Guy Bordin

Recent developments in quantification methods for metallothionein

The metallothioneins (MT), a family of proteins with relatively low molecular weight (6-7 kDa), are characterised by the intrinsic presence of 20 cysteinyl groups in their structure, which confers unique metal binding properties to the molecule. Since MT are involved in biological roles, quantification of MT remains an important task. To date, a large number of determination methods have been developed. In this paper recent developments, from 1995 to the present, in methodology employed in quantification studies of total MT and MT polymorphism are described. Different fields were taken into consideration, such as (i) separation techniques and hyphenated systems, (ii) electrochemical methods, (iii) immunological methods and (iv) quantification of MT mRNA. The data presented are based on our own and published results. A brief overview of the use of metallothionein as a biomarker is included as a relevant example of the importance of MT quantification. Finally, general problems associated with determination and evaluation of obtained results within the above four topics are mentioned.

Identification and quantification of major bovine milk proteins by liquid chromatography

In the field of food quality, bovine milk products are of particular interest due to the social and economic importance of the dairy products market. However, the risk of fraudulent manipulation is high in this area, for instance, replacing milk powder by whey is very interesting from an economic point of view. Therefore, there is a need to have suitable analytical methods available for the determination of all milk components, which is currently not the case, especially for the main proteins. The detection of potential manipulations requires then a clear analytical characterisation of each type of bovine milk, what constitutes the goal of this work. The separation of the major milk proteinic components has been carried out by ion-pair reversed-phase HPLC with photodiode array detection, using a C4 column. The overall optimisation has been achieved using a statistical experimental design procedure. The identification of each protein was ascertained using retention times, peak area ratios and second derivative UV spectra. Quantification was based on calibration curves drawn using purified proteins. Major sources of uncertainty were identified and the full uncertainty budget was established. The procedure was initially developed using the skimmed milk powder certified reference material CRM 063R and then applied to various types of commercial milks as well as to raw milk. The method is able to separate and quantify the seven major proteins (K-casein, alphas2-casein, alphas1-casein, beta-casein, alpha-lactalbumin, beta-lactoglobulin B and beta-lactoglobulin A) in one run and also to provide precise determinations of the total protein concentration. These are important results towards the further development of a reference method for major proteins in milk. In addition, the use of a certified material reference is suggested in order to make comparisons of method performances possible.

Multiresidue determination of (fluoro)quinolone antibiotics in swine kidney using liquid chromatography–tandem mass spectrometry

New antibiotics were recently developed, among which are the (fluoro)quinolones. This paper presents an analytical method which allows the determination of 11 (fluoro)quinolones in swine kidneys: norfloxacin, ofloxacin, cinoxacin, oxolinic acid, nalidixic acid, flumequine, enrofloxacin, enoxacin, ciprofloxacin, danofloxacin and marbofloxacin. The procedure involves a rapid and efficient pre-treatment by solid-phase extraction (recoveries 83-98%), followed by the sensitive and selective determination of all compounds in a single run using LC-ESI-MS-MS. Multiple reaction monitoring (MRM) was used for selective detection of each (fluoro)quinolone. Quinine was selected as internal standard. The accuracy of the method, expressed as recovery, was between 89 and 109%; the repeatability had a maximum RSD lower than 15%. The limits of detection (LOD) were much lower than the respective Maximum Residue Limits (MRL)/4.

Validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of 11 (fluoro)quinolone antibiotics in swine kidney.

