Evaluation of the illegal use of clenbuterol in Portuguese cattle farms from drinking water, urine, hair and feed samples.

The recent discovery of clenbuterol contamination in Portuguese food led to the specific inspection of 16 cattle farms for beta-agonists, involving the analysis of a total of 486 samples (78 feed, 106 drinking water, 168 urine and 134 hair). The samples were screened for the beta-agonists: bromobuterol, cimaterol, clenbuterol, clenpenterol, clenproperol, hydroxymethylclenbuterol, mapenterol, salbutamol and terbutaline. Only clenbuterol was found in all analyzed matrices and the most likely method of illegal administration to animals was through drinking water. Of all samples analysed, 14.15% of drinking water were found positive in the range 0.03-3.80 mg l(-1) clenbuterol. Inclusion of hair samples in the Portuguese plan for clenbuterol residue control in live animals is discussed.

Levels of ochratoxin A in serum from urban and rural Portuguese populations and estimation of exposure degree.

Urban and rural population exposure to ochratoxin A (OTA) in central zone of Portugal was investigated in three places: Coimbra, Verride and Ereira. The analytical method proposed for the determination of ochratoxin A involved extraction with chloroform-orthophosphoric acid, cleanup through an immunoaffinity column (IAC), high performance liquid chromatography (HPLC) with spectrofluorimetric detection (FD) for separation and identification of ochratoxin A, and confirmation with HPLC-FD after OTA methylation in serum. The limit of quantification of the proposed method was 0.1 microg/L for serum and 0.05 microg/L for blood. OTA recoveries in serum ranged from 70.3% to 115.3% for levels at 0.25 microg/L and 0.5 microg/L, respectively, with a within-day RSD between 8.0% and 16.2%. Ochratoxin A serum levels were evaluated in an hundred and four donors from Coimbra city, Verride, and Ereira. The study revealed a frequency of detection of 100%. The ratio of ochratoxin A level in serum to whole blood was 2.0+/-0.7. The overall concentrations range from 0.25 to 2.49 microg/L, 0.14 to 1.91 microg/L, and 0.19 to 0.96 microg/L, for samples of Verride, Ereira, and Coimbra, respectively. The mean concentration and standard deviation were 0.78+/-0.53 microg/L, 0.44+/-0.31 microg/L, and 0.42+/-0.18 microg/L for the same samples. A significant difference was found in Verride population (P-value=0.000). Levels of OTA are clearly higher in males from rural areas than in females. For all samples, a significant difference was found in Verride male population (P-value=0.014).

A comparative study of extraction apparatus in HPLC analysis of ochratoxin A in muscle.

Ochratoxin A (OTA) is a secondary fungal metabolite produced by several moulds, mainly by Aspergillus ochraceus and by Penicillium verrucosum, that occurs in meat products. The aim of this work was to optimize an efficient extraction procedure for the determination of OTA in muscle tissue in order to assess its occurrence in meat samples. Three different apparatus, a Waring blender, a switching apparatus, and an ultrasonic processor, were evaluated to verify the efficiency of extraction. The analytical methods proposed involve the extraction with chloroform-orthophosphoric acid, cleanup through an immunoaffinity column, high-performance liquid chromatography/fluorescence detection for separation and identification of OTA, and confirmation with liquid chromatography/FD after methylation of OTA in muscle tissue. The limit of quantification of the proposed method was 0.04 microg kg(-1). Recoveries of OTA, using switching apparatus, ranged from 90.3 to 103.2% for chicken muscle spiked at 2.4 and 0.48 microg kg(-1), respectively, with a within-day relative standard deviation of 17 and 15.3%. The proposed method was applied to 38 chicken, swine, and turkey muscle samples and the presence of OTA was confirmed in five samples. Finally, the estimated daily intake of OTA in this study was between 23 pg kg(-1) body weight per day for swine samples and 18 pg kg(-1) body weight per day for turkey samples.

Organochlorine pesticide residues in European sardine, horse mackerel and Atlantic mackerel from Portugal.

This paper reports the results for the surveillance of nine organochlorine pesticides (HCH isomers (alpha, beta, e, gamma), p,p'-DDD, p,p'-DDT, p,p'-DDE, p,p'-DDD, HCB and aldrin) in muscle of three fish species, European pilchard (Sardina pilchardus), Atlantic horse mackerel (Trachurus trachurus) and Atlantic mackerel (Scomber scombrus). Analytical methodology included n-hexane extraction, clean-up with 2% deactivated Florisil, and quantification with gas chromatography-electron capture detection (GC-ECD). The highest mean concentrations were found for p,p'-DDT in sardine and mackerel at levels of 30.1 and 109.9 microg kg(-1), respectively, and for p,p'-DDD in horse mackerel at 51.9 microg kg(-1). Three species had higher levels for S-DDT than S-HCH. The estimated daily intake of organochlorine pesticides in the three species showed that in sardine, the highest EDIs were found for aldrin, at 1.8 ng kg(-1) bw day(-1), which represents 1.8% of the acceptable daily intake (ADI), and for ss-HCH, at 4.0 ng kg(-1) bw day(-1), representing 0.4% of ADI. Lowest values were found for Atlantic mackerel. Statistical analysis to determine the differences in mean concentrations of pesticides between species, and any correlation between groups of residues related with each one of the species, was undertaken.

