Wolfgang Buscher

Interaction of thimerosal with proteins—ethylmercury adduct formation of human serum albumin and β-lactoglobulin A

The interaction of thimerosal, an ethylmercury-containing bactericide and fungicide used as preservative in vaccines and other drugs, with free thiols in proteins has been investigated using gradient reversed phase liquid chromatography (LC) with inductively coupled plasma mass spectrometry (ICP-MS) and electrospray mass spectrometry (ESI-MS) detection. As model proteins, β-lactoglobulin A (18.4 kDa) from bovine milk and human serum albumin (66.5 kDa) have been used. Physiological conditions upon an intravenous injection of thimerosal-containing drugs were mimicked. The formation of ethylmercury–protein adducts was proved and the identification of the binding site of ethylmercury, a free thiol residue in the peptide T13 was achieved after tryptic digestion of β-lactoglobulin A.

Detoxification of mercury species—an in vitro study with antidotes in human whole blood

To investigate the effects of mercury species intoxication and to test the efficiency of different commonly applied antidotes, human whole blood and plasma surrogate samples were spiked with inorganic mercury (Hg2+) and methylmercury (MeHg+, CH3Hg+) prior to treatment with the antidotes 2,3-dimercaptopropan-1-ol (British Anti Lewisite), 2,3-dimercaptosuccinic acid (DMSA), and N-acetylcysteine (NAC). For mercury speciation analysis in these samples, liquid chromatography was coupled to either inductively coupled plasma mass spectrometry (ICP-MS) or electrospray ionisation time-of-flight mass spectrometry (ESI-TOF-MS). Adduct formation between mercury species and physiological thiols (cysteine and glutathione) was observed as well as the release of glutathione under treatment with the antidotes DMSA and NAC.

Liquid chromatography with complementary electrospray and inductively coupled plasma mass spectrometric detection of ferrocene-labelled peptides and proteins

Succinimidylferrocenyl propionate (SFP) is introduced as labelling agent for amino functions in peptides and proteins. The resulting derivatives are characterised by considerably lower polarity compared with the native analytes and can thus be well separated by means of reversed phase liquid chromatography (RP-LC). The reaction products are characterised by electrospray ionisation mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS). A further advantage of the method is a simple and straightforward derivatisation protocol. Different basic and acidic model proteins as lysozyme, ß-lactoglobulin A and insulin were derivatised using SFP. Furthermore, the first dual-labelling strategy of thiol and amino groups with ferrocene-based reagents is presented. Whereas the amino groups were derivatised with SFP, the thiol groups were functionalised by reaction with ferrocenecarboxylic acid(2-maleimidoyl)ethylamide. Again, LC/ESI-MS is a suitable tool to characterise the modified peptides and proteins.

In vitro study of thimerosal reactions in human whole blood and plasma surrogate samples

Because of its bactericidal and fungicidal properties, thimerosal is used as a preservative in drugs and vaccines and is thus deliberately injected into the human body. In aqueous environment, it decomposes into thiosalicylic acid and the ethylmercury cation. This organomercury fragment is a potent neurotoxin and is suspected to have similar toxicity and bioavailability like the methylmercury cation. In this work, human whole blood and physiological simulation solutions were incubated with thimerosal to investigate its behaviour and binding partners in the blood stream. Inductively coupled plasma with optical emission spectrometry (ICP-OES) was used for total mercury determination in different blood fractions, while liquid chromatography (LC) coupled to electrospray ionisation time-of-flight (ESI-TOF) and inductively coupled plasma-mass spectrometry (ICP-MS) provided information on the individual mercury species in plasma surrogate samples. Analogous behaviour of methylmercury and ethylmercury species in human blood was shown and an ethylmercury-glutathione adduct was identified.

New torch design for inductively coupled plasma optical emission spectrometry with minimised gas consumption

A new torch design for inductively coupled plasma optical emission spectrometry was developed. The torch was made of one quartz tube with a ball-shaped excitation zone. Under optimised working conditions of the 27.12 MHz Ar plasma with 1.1 kW RF power, the total argon gas flow could be reduced to only 0.6 L min−1 with external air cooling integrated in the fastening of the torch. Fundamental studies of selected element emissions, emission profiles, the effect of auxiliary plasma and sample carrier gas flow rates were carried out. The attainable excitation and rotational temperatures in the new torch were determined at different sample carrier gas flow rates. For 1.1 kW RF power and total gas flows between 50–800 mL min−1 they were found to be in the order of 6000–10 000 K for the excitation temperatures and 3700–4100 K for the rotational temperatures. The new torch was coupled with an ultrasonic nebuliser and detection limits between 0.03–2.9 μg L−1 could be determined for the elements Ba, Li, Mg, Ca, Cd, K and Al. The new torch delivered promising results that raise hopes of having a new generation of torches allowing low argon consumption.

