National Veterinary Institute, Department of Chemistry, SF-00101, Helsinki, Finland A liquid chromatographic method is described for the determination of biogenic amines found in dry sausages: tryptamine, phenylethylamine, putrescine, cadaverine, histamine, serotonin, tyramine, spermidine, and spermine. Amines were extracted with perchloric acid solution and derivatized with dansyl chloride. After derivatization, ammonia was added to remove an interfering peak near cadaverine. Liquid chromatographic separations were performed by using a Spherisorb ODS2 column and an ammonium acetate-acetonitrile gradient elution program. The limits of determination of the individual amines were 1-5 mg/kg. This method is also applicable to detection of amines in other food samples.
A microbiological method was developed for group level identification of antibiotics in incurred kidney and muscle samples from cattle and pigs. The method was composed of six test bacterium-plate growth medium combinations and the result was recorded as a profile of growth inhibition zones. The sample profiles were compared to two sets of references: one constructed with standard antibiotic solution profiles, and the other with these combined with profiles of microbiologically and chemically identified residues from incurred samples. The algorithm employed in profile comparison located the minimal sum of absolute pairwise differences over the tests, with the addition of a number of experimentally observed intra-test criteria. Chemical identification and quantitation of incurred residues was based on liquid chromatography. The method identified penicillin G as a penicillinase sensitive penicillin, enrofloxacin and ciprofloxacin belonging to fluoroquinolone group, and oxytetracycline belonging to tetracycline group. Each of these residues was microbiologically identified below the Maximum Residue Limit (MRL) for kidney tissue. Combining sample profiles with the standard reference data set did not enhance the resolution. Microbiological and chemical identification test results were in good agreement. The results of this study show that a microbiological identification method is a useful tool in preliminary characterisation of antibiotic residues in animal tissues.
Incurred penicillin G, oxytetracycline, enrofloxacin and ciprofloxacin residues in bovine and porcine muscle and kidney samples were analysed by microbiological and chemical methods, the former using Bacillus subtilis BGA as a test organism on agar media of pH 6, pH 7.2 and pH 8 and the latter using liquid chromatography. Least squares fits between the logarithms of the chemically obtained concentrations of the antimicrobials and the widths of the inhibition zones were used to estimate the inhibition zone widths corresponding to the maximum residue limit concentrations. In vitro sensitivities were determined with standard antimicrobial solutions. The results indicate that if B. subtilis BGA is used as a test organism, muscle tissue cannot be used as test material for screening oxytetracycline, enrofloxacin and ciprofloxacin residues on the plates used in this study, while penicillin G can be screened from muscle tissue. Because of the numerous factors causing or increasing variation in the analysis, the inhibition zone caused by a given antibiotic concentration cannot be predicted precisely. Therefore, a positive agar diffusion test needs to be confirmed chemically. If a kidney sample gives a positive agar diffusion test result, the antimicrobial concentration in a muscle sample from the same carcass should be checked chemically.
A method for the determination of malachite green and its major metabolite leucomalachite green in rainbow trout muscle is reported with limits of detection of 0.8 and 0.6 microg kg(-1), respectively. Residues were extracted with an acetonitrile-acetate buffer mixture and partitioned into methylene chloride. Clean-up of the extracts was performed on alumina and propylsulfonic acid solid-phase extraction columns using the automated solid-phase extraction system. The chromatographic separation of malachite green and leucomalachite green was achieved on a Chromspher 5B column using an acetonitrile-acetate buffer mobile phase. Leucomalachite green was converted to malachite green by post-column oxidation before spectrophotometric detection at 600 nm. The mean recoveries of malachite green and leucomalachite green from control rainbow trout muscle spiked at 2-50 microg kg(-1) were 65% (range 63.4-65.9%, relative standard deviation 3.9-16.1%) and 74% (range 58.3-82.6%, relative standard deviation 3.3-11.4%), respectively. Qualitative confirmation of the determined residues was performed with liquid chromatography coupled with tandem mass spectrometry detection with limits of detection of 2.5 and 1 microg kg(-1) for malachite green and leucomalachite green, respectively.
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