PCBs and PCDD/FS in fish and fish products and their impact on the human body burden in Belgium.

The concentrations of marker PCBs (28, 52, 101, 118, 138, 153, 180) in fish have been assessed with GC-MS: an average concentration of 540 ng-PCB g(-1) fat (5.02 ng-PCB g(-1) wet weight) was observed. The average concentration of PCDD/Fs, assessed with the CALUX bioassay, amounted to 64 pg-CALUX-TEQ g(-1) fat (0.58 pg-CALUX-TEQ g(-1) wet weight) and that of PCDD/Fs + dioxin-like PCBs amounted to 131 pg-CALUX-TEQ g(-1) fat (1.18 pg-CALUX-TEQ g(-1) wet weight). Results of the PCB congeners analyses show that PCB-153 is the most abundant congener in almost all samples, with also main contributions of the 138- and 180-congeners. For some species such as the sand sole and lemon sole, a fairly constant PCB content, independent of the fat percentage, was observed. For a second group of species such as whelks, cod, and whiting, a positive correlation was observed between their PCB concentration (ng g(-1) fat) and their % of fat. The relationship between marker PCBs and PCDD/Fs concentrations, when plotted on a log scale, fits a straight line (correlation coefficient r = 0.83). With our results on fish and literature data for other food products, intake of marker PCBs and PCDD/Fs could be calculated for the adult population in Belgium (19-60 years old). The Total Daily Intake (TDI) of marker PCBs (ng-PCB day(-1)) ranges between 1690 and 2210. The TDI of PCDD/Fs (pg-CALUX day(-1)) ranges between 80.5 and 122, that of PCDD/Fs + dioxin-like PCBs amounts to 151. When PCDD/Fs in fish are assessed with GC-HRMS, the TDI can be lower. The relative importance of fish regarding marker PCB intake amounts to 15-19%, while regarding PCDD/Fs intake it amounts to 34-51%. Using TDI, the body burden evolution of marker PCBs and PCDD/Fs, with age has been calculated.

Calculation of the decision limit (CCalpha) and the detection capability (CCbeta) for banned substances: the imperfect marriage between the quantitative and the qualitative criteria.

Initially in the Decision 2002/657/EC the criteria for the calculation of the decision limit (CCalpha) and the detection capability (CCbeta) have been estimated as purely quantitative (alpha-error is 1% and beta-error is 5%). In 2004, the European Commission has issued a document to provide guidance for the interpretation of the 2002/657/EC. In this document it is mentioned that also qualitative criteria should be fulfilled. Therefore, the calculated CCalpha and CCbeta must be verified by using fortified samples. The method should be able to detect/identify the target component in 50% of the cases at CCalpha and in 95% of the cases at CCbeta. Analytical methods for the analysis of nitroimidazoles, nitrofurans and corticosteroids with LC-MS/MS have been validated by fortifying blank samples below and above the MRPL. CCalpha and CCbeta were calculated using the ISO 11843 approach. In addition, the frequency of methodical compliance for the qualitative criteria was determined at each concentration level. It was observed that at the calculated CCalpha and CCbeta levels the qualitative criteria were not fulfilled. It was concluded that the detection capability of the analytical method should be calculated by using decreasing fortification levels at and below the MRPL. A protocol validating methods for banned substances by limiting the number of samples is presented and the qualitative criteria for the assessment of CCalpha and CCbeta were verified based on the same set of data without the need of performing additional validation experiments.

A downscaled multi-residue strategy for detection of anabolic steroids in bovine urine using gas chromatography tandem mass spectrometry (GC-MS3).

Within the scope of the European Community member states' residue monitoring plan, illicit administration of anabolic steroids is monitored at slaughterhouse level as well as on living animals. At farm level, urine is one of the target matrices to detect possible abuse of anabolic steroid growth promoters. Optimisation of the routinely applied analysis method resulted in a procedure for which high performance liquid chromatographic (HPLC) fractionation prior to GC-MS(n) analysis was no longer required. Analytical results could be obtained within 1 day and only 5 mL urine was needed to carry out the screening procedure. Using the downscaled methodology, all validation criteria described in the European Commission document 2002/657/EC could be fulfilled, and the minimum required performance limits (MRPLs) established for anabolic steroids in urine, could be achieved. A higher GC-MS technique's specificity was achieved by detecting the steroids using GC-MS3. Nevertheless, it was decided to screen routinely sampled urine with GC-MS2 whereas GC-MS3 was applied to confirm the presence of anabolic steroid residues in suspected sample extracts.

