Combining Isotope Dilution and Standard Addition-Elemental Analysis in Complex Samples.

A new method combining isotope dilution mass spectrometry (IDMS) and standard addition has been developed to determine the mass fractions w of different elements in complex matrices: (a) silicon in aqueous tetramethylammonium hydroxide (TMAH), (b) sulfur in biodiesel fuel, and (c) iron bound to transferrin in human serum. All measurements were carried out using inductively coupled plasma mass spectrometry (ICP-MS). The method requires the gravimetric preparation of several blends (bi)-each consisting of roughly the same masses (mx,i) of the sample solution (x) and my,i of a spike solution (y) plus different masses (mz,i) of a reference solution (z). Only these masses and the isotope ratios (Rb,i) in the blends and reference and spike solutions have to be measured. The derivation of the underlying equations based on linear regression is presented and compared to a related concept reported by Pagliano and Meija. The uncertainties achievable, e.g., in the case of the Si blank in extremely pure TMAH of urel (w(Si)) = 90% (linear regression method, this work) and urel (w(Si)) = 150% (the method reported by Pagliano and Meija) seem to suggest better applicability of the new method in practical use due to the higher robustness of regression analysis.

Comparison of potential higher order reference methods for total haemoglobin quantification-an interlaboratory study.

The total haemoglobin (Hb) concentration in blood is one of the most frequently measured analytes in clinical medicine because of its significance for evaluating the health state of a human. The spectrophotometric cyanmethaemoglobin (HiCN) method is the internationally accepted conventional reference method to determine this biomarker. It is frequently used in clinical routine diagnostics but is not traceable to the International System of Units and thus does not meet highest metrological demands. A further critical issue is the toxicity of the necessary potassium cyanide. Different methods to solve these problems are reported here. They all were validated against the HiCN method in an interlaboratory comparison by measuring the total Hb concentration present in the certified reference material JCCRM 912-2M. Methods considered were the spectrophotometric alkaline haematin detergent (AHD) method as well as several isotope dilution (ID)-based approaches. The latter include inductively coupled plasma mass spectrometry (ICP-MS), species-specific (SS) ICP-MS, organic MS and Raman spectrometry. Graphical abstract ᅟ.

Influence of cimetidine and its metabolites on Cisplatin--investigation of adduct formation by means of electrochemistry/liquid chromatography/electrospray mass spectrometry.

Cimetidine has been studied as an additive in cancer chemotherapy. It is claimed to reduce the side effects of Cisplatin. This study focuses on possible interactions between Cisplatin and cimetidine on the molecular level. Due to the fact that cimetidine is metabolized in the liver, interactions between its metabolites and Cisplatin are also investigated. By means of LC/ESI-MS, Cisplatin-cimetidine adducts were detected. In a second step, the metabolism of cimetidine was simulated by electrochemical oxidation. These results were compared with microsomal incubations of cimetidine using rat and human liver cell microsomes. Because the two methods showed a correlation, the electrochemical approach was further used to investigate Cisplatin's interactions with metabolites of cimetidine. However, notable interactions that might take place in the human body could neither be observed for pure cimetidine nor for its metabolites. Finally, the impact of cimetidine on Cisplatin-protein interactions were studied using the model protein ß-lactoglobulin A. In the presence of cimetidine, the affinity of Cisplatin towards the model protein appears to be increased.

The interaction of platinum-based drugs with native biologically relevant proteins.

This study focuses on the identification of the products that are formed upon binding of therapeutically relevant platinum complexes to proteins like ß-lactoglobulin A (LGA), human serum albumin (HSA), or human hemoglobin (HB). The respective proteins were incubated with the platinum-based anticancer drugs cisplatin, carboplatin, and oxaliplatin. LGA was selected as the model protein in addition to the two most abundant blood proteins HSA and HB. In case of the model protein, the effect of free thiol groups on the affinity of cisplatin, carboplatin, and oxaliplatin was investigated by means of liquid chromatography electrospray ionization time-of-flight mass spectrometry (LC/ESI-ToF-MS). The reduced form of LGA, which contains four free thiol groups more than the native LGA, shows a much higher affinity to the platinum-based drugs. By means of liquid chromatography coupled to inductively coupled plasma mass spectrometry, the reaction behavior of the platinum-based drugs towards HSA and HB was investigated under different conditions considering the chloride concentration (4 or 100 mM) and the incubation time (24 and 48 h). In case of carboplatin, less than 6 % protein-bound platinum was detected. However, both cisplatin and oxaliplatin display a high affinity to the proteins investigated. Further information was obtained by means of LC/ESI-ToF-MS. In case of oxaliplatin, the complex [Pt(DACH)](2+) (DACH=C(6)N(2)H(14)) was identified interacting with HSA and HB. For cisplatin, different results were observed for the two proteins. The complex [Pt(NH(3))(2)Cl](+) interacted predominantly with HSA and [Pt(NH(3))(2)](2+) with HB.

Simulation of the oxidative metabolism of diclofenac by electrochemistry/(liquid chromatography/)mass spectrometry.

Diclofenac is a frequently prescribed drug for rheumatic diseases and muscle pain. In rare cases, it may be associated with a severe hepatotoxicity. In literature, it is discussed whether this toxicity is related to the oxidative phase I metabolism, resulting in electrophilic quinone imines, which can subsequently react with nucleophiles present in the liver in form of glutathione or proteins. In this work, electrochemistry coupled to mass spectrometry is used as a tool for the simulation of the oxidative pathway of diclofenac. Using this purely instrumental approach, diclofenac was oxidized in a thin layer cell equipped with a boron doped diamond working electrode. Sum formulae of generated oxidation products were calculated based on accurate mass measurements with deviations below 2 ppm. Quinone imines from diclofenac were detected using this approach. It could be shown for the first time that these quinone imines do not react with glutathione exclusively but also with larger molecules such as the model protein ß-lactoglobulin A. A tryptic digest of the generated drug-protein adduct confirms that the protein is modified at the only free thiol-containing peptide. This simple and purely instrumental set-up offers the possibility of generating reactive metabolites of diclofenac and to assess their reactivity rapidly and easily.

