Maria Irene Noronha da Silveira

Food poisoning by clenbuterol in Portugal

This paper describes the occurrence of four cases of acute food poisoning, involving a total of 50 people, due to the ingestion of lamb and bovine meat containing residues of clenbuterol. Symptoms shown by the intoxicated people may be generally described as gross tremors of the extremities, tachycardia, nausea, headaches and dizziness. Analytical methodology developed for the determination of clenbuterol in meat, liver and blood samples is described. Procedures are described which should be followed when the described symptoms are evident in a group of people who have ingested contaminated meat, and particularly liver of ruminants.

Control of the illegal use of clenbuterol in bovine production

This study is based on a plan of collecting different matrices (hair, eye, muscle, liver and kidney) in order to define a strategy for the control of the illegal use of clenbuterol in bovine production. Of all matrices utilised, hair is recommended for the analytical control of clenbuterol in living animals, due to its being permanently available and easy to collect. The eye, or rather the retina, is the matrix which gives the most trustworthy result, after the animal slaughter, and the one that best helps in the determination of the illegal use of clenbuterol in a perspective of gradual food safety improvement.

Detection, accumulation, distribution, and depletion of furaltadone and nifursol residues in poultry muscle, liver, and gizzard.

Nitrofurans were broadly used as an extremely effective veterinary antibiotic especially in pig and poultry production farms. Because of fears of the carcinogenic effects on humans, the nitrofurans were banned from use in livestock production in many countries, including the European Union. The present study examines the accumulation, distribution, and depletion of furaltadone and nifursol and of their tissue-bound metabolites [3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) and 3,5-dinitro-salicylic acid hydrazine (DNSAH), respectively, in poultry edible tissues (muscle, liver, and gizzards) following administration to chickens of therapeutic and subtherapeutic concentrations of both compounds. Nitrofurans determination was performed by high-performance liquid chromatography-diode array detection and liquid chromatography-tandem mass spectrometry, respectively, for feeds and for poultry tissues. Furaltadone and nifursol, in very low concentrations, were found in samples of muscle, liver, and chickens gizzard collected from slaughtered animals after 5 weeks of treatment and no withdrawal time period. When a withdrawal time period of 3 weeks was respected, no detectable nitrofuran parent compounds was observed in all of the studied matrices. For AMOZ, concentrations of 270 μg/kg in meat, 80 μg/kg in liver, and 331 μg/kg in gizzard were determined after administration of a medicated feed with furaltadone (132 mg/kg), 3 weeks after withdrawal of treatment. For DNSAH, the concentration values obtained are much lower than those observed for AMOZ. For meat, liver, and gizzard, DNSAH concentrations of 2.5, 6.4, and 10.3 μg/kg, respectively, were determined, after administration of a medicated feed with nifursol (98 mg/kg), 3 weeks after withdrawal of treatment. The gizzard could be considered a selected matrix for nitrofuran residues evaluation in poultry, due to its capacity of retaining either nitrofuran parent compounds or metabolites in higher concentrations, regardless of the administered dose or of the respected withdrawal time period.

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 (α, β, e, γ), 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 µg kg−1, respectively, and for p,p′-DDD in horse mackerel at 51.9 µg kg−1. Three species had higher levels for Σ-DDT than Σ-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 β-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.

β2-adrenergic agonist residues : simultaneous methyl- and butylboronic derivatization for confirmatory analysis by gas chromatography-mass spectrometry

A derivatization procedure for confirmatory residue analysis of beta2-agonists is described. Methyl (MBA) and butyl (BBA) boronic acids are simultaneously used for the derivatization of tulobuterol, mabuterol, mapenterol, salbutamol, clenproperol, clenbuterol, clenpenterol and bromobuterol by GC-MS determination. A temperature of 55 degrees C during 60 min was selected as optimal temperature-time condition for simultaneous MBA and BBA beta2-agonists derivatization. It was also observed that stability of boronic derivatives was maintained at -20 degrees C over a period of four days. The proposed methodology was tested in urine and it could be applied for confirmatory residue analysis of clenbuterol-like compounds.

Determination of furaltadone and nifursol residues in poultry eggs by liquid chromatography-electrospray ionization tandem mass spectrometry.

