06 microg/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 microg/L. Human milk concentrations of Co (median: 0.19 microg/L), Fe (380 microg/L), Mn (6.3 microg/L), Ni (0.79 microg/L), and Se (17 microg/L) were at the low end of the corresponding reference ranges. Concentrations of Cr (24.3 microg/L) in human milk were five times higher than the high end of the reference range. For Al (67 microg/L), As (6.7 microg/L), and V (0.18 microg/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.