Study of the degradation of butyltin compounds in surface water samples under different storage conditions using multiple isotope tracers and GC-MS/MS.

The degradation of butyltin compounds in surface water samples under different storage conditions has been studied. A triple spike solution, containing monobutyltin (MBT), dibutyltin (DBT) and tributyltin (TBT) labelled with a different tin isotope, was added to the sample to calculate the extent of the interconversion reactions among butyltin compounds. Real surface water samples (river water) were collected and stored in glass, polypropylene or polytetrafluoroethylene (PTFE) containers. The presence of light, addition of acetic acid, storage temperature (22, 4 or -18 °C), and the influence of a filtration step were evaluated. Moreover, Milli-Q water with and without the addition of a high concentration of humic acids was prepared in parallel and the results compared to those obtained from the real samples. The water samples were analysed by gas chromatography-tandem mass spectrometry (GC-MS/MS) in selected reaction monitoring (SRM) mode at two different storage times (2 weeks and 4 months after its preparation) to carry out both a short- and a long-term stability study. The lowest butyltin degradation was obtained when the samples were stored at -18 °C in the dark. Under these conditions, both TBT and DBT showed negligible dealkylation factors after 2 weeks. After 4 months, DBT dealkylation to MBT increased up to 19 % but TBT degradation was not observed.

Determination of ultratrace levels of tributyltin in waters by isotope dilution and gas chromatography coupled to tandem mass spectrometry.

The current EU legislation lays down the Environmental Quality Standards (EQS) of 45 priority substances in surface water bodies. In particular, the concentration of tributyltin (TBT) must not exceed 0.2ngL(-1) and analytical methodologies with a Limit of Quantification (LOQ) equal or below 0.06ngL(-1) are urged to be developed. This work presents a procedure for the determination of ultratrace levels of TBT in water samples by Isotope Dilution and GC-MS/MS operating in Selected Reaction Monitoring (SRM) mode which meets current EU requirements. The method requires the monitorization of five consecutive transitions (287>175 to 291>179) for the sensitive and selective detection of TBT. The measured isotopic distribution of TBT fragment ions was in agreement with the theoretical values computed by a polynomial expansion algorithm. The combined use of Tandem Mass Spectrometry, a sample volume of 250mL, the preconcentration of 1mL of organic phase to 30µL and an injection volume of 25µL by Programmed Temperature Vaporization provided a LOQ of 0.0426ngL(-1) for TBT (calculated as ten times the standard deviation of nine independent blanks). The recovery for TBT calculated in Milli-Q water at the EQS level was 106.3±4%. A similar procedure was also developed for the quantification of dibutyltin (DBT) and monobutyltin (MBT) in water samples showing satisfactory results. The method was finally implemented in a routine testing laboratory to demonstrate its applicability to real samples obtaining quantitative recoveries for TBT at the EQS level in mineral water, river water and seawater.

Species-specific isotope dilution analysis and isotope pattern deconvolution for butyltin compounds metabolism investigations.

A methodology for the study of the absorption and metabolism of butyltin compounds in laboratory animals using isotopically enriched species was developed. The method is based on the oral administration of 119Sn-labeled monobutyltin (MBT), 118Sn-labeled dibutyltin (DBT), and 117Sn-labeled tributyltin (TBT) to the animals and the measurement of both the concentration and isotopic composition of these compounds in the different tissues by GC-ICPMS. The degradation of butyltin compounds during their metabolism was computed using least-squares isotope pattern deconvolution, and their concentration was measured by reverse isotope dilution analysis using natural-abundance MBT, DBT, and TBT standards. Male Wistar rats were used as models to evaluate the proposed methodology. Preliminary toxicological results obtained with one rat indicate that TBT is highly absorbed (64.4%), and it is found in all organs with relatively high levels in stomach and intestines. The apparent absorption of DBT was 27.3% and was mainly found in liver, kidney, and intestines. However, a large proportion of the found DBT is formed from the degradation of TBT (approximately 40% of the found DBT in liver is degraded TBT). The apparent absorption of MBT was found to be 12.5%, and the originally administered MBT was mainly recovered in the feces. However, MBT was clearly detected in liver, kidney, stomach, intestines, and urine as degradation products of DBT and TBT. Although a significant variability from rat to rat is expected to be obtained, the analytical variability provided by this methodology is small enough to yield meaningful biological results. The results obtained demonstrate that the developed methodology is able to follow qualitatively, quantitatively, and simultaneously the specific metabolic pathways of different species of a given element.