Interlaboratory study based on a one-plate screening method for the detection of antibiotic residues in bovine kidney tissue.

A microbiological test based on one-plate, seeded with Bacillus subtilis BGA, is frequently used in Belgium as a screening method to monitor antibiotic residues in kidney tissue of slaughter animals. The procedure as described in the National Legislation recommends test agar pH 7.2 from Merck. Since this type of agar is no more commercially available, two kinds of agar medium are proposed on the market: niertest agar from Biotrading and Standard II nutrient agar from Merck. Niertest agar, which is actually used by a few laboratories, is a ready-to-use medium manufactured from test agar pH 7.2 from Merck. Its composition is identical to those of test agar pH 7.2. Standard II nutrient agar is the second alternative culture medium which is used by a higher number of laboratories. The equivalence of the standard II nutrient agar medium to both identical culture media (test agar pH 7.2 and niertest agar) was evaluated during a national collaborative trial. Results of this study as well as the experimental procedure are presented.

Open digestion under reflux for the determination of total arsenic in seafood by inductively coupled plasma atomic emission spectrometry with hydride generation.

A method for the determination of arsenic (As) in seafood by inductively coupled plasma atomic emission spectrometry with continuous hydride generation is described. Several analytical parameters have been investigated and optimised. The analytical features of the method (recovery, precision, accuracy and limit of detection) were calculated. Practical detection limit of 3.6mug/kg fresh weight for As has been reached. The precision of the method expressed as relative standard deviation (R.S.D.) was in the range of 2.7-3.7% and the recovery percentage ranged from 98.4 to 101.8%. The reliability of the developed method was checked by analysing several certified reference materials. A complete mineralization was obtained for arsenobetaine (AsB) containing reference material with a mixture of nitric and sulphuric acids followed by adding hydrogen peroxide in an open digestion system. This method can be applied to routine analysis without any risks of interferences.

Multi-residue analysis of tranquillizers in meat: confirmatory assays using mass spectrometry.

A rapid and sensitive multi-residue method was developed to attempt to confirm the presence of the beta-blocker carazolol and the tranquillizers acepromazine, azaperone, chlorpromazine, propionylpromazine and xylazine in pig muscle tissues. The procedure involves determination by liquid chromatography coupled with tandem mass spectrometry. The liquid chromatographic separation was performed on a Symmetry C18 column with gradient elution. A mixture of aqueous buffer, containing 0.01% m/v trifluoroacetic acid (pH 3.5), and acetonitrile at a flow rate of 0.4 ml min-1 was used as the mobile phase. The abundant parent ions [M+ H+] produced by positive electrospray ionisation were selected for collisional dissociation with argon. Fragment ions were recorded with daughter ion scan and multiple reaction monitoring. The analytes were identified unambiguously by assessing retention times and diagnostic ions in meat samples spiked from 50 micrograms kg-1 [maximum residue limit (MRL) for azaperone and azaperol] to 5 micrograms kg-1 (MRL for carazolol).

A one-plate microbiological screening test for antibiotic residue testing in kidney tissue and meat: an alternative to the EEC four-plate method?

A one-plate screening method is described for the microbiological detection of antibiotic residues by growth inhibition of Bacillus subtilis in agar medium, at pH 7, which contains trimethoprim for better detection of sulphonamides. beta-Glucuronidase is added to the samples to enable the detection of chloramphenicol residues. The sensitivity was determined for 16 antimicrobial substances frequently used in animal husbandry. A comparison was made with the sensitivity of the EEC four-plate test and existing maximum residue levels. The method has been used in Belgium for 5 years for the monitoring of antimicrobial residues in kidney tissue and meat of slaughter animals.

Immunoaffinity-chromatography purification of salbutamol in liver and HPLC-fluorometric detection at trace residue level.