The use of nitrofurans as veterinary drugs has been banned from intensive animal production in the European Union (EU) since 1993. The objective of the present study was to evaluate the accumulation and depletion of furaltadone and nifursol and their side-chain metabolites 5-methylmorpholino-3-amino-2-oxazolidinone (AMOZ) and 3,5-dinitrosalicylic acid hydrazide (DNSAH) in eggs after administration of therapeutic and subtherapeutic doses of the drugs to laying hens during three consecutive weeks. LC-MS/MS, with positive and negative electrospray ionization methods, was used for the determination of parent compounds and metabolites in yolk and egg white and was validated according to criteria established by Commission Decision 2002/657/EC. The decision limit (CCα) and the detection capability (CCβ) of the analytical methodology for metabolites were 0.1 and 0.5 μg/kg for AMOZ and 0.3 and 0.9 μg/kg for DNSAH, respectively. For the parent compounds, CCα and CCβ were 0.9 and 2.0 μg/kg for furaltadone and 1.3 and 3.1 μg/kg for nifursol, respectively. The data obtained show that the parent compounds are much less persistent than their side-chain metabolites in either yolk or egg white. Between the studied metabolites, AMOZ is the most persistent and could be detected in either yolk or egg white three weeks following withdrawal from treatment.

Clenbuterol Storage Stability in the Bovine Urine and Liver Samples Used for European Official Control in the Azores Islands (Portugal)

Clenbuterol is a well-known growth promoter, illegally used in farm animals, especially in cattle. Samples collected for the screening of beta(2)-agonist residues in Portuguese Azores Islands must travel through all the nine islands until they reach Azores Central Laboratory. If any suspicious sample is detected, it must be further transported to the National Reference Laboratory in Lisbon for confirmation. As a consequence of these circumstances, samples are submitted to different transport and storage times, as well as different temperature conditions and in some cases successive freezing and thawing cycles. As clenbuterol is the most detected beta(2)-agonist growth promoter in the Portuguese Residue Monitoring Plan, studies were conducted on the stability of this compound in incurred samples (bovine liver and urine) at +4, -20 and -60 degrees C over time. Samples kept at -20 degrees C were also analyzed over time after successive freezing and thawing cycles. The analyses of clenbuterol over time were performed by gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring (SIM). Clenbuterol in incurred urine and liver samples was significantly stable up to 20 weeks at -20 and -60 degrees C and after, at least, six consecutive freezings and thawings. At +4 degrees C, clenbuterol remained stable, at least until 12 weeks in urine and up to 20 weeks in liver.

Solid phase extraction (SPE) for multi-residue analysis of β2-AGONISTS in bovine urine

Solid phase extraction (SPE) for the treatment of bovine urine samples in the analysis of β2-agonist multi-residues by gas chromatography-mass spectrometry (GC-MS) was evaluated. Mabuterol, mapenterol, clenproperol, terbutaline, clenbuterol, salbutamol, clenpenterol, bromobuterol and hidroxi-metilclenbuterol (NA1141) were tested with five different mechanisms: adsorption, reverse phase, polar, ion exchange, and mixed phase, and eleven sorbents: diatomaceous earth, octadecyl (C18), octyl (C8), native silica (Si), diol (2OH), amine (NH2), trimethylaminopropyl (SAX), propylbenzenesulphonic (SCX), Bond Elut Certify® (C8+SCX), Clean Screen DAU® (CSDAU) and Multimode (C18+SCX+SAX). Recoveries of the nine β2-agonists from urine samples spiked at 20 ng mL−1 using metoprolol as internal standard were in ranges of 2.9% to 58.1%, of 12.0% to 60.7%, of 6.3% to 31.3%, of 3.3% to 56.8% and of 17.7% to 66.9%, respectively for adsorption, non polar, polar, ion exchange, and multiple extraction mechanisms. The coefficient...

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 β-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 β-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.

OPTIMIZATION OF DIPHASIC DIALYSIS PROCEDURE FOR CLENBUTEROL RESIDUES EXTRACTION IN BOVINE RETINA AND HAIR

Development of a simple, rapid, and accurate methodology for clenbuterol residue extraction in bovine hair and retina by diphasic dialysis was evaluated. After bovine hair and retina samples digestion, diphasic dyalisis was used as an extraction procedure using four organic solvents (dichloromethane, n-hexane, ethyl acetate, and diethyl ether) and five different buffers (acetate, phosphate, borate, carbonate, and citrate). The organic extract was evaporated to dryness under a nitrogen stream and the residue was derivatized with butylboronic acid. The results of GC-MS determination show that acetate buffer [sodium acetate 0.2M: acetic acid (95:5) pH ≈ 5.8] was the best choice for buffer dialysis, when dichloromethane and diethyl ether were elected as extraction organic diphasic dialysis solvents for hair and retina, respectively. Discussion of validation data show that diphasic dialysis could be a good, rapid, accurate, and economic extraction procedure to determine clenbuterol in bovine hair and retina samples.