A LC-MS-MS method has been validated for the simultaneous quantification of 11 (fluoro)quinolone antibiotics at the maximum residue level (MRL) in swine kidney. The studied compounds were danofloxacine, cinoxacine, ciprofloxacine, noxacine, enrofloxacine, flumequine, marbofloxacine, nalidixic acid, norfloxacine, ofloxacine and oxolinic acid. The method involves solid-phase extraction of these compounds followed by LC-MS-MS analysis using an electrospray ionisation interface. Limits of quantification < or = 50 microg/kg could be obtained in swine kidney, much lower than every MRL. The validation is discussed. This work was carried out in order to support the European Union policy on consumer health

Trace enrichment of (fluoro)quinolone antibiotics in surface waters by solid-phase extraction and their determination by liquid chromatography–ultraviolet detection

A new and simple analytical methodology for the simultaneous analysis of acidic and zwitterionic (fluoro)quinolones in surface waters at trace concentration level is presented. The method is based on the preconcentration of these analytes by a solid-phase extraction procedure and their subsequent quantification by liquid chromatography using ultraviolet detection. The breakthrough volumes of the selected (fluoro)quinolones in four different sorbents--C18, styrenedivinylbenzene (SDB), C18-cation-exchange and SDB-cation-exchange--have been evaluated and varied between 25 and 150 ml depending on the antibiotic and the sorbent used. An exhaustive study of the influence of sample pH on the preconcentration step has been carried out in order to find a suitable procedure for extraction of acidic and zwitterionic FQs in one single step. Under optimum conditions, it was possible to percolate up to 250 ml of water solution onto both C18 and SDB-cation-exchange cartridges with quantitative recoveries for all the analytes tested. However, matrix components of the surface water samples analysed negatively affected the recoveries of the analytes in the SDB-cation-exchange cartridge and thus, C18 cartridges were finally selected for the analysis of the (fluoro)quinolones in lake and river water. The limits of detection achieved with this procedure varied between 8 and 20 ng l(-1) proving its suitability for the determination of the (fluoro)quinolones in water samples at a realistic environmental concentration level.

Development of new analytical methods for selenium speciation in selenium-enriched yeast material

A sequential extraction allowing the discrimination of water-soluble and non-soluble selenium fractions has been developed to evaluate the availability of selenium (Se) in an Se-enriched yeast candidate reference material. The fractionation of selenium-containing compounds in the extracts was achieved on preparative grade 200 Superdex 75 and columns. It showed that water-soluble selenium is present in several fractions with a large mass distribution. Low-molecular- (< or = 10,000) and high-molecular-mass selenocompounds (range 10,000-100,000) were considered separately for further experiments. The analytical approach for low-molecular-mass selenocompounds was based onanion-exchange HPLC with on-line inductively coupled plasma (ICP) MS for quantitative analysis. Selenocystine, selenomethionine, selenite and selenate were quantified in the fractions isolated in preparative chromatography. The study revealed the existence of various unidentified Se species in yeast material. The Se-containing proteins in the yeast material have been further separated and selenium quantified by the combination of gel electrophoresis and electrothermal vaporization-ICP-MS. This new approach allows the separation of the proteins with high resolution by sodium dodecylsulfate-polyacrylamide gel electrophoresis and the sensitive determination of selenium in the protein bands.

2-Dimensional gel electrophoresis technique for yeast selenium-containing proteins—sample preparation and MS approaches for processing 2-D gel protein spots

A novel integrated approach is proposed for the analysis of intact yeast selenium-containing proteins purified by a gel electrophoresis technique. The strategy consists of three components: high-resolution two-dimensional gel electrophoresis (2-DE) for proteins, laser ablation-inductively coupled plasma-dynamic reaction cell-mass spectrometry (LA-ICP-DRC-MS) for selenium detection and protein characterisation by electrospray mass spectrometry (ion-trap (IT MS) and time-of-flight (TOF MS) instruments). High-resolution 2-DE is the method of choice for protein purification before their characterisation by mass spectrometry techniques. SDS-PAGE which is a denaturating technique is applicable in this case as the selenium is covalently bound to the proteins. LA-ICP-DRC-MS was used, as optimised in previous work for several elements, which also showed that the limit of detection (absolute LOD of 0.5 pg Se per crater or 70 ng Se g−1 gel) was low enough to detect Se in the protein spots. Protein spots were excised from 2-D gels, destained and extracted. Information on the mass of Se-containing proteins were obtained from IT MS and TOF MS. Measurements of the proteins were made with two different MS instruments in order to validate the results and obtain the highest accuracy and resolution on the molecular masses. Up to ten selenium-containing proteins were characterised in terms of molecular mass in the range 9–20 kDa. This strategy has particular application to the possible establishment of a 2-D reference map (Se content and molecular masses) for Se-containing proteins in yeast.