A method combining immunoaffinity-chromatography (IAC) and high-performance liquid chromatography (HPLC) for the analysis of Salbutamol in liver with a low quantification limit of 1 micrograms/kg has been developed. Salbutamol was extracted with 0.01 mol/L HCl and purified by IAC. The samples were analysed on a liquid chromatograph fitted with a C18 mu-Bondapak column. A fluorometer was used for the detection of salbutamol. Recoveries of 67-80% could be obtained.

Determination of levamisole and thiabendazole in meat by HPLC and photodiode array detection.

An HPLC method for the analysis of levamisole and thiabendazole has been developed with recoveries varying over 63-75%. The two anthelmintics are extracted from meat with ethyl acetate, purified by liquid/liquid extraction and analyzed quantitatively on a mu Bondapak C18 column. The optimum detection is achieved by means of a photodiode array detector at 240 nm for levamisole and at 300 nm for thiabendazole. The detection limits for both compounds in meat are 25 micrograms/kg and 5 micrograms/kg, respectively.

Salbutamol identification in liver and urine by high-performance thin-layer chromatography and densitometry.

This paper describes a rapid method for the identification of salbutamol in liver and urine. Salbutamol is extracted from liver with an acid solution, purified on Baker columns and eluted with methanol. After derivatization, salbutamol is detected on HPTLC plates as a blue spot. Urine samples are directly purified on the C18 columns and then the same procedure is followed as for the liver samples. Using this screening method, salbutamol can be semi-quantitatively determined at the micrograms/kg level.

Determination of thiabendazole residues in meat by HPLC using ultraviolet and fluorometric detection.

An HPLC method is described for the residue analysis of thiabendazole in meat. The recovery varies from 62 to 75%. Thiabendazole is extracted from the tissue using 3 mol HCl, eluted from the Extrelut-20 column with dichloromethane and then injected onto a C18 column. The optimum conditions for detection are described using ultraviolet and fluorescence spectroscopy. The sensitivity is such that thiabendazole can be determined at a level of 5 micrograms/kg meat. The absolute detection limit with fluorometry is 100 pg.

Cimaterol and clenbuterol residue analysis by HPLC-HPTLC in liver.

A method has been developed for the analysis of cimaterol and clenbuterol residues in liver, with detection limits of 0.25 micrograms/kg and 0.5 micrograms/kg, respectively. The recovery varied from 55% to 60%. After extraction, a clean-up procedure with Baker-spe C-18 columns was performed. The two chemical compounds of interest were eluted with methanol. Cimaterol and clenbuterol were quantitatively determined by high-performance liquid chromatography (HPLC) using an RP-Select B (5 microns) column and a post-column reaction procedure. The positive results were confirmed by high-performance thin-layer chromatography (HPTLC) as this technique reaches the same level of sensitivity as the HPLC method.

Clenbuterol residue analysis by HPLC-HPTLC in urine and animal tissues.

A method for clenbuterol residue analysis in urine and animal tissues has been developed. The detection limits are 0.25 micrograms/l and 0.5 micrograms/kg, respectively. The recovery in urine varies from 85% to 90% and in animal tissues from 70% to 74%. The beta 2-agonist was liberated from the tissues by an enzymatic digestion, purified on Chem Elut columns using alkaline conditions and extracted with 0.01 mol/l HCl. Clenbuterol was quantified by high-performance liquid chromatography (HPLC) on a RP-8 column and a post-column reaction procedure. High-performance thin-layer chromatography (HPTLC) was performed on silica gel 60 plates and clenbuterol visualized by means of the modified Ehrlich's TLC spray reagent. Since this method is sensitive, as is HPLC, it was used to obtain a confirmation and to exclude false positive results.

A two-dimensional high-performance thin-layer chromatographic screening method for sulphonamides in animal tissues.

A procedure for the identification of sulphonamides in edible animal tissues by two-dimensional high-performance thin-layer chromatography is described. Sixteen sulphonamides can be detected at an absolute level of 10 ng. The absolute detection limit of the sulphonamide standards is 1 ng. After extraction of the sulphonamides with chloroform/acetone, the acidified extract is concentrated and purified using a cation-exchange solid phase extraction column. The column is treated with ammonia vapour and the sulphonamides are then eluted with methanol. Blank samples of edible animal tissues spiked with sulphonamides (frequently used in Belgium) result in very good separation.