Capillary electrophoresis hyphenated to inductively coupled plasma-sector field-mass spectrometry for the detection of chromium species after incubation of chromium in simulated sweat

The presence of chromium in chromium-tanned leather represents a considerable health problem since it can lead to chronic allergic contact dermatitis. Apart from trivalent chromium (Cr(III)), which is used for tanning, leather often contains hexavalent chromium (Cr(VI)), resulting from the oxidation of Cr(III) during the tanning process. This study deals With the chromium compounds in simulated sweat when brought into contact with Cr(III) or Cr(VI) and with chromium-tanned leathers. A capillary electrophoresis (CE) method was developed, with inductively coupled plasma-sector field-mass spectrometry (ICP-SF-MS) for element-specific detection. Two different electrophoretic runs, applying once the positive and once the negative polarity mode, were necessary for the detection of positively and negatively charged chromium species. Although sometimes described in the literature, a pre-run derivatization of the chromium-species was not performed here to prevent species transformation. 50 mmol(.)L(-1) sodium phosphate at a pH of 2.5 was used as CE separation buffer and as make-up liquid for the CE-ICP-SF-MS interface. When applied to simulated sweat samples incubated with Cr(VI), this method showed that methionine is responsible for the reduction of Cr(VI) into Cr(III), which, at its turn, forms a complex with lactic acid. In the case of sweat plus Cr(III), the latter step was also seen. Applied to simulated sweat in contact with leather samples, the method developed showed the presence of the former species among a much more complex pattern

CE-ICP-SFMS for the detection of S and Zn in Aeromonas hydrophila Zn-beta-lactamase

Aeromonads are microorganisms that can cause both human and animal infections and is the known source of hospital-acquired infection. Some Aeromonas strains produce metallo-beta-lactamase enzymes, which are at the origin of beta-lactam resistance in members of this genus. The metallo-beta-lactamases are clinically relevant because of their ability to hydrolyse carbapenem antibiotics, and they also represent a relevant investigational model for studying molecular class B beta-lactamases because of their unique enzymological behaviour.(1,2) The Aeromonas hydrophila metallo-beta-lactamase contains Zn as enzymatic cofactor.(3-5) In this work, Zn bound to the metallo-beta-lactamase is separated from free Zn ions by capillary electrophoresis (CE) and the elements Zn and S are detected and quantified simultaneously by use of an inductively coupled plasma-sector field mass spectrometer (ICP-SFMS) operated at medium mass resolution. The goal of this investigation is to develop a method that allows a fast and accurate determination of the Zn/protein ratio. This ratio can be calculated from the Zn/S ratio. The quantification method is limited to proteins where the sulphur stoichiometry is known. The A. hydrophila Zn-beta-lactamase investigated in this work is known to contain 4 methionines and 1 cysteine. For proteins of which the primary structure is unknown, additional structural information is necessary. In this work A. hydrophila Zn-beta-lactamase is being used as proof of concept. Optimum conditions for CE, ICP-SFMS and their hyphenation were investigated. The use of the sodium salt of phytic acid as buffer additive is discussed into more detail

Capillary electrophoresis hyphenated to inductively coupled plasma-sector field-mass spectrometry for the stoichiometric determination of Zn bound to Aeromonas hydrophila Zn beta-lactamase

A method was developed for the determination of the stoichiometric Zn/protein ratio for the Aeromonas hydrophila metallo beta-lactamase AE036. Capillary electrophoresis (CE) was used to separate free Zn ions from the Zn bound to the metalloprotein, the only Zn-species which is biochemically relevant. An inductively coupled plasma-sector field-mass spectrometer (ICP-SF-MS), operated at a mass resolution of 3000, was used as an element-specific detector. If the primary structure of the protein and thus the number of sulfur-containing amino acids is known, as is the case for Aeromonas hydrophila Zn beta-lactamase, monitoring of both Zn and S with ICP-SF-MS permits the Zn/protein ratio to be determined. The method was optimised in order to obtain short migration times (less than 10 min) for the metallo beta-lactamase and for standards with bare fused silica capillaries. After optimisation, a 75 mmol L-1 bis-Tris background electrolyte buffered at a pH of 6.75 was used for the separation, a voltage of 30 kV was applied over a capillary with a total length of approximately 70 cm and the sample was introduced hydrodynamically at 1000 mbar s. Quantitative results (stoichiometric Zn/protein ratio of 1 and 2, before and after saturation of the enzyme with Zn, respectively) were obtained using external calibration with albumin as a S standard and ZnCl2 as a Zn standard. The method was also demonstrated to be precise with < 5% RSD on the Zn/protein ratio for n=3. An absolute limit of detection of 8 ng of A. hydrophila Zn beta-lactamase (calculated on the basis of sulfur) was obtained under these conditions