Trace metals in the marine bivalve Macoma balthica in the Westerschelde estuary (The Netherlands). Part 1: Analysis of total copper, cadmium, zinc and iron concentrations-locational and seasonal variations

Abstract Total concentrations of copper, cadmium, zinc and iron in the marine bivalve Macoma balthica have been measured every 2 months for 1 year by atomic absorption (AAS) and emission (ICP) spectrometry. In the sample treatment, the freeze-drying and the digestion procedure were studied in detail. Although the range of fluctuations for the four metals are different, they show a similar temporal pattern, with higher concentrations in winter and lower in summer, ranging from 16.8 to 32.1 μg·g −1 for Cu, from 0.19 to 1.13 μg·g −1 for Cd, from 377 to 692 μg·g −1 for Zn and from 506 to 1955 μg·g −1 for Fe. The relationship between metal content and body weight varies from a highly significant straight line correlation (for copper and zinc) to no correlation at all (for cadmium). Thus, copper and zinc appear to be largely controlled by biological processes, while cadmium depends mostly on environmental levels, iron fluctuations being dependent on both factors.

Development of a capillary zone electrophoresis–electrospray ionisation tandem mass spectrometry method for the analysis of fluoroquinolone antibiotics

The applicability of a capillary zone electrophoresis-electrospray ionisation tandem mass spectrometric (CZE-ESI-MS-MS) method for the separation of nine fluoroquinolones was investigated. Method optimisation involved systematic trouble-shooting starting with the type and duration of capillary pre-washing and conditioning, the choice of both the CE run buffer, MS sheath liquid, CE run potential, ESI spray voltage, sheath gas flow-rate, MS capillary voltage and CE capillary and MS capillary temperatures. Another extremely important factor was found to be the degree to which the CE capillary protrudes into the ESI chamber as well as whether or not sheath gas and spray voltage are employed during the CE injection or not. The importance of the latter has, to our knowledge, not been addressed elsewhere. Nine fluoroquinolones have been separated and detected in a single run by this technique.

Separation of metallothionein isoforms with capillary zone electrophoresis using an uncoated capillary column effects of pH, temperature, voltage, buffer concentration and buffer composition

Abstract Metallothionein (MT) isoforms, sulphydryl-rich proteins with a high affinity for both essential and toxic trace metals, are readily separated by capillary zone electrophoresis (CZE) with an uncoated polyimide-clad fused-silica capillary using Tris-borate buffer. The metallothionein samples investigated were rabbit liver MT, rabbit liver MT-1, rabbit liver MT-2 and horse kidney MT. The effects of temperature, buffer pH, buffer concentration, buffer composition and running voltage on the separation efficiency were investigated. An improved separation efficiency compared with published methods using uncoated fused-silica capillaries was obtained, allowing the separation of peaks which previously co-migrated. The running conditions used were voltage 11 kV, temperature 20°C and buffer 110 mM Tris-110 mM borate (pH 6.90). Under these conditions, horse kidney MT exhibited five putative isoform peaks and rabbit liver MT nine or ten peaks. Interestingly, the experiments indicated that rabbit liver MT contains one major component which is not found in either isoform MT-1 or MT-2, possibly indicating that the improved separation efficiency made it possible to detect a new group or structure. Based on this initial evaluation, CZE with an uncoated fused-silica capillary using Tris-borate buffer appears to be a very useful method for the separation of metallothionein isoforms.