The degradation of L-tyrosine to phenol and benzoate in pig manure : The role of 4-hydroxy-benzoate.

The formation of odorous compounds in piggery wastes was investigated. Phenol and para-cresol are generally encountered in these typically anaerobic environments. They are produced from L-tyrosine by microbial metabolism. Phenol is further converted to benzoate via para-carboxylation. The biochemical pathways were studied by feeding manure with miscellaneous metabolites at concentration between 5 and 20 mM. Metabolites were analyzed by gas chromatography (GC) and high-performance liquid chromatography (HPLC). Experiments were carried out at room temperature. The degradation of L-tyrosine to phenol, benzoate, and para-cresol was confirmed. 4HPPyrA and 4HPAA are not intermediate compounds in phenol production. It was shown that phenol was converted to benzoate without any production of 4HBA. Other experiments showed that 4HBA was decarboxylated to phenol, but not dehydroxylated to benzoate.When phenol was added in presence of benzoate (5 mM each) or alone at higher concentrations (10 or 20 mM), transient small amounts of 4HBA were observed (about 0.02 mM). Our experiments show that 4HBA is not an intermediate metabolite in the conversion of phenol to benzoate. The decarboxylation of 4HBA to phenol is probably the last step of another degradation pathway. This reaction is proposed to have a weakly reversible property, explaining 4HBA production.

GC-MS determination of anabolic steroids after multi-immunoaffinity purification.

For many years, EC regulations have prohibited the use of anabolic agents in food-producing animals. Multiple screening methods have been published, but some lack specificity and some are difficult to apply when screening for unknowns in surveillance programmes. This paper presents a new and powerful technique, combining multiresidue immunoaffinity chromatography and GC-MS, for the simultaneous identification and semi-quantification of various anabolic steroids in urine and faeces samples of bovine origin. It should reduce the cost, time and effort of screening by limiting the number of tedious clean-up steps and analyses required. A preliminary extraction step is applied to the individual biological specimens: solid-phase extraction followed by enzymatic digestion in the case of urine samples and a single liquid extraction step for faeces. This step is followed by a first clean-up step involving both a solid-phase column and a rapid RP-HPLC separation. The individual biological fractions (urine or faeces) are further purified on a multiresidue immunoaffinity chromatographic gel (MIAC-steroids-CER) so as to decrease interferences due mainly to background signals. A final trimethylsilyl derivatization is followed by the analysis of the biological samples by a sensitive and specific GC-MS procedure.

Multi-laboratory study of the analysis and kinetics of stanozolol and its metabolites in treated calves.

The European Union banned the use of anabolic steroids for cattle fattening in 1988. Analytical techniques able to detect trace amounts of the parent drugs and their metabolites are mandatory for the control of abuse. Stanozolol (Stan) is an anabolic steroid that is often found in injection sites and cocktails. However, it has never been detected in tissues (kidney fat, meat) or excreta (urine, faeces) taken during regulatory inspection. The difference between the structure of Stan and the other steroids (a pyrazole ring fused to the androstane ring system) is probably the cause of this phenomenon. In the multi-laboratory study described here, veal calves were treated with intramuscular doses of Stan. In the excreta of these calves the presence, absence and/or concentration of Stan and of its major metabolites 16 beta-hydroxystanozolol and 3'-hydroxystanozolol were determined. For the determination of these analytes the different laboratories used different extraction and clean-up procedures and also evaluated different analytical techniques such as GC-MS (negative chemical ionization) and LC-MS-MS. The aim of this investigation was to explore which analyte should be validated for veterinary inspection purposes.