Dichloroacetate metabolically targeted therapy defeats cytotoxicity of standard anticancer drugs.

PURPOSE: The observation that the orphan drug dichloroacetate (DCA) selectively promotes mitochondria-regulated apoptosis and inhibits tumour growth in preclinical models by shifting the glucose metabolism in cancer cells from anaerobic to aerobic glycolysis attracted not only scientists', clinicians' but also patients' interests and prompted us to further evaluate DCA effects against paediatric malignancies. METHODS: The effects of DCA on mitochondrial membrane potential (ΔΨm), cell viability and induction of apoptosis were evaluated in paediatric tumour cell lines and the non-malignant cell line HEK293. In addition, combinations of DCA with the standard anticancer drugs cisplatin, doxorubicin, and temozolomide were tested and intra- and extra-cellular platinum species analysed. RESULTS: DCA selectively induced phosphatidylserine externalisation and reduced ΔΨm in paediatric tumour cells compared to HEK293 cells, but DCA concentrations ≤ 10 mmol/L only moderately inhibited the growth of 18 paediatric tumour cell lines. DCA neither influenced the in vitro stability of cisplatin nor the cellular cisplatin uptake, but it abrogated the cytotoxicity of cisplatin in 7 out of 10 cell lines. DCA also affected the cytotoxicity of doxorubicin but did not influence the cytotoxicity of temozolomide. Despite phosphatidylserine externalisation, DCA failed to activate caspase 3/7 and, moreover, suppressed caspase 3/7 activation by cisplatin and doxorubicin. CONCLUSIONS: Our results indicate that apart from the intriguing effects of DCA on the glucose metabolism of cancer cells, the use of DCA for cancer treatment has to be evaluated carefully. Moreover, compassionate use of the orally available drug by patients with cancer themselves without medical supervision is strongly discouraged at present.

Speciation analysis of gadolinium chelates in hospital effluents and wastewater treatment plant sewage by a novel HILIC/ICP-MS method.

The behavior of Gd chelates used in magnetic resonance imaging (MRI) within the process of sewage treatment is widely unknown. Due to the varying toxicity of the particular Gd species [J. M. Idee et al. Fundam. Clin. Pharmacol. 2006, 20, 563-576], it is important to not only investigate total Gd concentrations, but the Gd species as well. This work describes a novel method for speciation analysis of the most important gadolinium chelates in wastewaters. This novel approach consists of coupling hydrophilic interaction chromatography (HILIC) with inductively coupled plasma mass spectrometry (ICP-MS). HILIC/ICP-MS exhibits high separation efficiency for the simultaneous separation of the five predominantly applied MRI contrast agents and the required selectivity and sensitivity for trace determination in wastewater samples. For the first time, the distribution of particular Gd chelate complexes was determined in hospital effluent, municipal sewage, and wastewater treatment plant (WWTP) samples. The data were compared with the total concentration of Gd as determined by ICP-MS. The active compounds of Multihance, Dotarem, and Gadovist were identified in local WWTP samples. Interestingly, the macrocyclic, nonionic compound Gd-BT-DO3A (Gadovist) was found to be the most abundant Gd complex in all investigated samples. This is in contrast to prevalent assumptions that linear ionic Gd chelates such as Gd-DTPA (Magnevist) would be the predominant species [G. Morteani et al. Environ. Geochem. Health 2006, 28, 257-264 and M. Bau and P. Dulski, Earth Planet. Sci. Lett. 1996, 143, 245-255]. Although contrast agent concentrations tend to be reduced during wastewater treatment, Gd-BT-DO3A was still found in WWTP effluents.

Quantification and excretion kinetics of a magnetic resonance imaging contrast agent by capillary electrophoresis-mass spectrometry.

A novel method for the analysis of Gadolinium-based contrast agents in complex clinical matrices is presented. Three commonly applied ionic contrast agents for magnetic resonance imaging were separated by CE and detected by ESI-MS. Blank urine samples were spiked with Dotarem (Gd-DOTA, Gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), Magnevist (Gd-DTPA, Gadolinium-diethylenetriaminepentaacetic acid) and Multihance (Gd-BOPTA, Gadolinium-benzyloxymethyl-diethylenetriaminepentaacetic acid) to determine the recovery rates. The figures of merit were determined with LODs as low as 2.0 x 10(-7) mol/L for Gd-DOTA, 5.0 x 10(-7) mol/L for Gd-DTPA and 1.0 x 10(-6) mol/L for Gd-BOPTA. The respective LOQs were 6.6 x 10(-7) mol/L for Gd-DOTA, 1.5 x 10(-6) mol/L for Gd-DTPA and 3.3 x 10(-6) mol/L for Gd-BOPTA. The linear working range comprised two orders of magnitude starting at the LOQ, with regression coefficients of R > or = 0.999 for all investigated analytes. Using this CE-MS method, Gd-DOTA was quantified in seven urine samples obtained at different times after delivery from a volunteer magnetic resonance imaging patient who was treated with Dotarem. Additionally, total Gd concentrations were determined by means of ICP-optical emission spectroscopy to validate the CE-MS data. To compensate for dietary dilution effects of the urine samples, creatinine was determined by HPLC with UV/Vis absorption detection. Gd-DOTA concentrations were normalized to urinary creatinine, illustrating the fast excretion kinetics of Gd-DOTA.