Analysis of doped luminescent lanthanide fluoride nanoparticles by low gas flow inductively coupled plasma optical emission spectrometry

Low-flow inductively coupled plasma optical emission spectrometry is applied for the analysis of water-soluble LaF3nanocrystals doped with different lanthanide ions (Ce3+, Eu3+, Ho3+, Tb3+) for the first time. Colloidal solutions of citrate-stabilized nanoparticles (mean size 24 nm) were directly introduced into the plasma and no significant differences to digested particles were observed. The low-flow approach reduced the argon consumption of conventional instrumentation by 95% and provided limits of detection (LOD) for rare earth elements in the low microgram-per-liter range (Eu: 0.08 µg L−1, Ho: 0.18 µg L−1, La: 0.36 µg L−1, Tb: 0.56 µg L−1, Ce: 3.14 µg L−1). Relative standard deviations (RSDs) in the range of 1–2.2% with pneumatic sample introduction were obtained. The analytical performance of the low-flow torch was assessed by direct comparison with a conventional torch. Recoveries in the range of 97–104% were observed for most elements.

Correcting distortion in a monochromatic imaging spectrometer for application to elemental imaging by glow discharge-optical emission spectrometry

A method to correct for instrumentally introduced image distortion in a monochromatic imaging spectrometer (MIS) was developed, evaluated and applied to surface elemental imaging in glow discharge-optical emission spectrometry. The method is based on fixed-spatial-frequency images, in which calibration points are localized to characterize the distortion at a given wavelength. In the MIS, a linear increase in image width with wavelength was found in the horizontal dimension, whereas no significant image distortion in the vertical dimension was observed. The ratio between the horizontal and vertical image widths was 18 ± 2% at 400 nm and 46 ± 2% at 750 nm. Correction of distorted images was performed computationally by means of bilinear interpolation. The geometric error, in terms of residuals of the interpolation, was calculated for every pixel in the reconstructed image (512 × 512 array) and typically found to be between 0.12 and 0.18 pixel. The developed method was applied to elemental imaging by glow discharge—optical emission spectrometry as a proof of principle. A glow discharge cell suitable for large-format elemental imaging (7.29 cm-diameter sputtering area) was used for imaging of circular brass samples. Significant differences between vertical and horizontal emission profiles from distorted emission maps (e.g. Zn I 481.05 nm: 30% difference between vertical and horizontal FWHM) could be successfully corrected (4.8% difference after correction) by the method presented here.

Pressure waves generated in a Grimm-type pulsed glow discharge source and their influence on discharge parameters

Gas pressure waves in both argon and helium direct current (dc) pulsed glow discharge (PGD) in a Grimm-type source were found and studied with an inserted microphone. First and second harmonics of pressure vibrations were found to be in good agreement with the resonant frequency (2.7 kHz) of the discharge cell. Formation of the gas pressure wave takes place during about 150 µs in edges of PGD. Significant influence of pressure changes on the plasma parameters such as electrical current and optical emission was found. Possible mechanisms responsible for the generation of pressure waves are discussed. In future studies, the results presented here should be taken into account, e.g. for discharge optimization, diagnostics, plasma simulations and corresponding plasma-analytical setups.

A new ion source design for inductively coupled plasma mass spectrometry (ICP-MS)

Based on the static high sensitivity inductively coupled plasma (SHIP) introduced by Buscher et al. (W. Buscher, A. Klostermeier, C. Engelhard, S. Evers, M. Sperling, J. Anal. At. Spectrom., 2005, 20, 308-314), a new torch design for ICP mass spectrometry was developed. The SHIP-torch, including the external air cooling system, was modified in order to allow its application as an ion source in a conventional ICP mass spectrometer. While the torch geometry was adjusted for its use in connection with the sampling interface, the principal shape of the torch remained the same as in the recently developed SHIP-OES-system. The plasma discharge was operated at 0.65 kW rf power with a total plasma gas flow rate of 1.95 L min−1 and the mass spectrum was investigated. A standard pneumatic nebuliser was used as sample introduction system and the ion signals of a multi elemental standard solution were measured for different sample carrier gas flow rates from 0.2 to 1.4 L min−1. Limits of detection were obtained for a number of elements. These results were compared to those achieved with the conventional ICP-MS setting.

Low gas flow inductively coupled plasma optical emission spectrometry for the analysis of food samples after microwave digestion.

In this work, the recently introduced low flow inductively coupled plasma optical emission spectrometry (ICP-OES) with a total argon consumption below 0.7 L/min is applied for the first time to the field of food analysis. One goal is the investigation of the performance of this low flow plasma compared to a conventional ICP-OES system when non-aqueous samples with a certain matrix are introduced into the system. For this purpose, arsenic is determined in three different kinds of fish samples. In addition several nutrients (K, Na, Mg, Ca) and trace metals (Co, Cu, Mn, Cd, Pb, Zn, Fe, and Ni) are determined in honey samples (acacia) after microwave digestion. The precision of the measurements is characterized by relative standard deviations (RSD) and compared to the corresponding precision values achieved using the conventional Fassel-type torch of the ICP. To prove the accuracy of the low flow ICP-OES method, the obtained data from honey samples are validated by a conventional ICP-OES. For the measurements concerning arsenic in fish, the low flow ICP-OES values are validated by conventional Fassel-type ICP-OES. Furthermore, a certified reference material was investigated with the low gas flow setup. Limits of detection (LOD), according to the 3σ criterion, were determined to be in the low microgram per liter range for all analytes. Recovery rates in the range of 96-106% were observed for the determined trace metal elements. It was proven that the low gas flow ICP-OES leads to results that are comparable with those obtained with the Fassel-type torch for the analysis of food samples.