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Probing metal-complexes with metallothioneins by reversed phase microbore chromatography and capillary zone electrophoresis coupled with inductively coupled plasma and electrospray mass spectrometry.

Four different hyphenated techniques: microbore reversed phase (RP) HPLC-ICP MS, CZE-ICP MS, RP HPLC-ES MS and CZE-ES MS were investigated for the characterization of metallothionein-metal complexes under neutral pH conditions. Particular attention was given to the differentiation between metallothionein and artifact signals, identification of mixed-metal complexes, and the validity of the molecular mass as the identification parameter of the different MT iso- and sub-isoforms. Despite the similar morphology of chromatograms and electrophoregrams mass spectrometry revealed different origin of the apparently corresponding peaks. The performance of the four above mentioned techniques was characterized using the example of rabbit liver MT-1 preparation. Reversed-phase HPLC with post-column acidification prior to ES MS was judged to be the most versatile technique for the characterization of metal complexes with metallothioneins but other techniques offer valuable auxiliary information.

Animals↗

Germanium dioxide as internal standard for simplified trace determination of bromate, bromide, iodate and iodide by on-line coupling ion chromatography-inductively coupled plasma mass spectrometry.

The use of elemental mass spectrometry as detection for ion chromatography allows sensitive determination of several bromine and iodine species at a reasonable time scale. Lowest concentrations observable are 66 ng L(-1) for bromate, 45 ng L(-1) for iodate, 74 ng L(-1) for bromide and 151 ng L(-1) for iodide. A major drawback of previous IC-ICP-MS applications is the high consumption of time and thus the running costs. The use of GeO2 as internal standard not only allows improved external calibration, but also semiquantitative determination of bromate, bromide, iodate and iodide without any calibration procedure. Furthermore, GeO2 can be used for all known types of anion exchange columns regardless of their construction principles. It is shown, that the analyte-to-GeO2 ratio of four bromine and iodine species was nearly constant over 4 months and almost independent from the ICP-MS instrumental settings. The quantification by means of the analyte-to-GeO2 ratio for samples taken from a bromate round robin test shows that the values obtained are in excellent agreement with calibration curve and isotope dilution results.

Bromates↗

[Speciation analysis of mercury by capillary electrophoresis on-line coupled with inductively coupled plasma mass spectrometry based on a micro mist nebulizer and a removable interface].

A method for mercury speciation analysis was developed by CE-ICP-MS with a Micro Mist nebulizer and a removable interface. Contamination of ICP-MS detector by the rising solution was avoided with the use of a removable interface during capillary rinsing. Baseline separation of methylmercury (MeHg) and inorganic mercury (Hg2+) was obtained by CE in a 45 cm x 75 microm i.d. fused-silica capillary at 22.5 kV, while a mixture of 30 mmol(-1) H3BO3 + 10% CH3OH (pH 8.7) acted as running electrolyte. The precisions (RSD, n=5) of migration time and peak area for the mercury species were in the ranges of 3.5%-3.8% and 2.3%-5.1%, respectively. The limits of detection (3sigma) of MeHg and Hg2+ were 47 and 48 microg x L(-1), respectively.

Boric Acids↗

A linear analog of atrial natriuretic peptide (ANP) discriminates guanylate cyclase-coupled ANP receptors from non-coupled receptors.

Atrial natriuretic peptide (ANP) contains a disulfide which is generally considered to be required for biological activity. A truncated linear ANP analog, des-Cys105,Cys121-ANP-(104-126) (referred to as analog I), that lacks the 2 cysteine residues of the parent peptide was synthesized. In competition binding studies using rabbit lung membranes, ANP-(103-126) and analog I displaced bound 125I-ANP-(103-126) from specific ANP binding sites 100 and 73%, respectively. The concentrations of ANP-(103-126) and analog I that produced 50% inhibition of radioligand binding to the membranes were 0.26 +/- 0.07 and 0.31 +/- 0.09 nM, respectively. Radioiodinated ANP-(103-126) and analog I were chemically cross-linked to binding sites on rabbit lung membranes, and the labeled membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. 125I-Analog I specifically labeled a 65,000-dalton protein and a 135,000-dalton protein which, under reducing conditions, dissociated into 65,000-dalton subunits. In contrast, 125I-ANP-(103-126) labeled specifically a nonreducible 135,000-dalton protein, in addition to the 65,000-dalton species and the reducible 135,000-dalton species. ANP-(103-126) (100 nM) stimulated rabbit lung particulate guanylate cyclase activity, whereas analog I, at the same concentration, had no effect on cyclic GMP production and did not antagonize the effect of ANP-(103-126). From these observations, we conclude that analog I is a selective ligand which binds to approximately 73% of the total ANP binding sites present in rabbit lung membranes. Unlike ANP-(103-126), analog I does not bind to the remaining 27% of the binding sites and does not activate guanylate cyclase. Binding to the cyclase-linked ANP receptor correlates with the specific labeling by 125I-ANP-(103-126) of the nonreducible 135,000-dalton membrane protein.

Animals↗