Validation of an analytical methodology for determination of oxytetracycline and tetracycline residues in honey by HPLC with fluorescence detection.

An analytical method for the determination of OTC and TC residues in honey was developed. Sample treatment involves an extraction in EDTA-McIlvaine buffer, followed by a solid-phase cleanup step. With regard to the cleanup procedure, different SPE cartridges were evaluated and the results presented. The method was validated according to the guidelines laid down by the 2002/657/EC European Decision parameters: decision limit (Cc alpha) and detection capability (CC beta) were 20 and 21 microg/Kg and 49 and 50 microg/Kg for OTC and TC, respectively, and recoveries of OTC and TC from spiked samples, at three fortification levels, were higher than 87% for both compounds. The analytical method was applied to 57 honey samples.

Survey of caffeine levels in retail beverages in Portugal.

The caffeine content of 85 retail beverage samples purchased from local supermarkets between 1995 and 2004 was determined. The potential intake of caffeine through the consumption of these beverages (but excluding coffee) was estimated for students of the University of Coimbra, Portugal. The caffeine content of the beverages ranged from 47.5 to 282.5 mg l(-1) for teas, from 20.1 to 47.2 mg l(-1) for tea extracts samples, and from 80.7 to 168.7 mg l(-1) for cola soft drinks. Caffeine was not completely absent from caffeine-free colas, and energy drinks had a far greater caffeine content than regular drinks, ranging from 21 to 2175 mg l(-1). Soft drinks were consumed by 72% of the individuals, although 14% of the survey participants did not drink any of the different types of the beverages studied. Contrary to expectations for this age group, no consumptions of energy drinks was reported. Daily caffeine intake was estimated to range from 4.7 to 200 mg day(-1), but with only 5% reporting a daily intake around 200 mg caffeine. Cola-type beverages were an important dietary source of caffeine for the population studied. Statistical differences in the caffeine intake between the male and female populations were found, with p = 0.014, being higher for the male population. Of the beverages studied, cola-type drinks showed statistical differences for the male population, p = 0.03, and tea showed statistical differences for female population p = 0.013, respectively.

Determination of organochlorine pesticide residues in honey from the central zone of Portugal and the Valencian community of Spain.

In this study nine organochlorine pesticide residues (alpha-, beta-, and gamma-hexachlorocyclohexane (HCH), hexachlorobenzene (HCB), aldrin, p,p'-DDE, p,p'-DDD, o,p'-DDT, and p,p'-DDT) in forty nine samples of honey collected from markets of Portugal and Spain during 2001 and 2002, respectively, were evaluated. For this evaluation, three analytical procedures were studied. The analytical procedure, based on LLE extraction with ethyl acetate followed by gas chromatography-electron-capture detection (GC-ECD) for quantification, and mass spectrometry (GC-MS) for confirmation, has been selected. Recoveries of spiked samples ranged from 68%, for beta-HCH, and 126% for p,p'-DDT, for fortification levels between 10 and 100 microg/kg, and 64%, for alpha-HCH, and 143% for gamma-HCH for fortification levels between 20 and 200 microg/kg. Limits of quantification, using GC-ECD, were from 0.01 and 0.10mg/kg, and limits of detection between 0.001 and 0.02 mg/kg. Fourteen Valencian samples were contaminated, containing residues of HCB or/and HCH isomers. The frequency of detection was 56% for Spanish samples. In Portugal, 23 samples were contaminated, what means 95.8%. In Spanish samples, concentrations range from nd to 0.03 mg/kg for HCB, and nd to 2.24 mg/kg for HCH-total. The mean concentration and standard deviation were 0.017+/-0.011 mg/kg for HCB, and 0.579+/-0.747 mg/kg for HCH-total, contributing the gamma isomer with the highest values. The samples from Portugal showed higher levels. Levels of HCB ranged from nd to 0.39 mg/kg. HCH-total ranged from nd to 4.86 mg/kg, and DDT-total from nd to 0.658 mg/kg. Mean concentration and standard deviation were 0.09+/-0.116 mg/kg for HCB, 1.357+/-1.30 mg/kg for HCH-total, and 0.143+/-0.193 mg/kg for DDT-total.