Preparation of trout liver microsomes for iron speciation in P-450 enzymes by AE-FPLC with ICP-(ORS)MS detection.

Cytochromes P-450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiological and xenobiotic compounds in eukaryotes and prokaryotes. The multiplicity of this group of enzymes has been widely studied by chromatographic techniques, mainly high-performance liquid chromatography (HPLC). Because these enzymes are membrane-bound proteins, sample preparation for chromatographic separation of P-450 enzymes requires a solubilization step. The sample-preparation procedures are critical, because detergents affect not only the efficiency of protein solubilization but also their further chromatographic resolution. Trout liver microsomes have been taken here as a model sample to investigate iron speciation in cytochrome P-450. Trouts were treated intraperitoneally with beta-naphthoflavone, a potent inducer of some P-450 enzymes, and a microsomal suspension containing 7.4+/-0.1 nmol mL(-1) P-450 enzymes was obtained by ultracentrifugation. Lubrol PX was selected as detergent for solubilization, resulting in about 90% solubilization recovery. The solubilized cytochromes P-450 were further separated by AE-FPLC, with UV detection, or coupled to ICP-MS with an octapole reaction system, ICP-(ORS)MS (monitoring Fe signals at masses 54, 56, and 57). A sampling procedure and chromatographic conditions are developed and were successfully applied to iron speciation in trout liver P-450 enzymes. ICP-(ORS)MS detection of P-450 enzymes is Fe-specific and so will give accurate information on the prosthetic group of the protein, which can constitute an advantageous alternative to classical methods for detection of these hemoproteins.

Brown trout as a sentinel organism for organic pollution in the field using catalytic and immunochemical assays of cytochrome P-450 1A.

The measurement in some living organisms of adequate biomarkers (e.g. cytochrome P-450) to assess the organic pollution in freshwater ecosystems is well established. However, the sensitivity of this approach depends on the analytical measurement method employed and on the chosen living organism for the biomonitoring. Three analytical methods were compared for measuring cytochrome P-450 1A levels in wild brown trout (Salmo trutta): a catalytic one, based on measurement of the ethoxyresorufin-O-deethylase (EROD) activity, and two immunochemical methods, namely, enzyme-linked immunosorbent assay (ELISA) and western blotting. The P-450 1A levels in those animals from a river located in an industrialized area (Trubia River, Northern Spain) and also from individuals living in a low-contamination reference area have been studied. Significant differences (Mann-Whitney U-test, P < 0.01) between rivers were found (ELISA and EROD assays), with the values for Trubia river being similar to those observed in laboratory experiments with well known P-450 1A inducers. However, no significant differences were observed in terms of sex and age. Western blot analysis confirmed the presence of a single band of 56 kDa (corresponding with P-450 1 A protein) in microsomes of fish caught in the Trubia river. On the other hand, and associated with the chemical analysis of PAHs in the waters of both rivers by SPME-GC-MS, high levels of naphthalene (P-450 1A inducer) in the contaminated river were found. In brief, a wide difference between basal levels and P-450 1A induction levels could be detected in trout living in natural field conditions using both EROD activity assay and immunochemical methods. Therefore, brown trout could constitute a good sentinel organism to biomonitor the exposure to PAHs in rivers using P-450 1A measurements.